Message from the President

The National Institute of Agrobiological Sciences (NIAS) conducts life science research on plants, animals and insects to facilitate the development of Japan’s domestic agricultural industry. The institute further seeks to resolve 21st Century global issues, including sustainable food production and supply and environmental issues, in order to play a leading role in social and economic transformation and to help create the new bioindustries that are expected to foster major economic growth. NIAS actively cooperates in biotechnology research and genome analysis with universities, private corporations and other independent administrative institutions for extensive and efficient promotion of advanced studies. Examples of major achievements accomplished by NIAS this year include: (A) complete sequencing of the rice genome and isolation and functional analysis of plant genes essential to the symbiotic nitrogen fixation system, representing the area of life science research based on genome biology, etc., (B) development of a novel method for producing coenzyme Q10, representing the field of innovative technology development for dynamic development of agriculture, forestry and fisheries, (C) a novel porous three-dimensional structure using silk fibroin, representing research aimed at creation of novel industries, (D) creation of a cloned pig that expresses the green fluorescent protein (GFP) through effective selection of transformed cells (development of model pig for organ transplantation), representing basic technology supporting the biotechnology field, and (E) establishment of cultivated rice core collection, representing the field of collection, evaluation, storage, propagation, distribution and information management of biological resources. We shall continue to strive to satisfy society’s expectations by designing and conducting advanced research in the life sciences field involving plants, animals and insects. We look forward to your continued support and understanding and welcome your frank comments on the NIAS research activities introduced in this annual report.

Teruo Ishige President National Institute of Agrobiological Sciences

- 1 - Annual Report 2005

Contents

Message from the President ...... 1 Organization ...... 4

Topics of Research in This Year

Genome and Biodiversity Research Division ・Construction of EST database covering 80% of silkworm genes and its EST microarray...... 5

Insect and Animal Sciences Division ・Novel method for production of transgenic cloned pigs: Results of electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin...... 7 ・Developmental characteristics of transgenic silkworm with over-expression of juvenile hormone esterase...... 9 ・Different physiological properties in a pool of mandibular closer motor neurons in insects...... 10 ・Visual and chemical phytomimesis in giant geometer...... 12 ・Recombinant production of modified termite cellulases in E. coli...... 14 ・Mode of action by which entomopoxvirus spindles enhance nucleopolyhedrovirus infection...... 16 ・Development of useable material from Hornet silk...... 18 ・A new process to form a porous 3D structure from silk fibroin...... 20 ・Development of the middle silk gland specific gene expression system for the production of recombinant proteins in the transgenic silkworm...... 22 ・Preparation of elastic silk sericin hydrogel from the cocoons of SERICIN HOPE silkworm...... 24 ・Breeding of the silkworm race “SERICIN FLAVO” which simultaneously secretes sericin with flavonol...... 26

Plant Science Division ・The completion of a high-quality rice genome sequence - a major milestone in cereal genomics...... 28 ・Production of coenzyme Q10 enriched rice seeds by engineering of isoprenoid side chain...... 29 ・A rice core collection selected based on DNA polymorphism...... 30 ・A new floral switch gene revealed a novel photoperiodic flowering pathway for short-day promotion in rice...... 32 ・Calcium-signal transduction cascade for cold tolerance and elongation in rice leaf sheath...... 35 ・X-ray crystallographic analysis of Irpex lacteus aspartic proteinase...... 37 ・Structure Genomics Studies of Rice Proteins...... 39 ・Cloning and characterization of a plastid localized ion channel gene crutial for nodule formation in Lotus japonicus...... 41 ・Improvement in yield characteristics of transgenic potato with a maize SPS gene...... 43 ・Development of transgenic rice seeds accumulating mGLP-1 that stimulates insulin secretion....45 ・Analysis of Mutations from Pollen Irradiation...... 47 ・New apple cultivar‘Houiku Indo’，a mutant resistant to alternaria blotch disease by gamma-ray irradiation...... 50

- 2 - Research Activities

Genome and Biodiversity Research Division ・Genome Research Department ...... 54 ・Genetic Diversity Department ...... 63 ・Genebank ...... 72

Insect and Animal Sciences Division ・Developmental Biology Department ...... 81 ・Molecular Biology and Immunology Department ...... 88 ・Physiology and Genetic Regulation Department ...... 92 ・Insect Genetics and Evolution Department ...... 99 ・Insect Biomaterial and Technology Department ...... 106 ・Insect Biotechnology and Sericology Department ...... 112

Plant Science Division ・Molecular Genetics Department ...... 121 ・Biochemistry Department ...... 130 ・Plant Physiology Department ...... 135 ・Plant Biotechnology Department ...... 143 ・Institute of Radiation Breeding ...... 152

List of Publication ・Original papers ...... 158 （Author Index）...... 180 ・Review ...... 185

International Meetings and Foreign Visitors ...... 188 Executive Members and Research Staff Members ...... 192 Members of NIAS Evaluation Comittee ...... 200

Financial overview ...... 201 Location and How to access ...... 202

- 3 - Organization

President Insect Nutrition and Matabolism Laboratory Vice President（Insect and Animal Sciences) Insect Neurobiology Laboratory Vice President(Plant Sciences) Insect Behavior Laboratory Auditor Animal Gene Function Laboratory Department of Research Planning and Coordination Animal Cell Biology Laboratory Research Planning Division Animal Neurophysiolory Laboratory Research Evaluation Division Animal Neuroendocrinnolory Laboratory Research Coordination Section Insect Genetics and Evolution Department Office for GMO Research and Development Insect-Plant Interactions Laboratory Technology Transfer Section Natural Enemies Laboratory Administration Section Symbiosis Laboratory Information and Public Relation Section Insect Patholory Laboratory Field Management Section Insect Genetics Laboratory Genome Resource Center Insect Molecular Evolution Laboratory QTL Genomics Research Center Insect Biomaterial and Technology Department Center for GMO Research and Information Biopolymer Characterization Laboratory Insect Gene Function Research Center Biomaterial Development Laboratory Department of Administration Biomimetic Laboratory General Affairs Section Insect Products Utilization Laboratory Accounting Section Insect Biotechnology and Sericology Department Facility Management Section Insect Cell Engineering Laboratory Insect Gene Engineering Laboratory Genome and Biodiversity Research Division Mass Production System Laboratory Director of Genome and Biodiversity Research Division New Silk Materials Laboratory Genome Research Department Sericultural Science Laboratory Plant Genome Laboratory Animal Genome Laboratory Plant Science Division Insect Genome Laboratory Molecular Genetics Department Bioinformatics Laboratry Functional Genomics Laboratory DNA Bank Applied Genomics Laboratory Genetic Diversity Department Epigenetics Laboratory Molecular Biodiversity Laboratory Gene Expression Laboratory Biosystematics Laboratory Gene Regulation Laboratory Evolutionary Dynamics Laboratory Biochemistry Department Germ Cell Conservation Laboratory Crystallography Laboratory Applied Microbiology Laboratory Biophysics Laboratory Adaptation Systems Laboratory Glycobiology Laboratory Biometrics Laboratory Membrane Biology Laboratory Genebank Plant Physiology Department Plant Genetic Resources Labolatory Photosynthesis Laboratory Microorganism Genetic Resources Laboratory Carbon Metabolism Laboratory Animal Genetic Resources Laboratory Development Biology Laboratory Genetic Resources Management Section Environmental Physiology Laboratory Disease Physiology Laboratory Insect and Animal Sciences Division Nitrogen Fixation Laboratory Developmental Biology Department Plant Biotechnology Department Developmental Mechanisms Laboratory Gene Design Laboratory Developmental and Differentiation Laboratory Plant Gene Engineering Laboratory Animal Genetic Engineering Laboratory Plant Cell Engineering Laboratory Embryonic Technology Laboratory Molecular Breeding Laboratory Insect Growth Regulation Laboratory Biosystems Laboratory Reproductive Biology and Technology Laboratory Institute of Radiation Breeding Molecular Biology and Immunolory Laboratory Mutation Genetics Laboratory Molecular Immunolory Laboratory Radiation Technology Laboratory Innate Immunity Laboratory Mutation Breeding Laboratory Experimental Animals Laboratory Physiology and Genetic Regulation Department Insect Life-Cycles and Physiology Laboratory

- 4 - Topics of Research in This Year おおおおおおおお Construction of EST database covering 80% of silkworm genes and its EST microarray

Kazuei Mita, Kimiko Yamamoto, Keiko Kadono-Okuda Genome Research Department

TobuildafoundationforthecompletegenomeanalysisofBombyx mori,wehave constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains more than 85,000 ESTs from 53 cDNA libraries derived from 19 different tissues, which are grouped into 17,615 nonredundant ESTs. Since the total number of Bombyx genes is estimated to be roughly 20,000, the present EST database will cover more than 80% of Bombyx genes. The comparison with FlyBase (Drosophila genes) shown in Table indicates the similar estimation. All sequenced ESTs are compiled into the

Table Silkworm ESTs categorized by gene ontology terms.

Bombyx EST database, named KAIKObase, which can be accessed at http://sgp.dna.affrc.go.jp. We have constructed EST microarrays containing 6,000 nonredundant ESTs for many functional studies as well as for genome analysis as a major application of EST database. We designed and synthesized 6,000 specific primers located approximately 500 base pairs

- 5 - downstream from the 5’ end of each cDNA to remove repetitive sequences from DNAs to be used for the microarray. 6,000 DNAs were amplified by PCR using specific primers, followed by spotting on glass slides (Fig. 2). EST microarray technology was successfully used to

Fig.2 Outline of 6000 EST microarray construction identify and isolate ecdysone-responsive genes, and to detect the changes of gene expression in wing discs during metamorphosis as a model to describe the gene cascade triggered by ecdysteroid hormone.

Fig. 1 Bombyx EST database, KAIKObase

- 6 - Topics of Research in This Year いいいいいいい Novel method for production of transgenic cloned pigs: Results of electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin

Satoshi Watanabe, Shun-ichi Suzuki, Daiichiro Fuchimoto, Takashi Nagai and Akira Onishi Developmental Biology Department

Itwaswidelyknownthattheviabilityandthenormalityofdonorcellsweremost important for the efficiency of the producing somatic cloned animals. Therefore, it was also known that we should avoid culturing donor cells for a long period to keep cellular condition. Because, the long term culture cause cellular aging and chromosomal abnormality and greatly reduce the efficiency of production of somatic cloned animals. However, for the production of transgenic cloned animal, we have to culture the donor cells for long period for the gene transfer and the subsequent selection of the recombinant cells. In general, it takes more than 14 days for the recombinant selection with protein synthesis inhibiting antibiotics G418, and theefficiencyoftheproductionofclonedanimalsisquitelow.Fromthesephenomena,we hypothesized that shortening of the donor cells for selection of recombinants might improve the efficiency of production of transgenic cloned animals. To confirm this, we decided to introduce transgene into the somatic cells freshly obtained from fetuses and to use puromycin as a selection drug to obtain the recombinant cells for shortening the culture period of the donor cells. Puromycin is an effective inhibitor of protein synthesis and puromycin N-acetyl transferase gene (pac), whose gene product catalyzes antibiotic puromycin has been widely used as a dominant selection marker in ES cell-mediated transgenesis. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene, pCAG-EGFPac (Fig. 1), carrying both EGFP cDNA and pac.

CAG-EG FP PGK-pac-p(A)

CMV CA EGFPac -I E EGFP pA p(A) pac PGK in tr o n Sal I Sal I

EGFP cDNA EG FPf2 EGFPr2

42 1 b p Fig.1 Schematic representation of the EGFPac transgene used to express the puromycin resistant gene (pac) and enhanced green fluorescent protein (EGFP) cDNA. pEGFPac was constructed by inserting the CAG-EGFP fragment into the pPGK-pac-p(A) cassette vector. Arrows indicate 5’ to 3’ orientation of the cDNA or gene. Two expression units [CAG-EGFP andPGK-pac-p(A)]werealignedinatail-to-tailmanner.

- 7 - This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 μg/ml), cultured for 7 days, and then screened for EGFP expression prior to somatic cell cloning (Fig. 2B). These cells were used for nuclear transfer as donor and the EGFP fluorescence was observed in the manipulated embryos (Fig. 2D). These embryos (1680) were transplanted into the oviducts of 14 foster mother sows. Four of them became pregnant and 9 piglets were delivered. Of the nine piglets, only one grew healthily after weaning (Fig. 2E). The surviving pig clearly

Fig.2 EGFP expression in porcine fetal somatic cells (A, B) surviving in the presence of 2 μg/ml of puromycin for 7 days after transfection with EGFPac and in blastocysts (C, D) derived from A B ABembryos transferred with nuclei of the EGFP-expressing cells. Cells and blastocysts were microscopically inspected for EGFP fluorescence under UV illumination. A and C, bright field; B and D, dark field. The L15-112 piglet derived from enucleated oocytes C D transferred with nuclei of EGFP-expressing somatic cells (E).

expressed EGFP fluorescence in a variety of tissues. The intense fluorescence was observed in the skin (including hair) and epithelial tissues. Our results indicate that puromycin is useful for the rapid selection of recombinant cells from non-cultured cells, and moreover, may confer the efficient production of genetically engineered domestic animals via nuclear transfer techniques. Especially, our method curtailed the period from the gene transfer to obtaining recombinant cells up to 9 days. The present study is the first to report on the usefulness of puromycin for production of EGFP transgenic piglets after somatic cell cloning and embryo transfer. However the reason of the postnatal lethality which observed also in non-transgenic clones is still remained. We must dissolve this problem to produce the transgenic cloned animal much more efficiently.

- 8 - Topics of Research in This Year いいいいいいい Developmental characteristics of transgenic silkworm with over-expression of juvenile hormone esterase

Takahiro Shiotsuki, Anjang Tan, and Toshiki Tamura Developmental Biology Department

Insect juvenile hormones (JHs) have important roles in insect physiology, especially development and metamorphosis, but studies of the molecular mechanisms of the JH have not been progressed yet. Hydrolysis of the methyl ester of JH by a JH-specific esterase is a key pathway for the degradation of JH. We generate transgenic silkworm strains that overexpress JH esterase using the GAL4/UAS system. The JH esterase activity in the hemolymph of 2nd and 3rd instar of the transgenic line is 8 to 10 times higher than that of middle stage of the 5th instar as the highest activity through the whole stage of wild-type. The overexpression of JH esterase from the embryonic stage resulted in larval-pupal metamorphosis after the third stadium, two stadia earlier than that observed in wild-type insects. This precocious metamorphosis indicates that JHs are not critical for normal development of embryo or larva before the second molt in Lepidoptera. The transgenic approach developed in this study allowed us to dissect the function of key physiological events that occur from embryogenesis. These types of studies were only possible in later larval stadia using physical techniques such as allatectomy or the application of JH analogs. We believe that our experimental system will allow further pioneering studies in insect endocrinology and physiology.

Fig. Effect of over-expression of JHE in silkworm. Left: larval-pupal intermediate; ceneter: precocious metamorphosis after 3rd insta; right: control pupae metamorphosed after 5th instar. Bar: 1 cm.

- 9 - Topics of Research in This Year いいいいいいい Different physiological properties in a pool of mandibular closer motor neurons in insects

Ken Sasaki, Kiyoshi Asaoka Physiology and Genetic Regulation Department

In most insect muscles, no more than 1-2 excitatory motor neurons control the force, but a mandibular closer muscle is controlled by 6-12 motor neurons and the subdivision of function among these neurons has been controversial. In this study, morphological and physiological properties of the mandibular closer muscle and its motor neurons were investigated in the silkworm, Bombyx mori, in order to determine whether a large pool of mandibular closer motor neurons in an insect consists of different functional groups. The musculature of the mandible consists of an opener and a closer muscle; the latter is immense, occupying almost all of the head. The mandibular closer muscle is innervated by the branches of the mandibular nerve from the mandibular neuromere of the suboesophageal ganglion. Backfilling with cobalt-lysine complex solution from closer nerve branches showed that the mandibular closer muscle of B. mori was innervated by 11 motor neurons with large somata and 2-3 unknown neurons with small somata (Fig.1A,B).

Fig. 1 (A) Ventral view of the suboesophageal ganglion (SOG) of the caterpillar, Bombyx mori, showing somata of closing motor neurons backfilled from mandibular nerve branches in the closer muscle. (B) Dorsal view of SOG showing backfilled dendrites. (D) and unknown small somata (S) positioned anterodorsally.

Intracellular recordings were made from the somata of the closer motor neurons, while muscle potentials were recorded extracellularly from the mandibular closer muscle at the same time (Fig.2). All 14 motor neurons that we identified could be physiologically divided

- 10 - Fig. 2 Typical recordings showing two types of mandibular closer motor neurons. Injection of depolarizing current into soma of each motor neuron evoked a sequence of spikes and matching muscle potentials. into two groups, the “fast” and the “slow” types. The fast type neurons (5 neurons) showed higher threshold for initiating soma spikes by intracellular current injections (4- or 5-nA). The slow type neurons (9 neurons) evoked spikes by lower current injections (1- or 2-nA). The motor neurons showed 1:1 relationships with the closer muscle activities during current injections and the slow type spiked relatively more frequently than the fast type. The fast motor neurons usually evoked relatively larger muscle potentials and produced fast phasic mandibular movements, whereas the slow neurons evoked smaller potentials and produced slow tonic mandibullar movements. The slow neurons spiked while the mandible was held in a fully closed position, whereas the fast neurons did not. The slow neurons did not spike while the mandible was free to move. We demonstrated here for the first time in insects the different cellular properties among members of a mandibular motor neuron pool in the caterpillar, B. mori.Thefastmotor neurons may contribute the chewing movements to grind a piece of the leaf by a strong force, whereas the slow motor neurons may be activated maintain the position of the mandible at a certain angle.

- 11 - Topics of Research in This Year いいいいいいい Visual and chemical phytomimesis in giant geometer

Toshiharu AKINO Physiology and Genetic Regulation Department

Avoiding ant attack Some insects evolve deceptive strategies in response to predation. They are camouflaged by a combination of visual signals including colour, shape, and movement to mimic other animals and plants. This mimetic camouflage may help insects to avoid predation by visually hunting predators such as birds. By contrast, chemical camouflage is effective in avoiding predation by animals (e.g. ants) that use chemical cues when searching for prey. We found that twig-like catepillars of the giant geometer Biston robustum (Geomteridae) used a combination of visual and chemical mimesis, which were associated with their food plant species. We have observed that predatory ants, incluiding Formica japonica, Lasius niger and Crematogaster tsushimae, are ignore the caterpillars even after walking on them (Fig. 1).

Fig. 1 Caterpillars of the giant geometer. Formica japonica worker ant walking on the caterpillar (top) Caterpillars fed on cherry (middle) and chinquapin (bottom)

Chemical phytomimesis The polyphagous caterpillars fed on the cherry, Prunus yedoensis, the chinquapin Castanopsis cuspidata, and the camellia Camellia japonica. In laboratory we confirmed that neither hatchlings nor the 2nd instar grew up on chinquapin and camellia. Presumably, the caterpillars will grow up on cherry and then move to other food plants if necessary. Gas chromatography-mass spectrometry revealed chemical resemblance of the cuticular surface waxes between the cherry twigs and the caterpillars fed on them (Fig. 2). It was

- 12 - also confirmed between the caterpillars and the respective host plant twigs. Through host exchange experiments, we confirmed that the caterpillarswereabletoadjusttheirmimicryto suit their host plants.

Fig. 2 Gas chromatograms of the surface chemicals of cherry twig (top) and the caterpillar fed on cherry (bottom). Chemical components and their relative ratio were almost identical between the caterpillar and the twig.

Behavioral concealment Analyses of the behavioral patterns suggest that the late instars would be nocturnal feeders. This habit is presumably adaptive because few birds have nocturnal vision. The caterpillars fed on a leaf intermittently during the nighttime, and occasionally chewed the leafstalk to get off the remained leaf after having consumed it, and then proceeded to feed on the reminder of the leafstalk left on the branch. This resulted in having to discard the leaf remnant with the feeding mark on it. Such leaf clipping behaviour is presumably a countermeasure to avian predators and thwart the host plant’s defenses.

Technical terms ★ Mimetic camouflage is the method which allows animals to harmonize with their surroundings. Camouflaged animals are usually unrecognized from the environment for the predator. ★ Twig-like catepillar is characterized by a combination of colour, the presence of horns and marking on the body that look like buds and scars on the twigs, the absence of abdominal legs except for a hind pair to grasp the twig, and the habit of resting in an unusual position with the body stretching out at an angle from the branch. ★ Cuticular surface waxes of insects mainly consist of hydrocarbons. They prevent body dehydration and are known to play semiochemical functions in several contexts of insect biology, involving species- and mate-recognition.

- 13 - Topics of Research in This Year いいいいいいい Recombinant production of modified termite cellulases in E. coli

Hirofumi Watanabe, Ni Jinfeng, Motomi Takehara Insect Genetics and Evolution Department

Recombinant production of termite endogenous cellulases (termite EGs, predicted consisting of 432 amino acids in all known sequences to date) in microbes such as E. coli and baker’s yeast has been difficult, and this problem is not solved by changing eukaryotic codon usages in the termite EG cDNAs into prokaryotic ones. We predicted that the cause of the problem would be found in the amino-acid sequence of termite EG.s available for us. It would be possible to exploring diverse termite species (around 2,600 species) to seek an accidentally-adaptable termite EG gene to the over-expression systems, but instead, we performed family shufflings (random homologous recombination) of available termite EG cDNA to obtain varieties of possible termite EG-like sequences. Four termite EG cDNAs from Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus and C. acinaciformis were randomly digested into short fragment by DNAseIandtheywererecombinedbyPCRcycles(heatdenaturingtoproducesinglestrand DNA, cooling to recombine the fragments randomly with their homologues, and DNA polymerase reaction to fill gaps in the recombinants) to produce chimeratic cDNA libraries. The chimeratic cDNAs were cloned into a normal (week) expression plasmid vector and their E. coli transformants were screened by halo assays to recover correctly reconstructed clones, which produce functional chimeratic EGs. Following second and third selections using screened clones as parents of the next generations, 134 clones (2.51%) were obtained from 5,330 screened clones. The screened clones were subcloned into an over-expression plasmid vector (pQE30), and the efficiency of the over-expression and the enzymatic activity of soluble extract of transformants (E. coli) wereinvestigatedonebyone.Asaresult,weobtaineda clone A18, which shows enhanced expression in E. coli (Fig 1).

Fig 1 A halo assay of termite EG transformed E. coli.Thesizeofthe halo indicates cellulase activity. 1.The transformant of native cDNA of R. speratus.，2. A18 modified cDNA transformant，3. Another modified cDNA transformant.

- 14 - The transformant of A18 produced recombinant termite EG into the soluble fraction of the host (E. coli) cells. The produced enzyme has a 6xHis tag at its N-terminus, by which it was purified using a His-tag affinity purification system (Fig 2). It was predicted that the recombinant termite EG kept basic higher structure of native termite EGs by the comparison of amino-acid sequences.

M 1 2 Fig 2 kDa 200 SDS-PAGE analysis of modified termite EG (A18) produced by the E. coli A18 transformant. M. molecular weight markaers.,1. 116 Crude extract of A18 modified cDNA transformant, 2. 66 Affinity-purified A18 modified termite EG.

45

31 22 14 7

The specific activity of the modified termite EG (A18) was 80-500 units (mmole of reducing sugar/min)/mg of protein, which was equal to and above that of the native purified-eyzme from R. speratus (80 units/mg), while A18 kept other basic eyzymatic properties of the native enzyme.

- 15 - Topics of Research in This Year いいいいいいい Mode of action by which entomopoxvirus spindles enhance nucleopolyhedrovirus infection

Wataru Mitsuhashi, Kazuhisa Miyamoto Insect Genetics and Evolution Department

Insect viruses such as nucleopolyhedroviruses (NPVs) and granuloviruses，which are pathogenic to pest insects, are potential agents for insecticides, because they are safe to men and livestocks, and show less environmental stress. They have drawbacks, however, such as high production costs and generally inferior field performance compared to chemical insecticides. To overcome these defects, materials enhancing strongly the virus-infections have long been screened and studied for the use of them as co-agents of the insecticide. Many entomopoxviruses (EPVs) form proteinaceous structures called spindles in the infected insect cells. The spindles do not contain virus particles. Those of some EPVs are known to enhance drastically some NPVs-infection. Thus, these proteinaceous bodies seem to be potential synergists of bio-control agents such as NPVs. However, mode of action by which EPV spindles enhance NPV infection has remained unclear. Spindles of Anomala cuprea EPV (AcEPV), a coleoptran EPV, were orally administered to silkworm (Bombyx mori) larvae, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles was also observed by the histological sectioning of the midgut (Fig.1). However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly (Fig.2). On the other hand, the pores on the PM, through which BmNPV virions can easily pass from the endoperitrophic side of the PM to the ectoperitrophic one like those on the PMs of some Noctuide caterpillars, were not observed by scanning electron microscopy.

AB Fig. 1 Histological sections of the PM of B. mori 3rd instar larvae. Bar indicates 100 μ m. A. Cross-section of a midgut after larva was fed 2.5 x 106 spindles. The PM is not detected. B. Cross-section of a midgut after larva was fed sterile distilled water as a substitute for spindles. The PM can be detected. Arrows indicate the PM.

- 16 - These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reach the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.

Fig. 2 Dissected peritrophic membrane (PM) of B. mori 3rd instar larvae A. The PM is short because of partial disappearance, after larva was fed 3 x 106 spindles. B. The PM of larva not fed spindles. That in full length is obseved.

- 17 - Topics of Research in This Year いいいいいいい Development of useable material from Hornet silk

Tsunenori Kameda Insect Biomaterials and Technology Department

Some kinds of hornet build large nests. Vespa simillima xanthoptera Cameron is one of most common hornets in Japan. This hornet is a social insect that builds a new nest each summer (see above facial portrait). The largest nests are over 1 m in diameter. The hornet’s mature larva (5th instar) transforms into the pupa after enwrapping itself in a silk weave (hornet silk, [Fig.1(A)]) comprised of protein fibers. Inside each large nest, it can be seen a very large volume of hornet silk caps (cocoon). This gives the silk of V. simillima xanthoptera Cameron a quantitative edge in terms of its potential availability as a material. However, no studies have ever tried to produce useable materials from the hornet silk. In this study, we tried to establish a suitable method for purification and the processing conditions of the silk of the hornet V. simillima xanthoptera Cameron as a new natural material. Inside the nest of the hornet, the cocoons adhere to the plant fibers, because the paper-like cell walls in the nest are made of chewed wood fibers. We found that the hornet silk in the native state could be dissolved at room temperature in aqueous 7.2 M and more concentrated lithium bromide within 15 min. In contrast, the wood fiber was insoluble in this solution. We used this difference in solubility to extract the hornet silk from the nest. Cocoons were dissolved in LiBr solution and removed the insoluble residue. Then, the LiBr solution of hornet silk was dialyzed in distilled water. After dialysis, the gelled hornet silk was obtained [Fig.1(B)]. The gelled hornet silk was freeze-dried, and a purified powder of hornet silk was obtained [Fig.1(C)]. Moreover, the gelled hornet silk was freeze-thawed, and a sponge-like material of hornet silk with porous 3D structure was obtained [Fig.1(D)]. Furthermore, we found that the hornet silk could be dissolved at room temperature in some halogenated organic solvents, such as trifluoroacetic acid (TFA) within a few minutes, dichloroacetic acid (DCA) within a few hours, and hexafluoroisopropyl alcohol (HFIP) within a few days. We were the first to discover that evaporation of the HFIP or TFA solutions yielded a flexible transparent film from the hornet silk. The transparency of the film prepared varied with the type of halogenated organic solvent used, probably because of differences in volatility. The transparency of the film prepared in HFIP solution was best. A film prepared from HFIP solution is shown in Fig.1(E). Furthermore, artificial fiber of hornet silk was successfully obtained from HFIP solvent system [Fig.(F)].

- 18 - Fig. Photographs of cocoon (A), gel (B), purified powder (C), sponge (D), film (E) and artificial fiber (F) of silk of the hornet Vespa simillima xanthoptera Cameron. The upper white cap in (A) is hornet silk, which covered the top of the burrow of the pupating larva, whereas, the downside brownish materials are plant fibers.

- 19 - Topics of Research in This Year いいいいいいい A new process to form a porous 3D structure from silk fibroin

Yasusshi Tamada Insect Biomaterial and Technology Department

Silk have been used as an excellent fabric material in clothing area. Recently new application technology of silk in wide variety area such as food, cosmetic, and medical fields, have been studied. The shape of silk material to be used in those fields is fiber, film, powder, gel, and solution. A porous 3D structure (spongy structure) is important shape to be suitable in various applications, however the utilization of silk spongy material is still unexplored. Because the technology to form a spongy structure having good mechanical properties with good reproducibility was not developed. A new process was found to form the fibroin spongy structure with both good porous structure and mechanical properties (Fig. 1). The process

Fig1 Fibroinspongymaterialformedbythenewprocess. (left: the external appearance, right: pore structure by SEM) very simply conducts freeze-thaw treatment of a fibroin aqueous solution in the presence of small amount of water-miscible organic solvents. This process requires no freeze-drying or repeated freeze-thaw treatment, no crosslinking chemicals, nor other materials. Indispensable conditions of this process are the addition of small amounts of solvents, freezing treatment, and freezing duration. The process will have advantages in the industrial production with good reproducibility. Almost all water miscible solvents were effective to form the sponge structure from the fibroin aqueous solution in the process. Compressive modulus and tensile strength of the fibroin sponge can be controlled by selecting the conditions of the process, such as fibroin concentration (Fig. 2), and solvent kind and concentration. Any significant decrease of the mechanical properties and any deformation of the fibroin sponge by autoclaving treatment were not observed. The fibroin sponge can be sterilized by autoclaving. MC3T3 cells proliferated well in the fibroin sponge for about 3 weeks. This result indicates that the fibroin

- 20 - sponge has cell compatibility and is applicable to cell support for tissue engineering scaffold.

Fig.2 Influence of fibroin concentration on compressive modulus of fibroin sponge. Coll: collagen sponge

- 21 - Topics of Research in This Year いいいいいいい Development of the middle silk gland specific gene expression system for the production of recombinant proteins in the transgenic silkworm

Keiro Uchino, Isao Kobayashi, Hideki Sezutsu, Toshio Kanda, Toshiki Tamura Insect Biotechnology and Sericology Department

Development of the production system of recombinant protein in the transgenic silkworm is very important and urgent problem. The system was recently constructed in the posterior region of the gland. However, the system possessed disadvantage of difficulty to extract proteins. The middle part of the silk gland has been also recognized as one of the suitable organs for the production of the recombinant proteins because the part possessed the ability to produce more than 100 mg proteins. However, the production in the part has been unsuccessful because the promoter used to construct the system does not possess enough activity. In addition, the recombinant proteins produced in the middle part possess an advantage in the solubility for water. That is, the proteins in the middle part are predicted to dissolve easily in water. To develop the system, we performed the amplification of promoter activity of the introduced gene in the middle part of silk gland using yeast GAL4/UAS gene regulation system. The strain inserted GAL gene under the control of sericin 1 gene promoter region was first obtained using newly constructed vector (Fig1, A) and then mated with the strain with UAS/GFP gene (Fig.1 B). The resultant larvae with both gene showed the production of When the strain was crossed with Nd-s mutant strain, the production the recombinant protein was more prominent (Fig.2 A) and easily extracted compared with that in the normal strain (Fig.2B). A large amount of the protein is accumulated in the middle part of silk gland without fibrous proteins. Importantly, the protein accumulated in the gland can be extracted much easily in water. The results suggest that production and extraction of recombinant proteins become much easy and reliable compared to the previous system using the posterior part of silk gland. We expect that the system will apply for the production of useful proteins, including for the medicines. the large amounts of recombinant protein, GFP, in the middle part of silk gland.

- 22 - Fig.1 Structure of the vector with the GAL4 gene under the control of sericin 1 gene promoter（A） and construction of strain with Ser1GAL4 and UASGFP with the mating of SerGAL4 and UASGFP strains(B).

Fig.2 Expression of GFP gene in the silk gland of the larva at the 5th instar (A) and GFP protein extracted the middle part of silk gland of the larvae crossed with Nd-s mutant strain (B).

- 23 - Topics of Research in This Year いいいいいいい Preparation of elastic silk sericin hydrogel from the cocoons of SERICIN HOPE silkworm

Hidetoshi Teramoto, Ken-ichi Nakajima, Chiyuki Takabayashi Insect Biotechnology and Sericology Department

Sericin is adhesive silk proteins synthesized exclusively in the middle silk glands of silkworms, Bombyx mori. Sericin contains many hydrophilic amino acids, which lends it high hydrophilicity and sensitivity to chemical modification. Moreover, recent studies have revealed unique characteristics of sericin, such as induction of heterogeneous nucleation of apatite and enhanced attachment of primary cultured human skin fibroblasts. Sericin is therefore anticipated as a novel naturally-occurring biomaterial. In this study we developed a preparation method for elastic sericin hydrogel. Although the gelling properties of sericin have been a matter of interest, elastic hydrogel made exclusively of sericin has never been reported. We attribute the weakness of hitherto known sericin hydrogels to the denaturation of sericin during extraction. Hence we used the cocoons of SERICIN HOPE silkworm as the sericin source. SERICIN HOPE cocoons consist almost exclusively of sericin (>98%), which facilitates obtaining intact sericin close to the native state. Sericin solution prepared from SERICIN HOPE cocoons exhibited distinct bands of three main sericin components by SDS-PAGE analysis ( Fig. 1, lane 1 ), showing that the solution

Fig. 1 The SDS-PAGE patterns of sericin solution. Lane 1, without heat treatment; lane 2, boiled for 20 min; lane 3, autoclaved at 121°C for 20 min.

contained intact sericin. Gelation was induced by the addition of alcohol. We added several kinds of alcohols and found that ethanol excels in the gelation of sericin. Sericin solution was mixed

- 24 - with ethanol and placed in a refrigerator overnight. An elastic sericin hydrogel was then obtained (Fig. 2A). When sericin solution was boiled for 20 min before the addition of ethanol, only a fragile hydrogel was obtained (Fig. 2B). Although the SDS-PAGE pattern of the boiled sericin solution still exhibited clear protein bands, each band became slightly fuzzy (Fig. 1, lane 2), which suggested slight decomposition of sericin molecules. When sericin solution was autoclaved at 121°C for 20 min, the protein bands of the main sericin components disappeared (Fig. 1, lane 3), showing significant decomposition of sericin molecules. Such a denatured sericin solution no longer formed hydrogel. These results indicate that the properties of sericin hydrogel are sensitive to the denaturation of sericin and that the formation of elastic hydrogel requires intact sericin.

Fig. 2 Sericin hydrogels prepared by the addition of ethanol into sericin solution (A) without heat treatment and (B) boiled for 20 min.

The reported sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be of use as a drug carrier or as a scaffold for tissue engineering.

- 25 - Topics of Research in This Year いいいいいいい Breeding of the silkworm race “SERICIN FLAVO”which simultaneously secretes sericin with flavonol

Toshio Yamamoto, Keisuke Mase, Tetsuya Iizuka, Takako Miyazima, Kenichi Nakajima, Hidetoshi Teramoto, Yasumori Tamura Insect Biotechnology and Sericology Department

Sericin is attracting attention as a natural material because of its high hydrophilicity or affinity to human skin. It is generally extracted from cocoons and raw silk using an alkaline agent. However, native sericin may now be directly used due to the development of the silkworm race, “SERICIN HOPE”. Its sericin cocoon, which we named “VIRGIN SERICIN”, is easily gelled and emulsified and can be used without decomposing. Green cocoons had already been known to contain flavonol, a type of polyphenol substance which has clear anti-oxidation and anti-bacterial activities. To add the flavonol functions to VIRGIN SERICIN,wecrossedSERICIN HOPE with the green-cocoon strain “Daizo”. The offspring were genetically fixed on the character of the green sericin cocoon, and were selected based on the following economic traits: amount of cocoon shell, degree of cocooning and survival rates. This led to the development of a new silkworm strain that simultaneously secretes sericin and flavonol without fibroin fiber (Fig. 1).

Fig.1 Cocoons of SERICIN HOPE (left) and SERICIN FLAVO(right).

This new race spins a high ratio of green sericin cocoons, each of which contains about 4.1mg of flavonol. We named this race “SERICIN FLAVO”. As these two characters, secreting sericin and flavonole, are controlled by the respective dominant genes, it is possible to release them as an F1 hybrid race which can be reared easily by ordinary sericultural farmers. Additionally, as with case of SERICIN HOPE, the amount of sericin and flavonol per larva of SERICIN FLAVO was increased by about 15% by crossing with a common race (Table 1). Furthermore the green sericin cocoon shows higher anti-oxidation (Fig.2) and anti-bacterial

- 26 - activity (Table 2). A shielding effect against ultraviolet rays was found not only in UV-B but also in the UV-A region. These flavonol functions are also active in the gel state. As the gel is easily emulsified with oil like SERICIN HOPE, it shows promise as an advanced cosmetic material. Table 1 Productivity of sericin and flavonol of SERICIN FLAVO.

Table 2 Anti-Bacterial activity of SERICIN FLAVO cocoon.

Fig.2 DPPH radical scavenging activity in cocoons from SERICIN HOPE and SERICIN FLAVO.

- 27 - Topics of Research in This Year いいいいいい The completion of a high-quality rice genome sequence - a major milestone in cereal genomics

Takashi Matsumoto, Jianzhong Wu, Yuichi Katayose, and Hiroshi Mizuno Genome Research Department

In December 2004, the International Rice Genome Sequencing Project (IRGSP) has completed the high-quality genome sequencing of the japonica rice variety, Nipponbare. The NIAS through the Rice Genome Research Program (RGP) has been playing a crucial role since this project was initiated in 1998, generating more than half of the sequence data, contributing a great deal in subsequent analysis and facilitating immediate release of relevant information in the public domain. The entire genome sequence was obtained using the map-based clone-by-clone genome sequencing strategy which means that every clone sequenced can be associated with a specific position in the genetic map. The completed sequence of the 12 rice chromosomes consists of 370 mega (million) nucleotide bases, which correspond to more than 95% of the entire rice genome. We have predicted a total of 37,544 protein genes in the genome, of which 29% exist in tandem arrays. A variety of known functional domains were detected within 60% of these gene products. The accurate genome sequence also elucidates the position and the nature of several important components of the genome, such as transposable elements, centromere, telomere, organellar DNA insertions, non-coding RNAs and ribosomal RNA genes. Rice is the first monocot plant to be completelysequencedandthesecondplantgenome since the sequence of Arabidopsis thaliana was elucidated in 2000. The complete rice genome sequence will serve as the fundamental tool in understanding rice biology including the function of a wide array of genes that comprise the rice plant. Moreover, this breakthrough will have a vast implication in agriculture because rice is a staple for about half of the world population. The sequence information can be greatly beneficial in identifying DNA markers for tagging agronomic traits, isolating genes of biological importance and dissecting the regulatory sequences which control gene expression. This will aid breeders to develop better rice varieties with increased productivity, disease resistance and tolerance to environmental stress. Recent studies on comparative genomics revealed the highly conserved gene order and content in the grass family. Because rice has the smallest genome size, rice is considered a model cereal plant and the rice genome sequence would be indispensable in understanding the genomes of other crops such as maize, barley, sorghum and wheat. Therefore the availability of a high-quality map-based rice genome sequence will open a new era of cereal genomics by promoting extensive studies in functional genomics, maximum utilization of crop diversity, and molecular biology-based breeding.

- 28 - Topics of Research in This Year いいいいいい Production of coenzyme Q10 enriched rice seeds by engineering of isoprenoid side chain

Sakiko Takahashi and Koh-ichi Kadowaki Genetic Diversity Department

Coenzyme Q (CoQ) is ubiquitous in a wide variety of organisms and plays an indispensable role as a component of the respiratory chain. CoQ is consisted from benzoquinone moiety and isoprenoid side chain, and the length of side chain varies naturally dependingontheorganisms.Forexample,humanandricehaveCoQ10andCoQ9, respectively. CoQ10 is used for medication of congestive heart failure in several countries and has recently attracted attention regarding its function as an antioxidant for human, and is sold as food supplements as well as cosmetics in commercial. To modify the length of side chain of CoQ, we introduced a decaprenyl diphosphate synthase gene (ddsA)fromGluconobacter suboxydans into rice cells using Agrobacterium-mediated transformation. In leaves and seeds of transgenic rice plants, CoQ10 was predominantly accumulated, while indigenous CoQ9 was trace level (Fig.). Thus, by expression of DdsA protein, we first enabled to modify the length of the side chain of CoQ in plants, and succeeded in production of CoQ10 in rice seeds. Since many major crops such as wheat, maize and sugarcane produce CoQ9 like rice, our method would be applicable to those crops. CoQ10 is stable against heat below 240°C, therefore, there is less concern about degradation of CoQ10 by cooking such as boiling. Daily uptake of CoQ10 would be easier by eating CoQ10-enriched staple foods than taking CoQ10 tablets. This is a novel method to produce edible CoQ10 in staple food in which CoQ10 is naturally not produced.

Fig. HPLC analysis of the length of side chain of CoQ. CoQs were extracted from leaves (A, B) and seeds (C, D). Wild type plant, A and C; transgenic plant, B and D. CoQ6 (internal control), CoQ9andCoQ10areindicatedbyarrows

- 29 - Topics of Research in This Year いいいいいい A rice core collection selected based on DNA polymorphism

Kaworu Ebana, Yoichiro Kojima ＊, Shuichi Fukuoka, Makoto Kawase and Kazutoshi Okuno Genebank ＊ Toyama Agricultural Research Cente

Rice, Oryza sativa L., is growing under diverse environmental conditions worldwide. Rice germplasm collections from a wide range of geographical origins have sustained their genetic diversity and adaptability to various biotic and abiotic constraints. The NIAS Genebank has conserved more than 32,000 long-term accessions of rice involving local landraces and exotic germplasm from all over the world. To encourage a use of genetic resources and to avoid duplicates for screening, we have focused on generating a core collection which consists of a limited set of accessions and represents the genetic variation derived from an entire rice germplasm collection. Recently, we generated two core collections of rice based on molecular diversity analysis derived from world and local rice germplasm collections conserved at the NIAS Genebank. Two different kinds of rice core collections are summarized as follows. A total of 332 accessions were chosen based on their geographical distribution from whole accessions and analyzed for their genotypes at 184 RFLP marker loci over 12 rice chromosomes. The accessions were divided into clusters in the dendrogram and one accession with the most variable allele combinations from each cluster was selected as a represent accession of core collection. This world rice core collection, which consists of a total of 66 varieties, maintains 90% of alleles detected at RFLP loci. We chose 236 Japanese local varieties from 2000 germplasm collections for molecular diversity analysis using SSR markers. Finally, we selected 35 accessions involved in a Japanese rice core collection. The Japanese rice core collection maintains 94% of genetic variation identified by SSR markers detected in the original population. Both of WRC and JRC cover phenotypic variations of several morphological and physiological traits comparable to its original population. These core collections are useful and convenient bio-resource for identification of genetic variation and for mining alleles. The core collections will be distributed to users according to the regulation of MTA and guidelines. URL and e-mail address of NIAS Genebank are as below, http://www.gene.affrc.go.jp/plant/Distrib/DistIdexJ.html,[email protected] .go.jp)

- 30 - World Rice Collection(WRC) Japanese Rice Landrace collection (JRC)

Initial population：236 accessions Initial population： 332 selected based on passport data from accessions selected 2,000 Genebank accessions ↓ geographically from 32,000 32 SSR marker loci，247alleles Genebank accessions ↓ 35 accessions

Fig. Variation detected in the core collection and the original population. Dark color shows the core collection and light color shows the original population.

- 31 - Topics of Research in This Year いいいいいい A new floral switch gene revealed a novel photoperiodic flowering pathway for short-day promotion in rice

Takeshi Izawa, Masahiro Yano

Molecular Genetics Department

Flowering plants are largely categorized into short-day and long-day plants. In some species, no floral response to photoperiods is observed, which makes another group, day-neutral plants. In 1920, Garner and Allard reported that many flowering plants recognize the day-length to determine flowering-time and set seeds at appropriate seasons. Recent studies in a short-day plant, rice and a long-day plant, Arabidopsis thaliana revealed that plants utilize an evolutionarily conserved flowering pathway to establish opposite photoperiodic responses. The CONSTANS (CO) gene in the long-day plant, Arabidopsis, and its ortholog Heading date1 (Hd1), in the short-day plant, rice, control mRNA expression of FT-group floral inducer genes and play a central role in the photoperiodic control of flowering (Izawa et al., 2002, 2003). CO induces FT mRNA in long-days, whereas Hd1 inhibits and induces mRNA expression of rice FT orthologs (Hd3a etc.) in long-days and short-days, respectively. Therefore, the transcriptional control of FT-group genes by CO/Hd1 is one of key steps that determine the photoperiodic response of flowering in these plants. Here, we have cloned a novel gene, termed Early heading date 1 (Ehd1), which is a major QTL between O. glaberrima and a japonica cultivar, Taichung 65 (T65), and plays a key role in photoperiodic flowering of rice (Fig.1, 2; Doi, Izawa et al., 2004). Ehd1 encodes a B-type response regulator (Fig.3) whoseorthologmaynotexistinArabidopsis.Inaddition,inthestepsofQTLcloningofEhd1, we happened to demonstrate that Ehd1 can function without the functional Hd1 gene since T65, one of the parents, was identified to be deficient in both Hd1 and Ehd1(Fig.1). This suggests that the Ehd1 pathway can be independent from the conserved Hd1/CO photoperiodic flowering pathway. Furthermore, we have shown that Ehd1 controls some FT-like and MADS-box gene expression, indicating that the Ehd1 pathway is also integrated into the conserved FT (or its orthologs) gene expression. There results show that rice flowers as a short-day plant, in which several day-length signals are integrated into gene expression into floral integrator genes such as Hd3a through both the evolutionarily conserved floral pathway mediated by Hd1 and the unique floral pathway mediated by Ehd1.

- 32 - A 1 2 3 4 56 7 8 9 101112 Hd1 Hd1-Nip = 98.9d hd1-T65 = 94.3d F = 10.1 (P<0.01)

Ehd1 Ehd1-Nip = 93.1d ehd1-T65 = 100.3d F = 30.5 (P<0.001)

B Exon 1 Exon 2

Zn finger CCT motif

ATG TGA Nipponbare (Hd1) 36b TAG 1901bp ATG T65 (hd1) *p

CD150

100

50

0 NIL T6 NIL T65 10L14D5 15L9D Fig. 1 Genetic analysis of Ehd1 and Hd1.(A) Major quantitative trait loci (QTLs) identified in 89 recombinant inbred lines derived from a cross between T65 and Nipponbare. A summary of single-point QTL analyses at the Hd1 and Ehd1 loci is shown to the right of the rice linkage map. (B) Allelic variation in Hd1. The genomic sequences of the functional Nipponbare and defective T65 alleles are shown schematically. The triangles and asterisk indicate insertions and a nucleotide substitution, respectively. T65 has a 1901-bp insertion in the exon 2 that results in a premature stop codon (TAG) ahead of the CCT motif. (C) Days to heading of NIL (Ehd1-gla) and T65 under short-day (10L:14D) and long-day (15L:9D) conditions. (D) Photograph of 80-day-old plants of NIL (Ehd1-gla) (left) and T65 (right) grown under SD (9L:15D).

- 33 - A KpnI 7.6kb 6 Fig. 2

4 Complementation test of Ehd1. （A）Days to

heading of T0 transformants under SD (10L:14D) 2 conditions. Open bar, empty vector; solid bar, 0 vector with the genome fragment. Only plants transformed with the 11.5-kb BamHI fragment 6 BamHI 11.5kb showed promotion of flowering. (B) Days to

4 heading of two T2 lines homozygous for the

2 Ehd1-Kas transgene (1-16, 5-6), NIL (Ehd1-gla) and T65 under conditions of SD (9L:15D) and LD 0 (14.5L:9.5D). Day to heading after transplanting B 1-16 55.5 1.7 5-6 53.4 2.6 T65 94.2 3.1 0 5 100 (d) Days to heading0 after sowing

A B ATG TAG 11 123197255 5' 3' 1 34 Receiver GAR 1 1kb P

C GA(ehd1-T65) K Ehd1-Kas 197 GKSRLTWTTQLHRQFIAAVNHLG.EDKAVPKKILGIMKVKH LTREQ VASHLQKYRMQLKK Golden2 183 RKVKVDWTPELHRRFVQAVEQLG.IDKAVPSRILEIMGTDC LTRHNIASHLQKYRSHRKH ARR1 236 KKPRVVWSVELHQQFVAAVNQLG.VEKAVPKKILELMNVPG LTRENVASHLQKYRIYLRR Psr1 21 PKPRLRWTTELHERFVDAVTHLGGPEKATPKTIMRVMGVKG LTLYH LKSHLQKFRLGKQP

Fig. 3 Structure of Ehd1.(A) Alignment of GARP domains of Ehd1-Kas, Golden2 (AAG32325), ARR1 (T51246), and Psr1 (AAD55941). The position of amino-acid variation in T65 (G to R) is indicated by an asterisk.

- 34 - Topics of Research in This Year いいいいいい Calcium-signal transduction cascade for cold tolerance and elongation in rice leaf sheath

Setsuko Komatsu, Arun Sharma, Abbasi Fida Molecular Genetics Department

Calcium is a ubiquitous signaling molecule and changes in cytosolic Ca2+ concentration are involved in plant responses to various stimuli, including environmental stresses and plant hormones. Increasing evidence shows that Ca2+-dependent protein kinases (CDPKs) are also involved in environmental stress response and plant hormone signaling. To identify the crosstalk between environmental stress response and plant hormone signaling, Ca2+-signal transduction cascade in rice seedling was analyzed using rice seedling. Rice CDPK13 was cloned from rice seedlings and its transcript shown to accumulate in response to cold stress and gibberellin (GA) treatment. The height of antisense CDPK13 transgenic rice lines was shorter than that of vector control, and the expression of CDPK13 was lower in dwarf mutants of rice than in their wild type. On the other hand, sense CDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control, and the expression of CDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. Using immuno-precipitation system, calreticulin was detected as interacting protein to CDPK13. To identify proteins that are regulated by the GA3 response in leaf sheath elongation in rice, proteins extracted from leaf sheath treated with GA3 were analyzed by the differential display proteome approach. Calreticulin was also identified as a responsive protein catalyzed by phosphorylation in GA signaling. In addition, calreticulin was phosphorylated by cold stress. These results indicate that CDPK13 and calreticulin might be important signaling components in response to GA and cold in rice. Furthermore, using the screening of calreticulin-interacting proteins through yeast two-hybrid system, two novel proteins were identified in rice. cDNAs that showed interaction with calreticulin from a rice suspension culture cell cDNA library and leaf sheath cDNA library were identified as calreticulin-interacting proteins and named CRTintP1 and CRTintP2, respectively. CRTintP1 contains a nuclear localization signal site and studies on cellular localization using CRTintP1::GFP validated its nuclear localization. The expression of CRTintP1 increased in response to cold stress, indicating that it is a stress-responsive gene. On the other hand, using in situ hybridization system, CRTintP2 was expressed particularly in the shoot apical and nodal apical meristem, which are important in leaf sheath elongation. The average height of the various antisense CRTintP2 transgenic rice lines was 50% of that of

- 35 - the vector control. These results suggest that the possible element involved in controlling stress-responsiveness and leaf sheath elongation, and cold tolerance and GA-dependent elongation may be regulated through distinct signaling pathways that crosstalk at the level of CDPK13, calreticulin and CRTintP1/CRTinrtP2.

Cold stress Gibberellins Abcisic acid Calcium Calcium dependent protein kinase Phosphorylation Calcium binding protein ∥ C S S Calreticulin cDNA library from callus cDNA library from crown C AS AS OsCDPK13 OsCDPK13 Binding protein Binding protein （OsCRTintP1） （OsCRTintP2）

Nucleus Crown Ubiquitin domain Histisine-rich Stress response GA/BL response

Cold response Leaf sheath elongation C S AS Calreticulin C S AS C S AS OsCRTintP1 OsCRTintP2

Figure 1. Analysis of the proteins interacting to calreticulin. The immuno-precipitation system was used to identify calcium dependent protein kinase. The yeast two-hybrid interaction-cloning system was used to identify novel calreticulin interacting proteins (CRTintPs).

Fig. Analysis of the proteins interacting to calreticulin. The immuno-precipitation system was used to identify calcium dependent protein kinase. The yeast two-hybrid interaction-cloning system was used to identify novel calreticulin interacting proteins (CRTintPs).

- 36 - Topics of Research in This Year いいいいいい X-ray crystallographic analysis of Irpex lacteus aspartic proteinase

Zui Fujimoto Biochemistry Department

Aspartic proteinase is a proteolytic hydrolase with two catalytic aspartic residues, has an acidic optimum pH, and is often referred to as “acid proteinase” or “carboxyl proteinase.” ILAP, an aspartic proteinase from Irpex lacteus (Hymenomycetes, Basidiomycota,akindof mushroom (E.C. 3.4.23.29)) has a high milk-clotting activity in relation to proteolytic activity (MCA/PA) and has been expected to become a good rennet substitute. We previously succeeded in purifying and crystallizing ILAP (Fig. 1). In order to obtain the structural basis of

Fig. 1 Typical crystal of ILAP.

N-domain

this enzyme for future protein engineering studies and rational design for industrial use, the crystal structure of ILAP in complex with pepstatin (a six-amino-acid peptide-like inhibitor) was determined pepstatin at 1.3 Å resolution. ILAP is a pepsin-like enzyme, and its structure is composed of anti-parallel b-sheets with N-terminal and C-terminal domains of about the same size (referred to as N-lobe and C-lobe, Fig. 2). The catalyticsiteisintheformofacleftlocatedbetween C-domain the two lobes, and two aspartic acids face each other to make a catalytic dyad. The structure and interaction Fig. 2 Ribbon model of the crystal pattern around the catalytic siteare conserved, in structure of ILAP in complex with agreement with the other aspartic proteinase/inhibitor pepstati

- 37 - complex structures reported previously. The high resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the

P1 or P1' site, and also in the P4 and/or P3 site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P3 and P4 site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P4 site (Fig. 3).

Fig. 3 Stereo view of the pepstatin binding structure of ILAP. Bound pepstatin, two catalytic aspartates are shown in orange and red. The hydrophobic pockets (P3 and P4) are indicated.

- 38 - Topics of Research in This Year いいいいいい Structure Genomics Studies of Rice Proteins

Toshimasa Yamazaki, Etsuko Katoh, Rintaro Suzuki, Zui Fujimoto, Mitsuru Momma, Toshiyuki Wako Biochemistry Department

In the Rice Genome Project, functions of rice genes have been analyzed systematically using various biological approaches, molecular genetics including mutant panel strategy based on transposon-induced mutagenesis, map-based strategy and microarray of gene-expression monitoring system. Since further understanding of gene functions requires a detailed knowledge of three-dimensional structures of proteins and their interactions with other biomolecules, we have carried out structure determination of various rice proteins in collaboration with 8 universities. We selected 135 target proteins and protein fragments based on potential importance in plant biology and biotechnology. To improve expression and solubility of proteins in Escherichia coli, we developed seven expression vectors in combination with several N-terminal fusion protein tags and protease sites which are adapted to the Gateway expression system. Of 135 targets, 131 (97%) could be expressed and 72 (53%) were soluble in the presence of the fusion protein tags. Finally, 38 proteins and protein fragments were obtained in mature forms suitable for solution NMR or/and X-ray crystallographic studies. We have successfully determined three-dimensional structures for 14 proteins by X-ray crystallography and for 9 proteins by NMR spectroscopy. Detailed examination of these structures allowed us to investigate the molecular mechanisms of the protein functions. Figure shows the NMR solution structure of the hinge-PAS1 fragment (S601-H740) of the rice phytochrome A (PHYA), the red and far-red light-sensing photoreceptor. The hinge-PAS1 fragment was selected as the target because the majority of loss-of-function missense mutations are observed within this region. These missense mutations are located on the solvent exposed surface of the b-sheet, suggesting that this surface is important in regulating the phytochrome A function through protein-protein interactions between subunits of the photoreceptor or binding to downstream signaling components. Similar results were obtained for phytochrome B (PHYB). However, PHYA and PHYB have distinct mechanisms for dimerization which is essential for the phytochrome function.

- 39 - Fig. Ribbon representation of NMR solution structure of the hinge-PAS1 fragment (S601-H740). Residues whose replacements lead to loss of function are highlighted.

- 40 - Topics of Research in This Year いいいいいい Cloning and characterization of a plastid localized ion channel gene crutial for nodule formation in Lotus japonicus

Haruko Imaizumi-Anraku, Yasuhiro Murakami, Shinji Kawasaki Biochemistry Department

Although nodule formation is indispensable for symbiotic nitrogen fixation by legume plants and rhizobia, hitherto its detailed process has not been known. Recently, by the advent of functional genome analysis strategies, some components on the process of signal transduction for nodulation have been cloned from some nodulation mutants, those having defecits in one of the elementary processes of the nodulation. For this progress, the model legume plant Lotus japonicus, a familiar yellow-flowered wild plants on roadsides, has played a great role, because rice and Arabidopsis can not make nodules. We have cloned and characterized functions of a pair of genes functioning at the early step of nodulation, CASTOR and POLLUX, from a set of mutants of Lotus japonicus completely lost in nodulation and mycorrhiza-forming abilities. These genes were named from their close homology, but they are located on different chromosomes (No. 3 and 6, respectively) on the non-homologous backgrounds, and defects of any gene deprive the nodulating abilities from the plants. For their cloning, our high quality original Lotus genome library played an important role, and the library has contributed for cloning other two genes on the early nodulation process, and a gene regulating the density of nodules. Ca-spiking is known as an important initial signaling phenomenon occurring around root hair nuclei, only 20min after application of nM order of Nod-Factor, the host-species specific signaling material from rhizobia, the mutants castor/pollux do not show this spiking. Therefore, the proteins coded by CASTOR/POLLUX are thought to be at the very early steps of the signal-transduction for nodulation. These proteins showed homology to the Ca2+-controlled K+ channel of a methane generating thermophilic bacteria, Methanobacterium thermoautotrophicum, andhavingtheCa2+ controlled ion-conduction regulating (RCK) domains, suggesting its important role in generating Ca2+ spiking. Probably, the two proteins construct the channel as a hetero-dimer. The minute differences of amino acids in the channel gateway, suggest some modification of the conducting ions (Fig. 1). The fluorescence labeling of the proteins indicated complete localization to the plastid (Fig. 2). This is a rather novel function for plastids, the organelle hitherto considered as rather specialized for metabolic factories.

- 41 - Fig.1 3D-modeling of the corresponding channel facing parts of CASTOR protein (right) after the X-ray analysis of the potassium ion channel of Methanobacterium thermoautotrophiocum (MTHK) protein (left). In spite of their overall similarity, the differences in critical amino acids in the gateway may differentiate the specificity of the ions passing the channel.

Fig.2 Intracellular localization of CASTOR/POLLUX proteins, each labeled with green fluorescence (Ag, Bg), and plastid localizing red fluorescence protein, as a reference (Ar, Br). The two signals overlapped completely as seen in the yellow merged signal (Am, Bm). The extending strand-like structure is plastomule.

- 42 - Topics of Research in This Year いいいいいい Improvement in yield characteristics of transgenic potato with amaizeSPSgene

Ken Ishimaru Plant Physiology Department

Potato is important cultivated crop cultivated in Europe, North America and Asian many countries. The improvement in potato yield is important with world level food production. Sucrose-phosphate synthase (SPS) is one of the key enzymes in the synthesis pathway of sucrose that is a major transportable photoassimilate. In the storage form of carbohydrates in leaves, the ratio of sucrose to starch content depends on plant species. Potato (Solanum tuberosum) mainly accumulates starch in leaves. To elucidate whether the increasing in SPS activity could affect sucrose synthesis and consequently yield characters, we produced and analyzed transgenic potato plants (cv. May Queen) overexpressing with a maize SPS gene. The tuber weight increased in accordance with SPS activity in transgenic potatoes cultivated in Wagner pots (1/2,000 are), and it was it to 1.5 times of control in the transgenic plant that SPS activity was the highest (tentatively named Ag1203). In the environmental condition that was similar to normal cultivation (isolation filed), Ag1203 was cultivated to clarify characteristic. SPS activity in leaves of Ag1203 increased to 3.8 times and the translocation rate of a carbohydrate from leaves increased to 1.2 times of control. In addition, as for Ag1203, leaf senescence rate became significantly slow in comparison with control. Between Ag1203 and control, there was no difference seen in general morphology and productivity of the allelochemical for a neighboring plant and microbial flora. The total yield per a plant or average weight of tubers in Ag1203 was 1.2 times as high as that in control (Fig.). These results suggested that the elevated SPS activity in leaves could affect sucrose synthesis and eventually lead to increase yield in the transgenic potato The concentration of sucrose is related to sweetness and the taste in potato. In potato, the carbohydrate in a tuber is mainly starch and little sucrose. In transgenic potato (Ag1203), the sucrose contents in tubers increased to 2.1 times comparing to control. The tuber of Ag1203 is supposed to be sweetish and increase the quality.

- 43 - Taken together the results in this study, the increase in SPS activity could improve both in quantity and quality in yield character of potato plants. It is thought that SPS activity of leaves may be available as the indicator of translocation competence in breeding of higher yield in potato plants.

Fig. Yield per a plant and average tuber weight in transgenic potato (Ag1203) and control (May Queen). Data are means of 30 plants. The number in parenthetic is given by the relation to control.

- 44 - Topics of Research in This Year いいいいいい Development of transgenic rice seeds accumulating mGLP-1 that stimulates insulin secretion

Hiroshi Yasuda, Lijun Yang and Fumio Takaiwa Plant Biotechnology Department

Diabetes is one of the serious life-style related diseases, and the number of diabetics are estimated 7.4 millions, including a preliminary condition runs up to 16.4 millions in Japan. Glucagon-like peptide-1 (GLP-1) is a potent blood glucose-lowering hormone that stimulates the secretion of insulin from pancreatic ß-cells, depending upon blood glucose concentrations. Therefore, the GLP-1 has been thought of a candidate for therapeutic agent of type II diabetes. We tried to develop transgenic rice seeds to accumulate large amount of the peptideinricegrain,inordertoimproveortreatthediabetesthroughadailydiet. Firstly, the gene coding for modified GLP-1 (mGLP-1), which is resistant to trypsin digestion, was synthesized using codons that are preferentially used in rice seed storage protein genes. The glutelin GluB-1 signal peptide sequence and the KDEL endoplasmic reticulum retention signals were attached to the N- and C-termini of the mGLP-1 peptide. This synthetic gene was expressed under the control of the rice storage protein glutelin 2.3kb GluB-1 promoter. However, the peptide did not accumulate in the seed due to gene silencing. To avoid gene silencing, the mGLP-1 peptide was expressed as a part of rice seed storage protein, 26kDa globulin. The mGLP-1 gene was inserted into the variable region of 26kDa globulin gene by replacing the junction sites between globulin and mGLP-1 with trypsin cleavage sites. Transgenic rice plants were generated using MAT-vector system and total seed protein were analyzed by SDS-PAGE gel. As shown in Fig. 1, the modified Fig. 1 SDS-PAGE analysis of total proteins in transgenic rice seed expressing fusion protein, mGLP-1/ globulin, and non-transgenic rice seed. (A) Total seed proteins extracted from a mature seed were separated by SDS-PAGE. The arrow indicates the fusion protein ‘mGLP-1/globulin’. (B) Western blot analysis was performed with

anti-GLP-1 antibody. Lane 1-7, T0 seed of transgenic rice plant using MAT-vector system. Lane 8, control seeds (non-transgenic rice).

- 45 - globulin containing the mGLP-1 could be detected as a visible band, and the highest accumulation level of the mGLP-1 was estimated 50 μg per grain. The seed extract treated with trypsin to release mGLP-1 from the globulin, stimulated insulin secretion from a mouse pancreatic ß-cell line (Table).

Table Stimulation of insulin secretion in mice pancreatic ß-cells by trypsin-treated seed extract from transgenic rice expressing fusion protein ‘mGLP-1/globulin’

As a other approach, five tandem repeated mGLP-1 gene (mGLPx5) was directly expressedunderthecontrolofthericeglutelin2.3kbGluB-1 promoter. The individual mGLP-1 is linked by arginine residue as a trypsin target site. When the construct was introduced into rice, transgenic rice plant accumulated about 180 μg mGLP-1 in the seed at the highest. Oral administration of the transgenic seeds to mice significantly suppressed elevating blood glucose level compared with non-transgenic rice seeds (Fig. 2).

Fig. 2 Blood glucose lowering activity in mice by oral administration of transgenic rice seeds expressing mGLPx5 peptide. Open, red and blue circles indicate oral administration of control seeds (non-transgenic rice), transgenic rice seeds expressing mGLPx5 and subcutaneous administration of synthetic mGLP-1 peptide (20 μg / kg), respectively. Horizontal axis indicates times after administration.

- 46 - Topics of Research in This Year いいいいいい Analysis of Mutations from Pollen Irradiation

Makoto Kusaba and Minoru Nishimura Institute of Radiation Breeding

An early genetic study showed that most radiation-induced mutations are not transmitted to progeny. In recent molecular studies in plants, M2 plants or their progeny, which contain only transmissible mutations, have mainly been analyzed, but the early results imply that these studies are insufficient as comprehensive descriptions of radiation-induced mutations. To study radiation-induced mutations caused by low-LET g-rays and high-LET carbon ions at the molecular level, we used the pollen irradiation method（Fig.） and the plant Arabidopsis thaliana to study various mutations, including nontransmissible mutations. This analysis revealed that most mutants induced with irradiation with g-rays (150 － 600 Gy) or carbon ions (40 － 150 Gy) carried extremely large deletions of up to ＞ 6Mbp,themajority of which were not transmitted to progeny（Table). Mutations containing 1- or 4-bp deletions, which were transmitted normally, were also found. Comparison of the deleted regions in the mutants showing various manners of transmission suggests that the nontransmissibility of the large deletions may be due to the deletion of a particular region that contains a gene or genes required for gamete development or viability. This means that most mutations induced by ionizing radiation have not been used in the mutation breeding of seed-propagated crops. However, 'nontransmissible' mutations can survive over generations in vegetatively propagated crops and have probably contributed to the genetic improvement of such crops.

- 47 - Fig.

Principle of the experiment. Pollen from Arabidopsis thaliana Col plants (M1) was irradiated and used to fertilize Ler plants carrying a recessive marker mutant gene (a). The resultant M2 plants were screened for the marker phenotype. These mutants were analyzed by using molecular markers around the marker locus that distinguish the Col and Ler genomes. This enables the deleted regions of the Col genome caused by pollen irradiation to be determined in the M2 plants. Col chromosomes are solid, Ler chromosomes shaded, and deleted regions open.

- 48 - Table Sizes of large deletions observed in the mutants induced by g-ray or carbon ion irradiations. A. g-ray/GL1 Irradiation dose (Gy) 150 300 600 No. of mutants a 9286 Max (Mbp)b >4.5 >6.0 >4.5 Min (Mbp)c 1 0.7 0.08

Ave (Mbp)d 2.2 2.7 1.8

B. Carbon ions/GL1 Irradiation dose (Gy) 40 150 No. of mutants a 39 Max (Mbp)b 1.1 >4.5 Min (Mbp)c 0.5 0.5 Ave (Mbp)d 0.7 2.5 a Number of mutants with a large deletion.

b Size of the largest deletion.

c Size of the smallest deletion among the large deletions. d Averagesizeofthelargedeletions.

- 49 - Topics of Research in This Year いいいいいい New apple cultivar‘Houiku Indo’， amutantresistantto alternaria blotch disease by gamma-ray irradiation

Yuji Ito, Tetsuo Masuda, Toji Yoshioka Institute of Radiation Breeding

Alternaria blotch, a serious apple disease in Japan, causes dark brown or black necrotic spots on apple leaves, branches and fruit, resulting in abnormal leaf fall in the summer, declining tree vigor, growth defects in fruit and poor bud formation that decrease fruit production and commercial value. The high cost of fungicide application 10 times or more a year has necessitated the development of commercial cultivars resistant to this disease. AnoldJapaneseapplecultivar‘Indo’ishypersensitivetoAlternariablotchdisease.It shows typical symptoms of necrotic lesions on the leaves inoculated with the filtrate derived from a pathogenic fungi culture medium solution（crude AM-toxin）.Hypersensitiveand resistant cultivars can be clearly distinguished by the treatment of crude AM-toxin inoculated to leaf sections harvested from in vitro cultured shoots. In 1992, in vitro plantlets of‘Indo’were irradiated with gamma-rays at a total exposure level of 80 Gy at a dose of 5.0 Gy/h in the gamma room of the institute of radiation breeding,

NIAS. A mutant shoot insensitive to crude AM-toxin was selected in the VM6 generation. The mutant was rooted, habituated and grown by top-grafting onto mature apple trees in the experimental field. Following the AM toxin treatment and an Alternaria blotch spore inoculation test, resistance was confirmed in this mutant (Fig.1 ) The degree of Alternaria

Fig.1 Response of purified AM-toxin on the leaves grown under field conditions. ‘Tsugaru’, ‘Fuji’, ‘Houiku Indo’, ‘Indo’and ‘Starking Delicious’ (Left to right).

blotch disease resistance of the mutant was slightly stronger than that observed in the cultivar ‘Fuji’. A survey of mutant for features other than resistance to AM toxin showed no marked difference from the original cultivar‘Indo’in flower, fruit form or fruit quality, and mutant seeded normally under field conditions from 2000 to 2003 (Fig. 2) . Though the decline of

- 50 - Fig.2 Fruits bearing shoots of ‘Houiku Indo’.

pollen fertility was observed in mutant, normal fruit set was confirmed in the pollination test using the pollen of mutant. This new mutant was assigned the name, ‘Houiku Indo’and cultivar registration was applied for under the Seeds and Seedlings Law of Japan on December, 2004. ‘Houiku Indo’can be cultivated under the same frequency of fungicide application as ‘Fuji’. It is expected to be cultivated as the revival of an old popular apple cultivar in apple production area of Japan . Induction of resistant mutant to Alternaria blotch disease by gamma-ray irradiation will be applicable to other susceptible apple cultivars with slight modification.

- 51 - Research Activities

Genome and Biodiversity Research Division

The National Institute of Agrobiological Sciences (NIAS) is composed of three divisions: (1) the Genome and Biodiversity Research Division, (2) the Insect and Animal Sciences Division, and (3) the Plant Science Division. The research field of the Genome and Biodiversity Division networks with the Insect and Animal Science Division and Plant Science Division. Therefore, research data is shared to maximize research efficiency. In the Genome and Biodiversity Division, there are 3 departments: (1) the Genome Research Department, (2) the Genetic Diversity Department and (3) the Genebank. The overall research output of these three departments is transmitted to the basic and applied scientific community. As a result, the Genome and Biodiversity Research Division contributes to science, agriculture and social life by distributing useful bio-resources and scientific results internationally. The Genome Research Department focuses on genome analyses of three major agricultural organisms, namely, rice, pig and silkworm. Our institute played a leading role in elucidatingthecompletesequenceofthericegenomeincollaborationwiththeInternational Rice Genome Sequencing Project (IRGSP). In addition, we have also successfully sequenced the mitochondrial genome of rice. Thus we have made significant contributions in elucidating the nuclear, chloroplast and mitochondrial genomes of rice. These research outputs are tightly linked to several important projects on rice functional genomics. In order to provide the scientific community with access to the biological resources generated from various genome related projects, the Rice Genome Resource Center (RGRC) was established in 2003. Detailed information can be obtained via the web at (http://rgp.dna.affrc.go.jp/ and http://rgrc.dna.affrc.go.jp/). Genetic erosion caused by various environmental, socio-economical and ecological changes necessitates research on genetic resources to maintain the genetic diversity of the biological world. In the Genetic Diversity Department, basic research on plant, microbe and animal diversity is conducted. The results of the research have contributed to rational and efficient methods for the classification, characterization, preservation and discovery of new biological resources. To accelerate research on genetic resources, NIAS also is the center of the MAFF Genebank system for agricultural genetic resources in Japan. The genebank has the responsibility for conserving and utilizing plant, microorganism and animal genetic resources and their DNA. The Genebank system involved the close linkage between NIAS and other sub-genebanks across Japan. Distributed genetic resources are used for research and breeding

- 52 - purposes under the regulation of Convention on Biological Diversity and International Treaty. Information related to genebank materials can be found at (http://www.gene.affrc.go.jp/ and http://www.dna.affrc.go.jp). An international workshop on genetic resources was held in conjunction with the 10th International Congress for Culture Collections in Tsukuba in 2004. The theme of the workshop was “Genetic and functional diversity of agricultural microorganisms” and two symposia and a poster session were included. Also the training course entitled “Identification and preservation of plant pathogens” was organized for Southeast Asian microbiologists in the schedule. This was a good opportunity to stimulate the development of global and national networks of culture collections and the improvement of the collection quality. The research activities of the Genome Research Department, the Genetic Diversity Department and Genebank are described below.

- 53 - Genome Research Department

Introduction The Genome Research Department focuses on genome analysis of major agricultural organisms, namely, rice, pig and silkworm, which are coordinated as genome research projects of our institute. The Rice Genome Research Program (RGP) aims to elucidate the structure of the rice genome using the map-based clone-by-clone sequencing strategy and characterize the genes that comprise the rice plant. The Animal Genome Research Program (AGP) aims to develop DNA markers for QTL analysis, construct a catalog of porcine genes and facilitate traceability in Japanese pork market. The Silkworm Genome Research Program (SGP) aims to characterize the silkworm genome by EST analysis, genetic and physical mapping, whole genome shotgun sequencing and expression profiling. Comprehensive analysis of genome sequence data, development of analysis tools and construction of genome databases are facilitated by the combined efforts of the DNA Bank and the Genome Informatics Laboratory. In addition, the DNA Bank also provides support to these research activities by developing an informatics infrastracture that facilitates the collection, storage and retrieval of DNA information from the public domain. Access to biological materials generated from these genome projects and other genome related projects of the institute is provided by the DNA Bank and the Rice Genome Resource Center (RGRC). The following reports summarize the major research topics and achievements of the six laboratories of this department.

Completion of Rice Genome Sequencing One of the most significant achievements in genome research is the completion of the high-quality genome sequence of the japonica rice cultivar Nipponbare. The collaborative efforts of the Rice Genome Research Program (RGP) and other participating groups of the International Rice Genome Sequencing Project (IRGSP) led to the completion of rice genome sequencing in December 2004 (See topic of the year). The RGP is in-charge of sequencing six of the 12 rice chromosomes. The completed sequence of the 12 rice chromosomes corresponds to 98.9% of the euchromatic region or 95.3% of the entire genome. In the final phase of genome sequencing, RGP continued to fill the remaining physical and clone gaps, elucidate the terminal structure of rice chromosomes, complete the sequencing newly identified clones to “finished” quality level, and perform high-throughput annotation. Two clone gaps were newly filled after screening of a fosmid clone library. These clones were completely sequenced thereby reducing the gaps and unsequenced regions of the genome. So far, there are only 36 remaining gaps in the euchromatic region in the latest physical map of rice. Moreover, fosmid clones with telomere-specific repeat sequences, have

- 54 - been mapped at the terminal regions of the chromosomes. This indicates that the telomere regions have been covered and that our physical maps have almost reached the telomere ends of the chromosomes. In an attempt to clarify the actual size of the entire genome, the physical distance of the clone gaps was measured by Fiber-FISH analysis. Thus based on the complete sequence of the euchromatic genome and an estimated distance of the physical gaps including the centromeric and telomeric regions, the most probable size of the entire genome is about 390 Mbp. The overall statistics including the completed nucleotide sequence, estimated size of gaps on the chromosome arms, telomeric and centromeric regions, and coverage of the sequence for the six chromosomes assigned to RGP are shown in Table 1. Analysis of the genome sequence was performed through a combination of automated annotation using RiceGAAS (Rice Genome Automated Annotation System) and manual curation. We adopted a gene annotation procedure (http://rgp.dna.affrc.go.jp/genomicdata/AnnSystem.html)whichcombinedab initio gene predictions and the results of homology search with the full-length transcripts, ESTs, and known proteins in public databases. We have completed the manual curation of 25,622 genes from 1,789 PAC/BAC clone sequences for the six chromosomes. These results are submitted to DDBJ as a part of information for each sequenced clone, and also published in our integrated rice genome browser INE (http://rgp.dna.affrc.go.jp/giot/INE.html) and annotation database RAD (http://rad.dna.affrc.go.jp/). Genome-wide gene annotation of the nucleotide sequence of the 12 chromosomes was also performed to characterize the genome structure of rice. A total of 37,544 non-transposon protein genes were predicted from the rice genome of which 29% are clustered in tandem arrays (Fig. 1).

Table 1 Sequencing statistics for the six rice chromosomes assigned to RGP

- 55 - Fig. 1 Frequency of tandemly arranged genes in the rice genome.

Gene Prediction and Identification in the Completed Rice Genome Sequence With the completion of the genome sequence of Oryza sativa spp. japonica cv. Nipponbare, the next logical step is to analyze the genome sequence using gene prediction programs and identify all the genes that comprise the rice plant. We identified the transcribed genomic regions in rice by comparing the full-length cDNA sequence data with the IRGSP Build 3.0 pseudomolecules which correspond to the completed genome sequence. As a result, a total of 22,816 loci were determined. Among them, 2,271 unmapped loci may correspond to unsequenced genomic regions, which were estimated to be ~5% of the genome. We used our customized gene finding program GLocate to annotate the genome sequence. The program has been modified which resulted in improved gene prediction and ~30% faster performance. It could run in parallel on four PC servers so that calculation for the entire rice genome sequence could be finished in six hours. Both sensitivity and specificity of the programarecomparablewithFgenesh(Table2),whichisconsideredtobeoneofthemost efficient ab initio gene-finding program for rice. GLocate is available through the WWW interface (Fig. 2). In combination with three other programs namely, Fgenesh, Genscan and Genscan+, we could predict a total of 69,002 loci. Since these predictions inevitably contain a number of errors, we adopted only the loci that were supported by monocot ESTs. As a result, we could identify an additional 6,941 loci that had not been identified from homology search with rice full-length cDNAs. Among the automatically predicted gene models, a total of 29,757 loci showed evidence of expression. These loci were subjected to similarity search against protein databases and InterProScan. The inferred functions were manually evaluated in the First Rice Annotation Project Meeting (RAP1), an annotation jamboree held in Tsukuba on December 13-18, 2004. The results will be available soon through the RAP1 database.

- 56 - Table 2 Comparison of ab initio gene-finding efficiency Nucleotide level Exon level Sn (%) Sp (%) CC Sn (%) Sp (%) GLocate 89.00 85.60 0.82 74.10 69.10 Fgenesh 94.20 86.90 0.87 80.50 75.60 GeneMark.hmm 88.50 83.20 0.8 69.30 61.50 GlimmerM 67.70 77.70 0.63 41.70 50.00 RiceHMM 62.30 73.00 0.57 18.60 22.80

Fig. 2 The web interface of Glocate, a Hidden Markov Model (HMM)-based gene prediction program for rice.

Molecular evidence of inter-species hybridization in the genus Gallus Mitochondrial D-loop analysis has provided a phylogenetic tree for fowls including chicken (Gallus gallus var. domesticus), supporting the hypothesis that red junglefowl (Gallus gallus; subdivided into five subspecies, Gallus gallus gallus, Gallus gallus spadicius, Gallus gallus bankiva, Gallus gallus jabouillei,andGallus gallus murgi ) is the direct ancestor of chicken based on morphology and progeny production. The phylogenetic positions of chicken and other fowl species in the genus Gallus are considered to be of great importance in the maintenance and improvement of chicken breeds through introgression of genetic variations from wild type genomes. However, phylogenetic analysis based on the DNA sequences is not sufficient to conclude the phylogenetic positions of various fowl species in the genus. We determined the sequences of whole mitochondrial DNA (mtDNA) and two segments of the

- 57 - nuclear genome in several Gallus species except for Gallus varius (green junglefowl). The nuclear genome segments include intron 9 of ornithine carbamoyltransferase gene (OTC)and 4 chicken repeat 1 (CR1) elements. The phylogenetic analyses based on mtDNA sequences revealed that the two grey junglefowls (Gallus sonneratii) were clustered in a clade with red junglefowl and chicken, and that one grey junglefowl was located in a remote position close to Ceylon junglefowl (Gallus lafayetii). The analyses based on the nuclear sequences revealed that alleles of grey junglefowls were alternatively clustered with those of Ceylon junglefowl and with those of red junglefowls and chicken. The alternative clustering of red junglefowl and chicken alleles were also observed. These findings taken together strongly indicate that inter-species hybridizations have occurred between grey junglefowl and red junglefowl/chicken, and between grey junglefowl and Ceylon junglefowl.

Fine mapping of a quantitative trait locus (QTL) for number of vertebrae on pig chromosome 1 During domestication, the number of vertebrae of pigs increased as selection for body composition and growth made progress. Wild boars uniformly possess 19 vertebrae (thoracic and lumber) and commercial breed pigs have 21 to 23 vertebrae. Using F2 experimental families, we detected a QTL for the number of vertebrae on chromosome 1qter (Fig. 3a). The effect of the QTL was mainly additive so that the number of vertebrae increased by approximately 0.6 per allele. To perform fine mapping of the QTL, we constructed BAC contigs spanning a 2.5 Mb region in chromosome 1 around the peak position of the QTL and developed microsatellite markers (Fig. 3b). Genotyping of these markers for 188 pigs from improved commercial breed pigs (Landrace, Large White, Duroc and Berkshire) and 26 unimproved Asian breed pigs (Jinhua, Meishan and Japanese wild boar) revealed that commercial breed pigs had remarkably low genetic variation in six markers lying adjacent to each other (red triangles in Fig. 3b) in contrast to Asian pigs which had considerable genetic variation in the markers. We sequenced a 650 kb region containing these six markers, and isolated 13 novel microsatellite markers (large triangles in Fig. 3c). Genotyping of these markers showed that 10 markers within a 250 kb region (red triangles in Fig. 3c) were almost fixed in commercial breed pigs but showed variation in Asian breed pigs. This suggests that the lack of genetic variation in commercial breed pigs can be attributed to selection for improved meat production of pigs. A mutated allele, which induced an increase in the number of vertebrae and consequently an increase in body length, was selected and spread widely. Sequence analysis showed that the two genes were located in this region. We are currently screening for SNPs to clarify QTL variation among commercial breed pigs and Asian breed pigs.

- 58 - (a) 30 QTL analysis on chromosome 1

20 -ratio F 10

0 (b) 0 50 100 150 (cM)

2.5 Mb

(c)

0 100 200 300 400 500 600 (kb)

No. of alleles and allele frequency (Top 4) Commercial 4 7 214111311332 breed pigs 0.60 0.86 0.99 1.00 0.92 1.00 1.00 1.00 0.99 1.00 1.00 0.99 0.58 0.63 (188) 0.33 0.07 <0.01 0.05 0.01 0.01 0.22 0.37 0.07 0.03 0.01 <0.01 <0.01 0.20 <0.01 0.02 0.01 Asian 7455105248284102 breed pigs 0.28 0.42 0.45 0.27 0.21 0.22 0.75 0.68 0.52 0.94 0.34 0.39 0.48 0.67 (26) 0.15 0.33 0.19 0.27 0.19 0.22 0.25 0.26 0.20 0.06 0.32 0.28 0.14 0.33 015 022 017 018 013 022 004 011 013 026 011

Fig. 3 (a) QTL analysis for the number of vertebrae on pig chromosome 1. (b) BAC contigs for a 2.5 Mb region and positions of microsatellite markers (red and black triangles). In commercial breeds, six markers (red triangles) were almost fixed. (c) Development of 13 novel markers (large triangles) in a 650 kb region by sequence analysis. The number of alleles and their corresponding frequencies in 188 commercial breed pigs and 26 Asian breed pigs are tabulated below.

Genomic organization and alternative splicing of Bombyx lipophorin receptor genes Cell surface receptors, which belong to the low-density lipoprotein receptor (LDLR) family, regulate diverse biological functions including the binding and uptake of plasma lipoproteins. Three BAC clones were used to characterize the genomic organization of a lipophorin receptor from silkworm, Bombyx mori (BmLpR).FourisoformsdesignatedasLpR1, LpR2, LpR3 and LpR4 were identified by cDNA analysis (Fig. 4). The deduced amino acid sequence of each isoform showed five functional domains that were homologous to vertebrate very low-density lipoprotein receptor (VLDLR). The BmLpR gene consisted of 16 exons separated by 15 introns spanning over 122 kb and was at least three times bigger than the

- 59 - human VLDLR gene. BmLpR1 harbored an additional twenty-seven amino acids in the O-linked sugar domain resulting in an extra exon. All four isoforms seem to have originated from a single gene by alternative splicing, and were differentially expressed in tissue- and stage-specific manner. Surprisingly, one of the isoforms, LpR4, was specifically expressed in the brain and ventral nervous system (Fig. 5). Additionally, it has a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible central nervous system (CNS)-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a CNS-specific receptor variant from a core member of the LDLR family in invertebrates.

1 2 3 4 5 6-10 1112 13 14 15 16 A

25A14 03C02

~10 kb 13C23 B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 LpR 1 I II III IV V VI VIIVIII A B C OS T CY 151 bp 1144 bp 2727 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 LpR 2&3 15 I II III IV V VI VIIVIII A B C OS T CY 151 bp 1144 bp 2646 & 2649 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 LpR 4 I II III IV V VI VIIVIII A B C OS T CY 151 bp 1719 bp 2661 bp EGF Precursor SP Ligand Binding 5’ UTR Homology 3’ UTR

Fig. 4 (a) An overview of the genomic organization of the silkworm LpR gene showing the position of exons numbered 1-16 (not to scale) and the BAC clones used for genomic characterization. (b) Exon organization and functional domains of silkworm LpR isoforms. The positions at which introns interrupt the exon region are indicated by arrowheads and the exon numbers in between. The signal sequence (SP), domains, and untranslated region (UTR) are shown for all isoforms. The five domains are indicated as cysteine-rich repeats in the ligand binding domain (I-VIII), cysteine-rich repeats in the epidermal growth factor precursor domain (A-C), O-linked sugar domain (OS), transmembrane domain (T) and cytoplasmic domain (CY).

- 60 - Fig. 5 RT-PCR expression of silkworm LpR4. 1-3: Larva, pupa and adult brain; 4-6: Larva, pupa and adult ventral nervous system; 7-9: Larva, pupa and adult fat body; 10-12: Larva, pupa and adult ovary; 13-15: Larva, pupa and adult Malpighian tubule; 16: Larval silk gland; M: DNA size markers.

Biological resources and information for the post-genome sequencing era The major challenge for rice researchers today is to piece together the information generated from the genome sequencing effort and other genome-related projects and analyze the data experimentally based on available resources. The DNA Bank and the Rice Genome Resource Center (RGRC) provide support to researchers around the world by collecting, preserving and distributing biological materials and information from genome sequencing and associated projects on rice genome analysis. In terms of biological materials, the resources that have been available to the scientific community through the DNA Bank include rice ESTs, RFLP markers, genomic clones in YAC, and the PAC/BAC clones used as template for genomesequencingfromtheRiceGenomeResearchProgram(RGP).TheRiceGenome Resource Center provides access to the rice full-length cDNA clones, Tos17 insertion mutant lines and genetic analysis materials. Additionally, as the institute continues to focus on genome analysis of other agriculturally important organisms, the DNA Bank also provides access to pig ESTs from the Animal Genome Research Program (AGP). BAC clones from the Silkworm Genome Research Program (SGP) will be available soon. The DNA Bank also continues to provide an informatics infrastracture for the maintenance of genome databases and development of tools that can be used for analyzing genome data. The major databases, homology search and analysis tools for rice such as INE (Integrated Rice Genome Explorer), RiceGAAS (Rice Genome Automated Annotation System), GLocate (HMM-based gene prediction program for rice), RiceBLAST (Rice sequence database BLAST search), RAD (Rice Annotation Database), Rice Proteome Database, PLACE (PLAnt Cis-acting Regulatory DNA Elements Database) have been widely accessed. The database PEDE (Pig EST Data Explorer), KAIKO GAAS (Kaiko Genome Automated Annotation System), KAIKO cDNA (collection of silkworm partial cDNA sequences) are also important sources of information for swine and insect genome research. TheRiceGenomeResourceCenter,whichhasbeenestablishedtosupportfunctional

- 61 - and applied genomics research in rice, is now on its second year of operation. A total of 2,552 full-length cDNA clones and 1,053 Tos17 mutant lines were distributed to researchers all over the world from April 2004 to March 2005. There were 28 requests for materials for genetic analysis including individual lines and genetic populations. The rice microarray system established by the RGRC has also provided an efficient strategy for large-scale characterization of rice genes. A 22-K oligo microarray designed from the full-length cDNA collection is now widely used by researchers from various institutes and universities within Japan. Imaging analysis using MFP-3D atomic force microscopy indicates that each DNA spot in this array is nearly circular in shape at 90 mm 5 90 mm scale (Fig. 6). Futhermore, increasing the magnification to 1mm 5 1mm showed a clear boundary between the glass surface and the DNA spot. This suggests that hybridization of target genes can be performed with high reproducibility and enhanced sensitivity. The 22K rice oligomicroarray is commercially distributed by Agilent Technologies.

Fig.6 High resolution imaging of a

80 10 DNA spot in the 22-K oligo

60 5

µm microarray. 40 0 nm

-5 20

-10

0 0 20 40 60 80 µm 90 μm  90 μm 3D image

1 μm  1 μm 3D image

4 2 0

Height -2 -4nm Glass surface DNA probe

0.0 0.5 1.0 1.5 2.0 2.5 3.0 µm

- 62 - Genome Diversity Department

The objectives of the Genetic Diversity Department are to conduct basic research into plant, animal and microorganism diversity from the molecular to the population level. Such research contributes to the development of new and improved methods for classification, characterization and preservation of germplasm and discovery of new biological resources for use in agriculture and other industries. Current plant research includes molecular and biological characterization and evaluation of the genera Hordeum, Oryza, Saccharum, Triticum, Glycine and Vigna. Mechanisms in plants that confer cold hardiness are also being investigated. Microbiological research includes genome analysis of plant pathogens, biosystematics and functional proteomics of such organisms as fungi, yeasts and bacteria. Animal research includes the development of the methods to utilize various types of germplasm and to improve the genetic performance of domestic animals. In addition, to support the MAFF Genebank project, database management systems are a focus of research and development. Major research outputs of the department except for ‘Topics of Research’ during fiscal 2004 are described below.

Hybrids between cultivated and wild soybean found in natural habitats Wild and cultivated soybeans are sympatric across much of Japan so the possibility that gene flow occurs between them is suspected. However, the soybean crop complex does not consist of an obvious weedy ecotype in Japan. Field surveys to detect introgression of cultivated soybean genes in wild soybean populations were conducted from Akita prefecture in the north to Saga prefecture in the south of Japan. In both Akita and Saga prefectures individuals were detected that were clearly hybrid derivatives between cultivated and wild soybeans (Fig. 1). The sites where these individuals occur will enable the fate of introgressed genes from cultivated into wild soybeans to be monitored in their natural habitat. In addition, in many parts of Japan wild populations were found that had some characteristics, such as smooth, shiny black seeds and large leaves, that suggested gene flow from cultivated soybeanshadoccurredatsometimeinthepast.

Fig. 1 Site where hybrids between cultivated and wild soybeans were found in Saga Prefecture, Kyushu, Japan.

- 63 - Molecular elucidation of gene flow in the soybean crop complex in Japan Using 20 SSR (microsatellite) primers, one for each linkage group in soybean, 616 individuals from 77 populations were analysed. All samples were directly collected in the field and came from all parts of Japan where wild soybeans grow, except Hokkaido. All individuals analysed had a seed size in the range of wild soybeans. The analysis revealed that about 6% of individuals were putative hybrids between cultivated and wild soybean (Fig. 2). The results suggest that gene flow between cultivated soybeans is a common phenomenon in Japan but it does not lead to the establishment of morphologically recognisable intermediate ecotypes that are commonly found in other crop complexes. The analysis revealed 7 SSR markers that showed clear allelic differentiation between wild and cultivated soybean. These markers will be useful in the monitoring of gene flow, gene persistence and gene dispersal in the soybean crop complex. Fig. 2 Distribution of individuals constituting of wild and cultivated soybean in the plane of the first two principal coordinates. The axis explained 7.4 % and 5.7 % of total variation. Samples having cultivated soybean membership as revealed by genetic admixture analysis are indicated as open triangle (P > 0.25) or open square(0.25 > P > 0.01).

Phylogenetic analysis of Oryza species based on sequence variation of organelle simple sequence repeats (SSRs) and their flanking regions SSRs and their flanking regions in the mitochondrial and chloroplast genomes were sequenced in order to reveal DNA sequence variation and clarify phylogenetic relationships among species in the genus Oryza. A total of 50 accessions of Oryza were analyzed using seven mitochondrial and five chloroplast SSR loci equal to or longer than ten mononucleotide repeats. Many base substitutions and deletions/insertions were identified in the SSR loci as well as in their flanking regions. Of mononucleotide SSR, G/C repeats were more variable than A/T repeats. Results obtained by chloroplast and mitochondrial SSR analyses showed similar phylogenetic relationships among species, although chloroplast SSR were more informative because of their higher sequence diversity. The CC genome is suggested to be the maternal parent for the two BBCC genome species (O. punctata and O. minuta) and the CCDD species O. latifolia, based on the high level of sequence conservation between the diploid CC genome species and these allotetraploid species (Fig. 3).

- 64 - Fig. 3 Schematic tree of the genus Oryza based on the maximum parsimony tree calculated from 12 organelle SSRs and their flanking sequences. The numbers in parenthesis indicate the number of accessions analyzed.

Cleistogamy genes in barley Cleistogamy, a closed type of flowering with ensured self-pollination, is an important trait to study reproduction, gene flow, and disease control. However, little is known about this character. In our genetic study, cleistogamy showed a simple monogenic inheritance in all eight crosses between closed- and open-type flowering lines. Closed-type flowering was recessive in five crosses, while it was dominant in the remaining three crosses. The result suggested two cleistogamy genes, cly1 and Cly2.Boththecly1 and Cly2 loci were mapped closely at the same region in chromosome 2HL suggesting their tight linkage (Fig. 4). In this case, at least three genotypes were given: cly1cly2 (open type), cly1Cly2 (closed type) and Cly1Cly2 (open type). Epistatic interaction of Cly1 > Cly2 was hypothesized. Alternatively, three alleles at a single gene locus were suggested. This study pointed out that barley is a good model for studying cleistogamy in cereal crops.

- 65 - e07m34-2 ABC165 1.6 MSU21 Fig. 4 e11m19-3 5.1 2.7 cly1 Linkage maps of cleistogamy genes in HvCSG 1.6 ABC153 0.3 MSU21 ABC317 1.3 e11m19-3STS chromosome 2H of barley. A: Azumamugi x Cly2 Kanto Nakate Gold RILs map including the 5.6 5.5 recessive cleistogamy gene cly1. B: Misato 0.5 e07m34-1 e07m25-8-1 ABC153 Golden x Satsuki Nijo F2 map including the ABG613 dominant cleistogamy gene Cly2.

14.7 38.5

MWG866

4.4

0.5 ABA005 MWG2200 MWG2200

A B

Agrobacterium-mediated transformation of brome- grass cultured cells Smooth bromegass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperatures or abscisic acid (ABA). Fuctional analysis of genes involved in such cold hardiness mechanisms is important. However, there has been no report or information on transformation of smooth bromegrass unlike other grass species. Agrobacterium strain EHA105 carrying a binary vector that contained NPT II, GUS and GFP genes were co-cultivated for 3 days with bromegrass cells at the late log or early stationary growth phase (7-9 day-old). Putative transformants were identified by selection for geneticin resistance and by examining GFP fluorescence. This allowed the elimination of escapes and the selection of the cells that expressed target genes. PCR and Southern blot analyses confirmed integration of GUS and GFP genes into the genome. Transformants with various levels of expression were obtained (Fig. 5). The method should allow reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass.

Fig. 5 Transformed calli (A) selected on geneticin medium exhibited GFP

- 66 - fluorescence (B) and GUS activity (C). Characteristics and infection mode of Microbacterium sp., the causal agent of red stripe of rice The phenotypic characteristics of Microbacterium sp. causative of red stripe of rice are summarized as follows. The isolate grew well at 37 ℃ and in peptone-based medium with 2% NaCl but not in 6.5% NaCl. It possessed both catalase and oxidase; did not produce mixed acids nor acetoin; failed to reduce nitrate and to produce indole from tryptophan; had no arginine dihydrolase and urease; produced H2S and hydrolyzed gelatin. It was able to assimilate a variety of carbohydrates which included sugars and organic acids, and to produce acids from sugars and sugar alcohol. While it was able to assimilate glucose and produced acids aerobically, it failed to do so anaerobically. Histopathology showed that the bacterial cells ingressed through stomata and multiplied in the intercellular spaces of substomatal parenchymatous tissues (Fig. 6). It translocated directly from parenchymatous tissues to transverse vascular systems through spiral vessel walls. Thus, the infection mode of the bacterium is very similar to that of Clavibacter spp. of the same family.

Fig. 6 Electronmicrograph showing multiplication of the Microbacterium sp. in the intercellular spaces of leaf parenchymatous tissues of rice.

Newly described species of Fusarium within the Fusarium graminearum species complex, causal pathogens of head scab of wheat and barley By the phylogenetic analyses, Fusarium graminearum, a causal pathogen of head scab of wheat and barley, was found to be a species complex, consisted of several different species. Strains of F. graminearum s. lato., from various regions of the world were taxonomically and phylogenetically re-examined in a joint study of NIAS, Japan, NCAUR and CDL, ARS/USDA and Penn State Univ., USA. Based on GCPSR, nine phylogenetic species have been elucidated among the strains examined. Based on precise phenotypic analyses, only three individual and three pairs of species could be distinguished within the complex. New species descriptions were, therefore, prepared on the species-specific DNA nucleotide positions. Consequently, in addition to F. graminearum Schwabe s. str., eight new species of Fusarium were formally described: F. austroamericanum, F. meridionale, F. boothii, F. mesoamericanum, F. acaciae-mearnsii, F. asiaticum, F. cortaderiae and F. brasilicum. Fusarium graminearum s. str. and F. asiaticum O’Donnell, T. Aoki, Kistler et Geiser (Fig. 7) were found to be main causal pathogens of head scab of wheat and barley in Japan.

- 67 - Fig. 7 Disease symptom of head scab of wheat in Japan and a causal fungus, Fusarium asiaticum O’Donnell,T.Aoki, Kistler et Geiser.

Comparative genomics of xanthomonads We performed a comparative genomic analysis of Xanthomonas oryzae pv. oryzae (Xoo), X. axonopodis pv. citri (Xac)andX. campestris pv. campsetris (Xcc), with special reference to the genes responsible for pathogenesis within the xanthomonads. The chromosomes of Xac and Xcc have a high degree of co-linearity, with the exception of three major rearrangements. When we analyzed whole genome nucleotide alignment between Xoo and Xac,theresults showed partial synteny, but numerous inversions , rearrangements and deletions (Fig. 8). As the Xoo genome harbours a greater number and variety of IS elements than those of Xac and Xcc, genome-wide inversion, rearrangement and deletion might be expected to have occurred to a greater extent in the Xoo genome. In fact, most of the synteny break-points between the Xoo and Xac genomes were bounded by IS elements in Xoo. The numerous ISs might be important in strain or race differentiation in Xoo, because mobile elements can disrupt genes, enhance recombination, and introduce mutations.

Fig. 8 MUMmer comparison analysis of the chromosomes between Xanthomonas oryzae pv. oryzae T7174 vs. X. axonopodis pv. citri 306. species. The forward matches are displayed in red, and the reverse matches in green.

- 68 - A putative transcriptional regulatorMstu1involved in control of fungal development in the rice blast fungus Magnaporthe grisea APSES proteins are fungal transcriptional regulators defined by similarities in their basic loop-helix-loop DNA binding domain, and are involved in regulating an array of functions related to fungal development, such as conidial development, filamentous growth and fertility. We have cloned the gene of a novel member of APSES proteins from M. grisea and analyzed its function using gene knockout mutants. To study functions of the Mstu1 protein, we generated gene deletion mutants of Mstu1 by homologous recombination. The mutant mstu1 showed slow mycelial growth on both oatmeal agar and 5 x YG media. In mstu1,areductionin formation of conidiophores and conidia (by 90 %) was observed. After incubating the mutant conidia on plant epidermal cells for 48 hours, a reduction in penetration was observed. Results from spray and punch inoculation assays showed mstu1 was impaired in penetration but were capable of infecting plants through wounds (Fig. 9). As with other fungal APSES mutants, mstu1 was female sterile during sexual reproduction. Taken together, our results demonstrated that Mstu1 is a novel member of APSES proteins which is indispensable for the fungal infectious life cycle of M. grisea.

Fig. 9 Results of infection assays. Left panel: Rice leaves sprayedwiththewildtypestrainorthemstu1 mutant. The mstu1 mutant greatly reduced pathogenicity toward intact rice cells. Right panel: The wild type or mstu1 conidia were placed on wounds on rice leaves. Lesions were observed in leavesinoculatedwithbothofthewildtypestrain and the mutant.

Microbial hydrogen production from bread waste Microbial hydrogen production coupled with bio-degradation of various organic wastes such as food residues is an attractive process since it can generate renewable energy and reduce wastes at the same time. Our recent work is focused on the generation of hydrogen from bread waste. In addition, the production of biogas free from sulfur, corrosive of fuel cells, is another goal in our project. The small-scale experimental system so far tested could generate sulfur-free bio-hydrogen for over 6 months by controlling the fermentation processes. The system successfully decomposed and dissolved about 80 percent of bread waste within only a quarter of the conventional processing time. We are now working on further

- 69 - development of the system towards practical application and launched a pilot-scale reactor (Fig. 10). This technology may extend to the utilization of other raw materials such as agricultural and forestry wastes in future.

Fig. 10 30L bench scale reactor for hydrogen production from bread waste.

Production of blastocysts from porcine primordial oocytes following xenografting to nude mice Primordial follicles are a store for ovarian follicles and a potential resource of oocytes for medical, agricultural, and zoological purposes. Xenografting of ovarian tissue provides a potential method for maturation of oocytes in primordial follicles (primordial oocytes) of large mammals.Theaimofthisstudywastoimprovethematurationanddevelopmental competence of porcine primary oocytes developed within xenografts by treating the host mice with follicle stimulating hormone (FSH) to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of kidneys of ovariectomized nude mice. Continuous treatment of FSH enhanced the growth of antral follicles in the xenografts. The oocytes recovered from the xenografts had the ability to mature in vitro and reached blastocysts in vitro by 7 day culture. The present results provide a possibility that a combination of xenografting and an appropriate hormone treatment of the host mice enables us to produce blastocysts in vitro from primordial oocytes of large mammals. Fig. 11 Blacstocyst formation from the porcine primordial follicles after xenografting to nude mice A: before fixation; B: after fixation and staining

- 70 - Evaluating what is estimated on the basis of the restricted best linear unbiased prediction (R-BLUP) procedure Restricted best linear unbiased prediction (R-BLUP) was derived by imposing restrictions on multiple-trait BLUP. It is an efficient method for improving fertility with keeping characteristics of an animal genetic resource population. The R-BLUP procedure estimates what are defined as restricted breeding values (RBV). The concept of RBV was represented algebraically and geometrically. The RBV based on an R-BLUP procedure imposing the same restrictions on all animals should be the same as those imposing restrictions on only some animals. The set of RBV was represented as the (q - r)-dimensional restricted hyperplane with a specific RBV represented as the point of intersection between the restricted hyperplane and the r-dimensional vector space that parallels G0C0 and passes through the breeding value (BV), where q and r are the numbers of traits and restrictions, respectively, G0 is an additive genetic variance-covariance matrix for the q traits and C0 isaq ´ r restriction matrix with full column rank. The relationships between BV and RBV for two traits are illustrated with an example (Fig. 12).

Fig. 12 Geometric interpretation of the relationship between breeding values (BV) and restricted breeding values (RBV) for two traits.

- 71 - Genebank

Genebank Project The genebank project on plant, microorganism and animal genetic resources has been implemented in national and international collaboration with a large number of organizations in public and private sectors. NIAS coordinates as a center of the project activities including the exploration, collection, characterization, evaluation, preservation, rejuvenation and documentation of genetic resources. In the fiscal year (FY) 2004, international collaborative field studies were carried out in cooperation with relevant research institutes in the collaborating countries. The project dispatched scientists to explore wild rice genetic resources in Myanmar, to do feasibility study on apples, pears, and stone fruits genetic resources in Xinjiang Uygur Autonomous Region of China, to survey wild and cultivated legumes genetic resources in Papua New Guinea, to collect and lactic acid bacteria genetic resources in China. The exploration and collection of tartary buckwheat was planned but postponed to the FY 2005. Collaborative field study program in Xinjiang Uygur will start properly from FY 2005 until FY 2007. The perilla genetic resources collected in the collaborative field study program in Korea was studied using AFLP analysis for possible conservation on farm. Twelve internal exploration missions were organized to explore different genetic resources within Japan by the NIAS and partner institutes in the project: wild sugarcane genetic resources in Kagoshima, Japanese chestnut genetic resources in Nagano, wild pear and others in Yamanashi, Nagano, and Hokkaido, wild relatives of blueberry in Hokkaido in collaboration with the United State Department of Agriculture (USDA), Akita University, and Hokkaido Central Agricultural Experiment Station, tea plants in Mie, wild soybean in Tokushima, perilla in Tokushima and Kochi, and mulberry trees in Hokkaido, filamentous fungi for possible natural enemy against pests in Kagoshima, toxic filamentous fungi in Nagano, domestic fowl germplasm in Kochi, and cattle germplasm in Yamaguchi. A total number of genetic resources preserved at the NIAS Genebank and partner institutions is presently 233,002 accessions of plant genetic resources, 21,535 accessions of microorganism genetic resources and 909 accessions of animal genetic resources. In 2003, 4,142 accessions of plant genetic resources, 703 accessions of microorganism genetic resources and 17 accessions of animal genetic resources were distributed to internal and external users for basic and applied researches. About 10 % of genetic resources were distributed overseas. Passport and characteristics data of individual accessions are provided on the website, (http://www.gene.affrc.go.jp/). The 10th International Congress for Culture Collections (ICCC-10) was jointly held at Tsukuba by the World Federation for Culture Collections (WFCC) and Japan Society for Culture Collections (JSCC) in October 2004. NIAS Genebank, which is a member of JSCC,

- 72 - held NIAS International Workshop on Genetic Resources as a symposium of the Congress, “Genetic and Functional Diversity of Agricultural Microorganisms”, and also held a training course for “Identification and Preservation of Plant Pathogens” as one of ICCC-10 training courses at NIAS Genebank. NIAS co-sponsored Worls Rice Research Conference which was held in November, 2004 and the Genebank scientists organized a concurrent session on “The genus Oryza, its diversity, evolution and utility”.

Japan-Myanmar collaborative exploration of wild rice in North and West Myanmar Under the NIAS Genebank Project, we explored Myanmar for wild rice in collaboration with the Department of Agricultural Research, Myanmar from Nov. 10th to Dec. 10th, 2004 to study their genetic diversity and to preserve them in the Genebank for future utilization. Myanmar was not surveyed systematically for wild rice especially in the northern and western areas although it has been suggested to be located in the center of diversity of rice and rich in its wild relatives. We collected 102 samples of wild rice (93 Oryza rufipogon,6O. officinalis, and 3 O. granulate). O. rufipogon, the presumed wild ancestor of rice, was often found in disturbed habitats in lakes, swamp and canals. Many populations in deep water were floating, while those in shallow water formed root systems in the bottom soil. It is noteworthy that O. rufipogon showed a larger variation within and between populations than the two distinct types reported, a taller perennial plant with low seed set and a shorter annual plant with high seed set. We observed huge that populations of tall floating wild rice in Indawgyi Lake, Kachin State had almost fertile panicles, while shorter plants in Rakhaine State showed various seed set from almost sterile to moderate.

Fig. 1 A huge floating population of O. rufipogon grew in the IndawdIyi Lake, Kachin State.

- 73 - Fig. 2 Short O. rufipogon grew in swamp by the roadside in Rakhaine State.

Pathogenic fungi causing new diseases of polygonatum and hyacinth Pathogenic fungi causing new diseases of two liliaceous flowering plants were found. One is an anamorphic fungus causing a disease on polygonatum (Polygonatum falcatum), and the other is an ascomycete causing a disease on hyacinth (Hyacinthus orientalis). The former fungus brings leaf spot, leaf blight and/or defoliation of polygonatum (Fig. 3A). Acervuli with setae of the causal fungus frequently appeared on their lesions. In culture of the fungal isolates on potato dextrose agar under black light, acervuli 40-120 μmindiameterwhich resemble to those on host lesions were formed (Fig. 3B). Conidia were falcate, smooth, hyaline, unicellular and 16-22×3-4 μminsize(Fig.3C). Appressoriaformedincultureon potato carrot agar under black light were dark grayish brown, ellipsoid to irregular and 10-16 ×6-9μm in size (Fig. 3D). The fungus was identified Colletotrichum dematium based on their morphological and cultural characters, and the disease was named anthracnose, tanso-byô in Japanese. The latter fungus causes rot of leaves, peduncles and/or flowers (Fig. 4A). The causal fungus formed perithesia, conidiophores and conidia on synthetic low nutrient agar under black light. Perithesia were dark purple to black, subglobose, ostiolate, 180-280 μmin height and 160-240 μm in width (Fig. 4B). Asci were hyaline, clavate, 60-86 μminlength, 10-12 μm in width and with 8 ascospores (Fig. 4C). Ascospores were hyaline, fusiform to allantoid, 1-3 septate and 18-26 × 2-4 μm in size (Fig. 4D). Conidiophores were hyaline, smooth, monophialidic, 4-8 μminheight,2-3μm in width and sometimes branched (Fig. 4E). Conidia were hyaline, smooth, falcate with foots at bases, 3-6 septate, 28-48 × 3-5 μm in size and not in chains (Fig. 4E). The fungus was identified Gibberella zeae [anamorph: Fusarium graminearum] based on their morphological and cultural characters. Since the

- 74 - teleomorph, G. zeae, was confirmed on not the host plant but artificial cultural media, the disease was named Fusarium rot, fuhai-byô in Japanese. The fungal isolates with information about the two new diseases are important as reference isolates for diagnosis of the diseases, and moreover useful for construction of their control measures and development of mycological studies from viewpoints of taxonomy, distribution, bionomics and parasitism.

Fig. 3 Anthracnose of polygonatum. A: natural symptom. B-D: causal fungus, Colletotrichum dematium. B: acervuli; C: conidia; D: appressoria.

Fig. 4 Fusarium rot of hyacinth. A: natural symptom. B-E: causal fungus, Gibberella zeae [anamorph: Fusarium graminearum]. B: perithesium; C: asci; D: ascospore; E: conidiophore and conidia.

Genetic Relationships among Japanese and Indonesian native breeds of chicken based on microsatellite DNA polymorphisms There are more than 30 distinctive breeds in Japan. Japan was essentially isolated from the outside world from 1635 to 1854. In that period many unique breeds were developed for special plumage, crowing and cockfighting. We analyzed the genetic relationships among

- 75 - Japanese native breeds of chicken based on microsatellite DNA polymorphisms (Takahashi et al. 1998). Native chicken in Asian country also has many varieties. There are about more than 32 distinctive breeds (species) based on phenotypic performances of native chicken in Indonesia and are being raised under extensive traditional system. However, the genetic background of native chicken in Indonesia has not been studied. The purpose of this study is to define the genetic relationships among Japanese and Indonesian native breeds of chicken based on microsatellite DNA polymorphisms. Native chicken breeds of Japan and Indonesia and one European breed (White Leghorn) were clearly separated from each other. Two big groups that correspond to Japanese native chickens and Indonesian native chickens were recognized. One Japanese breed (Small Game) that is thought to be derived from Malay-type chicken introduced into Japan from Thailand in the 16th or 17th century for cockfighting was in the middle between the Japanese and Indonesian native groups, although bootstrap values were not considered significant. Our results reflect geographical and historical background of each breed. To expand this study, manymoreAsiannativebreedsshouldbeanalyzed. So,wearecollaboratingwithresearch institutes in Asia.

Fig.5 NJ dendrograms of Japanese and Indonesian native breeds and White Leghorn based on DA distance (Nei 1983) Red: Japanese native breeds (TSA: Tosa-Kojidori, NGY: Nagoya, HNA: Hinai-Dori, STM: Satsuma-Dori, GTK: Jitokko, KNP: Kinpa, KEY: Koeyoshi, SKK: Shoukoku, ONG: Onaga-Dori, GIF: Gifu-Jidori, TTK: Toutenkou) Blue: Indonesian native breeds (ARB: Arab, STL: Sentul, KDB:Black Kedu PLG: Pelung, KPG: Kampung, MRW:Merawang) Black: White Leghorn (WLW: weak egg shell line, WLS: strong egg shell line, Nirasawa et al., 1998) Numbers on the nodes are persentage bootstrap values from 1,000 replications of resampled loci.

- 76 - A “framework” chicken linkage map for QTL mapping in a Japanese resource population Inthelastdecade,threeresearchgroups,Compton(C)group (Comptonlaboratory, Compton, UK), East Lansing (EL) group (USDA-ARS, East Lansing, USA) and Wageningen (W) group (Wageningen Agricultural University, Wageningen, NL), have published three different linkage maps of the chicken genome using different reference populations. Groenen et al. (2000) integrated these maps and reported a linkage map that was composed of 51 linkage groups and 1889 markers, including 801 microsatellites, 552 amplified fragment length polymorphism (AFLP), 244 randomly amplified polymorphic DNA (RFLP). Although 1,277 microsatellite markers and 3.1 million single nucleotide polymorphisms (SNPs) were reported, it is still relatively deficient in number markers, especially when fine mapping quantitative trait loci (QTL) and distribution is considered. To alleviate this situation, we developed new microsatellite markers and mapped them on a linkage map.

A (CA)n-enriched library of chicken was constructed using the method of Takahashi et al. (2001) and microsatellite markers were isolated. These microsatellites were prefixed with ‘ABR’(NationalInstituteofAgroBiological Resources, which is former name of National Institute of Agrobiological Sciences), and mapped using a population developed from a cross between Japanese Game and White Leghorn, which is developed in Hiroshima University. As a result, 296 markers including 193 ABR,43MCW,31ADL,22LEI,3HUJ,2GCT,1UMA and 1 ROS markers were mapped on chromosomes 1-14, 17-21, 23, 24, 26-28 and Z. In comparison with the data at the web site of the chicken draft sequence and previous genetic maps, the loci of 15 markers have not previously reported, while chromosomal loci and the order on chromosomes of all but 3 of the other markers corresponded to markers on the draft sequence. The linkage map is a framework for QTL mapping in the Hiroshima resource population. The loci of 15 ABR markers are newly mapped, because the draft sequence has many gaps. The new markers in this study can not fill the gaps in the chicken draft sequence but they may allow improvements in the assembly of the available sequence data.

The linkage map was shown in the next page.

- 77 - Fig.6 Chicken linkage map based on micro-satellite markers. （To be continued the next page）

- 78 - - 79 - Insect and Animal Sciences Division

The Insect and Animal Sciences Division consists of the following six departments. The Developmental Biology Department is conduct ｓ research to elucidate the physiological functions of germ cell lines and the hormonal effect of growth regulation, and develops embryonic technologyas well. This research focuses on several invertebrates and vertebrates. The Molecular Biology and Immunology Department is concerned with the molecular events involved in the signaling for antibacterial peptide synthesis in insects and signaling of cytokines and T cell receptors in mammals, the functional analysis of macrophage/microglia, hematopoietic stem cells and T cell subsets, the genetic mechanisms underlying complex disease traits, and the development and improvement of transgenic animal technology. Major research on cytokine signaling in the mammalian immune system, innate immune system in insect, the transformation of spermatogenic cells in vivo and genetic analysis of multi-factorial are being conducted. The Physiology and Genetic Regulation Department aims to elucidate the mechanisms of unique physiological phenomena observed in insects and animals. The studies include analyses of neural functions including brain, metabolic regulation, physiological bases for adaptation to environment, perception and function of behavior regulating chemicals, cell differentiation and the genes regulating them, in both invertebrates and vertebrates. The research activities of the Insect Genetics and Evolution Department are mainly focused on molecular and conventional genetics of insects including silkworm, biochemical analyses of insect-plant interaction, characterization and breeding of natural enemies, and basic and applied studies on insect-associated microbes including pathogens and symbiotes. The Insect Biomaterial and Technology Department is carries out research which aims to develop the biomimetic techniques of insects and their utilization such as the sugar reception of the flesh fly, and to clarify the characteristics of insect-born biomaterials for the development of new technologies. The major targets of the Insect Biotechnology and Sericology Department are the development of functional insect cell lines and transgenic insects, large-scale production of useful substances using the transgenic insect systems, and the development of new silk materials which are good for healthful and wealthy human life and which are based on the development of new sericultural technologies including breeding of characteristic new varieties of silkworms. The major research topics for 2005 from these six departments in the Insect and Animal Sciences Division are as follows:

- 80 - Developmental Biology Department

Gene silencing by RNA interference (RNAi) in the sawfly, Athalia rosae RNAi is a powerful method for gene function analysis in non-model species with little genetic background. We demonstrated that double-stranded RNA (dsRNA)-mediated interference was effective in the sawfly. Microinjection of the synthesized dsRNA targeting the sawfly white gene that encodes an ATP-binding cassette (ABC) transporter engaged in eye pigmentation caused decrease of the transcripts and resulted in the white mutant phenocopy in the embryonic eye pigmentation (Fig. 1). The effect occurred in a dose-dependent manner. Although the gene silencing activity of dsRNA was transient and not inherited by the next generation, the method serves as a tool to control gene expression especially in the early embryonic development.

Fig. 1 Effects of dsRNA-injection targeting transcripts of the sawfly white gene.

Differential nerve development during embryogenesis and regeneration in the oligochaete annelid Enchytraeus japonensis Enchytraeus japonensis reproduces asexually by dividing the body into several fragments that then regenerate to complete individuals in 4-5 days. Such large-scale regeneration, however, occurs only in some invertebrates. To better our understanding of why regeneration is so limited in many animals, despite their ability to undergo embryonic development from the single cell of a fertilized egg, comparisons were made between regeneration and embryonic development of E. japonensis using immunohistochemistry with an antibody against

- 81 - acetylated tubulin that visualizes nervous system development. The analysis revealed that the ways of central nervous system development differ between embryogenesis and the regeneration (Fig. 2), suggesting that regeneration is not a simple reiteration of embryogenesis but involves different regulatory mechanisms. The study provides a basis for the elucidation of mechanisms that are unique and crucial to regeneration.

Fig. 2 Schematic illustration of differential development of the central nervous system during embryogenesis and regeneration in E. japonensis. In embryogenesis, anlagen of the brain (b, orange) and ventral nerve cord (vnc, green) appear first, then the circumesophageal connectives (cec, yellow) develop; whereas in regeneration, the ventral nerve cord and circumesophageal connectives develop first and the brain appears last.

Trace of donor primordial germ cells in recipient embryos Primordial germ cells (PGCs) are the progenitor cells of spermatozoa or ova. In chickens, PGCs or their precursors are mostly scattered in the central disc of the area pellucida and located on the ventral surface of the epiblast at stage X. They translocate gradually to the dorsal side of the hypoblast at stages XI-XIV and are then carried anteriorly to the germinal crescent region. When the blood vascular system develops, PGCs enter the blood vessels and circulate temporarily throughout the blood vascular system, finally migrating to the germinal ridges and differentiating to spermatogonia in the testes or oogonia in the ovary. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-day incubated embryos (Fig. 3). Freshly collected primordial germ cells were also transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism

- 82 - in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-day incubated embryos by PCR (Fig. 4). This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.

Fig. 3 Expression of GFP gene in the gonads of 6-day incubated embryos. GFP-positive cells are scattered throughout the right and left gonads of embryos.

Fig. 4 PCR analysis of gonads of chimaeric chicken embryos. W: WL (positive control), B: BPR (positive control), N: negative control, B: blood cells, G: gonads

Higher expression of human DAF in transgenic cloned pigs Because organs of pigs are biologically and anatomically similar to human organs, porcine organs are expected to use as replacement for human organs, which are chronically in short supply. The major problem with xenotransplantation of porcine organs into humans is the hyperacute rejection caused by the reactions involving natural human antibodies and the complement system. It is considered that natural antibodies against 1,3Gal epitope on pig cells is the main cause of hyperacute rejection. Recently, the overseas companies succeeded to produce 1,3GT knocked out pigs by somatic cell cloning for elimination of 1,3Gal epitope.

- 83 - The kidney and heart from 1,3GT deficient pigs were transplanted into baboons, however, the survival period of organs was limited against expectations. To gain longer survival period after pig-to-primate transplantation, the additive effects by combination with other molecules will be necessary. One of the candidate molecules for extension of survival in xenotransplantation is human decay accelerating factor (hDAF). The hDAF is one of the human complement regulatory proteins. The longest survival of pig organs expressed hDAF in baboons was longer than 3 months, however, there were many rejections and long-term survival cannot be expected. There is a possibility that the survival period after transplantation depends on the level of hDAF expression, because the conventional transgenic pigs show only 1to 3 times higher expression than that in human. Increasing the level of expression of hDAF in transgenic pigs is expected for longer survival. In the present study, we separated and collected hDAF cells with high expression level by FACS (fluorescent-activated cell sorting) after gene transfer. The separated cells were cultured and used as a nucleus donor. 1711 of nuclear transferred embryos at 2-8 cell stage were transferred to 9 synchronized recipient pigs, and one recipient was delivered of two piglets (Fig.5). Both piglets showed more than 30 times higher expression of hDAF than that in human by FACS analysis (Fig.6). The cells from the transgenic pigs suppressed human complement enough in in vitro analysis. Such a high level of hDAF in transgenic pigs was never reported before. A combination of FACS with nuclear transfer has much superiority to produce transgenic pigs with desired expression level of gene.

Fig. 5 Transgenic cloned piglets expressing hDAF.

Fig. 6

Non-transgenic Comparison of hDAF expression by Transgenic pig cells pig cells FACS analysis. Human cells (HAEC)）

Human cells （HUVEC）

- 84 - Regulation of Silkworm Larval Development The regulatory mechanisms on insect development, such as ecdysis and metamorphosis are being studied on the silkworm at Insect Growth Regulation Laboratory. Recent progresses are as follows; (1) Occurrence of phosphorylation of MAP kinase by ten minutes incubation of silkworm cell line with a JH analog, methoprene, suggested the existence of the membrane receptor. We generated transgenic silkworms that overexpress JH esterase, and a precocious transformation was induced by the overexpression (Fig.7). (2) A new prothoracicostatic factor, Bommo-myosuppressin (BMS) and a specific receptor for BMS were identified in Bombyx mori. Strong prpthoracicostatic activity of BMS and high expression of BMS receptor in the prothoracic glands suggested that BMS functions as a prothoracicostatic hormone and plays an important role in controlling insect development. (3) Three juvenile hormone inducible genes were identified from a silkworm cell line using cDNA microarray. The ecdysone inactivating enzyme was identified from an entomogenous fungus, Nomuraea rileyi. (4) Mass spectrometry of proteins from all organs of the silkworm was proceeded, an establishment of the database is under construction. A series of proteins with the small amount were also applied for TOF-MS to be identified. (5) The cell number and polyploid level in silkworm dorsal epidermis of 5th segment from 3rd instar to pupa were measured by flowcytometer. The 8C cells became the most abundant in 3rd and 4th instar but the 16C cells became major in 5th instar. The cell number increased gradually during feeding period and decreased during molt. This decrease of epidermal cell number during molt might not be caused by apoptosis because of little caspase-3 activity. Relationships between ecdysteroid titer and ploidy of epidermis in the penultimate stadium larvae of the common armyworm, Pseudaletia separata, which classified into Day2 and Day3 type. Ploidy of Day2 type increased when ecdisteroid titer was rising, but, that of Day3 type increased before rise of ecdysteroid titer. This result suggested that ploidy of epidermis is not affected by rise of ecdysteroid titer directly.

Fig.7 Pupae of the ransgenic line precociously metamorphosed after 3rd instar with JHE-overexpression (right) and of the wild-type normally metamorphosed after 5th instar (left).

- 85 - New member of placental prolactin family protein: bovine prolactin-related protein VI (PRP-VI) Placenta produces an array of proteins during the gestation. Some of these proteins those have structural and functional similarities to pituitary prolactin (PRL) are called the placental PRL family proteins. Of these proteins, placental lactogen (PL) is commonly expressed across the species. However, in cows the member of placental PRL family expanded. Bovine PL, which has PRL-like bioactivity, have been known as a classical member, whereas other PRL-related proteins (PRP-I to -VI) are categorized as non-classical members of placental PRL family. We identified a new member of bovine PRP (PRP-VII) for the first time in thirteen years following the last reporting of PRP-VI. Cloned PRP-VII cDNA sequence contains a 929-nucleotide open reading frame. The cording sequence was composed of 717 nucleotides corresponding to the protein of 238 amino acid residues. The deduced amino acid sequence of PRP-VII was 63% homologous to that of PRP-I and 36% homologous to that of PL. The PRP-VII had a consensus sequence of N-linked glycosylation, Asn-X-Ser/Thr at the position of 60-62 amino acid residues. We submitted this sequence to the DNA Data Bank of Japan (DDBJ/GenBank Accession # AB187564). RT-PCR analysis revealed that the PRP-VII expressed only in the placenta. The results of quantitative real-time RT-PCR analyses showed that the expression of PRP-VII mRNA commenced before Day 27 and remained beyond Day 250 of gestation. The localization of PRP-VII message was found in trophoblast binucleate cells in Day 60 placentome by using in situ hybridization (Fig. 8). Recombinant PRP-VII protein was generated in HEK 293 cells as 29 kDa protein (Fig. 9). We identified PRP-VII, a new member of bovine PRL family protein from placental tissues. Although eight bovine PRL family genes have been reported, the biological functions of these proteins are almost unknown. In rodents, some non-classical member of PRL family protein interacts with immune-associating cells resulting in the attenuation of local immune responses at the feto-maternal interface. However in bovine, the role of PRL family protein on the immune system remains to be elucidated. In conclusion we added a new member of bovine PRL family gene. It has been shown that this gene was successfully translated in mammalian cells to express mature secreted protein. The PRP-VII is a new candidate protein for bovine implantation.

- 86 - Fig. 8 In situ hybridization of PRP-VII, PRP-I and PL. Scale bar =100μm on the upper and 20μm on the bottom.

er rk a la . M W C. Fig. 9 Recombinant PRP-VII . PRP-VII PRP-I PL-A M b b b N.

(A) 37 kDa expressed in HEK 293 cells. Anti-FLAG

25 kDa

(B) 37 kDa Anti-PRP-I

25 kDa

(C) 37 kDa Anti-PL

25 kDa

- 87 - Molecular Biology and Immunology Department

Animal and cell culture models for the study of mammalian immune system T cell receptor (TCR) signaling induces production of interleukin 2 (IL-2), accompanying with cytoskeletal rearrangement in these cells. Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for the Wiskott-Aldrich immunodeficiency syndrome, acts as an adaptor molecule in TCR signaling. To clarify the functional roles of the EVH1 domain of WASP in signaling pathway in T cells, we employ an intrabody strategy in which functions of the WASP-EVH1 domain are knocked down by single-chain antibodies (scFvs). When anti-WASP-EVH1 intrabody was expressed in the cytosol of T cells, the binding of the scFv intrabody to WASP resulted in inhibition of IL-2 synthesis upon TCR activation. Functional knockdown of WASP-EVH1 domain in TCR signaling was subsequently confirmed in scFv-transgenic mouse model, suggesting that the intrabody technology provides a powerful tool to study cellular functions in mammalian immune system. Microglia have important roles in development, homeostasis and pathogenesis in the brain. Microglia can respond to various extracellular signals such as neuronal injury or infection, and secrete several proteases, neurotrophic factors and various cytokines. Microglia support neuronal repair, yet in some pathological conditions, overactivated microglia produce neurotoxic substances and induce neuronal cell death. To understand the regulatory mechanisms of microglia in the brain, we have established several immortalized microglial cell lines from transgenic and gene-knockout mice by a retroviral vector containing human c-myc oncogene. Microglia established from prion protein-overexpressed transgenic mice were susceptible to the infection with several mouse-adapted scrapie strains, such as Chandler and ME7. Prions were persistently replicated in these microglia, suggesting that these cells provide a useful in vitro model system for prion research. In addition, we have established immortalized cell lines from fetal bovine brain tissue by transfecting SV40T antigen construct. These cell lines showed several characteristics of brain endothelial cells (Fig.1). Susceptibility of these bovine cell lines to the infection with BSE prion is now investigated.

- 88 - Fig.1 ABEndothelial characteristics of fetal bovine brain- derived cells immortalized by transfecting SV40T antigen construct. (A) Cobblestone morphology of the cells. (B) Expression of SV40T antigen by immunostaining. (C) Capillary-like tube CDformation on Matrigel. (D) Uptake of fluorescent dye-labeled low density lipoprotein. Bar=100 mm

Innate Immune System in Insect An antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was detected in the digestive juice of B. mori larvae. An antiviral protein having molecular mass of 24271 Da was purified from the digestive juice (Fig. 2). Partial N-terminal amino acid sequence of the protein was determined and cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 94% identity with B. mori serine protease so the protein was designated B. mori serine protease-2 (BmSP-2). Analysis of BmSP-2 gene expression showed that this gene is expressed in the midgut but not in the other tissues. In addition, BmSP-2 gen was shown to not be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that BmSP-2, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection. A cDNA encoding a Rel/NF-kB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reaction (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-kB homologue was designated A. dichotoma (A.d.) Rel A. The amino acid sequence of the of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% homology with Tribollum castanneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A was confirmed to bind specifically to the

- 89 - NF-kB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. TheactivityofA.d.RelAinmodulatingageneconstructoftheA.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing mutated NF-kB site, suggesting the importance of the interaction between the NF-kB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma. Fig. 2 Final HPLC purification profile

BmSP-2 100 of BmSP-2. BmSP-2 was eluted 0.3 with 49.5 acetonitrile/0.05% TFA by reverse-phase HPLC. The 80 arrow indicates the position of BmSP-2. 0.2 60

Absorbance at 280 nm 280 at Absorbance 40

0.1 (%) concentration Acetonitrile

20

0 0 51015 20 25 30 Retention time (min)

Genetic analysis of neonatal death with growth retardation in mice

Most F1-Dh/+ males, crosses between inbred DDD strain females and DH-Dh/+ males, die during the neonatal period. Affected F1-Dh/+ males exhibit a typical symptom of growth retardation (Fig. 3), with problems of urination and defecation. This seems to be caused by a rectovesical fistula formed between the rectum and the urinary bladder in conjunction with an imperforate anus (Fig. 4). The lethal phenotype does not occur in the reciprocal cross between DH-Dh/+ females and DDD males. Genetic mapping analysis of the loci underlying the lethality has revealed that the lethality is a consequence of synergism among three independent gene loci; that is, the DH allele at Dh locus on chromosome 1, the DDD allele at the Grdhq1 locus on the X chromosome, and a putative Y-linked locus in some inbred strains. With regard to the Y-linked locus, among the M. m. musculus Y, Y chromosomes from C57BL/6J and BALB/cA caused lethality, but Y chromosomes from C3H/HeJ and CAST/EiJ did

- 90 - not. In addition, among the M. m. domesticus Y, Y chromosomes from AKR/J, DDD, SJL/J, SWR/J, and TIRANO/EiJ caused lethality, but the Y chromosome from RF/J did not. Based on these results, it is possible to conclude that two distinct Y chromosomes coexist across the inbred mouse strains.

Fig.4 Rectovesical fistula: Inward view of vertically dissected specimen after Bouin's fixation.

Fig.3 Comparison of body size between growth -retarded Dh/+ (the lowermouse, 1.89 g) and normal littermate +/+ (3.79 g).

- 91 - P hysiology and Genetic Regulation Department

Odor coding by group of neurons in insect brain It has been thought that olfactory processing function in the insect antennal lobe (AL) is mediated by dynamic modulation of synchronized firing in groups of neurons. We electrophysiologically recorded spikes from small cluster of neurons and local field potentials (LFPs) recorded as sum of neural activity from AL and mushroom body (MB) simultaneously. Synchronization between spikes of AL neurons and LFPs in AL and MB observed odor-specifically. Odor-specific evoked spikes of presumptive AL projection neuron (PN) recorded in a fixed phase of the oscillation in the LFP of the AL. Similarly, odor-evoked spikes of the PN showed a fixed phase relationship with activities of the LFP of MB. These results indicate that various functional synaptic connections are formed in a group of neighboring AL neurons depending on odor quality. A group of PNs may encode information of specific odor by sending their synchronized spikes from AL to MB.

Fig. 1 Simultaneous recording of PN spikes (upper) and LFP in the MB (lower) during odor stimulation. Arrows indicate temporal correlation between the spikes and the synaptic activities.

Outbreaks of the migratory locust Locusta migratoria (Orthoptera: Acrididae) and control in China Outbreaks of the migratory locusts, Locusta migratoria L., occurred in the northwestern part of China. We investigated the processes leading to the outbreaks and successful control based on locusts collected in infested areas and information obtained from local experts and scientists. Swarms of adults landed on grazing and farming lands of Jiminay County, Altay Prefecture, Xingjian Uygur Autonomous Region in 2003 and 2004. In the spring of 2004, numerous nymphs that had developed black patterns appeared in Jiminay. They and migrants from north were sprayed with insecticides during the period from July 20 to August 6, 2004. As a result, further damage to crops and migration to other areas were prevented successfully. Morphometric measurements indicated that the migrants had reached the body dimentions

- 92 - typical for gregarious forms, i.e. the mean value of hind femur length / maximum head width ratio was 3.03 in females and 3.10 in males, and the mean value of elytron length / hind femure length ratios was 2.14 in females and 2.10 in males. In the laboratory, crowding alone failedtoinducegregariousmorphologyobservedinthefield.Weareinterestedinmaking ‘gregarious locusts’ in the laboratory.

Solitary phase Gregarious phase

Fig. 2 Typical pronotum shape of solitary and gregarious phase adults. Pronotum of solitary phase adults are convex whereas gregarious phase adults are concave.

Sterol nutrition of silkworms Sterol is an essential nutrient for insects. We had already shown that Samia cynthia ricini could grow in less dietary sterol than Bombyx mori and that sterol content of pupa was higher in Samia than Bombyx. Further analysis of sterol content of larval tissues of silkworms was carried out. As shown in Fig.3, sterol contents of midgut increased with amount of dietary sterol. Sterol contents of fat body were less than midgut, and sterol contents in Bombyx were less than Samia. These results indicate that sterol ingested by larva might be accumulated in midgut and then bring to pupa in Samia but not in Bombyx, and that sterol accumulation ability of Samia might be concerned with low nutritional requirement of sterol.

Fig. 3 Sterol contents of midgut and fatbody in two silkworms of Bombyx mori and Samia cynthia ricini

- 93 - Preparation of single-enantiomer semiochemicals using MαNP acid and M9PP acid A semiochemical that conveys information between the same species is called as pheromone. Various relationships have been reported between the chirality of insect pheromones and their biological activity. Therefore, enantiopure compounds are necessary for evaluating the biological activity of each enantiomer. MαNP acid and M9PP acid (Fig. 4) are useful for preparing enantiopure alcohols, because of their susceptivity to chiral separation and the advantage of not racemizing during condensation reactions and HPLC separation. MαNP acid and M9PP acid are also useful for determining the absolute configuration of secondary alcohols using the 1H-NMR anisotropy method.

Fig.4 Structures of novel chiral resolving agents.

MαNP acid and M9PP acid are recyclable, effective for HPLC separation and 1H-NMR anisotropy: (1) both the acids could resolve the alcohol enantiomers via esterification; (2) successive NMR analyses assigned the stereochemistry of MαNP and M9PP esters; (3) solvolysis of the esters yielded enantiopure alcohols and acids. The MαNP and M9PP moieties caused similar 1H-NMR anisotropy in the alcohol part of the esters. The chiral resolving ability of M9PP acid was slightly superior to that of MαNP acid in HPLC. Clearly, enantioresolution using MαNP acid and M9PP acid is useful for the small-scale synthesis of natural products and the preparation of single enantiomer agrochemicals and pharmaceuticals.

Visual and olfactory cues for mate orientation behavior in male white-spotted longicorn beetle, Anoplophora malasiaca Olfactory and visual cues were shown to mediate short-distance orientation in Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae). In a laboratory test, more than 90% (n = 42) of males walked straight upward when presented with an untreated surface with a 75° slope. When a freshly killed female was fixed at a short distance (10 cm ahead and 5 cm to left/right) from the starting point, 50% of males (n = 30) were oriented toward the female before direct contact (Fig. 5). Similar behavioral responses were observed when female extract was directly applied to the slope or to a glass rod model fixed on the slope. When black, white, and transparent colored rods with the extract were presented, the orientation response was significantly greater for black than to white

- 94 - and transparent rods, to which only a negligible response was observed.

Fig. 5 Percentages of orientation by male A. malasiaca on black, white, and transparent models treated with female extract (n = 20, Dose: 0.5 FE/model). Values were analyzed by a6x2χ2-test with Yates correction, and then compared by paired Fisher's exact test. Columns with the same letters are not significantly different at p = 0.01 (Fisher's exact test).

Lead nitrate-induced hypercholesterolemia in rats and mice Lead nitrate (LN) has been reported to induce development of hypercholesterolemia in rats. However, a mechanism for the development of hyperchoresterolemia remains unclear. Furthermore, species-difference in a heavy metal-induced toxicity is often observed. In the present study, we examined a species-difference between rats and mice in LN-induced hypercholesterolemia, and to further clarify a mechanism for the LN-induced hypercholesterolemia, wealso examined effects of LN on the gene expressions of hepatic cholesterogenic enzymes including HMG-CoA reductase (HMGR), a rate-limiting enzyme for cholesterol biosynthesis, in rats and mice. Level of total serum cholesterol was significantly increased at 12 h and 24 h after treatment with LN (100 ?mol/kg body weight, i.v.) in rats and mice, respectively, and the increased level was maintained up to 48 h (Fig. 6A). Gene expression level of hepatic HMGR markedly increased at 6-24 h after the LN-treatment in rats and mice (Fig. 6B). Likewise, hepatic gene expression levels of other cholesterogenic enzymes including squalene synthase and CYP51 also increased at 6-12 h. Further, hepatic gene expression of sterol regulatory element binding protein-2 (SREBP-2), a common transcription factor of cholesterogenic genes, increased at 6-12 h after the LN-treatment. The present findings demonstrated that there is no species-difference between rats and mice in LN-induced hypercholesterolemia and further suggested that the LN-induced hypercholesterolemia occurred through increases in gene expressions of SREBP-2, a transcription factor for cholesterogenic genes, and cholesterogenic enzymes including HMGR, a rate-limiting enzyme for cholesterol biosynthesis. Moreover, a system of LN-induced hypercholesterolemia would be useful for discoveringy of the drug and food showing anti-hypercholesterolemia effect.

- 95 - Fig. 6 Changes in level of total serum cholesterol (A) and hepatic gene expression of HMGR (B) in rats and mice.

Reconstruction of a Hard Connective Tissue utilizing a Pressed Silk Sheet as a Scaffold of Fibroblasts Various types of meshwork-incorporated scaffolds have been utilized for reconstructing organoids possessing the framework of a three-dimensional architecture with the mechanical strength, however conventional scaffolds are insufficient to reconstruct hard connective tissues such as ligaments and tendons. In this study, we noticed a pressed silk sheet prepared by adding the moisture to cocoon filaments, pressing them at high temperature of 120oC, and agglutinating the sericin of them because the silk thread has excellent properties in mechanical strength, safety for transplantation into human body, and water absorption. The observation of the pressed silk sheet by a phase-contrast microscope revealed that the diameter of each cocoon filament was about 15 micro-meter and the filaments formed meshworks with pore size axes of shorter than 200 micro-meter (Fig. 7-1). The mechanical strength of the silk sheet by measuring a maximum elastic force is about 4 times stronger than that of the cotton mesh. The scaffold composed of a silk sheet-embedded thin collagen gel membrane was prepared and fibroblasts were seeded on the both surfaces of the scaffold. Fibroblasts proliferated well on the scaffold and formed multilayers in the presence of ascorbic acid 2-phosphate, and some cells invaded into the collagen gel membrane framed by the meshwork of numerous strong silk filaments (Fig. 7-2). These data demonstrated the reconstruction of a hard connective tissue model.

- 96 - Fig. 7-1 Phase-contrast microphotographs of a pressed silk sheet. Fig. 7-2 Histological observations of a hard connective tissue model in low (A) and high (B) magnifications.

Electrical activity of the hippocampus in Landrace piglets Thehippocampusisknowntoplayakeyroleinmemorystorageandconsolidation following learning. However, neural activities of the hippocampus are not ascertained in pigs during behavioral tasks. The electrical activity of the hippocampus was therefore measured by radiotelemetry using male Landrace piglets, for studying relationships between neural functions in the hippocampus and the behavioral changes. During rest, slow waves with frequencies less than 10 Hz were often dominant in the electrical activity of the hippocampus. The amplitude was less than 200 V (Fig. 8). During rooting behavior, the electrical activity of the hippocampus was successfully recorded, but the slow waves were still observed. Temporal changes in the electrical activity are analyzed to reveal unique neural activities of the hippocampus in the rooting behavior, and then in operant conditioning tasks of the piglets.

Fig. 8 Electrical activity recorded by radiotelemetry in the left temporal hippocampus (A) when a piglet was at rest. A power spectrum by a Fast Fourier Transformation (FFT) algorithm analysis of (A) is shown in (B). Large peaks in the spectrum are present in the frequencies less than 10 Hz.

- 97 - Central melanocortin receptors are involved in the control of reproduction in goats Central melanocortin receptors, such as melanocoritn-3 (MC3-R) and -4 (MC4-R) receptors, and their endogenous agonist, α-melanocyte-stimulating hormone (α-MSH), and antagonist, agouti-related protein (AGRP), have recently become the focus of attention due to their profound influence upon feeding behavior. Because α-MSH, AGRP and melanocortin receptors are located in brain areas implicated in the regulation of gonadotropin releasing hormone (GnRH) secretion, we hypothesized that the central melanocortin system is also involved in the control of the GnRH pulse generator, an important center for reproduction. The GnRH pulse generator activity was electrophysiologically assessed at the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus of ovariectomized (OVX) goats. Any doses of MT II, a pharmacological MC3/4-R agonist, injected into the lateral ventricle significantly shortened MUA volley intervals. MT II-induced acceleration of MUA volley was partially blocked by pre-administration of SHU9119, an MC3/4-R antagonist. These results clearly demonstrate that the MC3/4-R is involved in maintaining the GnRH pulse generator activity in goats. The present findings also suggest that feeding behavior and reproductive function are simultaneously regulated by common neural substrates such as the melanocortin system.

Fig. 9 Percent changes (means ± SEM) in MUA volley intervals during 3 hrs after MT II, SHU9119, MT II with pre-treatment of SHU9119 or saline intracerebroventricular injection in OVX goats (n=4). Values with different letters are significantly different from each other (P<0.05). Figure is adapted from Matsuyama et al. (2005), Nuerosci Lett, 383, 289-294.

- 98 - I nsect Genetics and Evolution Department

The research activities of our department are mainly focused on molecular and conventional genetics of insects including the silkworm, biochemical analyses of insect-plant interaction, characterization and breeding of natural enemies, and basic and applied studies on insect-associated microbes, including pathogens and symbiotes.

Molecular and evolutional analyses of insect Organdy3.0 is likely to be an autonomous copy of Bombyx MITE-like transposon, Organdy. Identical Organdy3.0 copie weres isolated from three different sites. To elucidate genetic variation between regional strains of Bombyx mori,weisolatedDNA fragments that contain og gene encoding molybdenum cofactor sulfurase from 32 regional strains and analyzed these sequences. As result, we confirmed that genetic variation in og DNA sequences were classfield in the like groups. A magnitude of variation in og gene is larger than that found in Chicken genome wide survey. Phylogeographic analyses of chafer beetle were performed using nucleotide sequences of a 3 kb portion of the mitochondrial DNA obtained from the sugarcane green chafer beetle collected in the Ryukyu Islands and adjacent regions. The results indicated that individuals were roughly separated from local populations and that the distribution areas of several populations have expanded considerably in the recent years. In the brown planthopper, chemical induced carboxyl esterase (CE) level does not always correlate with the resistance level to insecticide, but even in such races, pre-induced level of CE seems to have some relations with resistance. We found that a 1-bp deletion in the Bmwh3 gene, an ABC transporter gene, causes white eggs, white eyes and translucent larval skin in w-3(oe) mutants.

Genetics and evaluation of silkworm stocks A total of 248 silkworm breeding stocks were maintained and characterized. On the home page of NIAS, data-base of silkworm breeding stocks is linked and opened to general use. In the study of intersex mutant of the silkworm, Bombyx mori,itwasfoundthatthe degree of intersex phenotype was able to enhance or reduce by phenotypic selection(Fig.1). To clone intersex gene of B. mori, a DNA fragment has been cloned by genetic subtraction. The similar sequence of the fragment was only found from BAC clones linking W chromosome

- 99 - which determins female character. The software, GENESCAN, has shown prospected exon-intron structure on the fragment. We could not show any relations between the tolerability of long-term storage tested previously and the depth of embryonic diapauses of B. mori. by correlation analysis.

Fig.1 Phenotype variation of intersex moths (Isx)ofBombyx mori. Normal female and male moths have seven and eight abdominal segments with scales, respectively. Intersex moths have the eighth-abdominal segment in various degrees between female and male.

Insect-plant interactions Previously we found that papain and ficin, the cysteine proteases in latex of papaya trees (Carica papaya) and wild fig trees (Ficus virgata), respectively, play crucial roles in the defenses of these plants against herbivorous lepidopteran larvae (see Annual Report 2004), we supposed that latex ingredients of other latex-exuding plants also have similar defensive roles. In order to investigate this possibility, we performed bioassays examining toxicities of leaves and latex of mulberry trees (Morus spp.) on larvae of Eri silkmoth (Samia ricini, Saturniidae) and cabbage moth (Mamestra brassicae, Noctuidae). When intact mulberry leaves were fed to the larvae, all the larvae died within 4 days without any increase in weight. However, larvae that were fed leaves that were cut into narrow strips and then washed with water to eliminate latex grew far better. Artificial diets containing mulberry latex also showed toxicities to these insects. These results indicate that latex play a defensive role again in mulberry trees against insect herbivory as in papaya and fig trees. We are now investigating the toxic factors in mulberry latex, and obtaining preliminary results indicating that certain inhibitors of metabolic enzymes in mulberry latex contribute to the toxicity of mulberry latex against caterpillars.

- 100 - Fig.2 A method for evaluation of resistance in rice to GRH. First-instar nymphs were released in a seedling in each test tube. Mortality counts were taken 3 days after insect release.

The green rice leafhopper (GRH), Nephotettix cincticeps has a laccase-type phenoloxidase (EC 1.10.3.2, p-diphenol:oxygen oxidoreductase) in salivary glands and discharges it as saliva during feeding. The laccase-type phenoloxidase is considered to play a role in coagulating salivary sheath and in detoxification of some phenolic substances in plants. To facilitate studies of the property and function of salivary laccase, we have cloned a cDNA for laccase from the salivary glands of GRH. The cDNA encodes a protein of 792 amino acids and, it contains a putative signal sequence, conserved histidines and cysteine that were necessary for binding copper. We have started to isolate insect resistant genes from rice a using map-based cloning method. The genomic region of a GRH resistant gene derived from ‘Nona Bokra’ was confirmedonchromosome5byexaminingthemortalityofnymphsonasetofKoshihikari/ Nona Bokra Chromosome Segment Substitution Lines (CSSL). The resistant gene was mapped within about 370 kb interval between two DNA markers near the centromere of chromosome 5 by the linkage analysis using F2 202 individuals.

Natural enemies In this financial year, we investigated the olfactory responses of two local populations of a predatory mite, Neoseiulus womersleyi (Acari: Phytoseiidae), collected from tea fields to the volatiles of tea plants infested with Tetranychus kanzawai (Acari: Tetranychidae). Chemical analysis showed that tea plants emitted five major herbivore-induced volatile compounds; 3-Hexen-1-ol acetate, (E)-β-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene, methyl salicylate, and a-farnesene, after being infested with T. kanzawai (Fig.3). Individuals of one population showed the significant preference for the volatiles of tea plants, whereas those of another population did not. The olfactory responses of each population to the volatiles of tea plant coincided with the responses to the volatiles of kidney bean plants. Also, we investigated the species composition of pirate bugs Orius spp. (Heteroptera:

- 101 - Anthocoridae) in a greenhouse where the commercial strain of O. strigicollis was used and the vicinity by multiplex-PCR as a species identification method. The results showed that the species composition of Orius spp. in the vicinity was not influenced by the releases. In addition, comparison of microsatellite markers of O. strigicollis between the greenhouse and the vicinity populations showed the existence of genetic difference between them, indicating relatively small number of released O. strigicollis escaped from the greenhouse.

Fig.3 Emission of volatiles by tea plants infested with 5 adult females of Tetranychus kanzawai for1,3,5,10,and24days.

Symbiotes Insect genomics cDNA clones, which were made from various tissues of the brown planthopper Nilaparvata lugens, were analyzed and 35,000 ESTs were totally accumulated. ESTs from the various tissues revealed many tissue-specifically expressed genes. The total ESTs were grouped into about 12,000 clusters. Oligonucleotide microarray, holding 22,000 probes, were made based on the clustering data, and were used for the gene expression analyses of the planthopper. Using the whole genome shotgun sequence database of silkworm Bombxy mori,the genes encoding JH biosynthetic enzymes were searched. Eight out of nine early JH biosynthetic pathway or mevalonate pathway genes were fully or partially identified in the database. Three copies of farnesyl diphospate synthase (FPS1-3) were found in the Bombyx genome. The expression level of FPS3 was almost specific to the corpora allata (CA), suggesting FPS3 works for JH synthesis. Six homologs of juvenile hormone acid

- 102 - methyltransferase (JHAMT), the final step enzyme in JH biosynthetic pathway, were also found in the Bombyx genome. These homologs were expressed in the peripheral tissues such as epidermis, testis and ovaries, but not in the CA, suggesting that the JHAMT homologs are involved in the regulation of JH titer in the peripheral tissues. Since FPS and JHAMT are single genes in the genome of Drosophila melanogaster, the presence of multiple FPS and JHAMT genes in B. mori indicates the presence of more complex regulation mechanisms of JH titer in B. mori.

Insect cellulases In order to obtained large amount of highly active termite endogenous cellulases (endoglucanases, EGs, in the glucoside-hydrolase family 9), we performed family shufflings (random homologous recombination) of four different termite EG cDNAs from Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus and C. acinaciformis.A chimerical cDNA library was made and clones adapted for over-expression in E. coli were screened. Selected clones were subcloned into an over-expression plasmid vector (pQE30) for second screening. One clone, after a His-tag affinity purification, showed enhanced expression in the bacteria. Thus, a modified cDNA adapted for over-expression in E. coli by a family shuffling combining only with cDNAs of eukaryotic origins.

Insect microorganisms & arthropod vectors Culicoides vectors Cattle often suffer with virus disease transmitted by haematophagous midges (Diptera, Ceratopogonidae). Life cycle and phylogenetic status of culicoides vectors are poorly understood and classification of species is complex. ITS sequences of rRNA genes were determined from various species for molecular discrimination of species and strains, and some species-specific sequences were obtained. Monitoring of occurrence of culicoides in Kagoshima and Okinawa by light trap revealed that Culicoides oxystoma, C. maculats and C. punctatus were dominant species.

Bacteria and endosymbiotes Enterobacter cloacae is often found in the gut of insects. We have identified two genes related with colonization of E. cloacae in the silkworm midgut. The two genes showed high identity with ManC and WaaL, respectively, which are involved in biosynthesis of lipo-polysaccharide. Themulberryleafhopper,Erythroneura mori, is a newly found vector of micoplasma

- 103 - disease of mulberry tree. Internal microflora of the pathogen vector, were investigated by determining sequences of 16S rRNA genes amplified from the leafhopper. E. mori harbored two intracellular symbiotes, Wolbachia and Rickettsia. Intracellular bacteria that cause reproductive alteration on insects and mite hosts were cultivated in the cell lines of silkworm. Interactions between Wolbachia and host cells and between CFB (Cytophaga-Flavobacterium-Bacteroides) bacteria and the host cells were studied in gene expression level by silkworm microarrays. Similar results are observed in two different arrays, cDNA microarray and 60-mer oligoarray. Wolbachia did not up-regulated any genes of host cells but CFB did some of genes related to host immune system.

Dicistroviruses Translation initiation mediated by the internal ribosome entry site (IRES) of dicistroviruses does not require initiator Met-tRNA. This suggests the possibility that mRNAs of eukaryotes might have more extensive coding regions than previously predicted, because the IRES elements determine the translation initiation site by virtue of their own tertiary structure formation. To test this hypothesis, we searched DNA databases using the pattern-matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of the IRES. Our search yielded several sequences, but their structures were suggested to be unstable in comparison to those of dicistroviruses, indicating that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator Met-tRNA, their structures are likely to be significantly distinct from those of dicistroviruses.

Insect pathogens Anomala cuptera entomopoxvirus(AcEPV) spindle, paracrystalin proteinaceous bodies, enhance nucleopolyhedrovirus(NPV) infection. This ability of the spindles did not decreased after the heat treatment, UV irradiation, dipping to the following germicidal agents, 3% formaldehyde, 70% ethanol and 0.2% benzalkonium chloride. These properties indicated that these bodies are suitable when using them as a synergist of the microbial pesticides derived from NPVs.. The susceptibility of 38 geographic races of the silkworm, Bombyx mori,toCry1Aband

Cry1Ac toxins of Bacillus thuringiensis were investigated. The wide variations on the LC50sof both toxins were observed in the larvae of geographic races. The ratio of the LC50 of Cry1Ab to the LC50 of Cry1Ac in each geographic race was similar in 37 races. These results suggest that the two toxins show the similar effect on the silkworm larvae. A susceptible silkworm race to Beauveria brongniartii showed high susceptibility to also other two species of entomopathogenic fungi, B. bassiana and Paecilomyces fumosoroseus.This

- 104 - race was considered to be weak in the defense mechanism against common factors of entomopathogenic fungi. The comparison of thickness of larval integument among resistant races and susceptible races showed that the thickness had no relation to their susceptibility. Myrothecium verrucaria isolated from Myrothecium leaf spot of mulberry produces toxic substances into the potato sucrose agar medium. Three toxins were purified from the crude toxic substances and identified as Verrucarin A, Verrucarin J and Roridin A respectively. These toxins showed weak growth inhibiting activity to the silkworm larvae.

- 105 - I nsect Biomaterial and Technology Department

Development of biosensors and related materials focusing on the immolization of chemical recognition molecules Liposome with various surface potentials was prepared by use of phosphatidylcholine and phosphatidylethanolamine. The surface potential could change to -38.7mV from -3.5mV by addition of 10mol% phosphatidylethanolamine. The Au-tip surface of surface plasmon imager, as the model surface, was modified by alkanethiols with carboxyl, amine, and hydroxyl group and the liposome solutions were flowed on the modified surface (Fig. 1). The liposome with higher negative potential adsorbed selectively on the amine surfaces. This result indicates that surface modification is effective to immobilize liposome on a solid surface.

Fig.1 Change of SPR image on various surfaces by flowing liposome solution. (left: before, right: after flow)

We prepared two kinds of extracellular matrix analogue oligosaccharide-silk fibroin (SF) conjugates, namely, lactose-SF conjugates (Lac-CY-SF) and chitooligosaccharide-SF conjugates (COS-CY-SF). Fibroblast attachment on Lac-CY-SF was comparable to that on SF while the attachment on COS-CY-SF was significantly lower than that on SF. Fibroblasts attached on Lac-CY-SF showed predominantly spread morphology whereas fibroblasts attached on SF showed round and spread shapes. Only round cells were observed on COS-CY-SF. Therefore, it is considered that Lac-CY-SF is more suitable as a substrate for fibroblast attachment than SF while COS-CY-SF is less suitable. We are developing a new type of biosensor that combines insect receptor proteins with a field effect transistor as a transducer. In this year’s study reported here, two types of lipid membrane support system, anisotropically etched silicon micro apertures and polyimide micropits, were fabricated. Optical microscopy and membrane capacitance measurements confirmed the formation of membranes on those microstructures. The possibility of real membrane potential measurement with this system was confirmed by measuring a simulated membrane potential.

- 106 - Fig.2 Lipidmembraneformed on silicon micro aperture. (Left: light microscopy, Right: fluorescent microscopy)

Fig.3 MOSFET chip bonded to an IC package

A technique to transform silkworm silkgland cells transiently was required for rapid evaluation of effectiveness of transfer vectors on its gene expression efficiency before generation of transgenic silkworm. For the purpose we constructed lipofection technique on silkgland cells. By injection of plasmid pHC-EGFP (expression vector of modified fibroin H chain - EGFP fusion protein) together with artificial lipid into 5th instar larvae resulted in transformation of silkgland cells. Transformation of silkgland cells was confirmed by expression of EGFP fusion protein into lumen of silkgland (Fig.4).

A B 1 2 34

Fig. 4 Transient expression of mH-EGFP in silkgland Fibroin solutions were fixed with 30 % EtOH and purified. Then purified fibroins of transfected (1,3) and normal silkworms(2,4) were observed under normal light (A) or EGFP filter (B). The former showed EGFP fluorescence (3) under EGFP filter light, while the latter didn't (4).

- 107 - Development of measurement and recording methods for obtaining bio-physical information on insects Microelectrodes for recording the action potentials of insects from their nerves and muscles have been developed. At first, I used silicon as the electrode material and fabricated microelectrodes by anisotropic etching and reactive ion etching. However, it was very difficult to control the shape and size of the microprobes of electrodes. In this year, I took an epoxy-based photoresist as the fabrication material. The results of recording the insect muscle potential with the epoxy-based microelectrodes were improved compared to those of silicon-based microelectrodes.

Fig. 5 Scanning electron micrograph of an epoxy-based microelectrode. Body and microprobes were made of epoxy resin. Gold wires were patterned in order to conduct the electrical signals from muscles or nerves. Microprobes were inserted into muscles when electromyographic signals were recorded.

Fig. 6 An example of recorded electromyograms. Arrows indicate the spikes of dorsal longitudinal muscles correspondedwithwingbeats of a silk moth.

Microelectrodes for recording the action potentials of insects from their nerves and muscles have been developed. At first, silicon was used as the electrode material and fabricated to construct a microelectrode by both anisotropic etching and reactive ion etching. However, it was very difficult to control the shape and size of the microprobes of electrodes. In this year, an epoxy-based photoresist was selected as the fabrication material (Fig.5). The results of recording the insect muscle potential with the epoxy-based microelectrodes were improved compared to those of silicon-based microelectrodes (Fig.6).

- 108 - Development of new sericological technology and new materials using sericulture related products Development of functional materials such as fine chemical using chitin and fibroin etc. The degree of polymerization of silk fibroin from biodegraded fibers and films was studied by high-performance-size exclusion chromatography (HP-SEC). HP-SEC pattern of biodegraded films was characterized by a shift of lower molecular weight ranges of the main chromatographic peak. Soluble degradation products of silk films consisted of a range of peptides widely differing in size, deriving from the amorphous sequences of the silk fibroin chains. Film fragments resistant to enzymatic degradation were enriched in glycine and alanine. All of these features can be related to the specific mechanism of the enzymatic attack, as well as to the number and accessibility of the available cleavage sites in the silk fibroin chains. The spherical microsphere was formed when theophylline, one of bronchodilator, and the dibutyryl chitin solution prepared from silkworm was mixed (Fig. 7). The dissolution rate of the theophylline was rapider, as the particle size of the microsphere was smaller. The usability as controlled release carrier of the chitin microsphere was indicated, because the releasability in neutral pH range was stable. Several fatty acids of which the index of unsaturation differs exists in the silkworm integment. The fatty acids were extracted only in the supercritical carbon dioxide without using organic solvents (Fig. 8). It was verified that the extracted volume is able to control by pressure and temperature. Deoxynojirimycin was detected in the supernatant and precipitation of a autoclaving extraction liquid of dead silkworm larvae as waste in the insect factory. The inhibitory activity for -glucosidase by DNJ of the supernatant and precipitation were stronger than dried mulberry leaf powder. It was indicated that the extraction liquid after sterilization was available as diabetes mellitus Characterization silk fibiroin and other biopolymers from chemical and physical respect and development of their applications to wound covering materials and others prevention material which are more effective than the mulberry leaf.

No theophylline Mixed with theophylline Fig. 7 The microspheres prepared from the chitin of waste cuticles derivation.

- 109 - Fig. 8 The liquid of the fatty acid extracted from the integment of silkworm larva by the supercritical carbon dioxide.

Human fibroblast fostering growth peptide was synthesized based upon amino acids composed of fibroin protein. And its properties of adhesivity and of fostering proliferation were examined using 3 types of cells that proliferate in the process of wound-recovering such as skin fibroblast, skin hardening cells, and vascular skin cells. Consequently, it has been revealed that two types of peptides, AASSVSSAASSRSYDYSRRNVRKN and GSSGFGPYVAHGGYSGYEYAWSSESDFGT, which derived from fibroin non-crystallization parts, possess properties of adhesivity and fostering proliferation. The amount of peptides is needed for fostering proliferation of peptide over 1000 times as much as fibroblast proliferation factor (FGF), so it is expected that the peptides will be utilized as wound-recovering material. Sericin A is the second richest component of the cocoon glue proteins. It distributes mostly in theflossandtheouterlayerofthecocoonandhaslowercrystallinitythansericinM,themain component of sericin.The primary structure of sericin A was determined by N-terminal sequences of enzymatically digested sericin A and homology search of the silkworm WGS database. The 86-residue repetitive sequence containing about 45mol% serine constitutes the core structure of sericin A, and it is considered to strongly control the property of the whole protein. Spider silk is one of the fibrous proteins, which have become the subject of extensive study as model polymer for high-performance fibers. Micro infrared spectroscopy and solid-state nuclear magnetic resonance spectroscopy were applied to get some insights into the molecular mechanism of elasticity in natural spider silk. These spectral data suggested that significant differences of molecular dynamics between the glycine and alanine residues in silk protein. In order to construct the high order structures arrangement, full sequence analysis of spider silk protein is in progress. We investigate the interaction between phospholipid membranes and silk fibroin recovered from the posterior silk gland of the silkworm and clarified that newly constructed fibroin, existing in the form of filaments, quickly penetrates phospholipid membrane without bursting them. We also have given special attention to the silk produced by hornets, and have tried to

- 110 - produce useable materials from it. In last year, we examined the dissolving ability of the hornet silk, and found that hornet silk could be dissolved in aqueous 7.2 M lithium bromide (LiBr) and hexafluoroisopropyl alcohol (HFIP). Through the use of aqueous LiBr and/or HFIP solutions of hornet silk, we were the first to discover the processing technique of purified powder, film (Fig. 9), gel, sponge and artificial fiber of hornet silk. In order to understand the mechanism of the fibroin spongy structure formation, X-ray diffraction and solid-state 13CNMRoffibroinspongeformedbyuseofvarioussolventswere performed. Both silk I and silk II type crystal structure was observed and the ratio was different by the kind of solvents. NMR results indicated b-sheet structure was formed during the process and the ratio of b-sheet structure in the fibroin sponge was influenced by both the amount and the kind of solvents. These results indicate that solvents would play an important role in formation of the fibroin spongy structure.

Fig. 9 Transparent film obtained by the formation of a cast upon drying of the hornet silk in the HFIP solution. To show the colorless and clearness, the photograph shows the film superimposing on the picture.

- 111 - I nsect Biotechnology and Sericology Department

Analysis of characteristics of improved Chinese silkworm races maintained as genetic resources On the improved Chinese races of the regional silkworm races maintained as genetic resources, we investigated the racial characteristics by the statistical analysis methods for correlations among many characteristics. The improved Chinese races are better in the mean values of cocoon weight, cocoon volume, cocoon filament length and raw silk percentage of cocoon as compared with the other regional silkworm races. The cocoon volume of the many improved Chinese races is smaller than that of complete elliptical type cocoons. The coefficients of variation of susceptibility (“Maneb”LC50) and lousiness of the improved Chinese races are greater than 30% as the other races. The bad lousiness is one of the characteristics of the improved Chinese races. The cocoon filament length of the improved Chinese races is about 350 meters long on the average in particular as compared with Chinese native ones, and the maximum is two times longer than the native ones. The raw silk percentages of cocoon of the native races are distributed in the narrow range of 6 to 14%, but those of the improved ones are distributed in thewiderangeof7to22%. On the whole, Chinese No.128, Chinese No.129, Chinese No.130 and Chinese No.131 are important from a viewpoint of filature in the Chinese improved races. The race of the largest size is Chinese No.23 and that of the smallest one is Chinese No.25. It is understood that these two races are useful for silkworm breeding concerned in the size of cocoon filament.

- 112 - Improvement and application of mapping systems in the silkworm, Bombyx mori Molecular linkage map has been improved and finally 330 EST-cDNA clones have mapped on 28 linkage groups. The methods for linkage analysis and mapping that were very efficient for the organisms without crossing over in one sex were developed. Scanning linkage analysis (SLA) is the method for linkage analysis using the same backcross segregants to know where genes are linked. BCMAP is the mapping method for the same individuals of the backcross segregants. These methods were introduced for making the molecular genetic map of EST-cDNA clones in the silkworm, Bombyx mori. The newly developed EST-cDNA clones were examined by Southern blot hybridization whether those showed effective RFLP or not. The clones showing effective RFLP were used for SLA and BCMAP. Finally new 70 clones were added to the map this year. The markers on the map and the methods were introduced to analyze many kinds of characters like resistant genes against BT-toxin, polyphagous gene and etc. The molecular markers on the map was also used to make homozygote of p and +p genes on the second chromosome. As shown on Fig.1, p/+ and +/+ were identified by using very closely linked RFLP marker. ← p/+ →← +/+ →

Fig.1 p/+ and +/+ tagged by RFLP

Analysis of insect culture cell in vitro gene expression sysytem Serum free-culture Bombyx mori cell line was established for the in vitro baculovirus gene expression vector system. This cell line can be cultured under a spinner flask system, and also can express a large amount of gene product by culturing in a medium contained a silkworm hemolymph. This will be commercialized after obtained a patent. As a characterization of cell line, new plant virus that was named as BmMLV was detected from some Bombyx mori cell lines. BmMLV is RNA virus that classified in Maculavirus, Tymoviridae family. This virus is transferred from cytoplasm to cytoplasm without coded on genome.

- 113 - .Bombyx mori cell line that was named as BmN4 accumulated lipids in a cytoplasm by treatment of lipid-differentiation-inductor medicines. BmFABP gene was cloned from genome. This has a high homology to aP2 gene of mammalian lipid cell differentiation marker. Promoter region and enhancer region were analyzed by construction of a deletion mutant gene. Atacin and lebocin promoter regulation mechanism was not the same. This was proved by analyzation of a cell that inoculated peptide glycan. This was shown by the evidence that Atacin activity was showed in high level in several cell lines. However lebocin promoter activity was not shown in some cell lines at all, or was shown a high level activity.

Fig.2 BmFABP promoter analysis FL;Full length

Utilization of transposon for the construction of transgenic insects and application for the analysis of insect genes To develop the technology related to the transgenic silkworm and apply for the production of useful materials, the TC/mariner family transposon minos has been studied as a vector of the transgenesis in the silkworm. The minos vector with green fluorescent protein gene under the control of the silkworm cytoplasmic actin gene promoter was used. When the vector and helper plasmid DNAs were co-injected into the silkworm embryos at early stage, many transgenic silkworms were identified in G1 generation. This suggests that the transposon minos is an efficient vector for the transgenesis in the silkworm. The effect of insertion sites on the gene expression was also investigated. Many lines with different insertion sites were examined and it was found that the expression patterns were affected by insertion sites when silkworm actin promoter was used for the experiment (Fig. 3). Similar phenomenon was observed in other promoters but the effect on the expression pattern was much less compared to the actin promoter. To identify genes that control the circadian behavior of fruit fly, we tried a gain-of-function screening using GAL4-UAS expression system. Screening of 2,500 UAS-insertion strains for effects on locomotor activity identified a number of strains that induce arrhythmic, short and long period of rhythms.

- 114 - Fig.3 Expression patterns of transgene of the transgenic silkworms lines. The differences of expression patterns were caused by the insertion into the different posiotions of genome. Number indicate line numbers of the transgenic silkworms.

Infection route of microsporidian in Lepidopteran insects When the host insects were infected with Nosema bombycis from silkworms or Nosema sp. from Antheraea pernyi, spores of microsporidia were optically detected in the feces, scale, and meconium medium. Small number of immature spores could be detected in the cocoon layers produced by silkworms infected with Nosema bombycis, whilealargenumberofmaturesporesweredetectedinAntherea pernyi cocoons infected with Nosema sp.. After the research of infection route, the spores detected in Bombyx mori cocoon are from the grown silkgland, and that in cocoon layers from Antheraea pernyi are linked to the meconium discharge process. The sericultural farmers should carefully handle the Antheraea pernyi cocoons from infected silkworms, since the spores bounded in the cocoon layers were quite infective, and easily diffused from the cocoons.

Development of a hypersensitive bioassay for a nucleopolyhedro- virus (BmNPV) We developed a hypersensitive bioassay for a nucleopolyhedrovirus (BmNPV) that used B. mori larvae previously treated with a chitin synthesis inhibitor. Peroral administration of a chitin synthesis inhibitor, Polyoxin AL wettable powder (Polyoxin AL), to the silkworm results in a disappearance of the intestinal peritrophic membrane, which implicates a loss in the silkworm of the intestinal barrier to pathogenic microorganisms. Such a larva is easily infected by a low dose of BmNPV ingested with a diet. We used the silkworm larvae that ingested Polyoxin AL for detection of the contamination by BmNPV in the dust collected at sericultural

- 115 - farmhouses. Dust samples were collected immediately after harvesting the cocoons at a mounting room of 15 sericultural farmhouses in Ibaraki Prefecture. The dust was divided into two aliquots and assayed for BmNPV by two methods, a conventional assay and a newly developed polyoxin assay. Conventional assay: a dust sample was mixed with 25 mM sodium pyrophosphate solution and stored at 4 ? for 24 h after it had been lightly shaken by hand. Then the supernatant was applied onto a diet and fed to 20 larvae at 2nd instar that had newly exuviated. Polyoxin assay: a dust sample was mixed with a 100 mM sodium pyrophosphate solution, and the mixture was shaken for 20 min on a shaker. A piece of diet was put in the muddy mixture to contaminate its surface with the dust and then served to 40 2nd instar silkworms that had ingested 0.5% Polyoxin AL in a diet for 24 h prior to this assay. In both the assays larvae were reared until they molted to the next instar or died of nucleopolyhedrosis. The number of the larvae that died of nucleopolyhedrosis is shown in Table 2. Many larvae (from 1 to 40, depending on the farmhouse they were raised) died of nucleopolyhedrosis in the polyoxin assay. On the other hand, only a few died of that disease in the conventional assay. Thus, a newly developed polyoxin assay is an extremely sensitive method for BmNPV detection and is superior to the conventional assay. The polyoxin assay should effectively be used to study the epidemiology of BmNPV.

Table 2 Number of silkworms that died of nucleopolyhedrosis to which the dust collected at sericultural farmhouses had been administered

* Forty (for polyoxin assay) andtwenty(forconventional assay) larvae were used in each of the assay.

- 116 - Breeding and utilization of a new silkworm race having special features Recoverable sericin -sheets were effectively produced by SERICIN HOPE silkworms spinning on a new flat-coccoon equipment. As the sericin can be used in its natural state without its high molecular substance decomposing, and it is easily gelled, we manufactured it for testing as a cosmetic material, such as emulsion (Fig. 4). This sericin, which we named NATURAL SERICIN, has higher binding activity to dye chemicals and a little anti-tyrosinase activity at the same level as ordinary sericin. Its solubility in hot water (60 ℃), NATURAL SERICIN is similar to that of ordinary sericin. However, a little variation of the solubility was found in silkworm genetic stocks, and some sericin components, especially low molecularity, seemed to be related with this character.

Fig.4 Differentforms of NATURAL SERINCIN a ; gel, b ; cream, c ; milky lotionm, d ; mousse

Basic study on silkworm race breeding From the results of molecular linkage analysis, the feeding behavior toward an artificial diet without mulberry leaf powder in J01 may possibly be controlled by several genes, except pph,. which controls the food preference of Sawa-J Pseudomonas aeruginosa of gram negative bacteria was inoculated into the silkworms orally for the investigation of bacterial resistance. When a high dose of bacteria was inoculated, an interracial variation of resistance to the bacteria was found, high for Daizo and low for Oha (Fig. 5). Reciprocal and backcrossing experiments between them suggested that a heterosis effect played a role in the resistance against bacteria.

- 117 - Fig.5 Accumulation mortality rate among silkworms races after the oral inoculation with pathogenic bacteris, P. aeruginosa.

Tea tree-type pruning method of mulberry with special reference to production efficiency of cocoon shell In order to establish an easy and efficient mulberry harvesting method, we examined usefulness of the tea tree-type pruning method that harvests mulberry leaves from each mulberry tree at three times a year． As a result, it is estimated that this new harvesting method is almost equal in mulberry yield, and a little better in cocoon weight and production efficiency of cocoon shell as compared with a conventional summer pruning method (Fig. 6)．

Fig.6

- 118 - The chemical modification of silk sericin and the development of characteristic silk products The relationship between molecular orientation and the secondary structure of silk sericin was investigated. Sericin films were artificially stretched after moistening with aqueous ethanol of various concentrations. The resulting molecular orientation was analyzed using polarized infrared spectroscopy. These analyses indicated that formation of aggregated strands among extended sericin chains induced by ethanol treatment is the key to generating molecular orientation. Sericin solution, prepared from the Sericin-hope cocoons, contains intact sericin and forms elastic hydrogels with the addition of ethanol. The sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be usable as a naturally occurring biomaterial. The silk fabric dyed by the extracts of Phellodendron amurense and Allium cepa was irradiated by ultraviolet ray. The antibacterial force became to be weak in accordance with fading, but it was confirmed that the antibacterial force improved after 20 hours. The texture of thin fabric woven with the silk threads of cocoons of “Hakugin” silkworms treated tri-molter, was more excellent than the ordinary silkworm race’s fabric on the values of the flexibility with soft feeling “SHINAYAKASA” and the crispness “SHARI” by KES system. ThenetrawsilkbecametobehardbyfixingsericinbyCyanuricChloride.Thistreated silk threads adapted for the weft of denim fabric. High bulk silk reeling machine had developed to reel the thick and bulky type silk threads. In this year, the thick and flat type silk reeling mechanism was introduced to the high bulk silk reeling machine. The reeling machine is able to reel the combination silk threads of high bulk type and flat type. It was examined to form the artificial skein and blood tube using cocoon filaments（Fig. 7）. The artificial skein was made by weaving single cocoon filament reeling up from a cocoon (Fig. 8), the artificial blood tube basement was made by knitting and winding cocoon filaments, the tube was degummed (dissolving sericin), and then put the fibroin solution on the tube.

Fig.7 Flexibletubemadeofcocoon Fig.8 Artificial skein basement weaved by usingingle filaments. cocoon filament reeled up from a cocoons as a warp and weft filament

- 119 - Plant Science Division

The Plant Science Division consists of the following five departments, and is actively engaged in multidisciplinary researches. Scientists at the Molecular Genetics Department are engaged in studies of the structure and function of plant genomes, genes, their products, and their networks, which are involved in various agriculturally important traits, and also the mechanisms regulating expression of these genes. Those at the Biochemistry Department are involved in researches on the three-dimensional structure of proteins, and structure-function relationships of proteins involved in response to hormones and other biotic signals in plant cells. Plant Physiology Department is engaged in analyses of molecular mechanisms of important physiological processes in plants including photosynthesis, morphogenesis such as leaf and floral organ development, the symbiotic process of nitrogen fixation, mechanisms of defense against plant pathogens, and tolerance against environmental stresses. The Plant Biotechnology Department is developing new techniques for next-generation plant biotechnology and also producing novel transgenic crops with superior traits which conventional breeding techniques can not produce. The Institute of Radiation Breeding is developing new technologies utilizing radiation for plant breeding, the creation of plant genetic resources through mutation induction, and the elucidation of gene expression mechanisms in plant mutants.

Major topics in each department are described the next page.

- 120 - Molecular Genetics Department

The research activities of this department are mainly focused on the analysis of the structure and function of rice genome, genes and their products, the development of tools and resources for functional analysis of rice genes, and the mechanisms regulating gene expression. Major topics in the fiscal year 2004 are described as follows.

Mapping and clustering of the alignment of full-length cDNA sequences revealed nearly 30,000 transcription units on the rice genome sequence Rice full-length cDNA project (Jan.2000- Sep.2003) has finally collected about 380,000 full-length cDNA clones and 580,000 EST sequences from 5’ and 3’ ends of the clones from more than sixty kinds of full-length cDNA libraries. From the part of these clones (ca. 170,000), 32,127 representative clones were completely sequenced and the data have been presented in KOME, Knowledge-based Oryza Molecular biological Encyclopedia site, http://cdna01.dna.affrc.go.jp/cDNA/ . Mapping of completely sequenced full-length cDNA clones (FL-cDNA) revealed, 30,495 mapped clones and 1632 unmapped clones, 30,359 clones were mapped onto the single position on the rice genome sequence, while 136 clones were mapped onto the multiple position (197 TU) among 30,495 clones. Single positions (Transcription units) are consisted of 13,505 TU with single cDNA clones and 6569 TU with more than two clones (total 20,586 TU). Clones on the 6569 TU showed alternative transcription start site, alternative splicing and alternative polyA additions (Fig. 1). Homology search using the PIR (Protein Information Resource) revealed 23,569 clones out of 32,127 clones have the PIR hit (BLASTX

- 121 - Finally nearly 30,000 expressed genes in rice were collected as full-length cDNA clones currently. Among them clones corresponding to 9400 TU remain to be sequenced. Gene models without relation to the transposable elements in ANE genes are currently about 6,000, these genes might be the missed gene in full-length cDNA collection. Considering these data, total number of rice expressed genes are about 36,000.

Fig. 1 Mapping and alignment result of 32,127 FL-cDNA clones to rice genome sequence Table 1 Highly duplicated genes in 32,127 FL-cDNA

Genome-wide identification of the rice calcium-dependent protein kinase genes All organisms use a network of signal transduction pathways to control their metabolism

- 122 - and to adapt to their environment. Among these pathways, calcium plays an important role as a universal second messenger. In plants, intracellular Ca2+ concentration is changed in response to various stimuli, including hormones, pathogens, light, and abiotic stresses. It has been reported that the plant-specific calcium-dependent protein kinases (CDPKs) from various plants are induced by a variety of stimuli. Therefore, CDPKs are considered to play important roles regulating downstream components of calcium signaling. Recently, a genome-wide analysis of Arabidopsis CDPKs identified 34 CDPK genes. Based on the result, it is thought thatCDPKsconstitutealargemultigenefamilyinhigherplants.However,todatethe biological function of most of the CDPKs, functional divergence in the CDPK gene family and their target proteins are still unclear. We conducted a genome-wide analysis of rice CDPKs and identified 29 CDPK genes and 8 other closely related kinase genes, (including five CDPK-related kinases (CRKs), one calcium and calmodulin dependent protein kinase (CCaMK), and two PEP carboxylase kinase-related kinases (PEPRKs)) (Fig. 2). The mRNA splicing sites of the rice CDPKs, CRKs,andPEPRKs (but not OsCCaMK)arehighly conserved, suggesting that these kinases are derived from a common ancestor. In the RNAgel blot analyses using RNA isolated from the basal parts including meristems, leaf blades, roots, and immature seeds, the rice CDPK genes showed diverse gene expression patterns, and the majority were preferentially expressed in a specific tissue. Expression of OsCPK9 waselevatedinseedlingsinfectedbyriceblast,indicatingthatthisgeneplaysan important role in signaling in response to rice blast treatment. Our genomic and bioinformatic analyses will provide an important foundation for further functional dissection of the rice CDPK gene family.

- 123 - Fig. 2 Phylogenetic relationships among CDPKs and its related kinases. Rice and Arabidopsis kinases are indicated by red and blue type, respectively.

Molecular characterization of a short grain mutant isolated by rice activation tagging We have generated 13,000 activation-tagging lines of rice. Several mutants including lesion mimic, stripe, dwarf, and short grain were obtained in the activation-tagging lines. This year, we analyzed the semi-dominant Short grain 1 (Sg1) mutant. We analyzed the genomic DNA of 24 T1 plants whether the T-DNA insertion linked to the grain phenotype. When the T-DNA insertion is heterozygous, the grain length is always short, and when it is homozygous, the grain length is always very short (Fig.3a), indicating that the Sg1 phenotype is linked to the T-DNA insertion. The Sg1 plant also shows semi-dwarf phenotype (Fig.3b) and the phenotype is partially restored by the addition of gibberellin. A full-length cDNA encoding ORF1 lies 1.4 kb downstream of the inserted T-DNA and expresses mainly in the young panicle of wild type. The cDNA does not express in the leaf blade of wild type but strongly expresses in those of T1 plants that show Sg1 phenotype. The deduced protein shows no homology with any known proteins. These results strongly suggest that transcriptional

- 124 - activation of this novel gene causes the Sg1 phenotype.

Fig.3 Seed and seedling phenotypes of Short grain 1 (Sg1)

Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene Rapid progress in studies on flower development has resulted in refining the classical “ABC model” into a new “ABCDE model” to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicot plants has been revealed. However, its conservation in monocot plants remains largely unknown. We show the conservation of E-function in rice by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1 and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1 cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123 (SEPALLATA1/2/3) and the petunia FBP2 (Floral Binding Protein 2), suggesting that OsMADS1 play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1 clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice.

- 125 - Heading date 5, encoding a putative subunit of a CCAAT-box-binding transcription factor, plays an important role in photoperiodic flowering in rice Fifteen quantitative trait loci (QTLs) for heading date (flowering time) of rice have been identified by using progenies derived from a cross between a japonica cultivar, Nipponbare, and an indica cultivar, Kasalath (Fig. 4a). Among them, four QTLs , which are involved in photoperiodic flowering, have been cloned by map-based strategy. Interestingly, these studies revealed a conserved feature in genetic control of flowering between rice (a short-day plant) and Arabidopsis (a long-day plant). For example, rice Heading date 1 (Hd1), that is an ortholog of Arabidopsis CONSTANS (CO), regulates transcription of Hd3a, that is corresponding with Arabidopsis flowering inducer FLOWERING LOCUS T (FT). Hd5, a QTL detected on the short arm of chromosome 8, was found to be involved in inhibition of flowering under long-day (LD) conditions, but no effect was observed under short-day (SD) conditions (Fig. 4b). To investigate the molecular mechanisms of photoperiodic flowering in rice, we performed map-based cloning of Hd5.

High-resolution linkage analysis of 2308 F2 plants defined a genomic region of 4.3 kb as a candidate for Hd5 and allowed us to focus one candidate gene, which showed high sequence similarity to a subunit of a CCAAT-box-binding transcription factor. Transgenic plants of Nipponbare with candidate genomic region of Kasalath exhibited a late flowering under LD conditions but no differences in flowering under SD conditions. This result clearly demonstrated that a candidate genomic region for Hd5 functioned to suppress flowering under LD conditions. Expression analysis revealed that there is no effect of Hd5 on the mRNA level of Hd1 and vice versa. However, mRNA level of Ehd1 (Early heading date 1), a rice flowering time gene recently cloned, was suppressed by Hd5 under LD conditions, resulting in suppression in mRNA level of Hd3a (Fig. 5). These results suggest that Hd5 plays an important role in inhibition of flowering under LD conditions through a transcriptional regulation upstream of Ehd1. At present, there is no report on the CCAAT-box-binding transcription factor subunit as a flowering time gene in Arabidopsis. Rice may evolve its unique photoperiodic flowering control using Hd5.

- 126 - AB 1723465 8 910 1112 140 NILHd 5 Hd 11 Hd3b 120 Hd9 Hd3a Hd5 Nipponbare Ehd1 Hd13 100 Hd8 Hd1 Hd4 80

Hd10 60 Hd 12 Hd2 Days-to-heading 40 Hd7 20 Hd6 0 SD LD NF Growth conditions

Fig. 4 A) Chromosomal location of QTLs for flowering time in rice. QTLs in ovals have been cloned. B) The effect of Hd5 on flwering time (days-to heading). A nearly isogenic line of Hd5 (NILHd5) and its isogenic control Nipponbare are used for analysis. SD: short-day conditions (10-h light, 14-h dark), LD: long-day conditions (14-h light, 10-h dark), NF: natural field conditions (Tsukuba, Japan). The Kasalath allele of Hd5 increased days-to-heading by about 14 days under LD and NF conditions. LD SD Fig. 5 5. 0 5. 0 The effect of Ehd1Nipponbare Ehd1 4. 0 4. 0 Hd5 on the NILHd 5 (x10-3) (x10-3) 3. 0 3. 0 expression of BQ BQ UBQ

U flowering time 2. 0 / / 2. 0 1 d

h genes, Ehd1 Ehd1

E 1. 0 1. 0 and Hd3a. 0. 0 0. 0 14.5h light 10h light Plant leaves 4. 0 4. 0 Hd3a Hd3a were sampled 3. 0 3. 0

(x10-5) (x10-3) every 2 hours

2. 0 2. 0 during a day. UBQ UBQ UBQ / / Sampling was

Hd 3a 1. 0 Hd 3a 1. 0 performed 6 0. 0 0. 0 14.5h light 10h light weeks after sowing under LD conditions and 3 weeks after sowing under SD conditions. Periods of light and darkness are indicated with white and black bars, respectively.

- 127 - The blue light impacts on photomorphogenesis and flowering-time in rice Compared with the extensive analysis of photoreceptor functions in photomorphogenesis and floral induction of a dicotyledonous plant, Arabidopsis thaliana, little is known on those of monocotyledonous plants. Here, we used a phytochrome-deficient and early-flowering mutant, se5, to elucidate photomorphogenesis and floral induction in rice, a monocotyledonous plant. Several kinds of light emitting diodes (LEDs), which have distinct monochromatic peaks, were used to monitor photomorphogenesis of rice seedlings. The results clearly indicate that additional blue lights can induce relatively proper de-etiolation responses of rice seedlings even in the se5 background (Fig.6). We have previously shown that phytochromes confer the photoperiodic flowering in rice (Izawa et al., 2000, 2002). Therefore, we next grew up rice plants under strong monochromatic red laser diode lights with blue LED lights as supplemental light sources to examine the effects of light quality for floral induction in rice. The results showed that the monochromatic red lights led to late flowering phenotypes even in se5 and only 1% addition of blue light resulted in significant promotion of flowering in both the wild type and se5 (Fig.7). These results indicate that rice is very sensitive to blue lights and blue light signals play pivotal roles both in photomorphogenesis and floral induction of rice. Flowering-time of hd1 mutants in rice, the counterparts of co mutants in Arabidopsis, under those conditions revealed partial contributions of Hd1 in blue- induced floral promotion and suggested the existence of another blue- induced floral genetic pathway(s). mRNA expression analyses of Hd3a, a floral switch gene in rice, also supported these results (Fig.8).

Fig. 6 Blue light effects in rice seedlings. From the left, WT (the wild-type, N8) in only red laser lights (R) (Black), WT with 1% blue LED lights in R (black-faint blue gradient), WT with 5% blue in R (black- light blue gradient), WT with 20 % blue in R (black-pale blue gradient), WT with 20 % blue in R (black-blue gradient), then a space, se5 mutant in only R (white), se5 with 1% blue in R (white-faint blue gradient), se5 with 5% blue in R (white - light blue gradient), se5 with 20 % blue in R (white-pale blue gradient), se5 with 20 % blue in R (white -blue gradient). Second sheath (2nd S),secondblade(2ndB),thirdsheath(3rdS),and3rdB,thirdblade(3rdB)weremeasured.

- 128 - Fig. 7 Blue light effects on flowering time in rice Filledbox(thewildtype,N8),openbox(se5).R;redlaserlight(R),RB1%;1%blueLED light in addition to R, RB5%; 5% blue LED light in addition to R; RB20%; 20% blue LED light in addition to R. A) Days to heading B) Leaf number at heading.

Fig. 8 Hd3a gene expression under red and blue LED light conditions. Relative folds for Hd3a/UBQ are shown. Dashed lines are for se5 mutants while solid lines for the wild type (N8).

- 129 - Biochemistry Department

Structural biology Crystallization and X-ray structural determination of proteins Crystallization is the first step required for X-ray protein structural determination. Suitable quality of large (>0.05mm in its smallest dimension) crystal enables collecting X-ray diffraction data, which provides the construction of 3-dimensional model of protein. The science of protein crystallization is still an underdeveloped field, and protein crystallization is mainly trial-and-error procedure. Proteins are slowly precipitated to become visible crystals from their solution containing special precipitant such as salts, polyethleneglycols of various polymerization degrees and organic solvents. For X-ray structural determinations of proteins, we have been crystallized several proteins, such as bacterial amylase, fructotransferase, fungal cellulase, chitin-binding protein of Streptomyces ABC-transporter, snake venom proteins, earth worm galactose binding lectin, viruses and their complexes with ligands (Fig. 1). These crystals have provided the suitable X-ray diffraction data for structural determination. Crystal of fructotransferase, which is a bacterial enzyme degrades inuline into oligosaccharides, belongs to the space group of R32 with unit cell parameters a=b=92.0Å c=229.8Å in the hexagonal axis. The phasing and initial model building were performed by multi-wavelength anomalous dispersion (MAD) using a selenomethionine-derivative crystal diffraction data at 2.3Å. The solved 1.4Å structure reveals a typical right-handed b-helix motif, as observed polygaracturonase and pectate lyase (Fig. 2).

a b c

d e f Fig.1 Examples of protein crystals. a:Bacterial fructotransferase, b:Earth worm galactose binding lectin, c:Fungal cellulase, d:Chitin binding protein of Streptomyces ABC-transporter, e:Star-shaped crystal of the chitin binding protein, f:Klebsiella a-amylase.

- 130 - Fig.2 Ribbon model of bacterial fructotransferase. The model shows a typical right-handed b-helix motif. (purple: a-helix, green: b-sheet).

Quantitative analysis of helix-coil transition of block copolypeptide,

Glu12-Ala12, by combined use of CD and NMR spectroscopy The a-helix is one of the fundamental structural motifs in proteins. The elucidation of a-helix formation energetics is therefore relevant for understanding protein folding mechanisms. The helix-coil transition has been extensively studied by theoretical and experimental methods. Nearly all the theoretical models utilize statistical mechanics to deal with an ensemble of conformations, and weights are assigned to each helical chain conformation based on the conformations of individual units. To incorporate cooperativity of the transition, two parameters are used to describe a helical segment: one is propagation parameter, which describes the free energy associated with adding a helical unit to a preexisting helical segment, and the other is the nucleation parameter, which is a correction applied to each helical segment to describe the energetic cost of nucleating the segment. In spite of the large number of experimental studies conducted using various types of polypeptides and oligopeptides, there is still much debate concerning the helix-forming propensities and the helix formation mechanisms. To further investigate helix-coil transition mechanisms,wehavequantitativelyanalyzedpH-andtemperature-inducedhelix-coil transitions of Glu12-Ala12 (EA) using CD and NMR spectroscopy (Table). The 750 MHz NMR spectra displayed excellent dispersion of the backbone amide proton signals, and enabled us to analyze conformational characteristics of all the residues in the EA peptide individually. The a-helix of EA is getting longer as decreasing pH: below pH 4, 21 residues of Glu3-Ala23 assume a continuous helix while only 8 residues of Glu12-Ala19 assume the helix above pH 7 (Fig. 3). The observation that the Glu block in EA forms a-helix is a sharp contrast to the result that Glu12, composed of only 12 Glu residues, is random coil under the same conditions, suggesting that the helical structure of the Glu block sequence easily propagates from the nucleated helix toward the N-terminus. The complete random-coil nature of Glu12 itself is

- 131 - attributed to the lower helix nucleation ability of the peptide. The important observation of the present study is that the helix-coil transition occurs stepwise, residue by residue, from both the N- and C-termini of the a-helix. No conformational equilibrium between the helical and random-coil states is detected for the residues in the central region of the helix. Quantitative analysis of temperature-induced helix-coil transitions at various pHs provides residual enthalpy changes for the transition, DHr = 0.86 kcal/res for Ala and 1.03 kcal/res for Glu. The transition energetics as well as the transition mechanism estimated in the present studies should provide a better understanding of the helix-coil transition.

Table Thermodynamic Parameters for Helix-Coil Transition of Glu12-Ala12

a ： The van’t Hoff enthalpy change DH and a midpoint temperature of transition Tm were determined by fitting the temperature dependence of CD at 222 nm with the two-state model. b ：The number of residues undergoing conformational change from a-helix to random-coil in a temperature range from 20 to 60 ºC, calculated using Tm and DH. c ： The residual enthalpy change calculated with a function of DHr =DH/DNH.

Fig. 3

Diagrams of the NMR data used to establish the helical structure of Glu12-Ala12 in 0.1 M NaCl solution at 20 ºC: pH 2.9 (A) and 7.4 (B).

- 132 - Proteomic analysis Based on the proteomic approach, several materials such as rice, silkworm, and bacteria, have been analyzed using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry or ion-trap mass spectrometry coupled with high-performance liquid chromatography after the separation by two-dimensional polyacrylamide gel electrophoresis or two-dimensional high-performance liquid chromatography. In addition to these conventional approaches, new methods have been developed for protein analysis, native two-dimensional electrophoresis to identify the protein interactions on gels, on-target sequential biochemical reactions for de novo sequences or identification of amino acid modifications after the measurements of proteins in a high-mass range, and the direct protein analysis which were extracted from a small tissue section (1 x 1 mm) using a mass spectrometry. Evidences of differential expression of proteins in the small tissues were showed by the help of hierarchical cluster analysis. Proteins were not only distributed with a gradient, but also they were specifically expressed in the part of tissue.

Molecular analysis of signal perception and transduction Induction of resistance against rice blast fungus in rice plants treated with apotentelicitor,N-acetylchitoheptaose N-acetylchitooligosaccharide is a fragment of chitin, which is a major component of cell wall of phytopathogenic fungi, exoskelton of Crustaceans, abundant on the earth and recyclable in the ecosystem. It also works as a potent elicitor in a variety of plants such as rice, barley, wheat, tomato, arabidopsis and soybean, inducing a set of defense responses including biosynthesis of phytoalexins, lignification, expression of defense-related genes and early responses such as membrane depolarization, production of reactive oxygen species, etc.. Because these responses are very similar to those induced in infected plants, it is very likely that pretreatment with N-acetylchitooligosaccharide can induce resistance against infection. In this study, we examined the effects of N-acetylchitooligosaccharide on the resistance in rice plants against infection with Magnaporthe grisea. Rice seedlings (Oryza sativa cv.Nipponbare, Blast resistance gene ++) were treated with N-acetylchitoheptaose at 10 mg/ml via root for 7 days and excised leaf sheath was inoculated with compatible M. grisea (P91-15B, race 001). Two days after inoculation, leaf sheath was observed microscopically (Fig. 4). It was noted that pretreatment with N-acetylchitoheptaose (GN7) conferred resistance in rice against infection with rice blast fungus, as shown by the inhibition of hyphal growth (panel L) and accumulation of fluorescent compounds (panel F). The extent of resistance by N-acetylchitoheptaose was comparable with that by saccharin, which is the active moiety of probenazol, a practical chemical inducer of resistance in rice against rice blast disease. It has been reported that a high-affinity binding site of N-acetylchitooligosaccharide, putative receptor for elicitor, occurs in the plasma membrane of rice leaf and root as well as

- 133 - suspension-cultured cells, and N-acetylchitoheptaose is likely to induce systemic resistance via root, because intact plants does not incorporate radiolabeled N-acetylchitoheptaose. These results show that N-acetylchitosaccharides are useful biomass for disease control.

Fig. 4 Growth of infection hyphae and accumulation of phenolic compounds. Leaf sheath was inoculated with M. grisea and incubated for two days. Representative observations of infection are shown. Arrows indicate appresorium.

Analysis of auxin-signaling pathway The plant hormone auxin regulates a wide range of plant growth and developmental processes including cell division, cell elongation and differentiation. This diverse effect of auxin on the plant may be due to multiple types of auxin receptors and auxin-signaling pathways. Recent studies support this possibility and indicate at least two types of auxin-signaling pathway; ABP1-dependent and ABP1-independent pathways. ABP1 is a soluble auxin-binding protein located mainly in the endoplasmic reticulum, and believed to function as an auxin receptor at the plasma membrane. To characterize the ABP1-dependent signaling pathway, photoaffinity cross-linking experiments were performed. A protein interacting with ABP1 was detected and identified. This protein is a plasma membrane protein. ABP1 may function on the cell elongation by regulating cell-wall loosening.

- 134 - P lant Physiology Department

The research activities of our department are mainly focused on the elucidation of molecular mechanisms of important physiological processes in plants. The major topics in fiscal 2004 are as follows.

Photosynthesis and carbon metabolism Proteomic analysis of six different organs of rice: comparison of enzymes involved in photosynthesis and primary metabolism To characterize photosynthesis in various organs of rice plants, soluble proteins from five green organs were compared by two-dimensional gel electrophoresis. Organs examined were leaf blade (LB), leaf sheath (LS), stem (St), rachis-branch including rachis (RB), and lemma + palea (LP). Root (Rt), a non-photosynthetic organ, was also examined for comparison. Immunoblotting, microsequencing or mass spectrometry identified 47 spots, which included spots of 9 and 14 enzymes involved in the Calvin cycle and carbon/nitrogen metabolism, respectively. Comparison of relative abundance of these spots indicated some unique features of green organs (Table ). The overall pattern of RB rather than LS resembled that of LB. The level of Rubisco activase on the basis of the Rubisco level was very low in RB. LP and St shared common features, namely, the abundance of the chloroplastic NADP-malic enzyme, the absence of the chloroplastic carbonic anhydrase, and very low levels of the chloroplastic glutamine synthetase 2, which could be indications of operation of the C4-like pathway in these organs.

Table Relative abundances of identified proteins in six organs of rice.

- 135 - Inheritance of C3-C4 intermediate photosynthesis, a mechanism to reduce photorespiration

C3-C4 intermediate plants show lower photorespiratory rate than C3 plants. We investigated the inheritance of C3-C4 intermediacy using various artificial hybrids between

Moricandia arveinsis (C3-C4) and cabbage (C3). It was demonstrated that the leaf anatomical traits of the C3-C4 intermediate such as the development of chloroplasts and mitochondria in the bundle sheath cells were inherited in the hybrids depending on the constitution ratio of the parent genomes. We are further investigating the biochemical and physiological traits of photorespiration for the reciprocal hybrids. It was unknown whether natural hybridization and subsequent hybrid speciation occurred between plants with different photosynthetic types. We found evidence showing that Diplotaxis muralis (Cruciferae) had been created by natural hybridization between D. tenuifolia

(C3-C4)andD. viminea (C3).

Isolation of rice mutants aberrant in vascular formation The vascular system of plants is a transport pathway for photosynthetic products, water, nutrients, and signal-transducing molecules, and is a possible target to improve the efficiencies of translocation and water transport in crops. We screened rice mutants that are impaired in the vascular patterning, and have isolated nine mutants that show the abnormal interveinal distance in either longitudinal or transverse veins (Fig. 1) and in both veins. These mutants would be useful for elucidating the molecular and genetic mechanisms to control the vascular formation in rice plants.

Fig. 1 Comparison of vascular patterns in leaf blades of wild-type and mutant rice plants. Note aberrant formation of transverse veins in the mutant. L, large longitudinal vein; S, small longitudinal vein; T, transverse vein.

- 136 - Responses to biotic and abiotic stimuli Three types of tobacco calmodulins characteristically activate plant NAD kinase at different Ca2+ concentrations and pHs We previously reported that three types of tobacco calmodulin (CaM) isoforms originated from 13 genes are differently regulated at the transcript and protein levels in response to wounding and tobacco mosaic virus-induced hypersensitive reaction (HR); wound-inducible type I and HR-inducible type III levels increased after wounding and HR, respectively, while type II, whose expression is constitutive and wound responsible, remained unchanged. Here, we show that these CaMs differentially activate target enzymes; rat NO synthase was activated most effectively by type III, moderately by type I and weakly by type II, and plant NAD kinase (NADK) was activated in the inverse order. Furthermore, we found that a suitable Ca2+ concentration differs by type (Fig. 2); type II activated NADK at lower Ca2+ of around 0.1 mM, which is the cytosolic concentration in unstimulated cells, type I did so at 1 - 5 mM, which is the increased Ca2+ concentration in stimulated cells, while type III did not at any Ca2+ level. NADK activation was highest over a pH range of 7.1 - 6.8 for which the cytosolic pH reportedly changed from 7.5 after being stimulated. Thus, tobacco CaMs, especially type I, effectively activate NADK in stimuli-induced conditions.

Fig. 2 Illustration on the effect of Ca2+ concentration and pH on NADK activation by NtCaMs. The NADK activity was enhanced under increased Ca2+ concentration and decreased pH in the cytosol. Crescendo bars indicate the levels of NADK activity.

Functions of chloride channels in rice We hardly know about intercellular behavior of chloride ions, a counter ion for Na+, under the high salt condition. The chloride channels (CLC) are the probable candidates for transport of chloride ions. We isolated loss of function mutants of OsCLC1 and OsCLC2 from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17,andraisedthe homozygote for each gene. Both mutants showed many different phenotypes from wild type, that is, inhibition of the growth in all developmental stages, retardation of flowering, low yield, salt sensitivity, etc. For instance, elongation of internode ?III-V is inhibited in both mutants, as

- 137 - shown in Fig. 3. It is obvious that the chloride channels play an important role in various steps of physiological events.

6 5 4 3 2 1 wild-type clc-1

clc-2

100 90 80 70 Panicle structure 60 first (top) internode 50 second internode 40 third internode 30 fourth internode fifth internode 20 sixth internode 10 0 wild-type clc-1 clc-2 Fig. 3 Inhibition of internodes elongation in clc mutants. The third to fifth internode of the mutant were shorter than that in the wild type, whereas the lower elongated internodes (first andsecond)weresimilarinlengthinbothmutantandwild-typeplants.

Distinct and cooperative functions of phytochromes A, B and C in the control of de-etiolation and flowering in rice We have isolated five alleles of phytochrome B (phyB) mutants (phyB-1 to phyB-5)and a phytochrome C (phyC) mutant (phyC-1) from rice. As reported in Arabidopsis,PHYCprotein levels were greatly reduced in etiolated seedlings of the rice phyB mutantsaswell.Sincewe had already obtained phyA mutants, we produced all combinations of double mutants to analyze the individual as well as cooperative functions of phytochromes in rice. When grown under constant far-red light (FRc), phyA mutants showed partially impaired de-etiolation and phyAphyC double mutants showed no significant residual phytochrome responses for the de-etiolation, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Among the single mutants, only the phyB seedlings exhibited a partial loss of sensitivity to constant red light (Rc) but still showed significant phytochrome responses mediated by Rc, such as coleoptile inhibition. However, the inhibitory effect by Rc was completely canceled in phyAphyB double mutants, while phyBphyC double mutants did not show any apparent decrease in sensitivity to Rc compared with phyB seedlings. These results indicate that phyA and phyB act in a highly redundant manner to control de-etiolation under Rc and that phyC has little effect on the Rc-mediated responses.

- 138 - Light-induced gene expression patterns in the phytochrome mutants accorded with their phenotypical responses. When grown under FRc, phyA seedlings still induced LHCB (Light-harvesting chlorophyll a/b binding protein)genesbutphyAphyC double mutants did not any longer, which means that phyC perceives FRc to induce LHCB genes in the phyA mutant background. In the pulse-irradiation experiments, phyB seedlings showed clear R/FR reversibility in the expression of LHCB genes. Even phyBphyC double mutant, in which phyA is the only active phytochrome, displayed clear R/FR reversibility. These results indicate that phyA as well as phyB is capable to mediate the low fluence response in rice etiolated seedlings. Both phyB and phyC mutants flowered equally earlier than wild type (WT) under long day (LD) conditions and phyBphyC double mutants flowered at the same time as phyB or phyC monogenic mutants. On the other hand, phyA mutation in the phyB or phyC background caused a further decrease in flowering time, whereas the phyA monogenic mutation had no effect on the flowering time under LD conditions. These observations suggest that both phyB and phyC are required for the perception of the LD photoperiod to delay flowering and that phyA acts downstream of the phyB and phyC to suppress the floral induction in response to the LD photoperiod. The floral initiation of Nipponbare (WT) was significantly enhanced under SD conditions compared to LD. While phyA mutantsfloweredslightlylaterthanWT,phyB mutants came into flower still earlier than WT even in the SD conditions. No significant effect of phyC was observed on the flowering under SD conditions. Unexpectedly, the phyAphyB double mutants flowered significantly later than WT and any phytochrome mutants. Thus, the apparent function of phyB in response to the SD photoperiod is different between in the presence and in the absence of phyA, that is, suppressing and promoting the flowering, respectively.

Epigenetic regulation Transgene-triggered, epigenetically regulated ectopic expression of a flower homeotic gene pMADS3 in Petunia pMADS3 is a class C floral homeotic gene of Petunia that is specifically expressed in stamens and carpels of developing flowers. We previously reported that introduction of a part of pMADS3 genomic sequence silenced endogenous pMADS3 (sil-pMADS3)intransgenic Petunia hybrida (Fig. 4). Here, we show that introduction of the same sequence triggers ectopic expression of endogenous pMADS3 in sepals, petals, and leaves (ect-pMADS3), accompaniedbyhomeoticconversionofthefloralorgansandalteredleafmorphologysimilar to those of an Arabidopsis curly leaf mutant (Fig. 4). The occurrence of the ect-pMADS3 phenotype depended on the presence of pMADS3 intron 2 in the transgenes (Fig. 4).

Occasionally, sil-pMADS3 and ect-pMADS3 phenotypes somatically interconverted. Some T1 progeny inherited their parent’s pMADS3 expression pattern, while others switched from sil-pMADS3 to ect-pMADS3 and vice versa. Both phenotypes occasionally occurred even after the transgenes were segregated away. RT-PCR analyses of ectopically expressed pMADS3

- 139 - transcripts indicated that the pMADS3 alleles were often differently regulated. Furthermore, reciprocal crosses with untransformed Petunia indicated that pMADS3 alleles other than the one ectopically expressed in T0 plants were sometimes expressed ectopically in T1 plants, the paramutation-like transmission of epigenetic regulation between alleles. We detected aberrant transcripts, including sense and antisense pMADS3 intron 2 sequences of heterogeneous molecular sizes, in the transformants irrespective of the pMADS3 phenotypes. This unique phenomenon could represent a new aspect of epigenetic gene regulation.

A. Fig.4 Transgene-triggered epigenetic pMADS3 genomic modulation of endogenous pMADS3. sequence intron2 A. pMADS3 genomic DNA. Large intron 2 (4 kb) contains several cis Putative Leafy CArG CCAATCA AAGAA binding site: T elements. Introduction of this sequence induced sil-pMADS3 and ect-pMADS3 B. pMADS3 phenotypes in petunia. B. pMADS3 expression and flower phenotypes in sil-pMADS3 and petal stamen pistil leaf sepal ect-pMADS3 plants. pMADS3 normally normal expresses in stamens and pistils. pMADS3 is silenced and stamens are

sil-pMADS3 petaloid in sil-pMADS3 flowers. Endogenous pMADS3 expresses ectopically and petals are staminoid in ect-pMADS3 flowers.

Desease resistance A strong field resistance QTL against rice blast was efficiently fine mapped with a newly developed residual heterozygocity analysis Resistance breeding is the most economically efficient and environment-friendly approach to defend crops from the diseases and some insects. Against the rice blast disease, most devastating disease of rice, several resistance genes which function as a single component have been discovered and cloned for rice breeding. However, most of them have been overcome by mutations of the pathogen fungus; a phenomenon known as breakdown of resistance. Field resistance is known to be recalcitrant against such resistance breakdown, and effective against almost all spectrum of races of the pathogens, but have not been fully utilized because their genetic structure must be analyzed by QTL. And for cloning of the component genes, reconstruction of their respective NILs has been needed before another series of fine genetic analyses.

- 140 - We have developed a novel system utilizing the RIL (recombinant inbred lines) used for the QTL analysis. By finding the lines that are heterogeneous only in a target gene region, but fixed in other QTL, and developing the selfed population of the following generations (Fig. 5). Applying this method to RILs of a cross between Kahei (a strong blast resistant, upland line) and Koshihikari (the standard elite line of Japan but very weak for rice blast), we have successfully narrowed the major resistant QTL in a region of 200kb in a short period.

residual heterozygote F6 population Marker of the target regon

Proliferation of the target hetero lines by self polination

Clear phenotype segregation on the homozygous background Analysis of phenotypes and genotypes of the region markers markers

610 10 5-(2)-1-① H H SSSSSS 54 30 31 11-(1)-3-① RRSSSSSS 93 40 41 14-(1)-7-① RRSSSSSS 66 41 42 14-(1)-8-① RRSSSSSS 11 51 52 17-(1)-3-① RRSSSSSS 15 52 53 17-(1)-4-① RRSSSSSS 27 55 56 17-(1)-9-① HHRRSSSS 69 34 35 12-(2)-3-① SSHHSSSS 82 45 46 15-(2)-4-① SSRRSSSS 11 1 1-(1)-9-① RRRRSSSS 17 5 5 3-(1)-5-① RRRRSSSS 29 8 8 4-(2)-1-① RRRRSSSS 10 11 11 6-(1)-3-① RRRRSSSS 18 13 13 7-(1)-3-① RRRRSSSS 33 17 17 7-(2)-5-① RRRRSSSS 46 28 29 10-(2)-10-① RRRRSSSS 62 32 33 11-(2)-6-① RRRRSSSS 65 33 34 12-(1)-2-① RRRRSSSS 77 36 37 13-(1)-6-① RRRRSSSS 86 46 47 15-(2)-8-① RRRRSSSS lines 94 48 49 16-(2)-4-① RRRRSSSS 23 54 55 17-(1)-7-① RRRRSSSS 20 61 62 18-(1)-10-① RRRRSSSS 858 59 18-(1)-1-① RRRRSSSS 12 59 60 18-(1)-2-① RRRRSSSS 16 60 61 18-(1)-9-① RRRRSSSS 35 65 67 19-(1)-10-① RRRRSSSS 24 62 63 19-(1)-1-① RRRRSSSS 28 63 64 19-(1)-3-① RRRRSSSS 32 64 65 19-(1)-7-① RRRRSSSS 59 71 74 21-(2)-1-① RRRRSSSS delimitation of the 36 73 77 22-(2)-8-① RRRRSSSS 40 74 79 23-(2)-1-① RRRRSSSS 44 75 81 F6-144-23-(2)-8-① RRRRSSSS 85 38 39 14-(1)-1-① RRRH?SSSS target gene region 38 26 27 10-(2)-7-① HHHHSSSS 57 23 24 10-(1)-8-① HHHHSSSS 61 24 25 10-(1)-9-① HHHHSSSS 88 94 102 9-(1)-3 SSSSSSRR 96 96 104 9-(1)-10 SSSSSSRR 75 83 90 6-(2)-6 SSSSSSSR 87 86 93 6-(2)-10 SSSSSSSR 84 93 101 8-(2)-9 SSHHHS/HHS 56 78 84 2-(2)-2 RRRRRRRS

Fig. 5 Scheme of the residual heterozygocity analysis of the recombinant inbred lines for positional cloning of QTL genes.

Symbiotic nitrogen fixation A novel Hist- mutant of Lotus japonicus that is defective in the bacterial infection and nodule organogenesis Leguminous plants form a specific organ “root nodule” to fix the atomospheric dinitrogen by symbiosis with soil bacteria Rhizobia. Nodule formation is triggered by mutual interaction between the host plants and microsymbionts and is programmed by the host plants. To clarify the plant genes involved in the nodulation program, molecular genetics for the plant mutants that are defective in symbiotic interactions with compatible rhizobium bacteria provides a powerful approach. Ljsym101 is a mutant derived from the regenerated plants of a model legume Lotus japonicus Gifu. Nodulation process in this mutant stops at the stage of the bumps. Inoculation with lacZ-labeled Mesorhizobium loti revealed that the infection frequency in the mutant was much lower than that in the wild type, and the infection thread formation is inhibited either on the surface of the root hairs or immediately after the penetration into the root hairs. Thus, Ljsym101 appears primarily to be a mutant with defect

- 141 - in the infection process. To identify the mutant gene, we employed a map-based cloning strategy. Fine mapping using 2,200 F2 plants focused the Ljsym101 locus in a 3 cM genetic distance on chr 5. A contig with TAC and BAC clones that covers the gene region was constructed by chromosome walking. DNA markers generated from this contig allowed showed us to delimit the Ljsym101 gene within about 100 kb region. To identify the mutation, direct sequencing of the mutant DNA fragments in this region is under going.

Microtubule dynamics in living root hairs as affected by Rhizobium nodulation signals The incorporation of a fusion of green fluorescent protein and tubulin-α 6from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs (Fig. 6). Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting "dynamic instability" as the main type of microtubule behavior in Lotus root hairs. Comparison of the four standard parameters of in vivo dynamic instability, i.e., the growth rate, the disassembly rate, and the frequency of transitions from disassembly to growth (rescue) and from growth to disassembly (catastrophe), revealed that microtubules in young root hairs were more dynamic than those in mature root hairs. Either inoculation with Mesorhizobium loti or purified M. loti lipochitin oligosaccharide signal molecules (Nod factors) significantly affected the growth rate and transition frequencies in emerging and growing root hairs, making microtubules less dynamic at a specific window after symbiotic inoculation. These results evidenced, for the first time, the biological significance of microtubule dynamics in the early signaling events leading to the establishment and progression of the Rhizobium/legume symbiosis.

Fig. 6 Time series of in vivo dynamic instability behavior of microtubules in root hairs of L. japonicus. Arrowheads point to growing microtubules, arrows show disassembling microtubules. Scale bar = 5

- 142 - P lant Biotechnology Department

The research activities of our department are mainly focused on the development of basic studies related to plant biotechnology as well as the generation of novel transgenic crops with superior traits which conventional breeding techniques can not produce. Major topics in 2004-2005 were as follows.

Genomic studies using rice genome resources "FOX hunting system " in rice Cloning and sequencing analyses of rice genome and full-length cDNAs (FL-cDNAs) demonstrated that more than 30,000 genes are expressed in rice. Functions of the genes, however, have not been elucidated in detail. To uncover functions of a large population of genes, gene-knockout approaches such as transposon or T-DNA tagging and also RNAi or antisense technology have been used in many cases through observation and analyses of loss-of-function phenotypes. These strategies may not always work efficiently, when a set of genes is composed of a multigene family and could confer redundant characters. Alternative approaches including activation tagging could enforce over-expression of endogenous genes and be complementary to gene-knockout ones. If we take advantage of a large set of FL-cDNA clones and could place each cDNA under the control of strong and constitutive gene promoter, large population of transgenic rice plants over-expressing cDNAs of our interests could be very useful for gene-function elucidation. This strategy, Full-length cDNA over-expresser gene (FOX) hunting system for random over-expression of a normalized FL-cDNA library, was worked out recently by Genomic Sciences Center, RIKEN. In collaboration with RIKEN, we have applied it to rice as an additional approach for analyzing gain-of-function phenotypes. pRiceFOX, which was constructed as a binary vector for rice FOX hunting, carries hygromycin resistance (HygR) gene driven by 35S promoter and restriction sites enabling directional cloning of FL-cDNAs under the control of maize Ubiquitin-1 (Ubi-1) promoter. A cDNA library in Agbrobacterium was made with approx. 15,000 cDNAs and pRiceFOX. The Agrobacterium library was then transformed into rice and HygR plants were selected and transferred to soil. Observation and analysis of phenotype(s) and identification of introduced cDNA(s) in approx. 3,500 independent transgenic rice plants are in progress.

Comparative genomic approach to agronomically important genes in wheat and barley A "finished" rice genome sequence with 99.99% accuracy came to us at the end of 2004 through the International Rice Genome Sequencing Project (IRGSP). In addition to the

- 143 - sequence, the Japanese Rice Genome Project (RGP) has provided much information such as full-length cDNAs, a high density EST map, rice insertion mutants, micro-arrays, several databases including proteome. All of this information is very useful and important for promoting wheat genomic research. Recently we started working on improving fusarium head blight (FHB) resistance in wheat using a comparative genomics approach in collaboration with three research institutes (NIAS, JIRCAS, and the STAFF Institute). To date we have identified three genomic regions on wheat chromosomes 5AL, 5BS, and 2DS associated with Type I resistance to FHB in a population of doubled haploid lines (DHLs) from the F1 cross of Sumai #3 and Gamenya. The QTLs on chromosome 2DS revealed risk factors for both Type I andTypeIIresistanceasanegativeeffectcontributedbySumai#3.Inthisstudy,wefocused on this QTL region of wheat chromosome 2DS, which has synteny with two rice chromosomes, 4 and 7. As a first step we have done in sillico mapping of wheat markers and ESTs on pseudo-molecules of rice chromosomes 4 and 7. About two-thirds of the markers or ESTs on wheat 2D could be assigned to the rice chromosomes, which shows the co-linearity between wheat 2D and rice chromosomes 4 or 7. Our in sillico mapping of wheat markers and ESTs restricted our target FHB QTL to the 6 Mb region of rice chromosome 4 (Fig.1). Several disease-related genes, not only classical disease resistance proteins but also genes for cytochrome P450s and ABC transporter-like multi-drug resistant proteins, were included in this chromosome region.

Fig.1 Comparative map between wheat 2DS and rice 4S chromosome.

- 144 - Gene targeting in higher plants Isolation and characterization of genes involved in homologous recombination Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination and recombinational repair in eukaryotes. Five Rad51-like proteins including Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are called Rad51 paralogs, have been identified from vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. To understand the role of Rad51 paralogs in plants, we isolated five RAD51 paralogous genes from Arabidopsis and characterized T-DNA insertion mutants. Expression of these genes were observed in flower buds and roots, and induced by genotoxic stresses such as ionizing irradiation. Arabidopsis RAD51B (AtRAD51B) mutantswereviableandfertile.Themutantswere moderately sensitive to -ray and hypersensitive to cisplatin suggesting AtRAD51B gene product is involved in the repair of double-strand DNA breaks via homologous recombination. Arabidopsis RAD51C (AtRAD51C) mutants grew normally during vegetative developmental stage, and were hypersensitive to cisplatin. The mutant plant produced aborted siliques and their anthers did not contain mature pollen grains (Fig. 2). Crossing of the atrad51C mutant with wild-type plant showed defective male and female gametogeneses as evident by lack of seed production. Furthermore, meiosis was severely disturbed in the atrad51C mutant. Our results indicated male and female sterility in atrad51C mutant plants, and that AtRad51C plays an essential role in meiotic recombination in Arabidopsis.

Fig. 2 atrad51C mutant plants are sterile. Opened flowers of wild-type (A) and atrad51C mutant plants (B). Siliques of wild-type (C) and atrad51C mutant plants (D). Siliques resulting from cross-fertilization between female wild-type and male wild-type (E) (as a control), and female atrad51C mutant plant and male wild-type (F). Anthers of wild-type (G, I) and atrad51C mutant plants (H, J). Anthers from 2 mm buds were stained with Alexander's solution (1969) (G, H) or stained with I2-KI (I, J). Tetrad of wild-type (K) and atrad51C mutant plants (L) stained with aniline blue. AtRAD51C expression was detected in only pollen mother cells by in situ hybridization (M). Black arrows indicate signals. No hybridization signal was detected when a sense probe was used (N).

- 145 - Gene targeting Precise modification of a plant genome via gene targeting (GT) is important for the study of gene function in vivo. We have developed an efficient procedure for targeted gene replacement by homologous recombination in the monocot rice. We have tried GT experiments in rice and succeeded to convert an endogenous acetolactate synthase (ALS) gene, which is involved in the biosynthesis of branched-chain amino acids in plants and the site of action for several herbicides, from herbicide bispyribac-sodium salt susceptible form to resistant form.

Enhanced homologous recombination and T-DNA integration in chromatin assembly factor-1 mutants in Arabidopsis thaliana Chromatin assembly factor-1 (CAF-1) is involved in nucleosome assembly after DNA replication and nucleotide excision repair. In Arabidopsis thaliana the FAS1, FAS2 and most likely MSI1 genes encode the 3 CAF-1 subunits p150, p60 and p48, respectively. These 3 proteins have been shown to form a complex with replication-dependent nucleosome assembly in vitro activity. Since in plant mutants lacking CAF-1 activity, histone assembly is expected to be reduced or delayed, we tested whether as a consequence genomic stability would be changed. In both, fas1 and fas2 mutants enhanced levels of DNA double-strand breaks (DSBs) were found and in planta assays of somatic homologous recombination (HR) indeed enhanced frequencies in the order of 40-fold increase in the mutants. Also the frequency of T-DNA integration, a specific example of non-homologous end-joining (NHEJ), was found elevated, although to a lesser extent. These data suggest that an increased accessibility to genomic DNA for proteins of the HR machinery as well as for T-DNA.

Molecular analysis of rice/blast interactions Plants activate self-defense systems to resist pathogens upon sensing them. There are several different classes of resistance: non-host resistance, variety-specific resistance mediated by R (resistance) genes, and basal (or general) resistance. Among these, basal resistance is not strong enough to avoid infection completely but understanding it at the molecular level is important to manipulate the durability of disease resistance. It is believed that basal resistance is established after plants perceive invading microbes through non-specific elicitors generated during infection process. One of them is fragmented chitin (N-acetylchitooligosaccharide), which derives from a common component of fungal cell walls. We have focused on the contribution of chitinolytic enzymes and their products, chitin elicitors, in rice/blast interactions. Previous studies revealed that chitin elicitor causes various defense-related cellular responses and induces blast resistance. Clarification of the signal transduction pathway starting with chitin elicitor will result in a better understanding of basal resistance lied in rice/blast interactions.

- 146 - This year we have investigated the EL5 gene, which is induced by chitin elicitor within 30 min in rice suspension-cultured cells. The EL5 gene encodes ubiquitin ligase (E3) containing a RING-H2 domain (Fig. 3A), which interacts with ubiquitin conjugating enzyme (E2), indicating that the EL5 contributes to proteolysis after the perception of chitin elicitor. Since mutations in the RING-H2 domain cause the loss of E3 activity in vitro, we overexpressed either the EL5 or its mutated genes in rice and characterized the transformants. Transgenic rice plants constitutively expressing the EL5 gene showed no phenotypic change, but unexpectedly, transgenic callus lines expressing the mutated EL5 genes, which lost E3 activity in vitro, were rootless on the regenerated medium (Fig. 3B). TheexpressionoftheEL5wasobservedinthebasalregionofshootswhererootsareformed and induced by auxin. Therefore, the EL5 seems to function at least both in the signaling pathway of chitin elicitor and root formation. Because the rootless phenotype is rather clear and stable, we will continue to analyze the functional mechanism of the EL5 using the indicative phenotype.

Fig. 3 Structure model of EL5 protein (A) and phenotype of transgenic rice plants expressing the EL5 gene (B) . TM, transmembrane motif; RING, RING-H2 finger domain. Transgenic rice plants are regenerated from embryonic callus. Left plant is expressing the wild type EL5 gene and the right rootless plant the mutated EL5 gene, which lost ubiquitin ligase activity in vitro.

Establishment of novel transgenic crops Transgenic rice plants for the treatment of Japanese cedar pollen allergy Immunotherapy using allergen-specific T cell epitope peptides is a safe and effective treatment for IgE-mediated allergic diseases. T cell epitopes derived from several allergens have been shown to possess therapeutic effects in both animal models and human clinical trials. Japanese cedar (Cryptomeria japonica) pollen is a major cause of pollinosis that elicits allergic disorders such as rhinitis and conjunctivitis in Japan. Two major allergens, Cry j I and

- 147 - Cry j II, have been isolated from the cedar pollen. Multiple domains of T cell epitope have been identified from the allergens. In addition, oral feeding to mice of a chemically synthesized T cell epitope peptide of Cry j II reduces levels of Cry j II-specific IgE and IgG antibody responses via a decrease in the production of allergy-associated IL-4 in mice. These results open new possibilities for the development of oral vaccination with T cell epitope peptides. We generated transgenic rice plants that express a synthetic peptide (7Crp) consisting of seven T cell epitopes of Cry j I and Cry j II (Fig. 4A). Under the control of the rice seed storage protein glutelin GluB-1 promoter, 7Crp peptide was specifically expressed and accumulated in the endosperm tissue at the level of 60 g 7Crp per grain (Fig. 4B). Oral feeding to B10.S mice of transgenic rice seeds expressing 7Crp before intranasal challenge with Cry j I allergen inhibited the development of allergen-specific CD4+ T cell proliferative responses. Furthermore, ongoing Cry j I-specific serum IgE responses were significantly suppressed in the group of mice orally fed with rice seeds expressing 7Crp. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.

Fig. 4 Expression of 7Crp in transgenic rice seed. (A) A schematic representation of the plasmid used for the expression of 7Crp. (B) Detection of 7Crp in transgenic rice seed. Lane 1: a non-transgenic rice seed Lane 2: a transgenic rice seed expressing 7Crp

- 148 - Transgenic crops expressing P450 monooxygenase The increased use of pesticides and herbicides on the agricultural scene has caused serious problems for plants, fish, insects, mammals, and sometimes to humans. These chemicals are usually removed from the environment by natural degradation and degradation by bacteria and plants. To decrease the load on the environment, the enhancement of degradation of these chemicals by plants should be as effective as the reduction in their usage. Phytoremediation is the use of plants and plant growth as a technique for detoxifying environmental polluted soils, sediments, and waters sites contaminated with organic and inorganic pollutants. Phytoremediation costs much less than physical and chemical remediation treatments and it prove to be a sustainable technology for bioremediation. It is considered that the over-expression of exogenous or endogenous genes is a good way to increase the remediation capacity of plants. Cytochrome P450 monooxygenase plays an important role in the oxidative metabolism of xenobiotics in higher plants as well as in mammals. The enzyme system on microsomes consists of many P450 species and a few NADPH-cytochrome P450 oxidoreductase molecules. Agrochemicals including herbicides were metabolized by P450 species, conjugated with glutathione or sugars, and compartmentalized into vacuoles, or cell walls, in plant body. The oxidation by P450 species is considered to be the limiting step of metabolism of foreign chemicals. We have introduced mammalian P450 species related to the metabolism of xenobiotics into the rice variety, Nippnbare. These mammalian P450 species have a high activity to metabolize various herbicides with different modes of action and in different chemical functional groups. These transgenic rice plants expressing human P450 species, CYP1A1 or CYP2B6 exhibited a remarkable cross-tolerance toward various herbicides. These transgenic rice plants also had an enhanced ability to metabolize them with different chemical structures owing to the introduced P450s (Fig. 5). These chemicals were supposed to be absorbed by the transgenic plants in the fields and metabolized rapidly into nonphytotoxic compounds. These transgenic rice plants should show not only the 100 herbicide tolerance but also high remediation Nipponbare ability towards various chemicals that are 90 2B6rice-1 80 2B6rice-2 widespread in agricultural environments such as 70 pesticides and industrial pollutants. 60

% 50 40 30 20 Fig. 5 Relative amount of metolachlor in the 10 culture medium of rice plants expressing 0 0123 CYP2B6. day

- 149 - Development of erected leaf rice by introducing the modified OsBRI1 gene To obtain full control of plant morphology is one of the final goals of crop breeding. The target of our study is to change the plant type of rice to the erected leaf type (lack of bending of leaf) by genetically modifying the brassinosteroids metabolic pathway. We devised a strategy using a newly-developed transformation method for a rice and employed the modified OsBRI1 gene fragment. We obtained the transgenic rice plants harboring the rice modified OsBRI1 gene driven by the constitutively expressing actin promoter. The transgenic rice plants showed erected leaf and semi-dwarfed phenotype (Fig. 6). The transgenic rice showed dwarfed plant height of about 95% of non-transgenic control rice (Oryza sativa japonica var. Don-to-koi). We performed the safety assessments of the transgenic rice to the environment in a closed and semi-closed greenhouse. Table 1 shows one of the data about production of allelochemical like substances. The soil on which transgenic rice had been grown did not affect the growth of successively cultivated plants on the same soil. Using these results, we evaluated the impact of the transgenic rice upon biodiversity, in terms of their competitive dominancy to wild organisms, production of toxic substance and crossing possibilities with wild plants. According to the results of the safety assessments, we concluded that the transgenic rice causes no particular impact to biodiversity.

Fig. 6 The morphology of whole plant (left) and flag leaf (right) in modified OsBRI1 gene introduced rice.

Efficient and publicly acceptable selection marker for genetic transformation The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. We propose a method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase

- 150 - (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. Therefore, our results indicated that the marker cassette system consisted exclusively of the host plant DNA (the self-cloned marker system) worked for the efficient selection in a monocot crop plant, rice. The self-cloned marker system presented here is expected to be publicly accepted, and can be potentially applied to other important crop plants for the production of transgenic plants.

- 151 - I nstitute of Radiation Breeding

The research activities of Institute of Radiation Breeding are focused on the development of new strains of seed-propagated, vegetatively propagated and woody crops through mutation by the application of various forms of irradiation. Mutations are induced by the following radiation sources: γ-rays irradiated in the Gamma Field, the Gamma Greenhouse and the Gamma Room. The institute is also involved in the development of new technologies for plant breeding mainly utilizing γ-ray and ion beam irradiation and chemical mutagens, including the elucidation of gene expression mechanisms in mutants. The institute provides irradiation service and cooperative research at the request of universities, private industries, prefectural experiment stations, and incorporated agencies of Ministry of Agriculture, Forestry and Fisheries. The major topics in fiscal 2004 are as follows.

Mapping of the EARLY YELLOWING 1 (EYE1) gene in rice Leaf senescence is a genetically regulated degeneration process to mobilize nutrients from senescing leaves to other tissues such as developing leaves and seeds. Leaf yellowing, which reflects degradation of chlorophyll, is a good indicator of senescence. Early-yellowing mutants are thought to be impaired in their regulation of senescence and could be useful material for studying the mechanism of senescence. A mutant line, NM67, which was obtained from cv. “Nihonmasari” treated with ethylenimine, showed early yellowing phenotype. Typically, in rice, leaf yellowing predominates in the late ripening period. However, NM67 begin yellowing from the tips of leaves about four weeks before heading. As they grow, the yellowing phenotype becomes severer, but they eventually develop mature and fertile panicles (Fig 1). Young plants of NM67 appear almost normal. This trait is considered to be conferred by a single recessive mutation, early yellowing 1 (eye1). In addition, the NM67 mutant line also have a mutant phenotype in the seed storage protein profile: low glutelin and high prolamin content. This trait was conferred by a dominant mutation, Low glutelin content 1 (Lgc1). Lgc1 is independent from eye1 and no low glutelin phenotype was observed in the eye1 line, which was selected from the F2 progeny between NM67 and Nihonmasari.

To determine the map position of EYE1,weanalyzedanF2 population from a cross between the eye1 homozygote (japonica) and cv. Kasalath (indica). Initial analysis using 39 eye1 homozygotes revealed that the EYE1 locus was located between the cleaved amplified polymorphism (CAPS) marker R663 and the simple sequence repeat (SSR) maker MRG5266 on chromosome 3 (Fig. 2) while Lgc1 is located on the long arm of chromosome 2. For further analysis, a CAPS maker OJ1212C05.2 was newly developed: the polymorphism between Nihonmasari and Kasalath was detected by DNA amplification with primer pair

- 152 - OJ1212C05.2F1 (5'-ATGATAATGCTGTTGCAAGAAATG-3') and OJ1212C05.2R2 (5'-CTGTCACTTACTGCAGATGCTTC-3') followed by digestion with HaeIII. Analysis using this maker and 75 eye1 homozygotes demonstrated that the genetic distance between EYE1 and OJ1212C05.2 was 0.66 cM. When the SSR maker C60465 was used, no recombinant was found. Thus, EYE1 is located near the centromere of chromosome 3.

Fig.1 Leaf yellowing of NM67 (A) and Nihonmasari (B).

Fig. 2 Linkage map of rice chromosome 3 showing the position of EYE1.

- 153 - Comparison between gamma ray and helium ion beam in sugarcane In a plant, radiation simultaneously induces various levels of mutation. Often, most of those mutations result in deletions. Generally, useful as well as unfavorable mutations are attributed to deletions and can vary with dosage and type of irradiation. Therefore, it is important to understand the optimum dosage and type of radiation necessary to generate useful mutations in a particular species. This step is critical in order to initiate an effective mutation breeding strategy It has been understood that in sugarcane, as the irradiation dose of gamma rays to a donor plant increases, the nuclear DNA content of the subsequent mutants are decreased. Increasing radiation dosage (up to an optimum point) also increase the mutation rate whereby mutations in quantitative characters such as stalk diameter and stalk length are also frequently induced. To determine the optimum doses of irradiation using a helium-ion beam, studies were initiated to determine the relationship of radiation dosage, mutation rate, nuclear DNA content and frequency of mutant quantitative characters in sugarcane. Using sugarcane variety, Ni 11, individual shoots were divided from a multiple shoot mass derived from a tissue cultured growing point. These cultured shoots were irradiated withgammaraysandwithhelium-ionbeam(4He2+, 100MeV). The survival rate was investigated 40 days following irradiation. Lateral shoots were separated from the stock as the vM2 generation. The dose response of the treatments on stalk diameter, stalk length, stalk number were investigated. An experimental field for the selection of mutants with hairless mutants was established separately. In the following year, five one-eyed-seedlings of hairless clones induced by helium ion irradiation were identified in the vM3 generation and were planted in the experiment field. Quantitative characters and nuclear DNA content were measured in each clone.

Nuclear DNA content decreased in the most of the clones obtained from the vM2 generation. Some of the survived clone lost more than 20% of their nuclear DNA. As the dose of helium ion radiation increased, nuclear DNA content decreased remarkably and its variationalsoincreasedincomparisonwithgammarays.NuclearDNAcontentof10Gy irradiated clones with the helium ion was equivalent to one of 40 Gy of gamma rays, and 40 Gy of the helium-ion beam was equivalent to 160 Gy of gamma rays, respectively (Fig. 3). The hairless mutation rate at 40 Gy utilizing the helium-ion beam irradiation treatment was almost equal to one dose of 200 Gy gamma ray irradiation (Fig. 4). A significant negative correlation (r=-0.97**) was found between the average hairless mutation rate and relative nuclear DNA content in each irradiation plot irrespective if the treatment utilized gamma rays or and helium-ion beam radiation (Fig. 5). Gamma rays and helium-ion beam radiations also showed the similar relationship that by increasing the irradiation dosage, and nuclear DNA content decreases and the hairless mutation rate rises. Though the induction frequency of the hairless mutation is high in higher radiation dosage plots, it is suspected that the observation of smaller stems (a quantitative trait) in the

- 154 - hairless mutants is a result of large chromosomal deletions or induced genome deficiencies. The optimum dose of helium-ion beam irradiation for sugarcane appeared to be 40 Gy when basing the conclusion on the relationship observed between adverse effect on quantitative characters and the frequency of induced hairless mutation.

1.45

1.40 Fig. 3 ガGammaンマ線 ray Effect on relative nuclear DNA 1.35 contents of sugarcane with 1.30 gamma ray and helium-ion Ｈｅイオン

相対核DNA量 Helium ion beam. 1.25 Relative DNA content

1.20

1.15 0 20406080100120140160 線量(Ｇｙ)Dose (Gy) 図１．ヘリウムイオンやガンマ線がサトウキビの核DNA量に及ぼす影響

Fig. 4 Relationship between the dose of gamma ray and helium-ion beam radiation, and the hairless mutation rate.

Fig. 5 Relationship between relative nuclear DNA content and hairless mutation rate.

- 155 - Identification of optimum density in pathogenic spores for a bioassay of tea anthracnose resistance Tea is an important cash crop, which supports the Japanese local economy. Anthracnose is one of the most serious diseases of tea, especially when it is cultivated in mountainous areas. Consequently, the breeding of anthracnose resistant tea cultivars are desired by the tea producing farmers. Mutation breeding of tea has been conducted to improve resistance to this disease as well as maintaining a cultivars desirable yield and quality characteristics. To initiate such a study, it is important for the tea breeder to establish efficient screening methods for the selection of resistant mutants following irradiation. Second, it is important to develop an appropriate artificial inoculation method. Typically, prior to inoculation, we scarify the abaxial leaf surface with a Phillips screwdriver and inoculate the wound with a suspension of conidiospores. Following inoculation, the espansion of the lesion is observed. However, this approach is not precise since the level of fungal infection is directly associated to conidiospores inoculum potential (with concentration and aging after culture) and leaf hardness. In order to improve the inoculation methodology, we needed to determine the optimum levels of inoculum concentration relative to inoculum potential and leaf hardness. From our investigations, we have developed a new bioassay method for identifying tea anthracnose resistance. First, the optimum conidium concentration of the inoculum is set at densities of 4×10 6 ~107 /ml for spores cultured for one month, 10７/ml for 1-3 months, 4×10７/ml for 3-5 months (Fig. 6). For fungi cultured for more than 6 months they are transferred to a new culture media and the concentrations are again set to the optimum densities discussed above. Second, with regard to leaf hardness, this variable component was standardized by utilizing a durometer and recording the reading in gram units. The optimum inoculum concentration to a scarified leaf was determined to be 4 x 106 ～ 107/ml if the durometer reading is (40 - 60 g); 4 x 107/ml if leaf hardness is indicated to be between 60 - 120 g; and 4×10７/ml if the leaf hardness index exceeds 120g (Fig. 7a, b). By standardizing the inoculum’s concentrations and applying an appropriate inoculum’s concentration relative to a leaf hardness index, we were able to obtain stable and consistent results and extend the practical analysis time for the assay. In addition, due to the efficiency of the method, we would decrease the number of leaves to be analyzed and evaluate a larger number of irradiated branches.

- 156 - Fig. 6 The changes of lesion formation under differences which passage of day after periodic transfer.

Fig. 7a The changes of lesion formation under differences which hardness of tea leaves and density of pathogenic spores (susceptible cultivar “Sayamakaori”.

Fig. 7b The changes of lesion formation under differences which hardness of tea leaves and density of pathogenic spores (relative resistant cultivar “Kanayamidori”.

- 157 - List of Publications

Original Papers Abbasi F, Onodera H, Toki 2004 OsCDK13, a calcium-dependent protein Plant 55:541-552 S, Tanaka H, Komatsu S kinase gene from rice, is induced by Molecular 1 cold and gibberellin in rice leaf Biology sheath Abbasi FM, Komatsu S 2004 A proteomic approech to analuze Proteomics 4:2072-2081 2 salt-responsive proteins in rice leaf sheath Arakaki N, Sadoyama Y, 2004 Mating behavior of the scarab beetle Applied 39(4):669- Kishita M, Nagayama A, Dasylepida ishigakiensis Entomology and 674 Oyafuso A, Ishimine M, Ota (Coleoptera: Scarabaeidae) Zoology 3 M, Akino T, Fukaya M, Hirai Y, Yamamura K, Wakamura S

Arakaki N, Kishita M, 2004 Precopulatory mate guarding by the Applied 39(3):455- Nagayama A, Fukaya M, Yasui male green chafer, Anomala Entomology and 462 4 H, Akino T, Hirai Y, albopilosa sakishimana Nomura Zoology Wakamura S (Coleoptera: Scarabaeidae) Asano T, Tsudzuki T, 2004 Complete nucleotide sequence of the DNA Research 11:93-99 Takahashi S, Shimada H, sugarcane (Saccharum Officinarum) 5 Kadowaki K chloroplast genome: a comparative analysis of four monocot chloroplast genomes Asano T, Tanaka N, Yang G, 2005 Genome-wide identification of the Plant and Cell 46(2):356- Hayashi N, Komatsu S rice calcium-dependent protein Physiology 366 kinase and its closely related 6 kinase gene families: comprehensive analysis of the CDPKs gene family in rice Bachmair A, Garber K, 2004 Biochemical analysis of long Methods in 260:73-82 7 Takeda S, Sugimoto K, terminal repeat retrotransposons Molecular Kakutani T, Hirochika H Biology Balgar-Venkobachar V, 2004 Studies on the relationship between 日本蚕糸学雑誌 73(1):47-56 Takabayashi C, Nakajima K, cocoon parameteres and raw silk 8 Turvekere-Hiriyanna S uniformity characteristics

Beninati T, Lo N, Sacch L, 2004 A novel alpha-proteobacterium Applied 70:2596- Genchi C, Noda H, Bandi C invades the mitochondria of ovarian Environmental 2602 9 cells of the tick Ixodes ricinus Microbiology

Bernardo AEN, Garcia RN, 2004 8S Globulin of Mungbean [ Vigna Journal of 52(9):2552 Adachi M, Angeles JGC, Kaga radiata (L.) Wilczek]: Cloning and Agricultural -2560 A, Ishimoto M, Utsumi S, Characterization of Its cDNA and Food 10 Mendoza EMT Isoforms, Expression in Escherichia Chemistry coli , Purification, and Crystallization of the Major Recombinant 8S Isoform Bosak N, Yamamoto R, 2005 A dense comparative gene map between Cytogenetic 108:317-321 Fujisaki S, Faraut T, human chromosome 19q13.3→q13.4 and and Genome 11 Kiuchi S, Hiraiwa H, a homologous segment of swine Research Hayashi T, Yasue H chromosome 6 Chen W, Zhu X, Nemoto T, 2004 Fetal heart rate monitoring from Animal Science 75:471-478 12 Kobayashi T, Saito T maternal body surface potentials Journal using independent component analysis Daimon T, Hamada K, Mita K, 2003 A Bombyx mori gene, BmChi-h , Insect 33:749-759 Okano K, Suzuki GM, encodes a protein homologous to Biochemistry 13 Kobayashi M, Shimada T bacterial and baculovirus chitinases and Molecular Biology Day RB, Tanabe S, Koshioka 2004 Two rice GRAS family genes Plant 54:261-272 M, Mitsui T, Itoh H, responsive to N - Molecular Ueguchi-Tanaka M, Matsuoka acetylchtooligosaccharide elicitor Biology 14 M, Kaku H, Shibuya N, are induced by phytoactive Minami E gibberellins: evidence for cross- talk between elicitor and gibberellin signaling in rice cells

- 158 - Doi K, Izawa T, Fuse T, 2004 Ehd1 , a B-type response regulator in Genes & 18(8):926 Yamanouchi U, Kubo T, rice, confers short-day promotion of Development 15 Shimatani Z, Yano M, flowering and controls FT-like gene Yoshimura A expression independently of Hd1

Fujimoto Z, Fujii Y, Kaneko 2004 Crystal structure of aspartic Journal of 341(5):1227 16 S, Kobayashi H, Mizuno H proteinase from Irpex lacteus in Molecular -1235 complex with inhibitor pepstatin Biology Fujimoto Z, Usui K, Kondo 2005 Crystallization and preliminary X- Acta 61(2):255- Y, Yasui K, Kawai K, Suzuki ray crystallographic studies of Crystallographic 257 T XynX, a family10 xylanase from a section F 17 Aeromonas punctata ME-1 Structural Biology and Crystallization Communications Fujino K, Sekiguchi H, Sato 2004 Mapping of quantitative trait loci Theoretical 108:794-799 T, Kiuchi H, Nonoue Y, controlling low-temperature and Applied 18 Takeuchi Y, Ando T, Lin SY, germinability in rice ( Oryza sativa Genetics Yano M L.) Fujishima-Kanaya N, Ito Y, 2004 The porcine homologues of six genes Animal 35(6):501- Suzuki K, Sawazaki T, located on human chromosome 8 ( RAB2 , Genetics 502 Hiraiwa H, Uenishi H, Awata CA3 , PTDSS1 , MATN2 , FZD6 , and 19 T SQLE ) assigned to porcine chromosome 4 by fluorescence in situ hybridization Fukaya M 2004 Effect of male body size on mating Applied 39(4):603- activity and female mate refusal in Entomology and 609 the yellow-spotted longicorn beetle, Zoology 20 Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae): Are small males inferior in mating? Fukaya M, Yasuda T, Akino 2004 Effects of male body size on mating Applied 39(4):731- T, Yasui H, Wakamura S, behavior and female mate refusal in Entomology and 737 21 Fukuda T, Ogawa Y the white-spotted longicorn beetle, Zoology Anoplophora malasiaca (Thomson)(Coleoptera: Cerambycidae) Fukaya M, Yasui H, Yasuda 2005 Female orientation to the male in Applied 40(1):63-68 T, Akino T, Wakamura S the white-spotted longicorn beetle, Entomology and 22 Anoplophora malasiaca by visual and Zoology olfactory cues Fukaya M, Akino T, Yasuda 2004 Visual and olfactory cues for mate Entomologia 111: 111- T, Yasui H Wakamura S orientation behaviour in male white- Experimentaris 115 23 spotted longicorn beetle, et Applicata Anoplophora malasiaca Fukui K 2004 Modeling the interactive effect of Plant 7:224-229 24 the photoperiod and temperature on Production shoot elongation of mulberry. Science Furukawa T, Ishibashi T, 2003 Characterization of all the subunits Plant 53:15-25 Kimura S, Tanaka H, of replication factor C from a Molecular 25 Hashimoto J, Sakaguchi K higher plant, rice ( Oryza sativa Biology L.), and their relation to development Furusawa T, Ohkoshi K, 2004 Embryonic stem cells expressing both Biology of 70:1452- Honda C, Takahashi S, platelet endothelial cell adhesion Reproduction 1457 26 Tokunaga T molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in Futani A, Tsuge S, Ohnishi 2004 Evidence for HrpXo-dependent Journal of 186:1374- K, Hikichi Y, Oku T, Tsuno expression of type II secretory Bacteriology 1380 27 K, Inoue Y, Ochiai H, Kaku proteins in Xanthomonas oryzae pv. H, Kubo Y oryzae Gomi T, Muraji M, Takeda M 2004 Mitochondrial DNA analysis of the Entomological 7: 183-188 28 introduced fall webwarm, showing its Science shift in life cycle in Japan Gotoh H, Matsumoto Y, 2,004 General Anesthesia of infant mice by Experimental 53:63-65 29 Imamura K isoflurane inhalation for medium- Animals duration surgery

- 159 - Goto T, Noda H, Fujita T, 2005 Wolbachia and nuclear-nuclear Heredity 94:237-246 Iwadate K, Higo Y, Saito S, interactions contribute to 30 Ohtsuka S reproductive incompatibility in the spider mite Panonychus mori (Acari: Tetranychidae) Haga K, Takano M, Neumann 2005 The Rice COLEOPTILE PHOTOTROPISM 1 Plant Cell 17(1):103- R, Iino M gene encoding an ortholog of 115 31 Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin Hamada Y, Suzuki J, Ohkura 2005 Changes in testicular and nipple Primates 46(1):33-45 S, Hayakawa S volume related to age and 32 seasonality in Japanese macaques (Macaca fuscata ), especially in the pre- and post-pubertal periods Harumi T, Sano A, Kagami 2004 Polymerase chain reaction detection Animal Science 75:p503-507 H, Tagami T, Matsubara Y, of single nucleotide polymorphisms Journal 33 Naito M in the chicken mitochondrial D-loop region Hashimoto K, Shibuno T, 2004 Isolation and characterization of Coral Reefs 23:485-491 Murayama-Kayano E, Tanaka stress-responsive genes from the 34 H, Kayano T scleractinian coral Pocillopora damicornis Hashimoto M, Kisseleva L, 2004 A novel rice PR10 protein, RSOsPR10, Plant and Cell 45:550-559 Sawa S, Furukawa T, Komatsu specifically induced in roots by Physiology 35 S, Koshiba T biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway Hashimoto M, Bar-on P, Ho 2004 β-synuclein regulates Akt activity Journal of 279(22):236 G, Takenouchi T, in neuronal cells: A possible Biological 22-23629 36 Rockenstein E, Crews L, mechanism for neuroprotection in Chemistry Masliah E Parkinson's disease Hashizume T, Horiuchi M, 2005 Effects of ghrelin on growth hormone Regulatory 126(1- 37 Nonaka S, Kasuya E, Kojima secretion in vivo in ruminants. Peptides 2):61-65 M, Hosoda H, Kangawa K Hatakeyama M, Sumitani M 2005 Preservation of a transgenic strain Insect 14(1):105- of the sawfly, Athalia rosae Molecular 109 38 (Hymenoptera) by artificial Biology fertilization using cryopreserved sperm Hatakeyama Y, Shibuya N, 2004 Structural variant of the intergenic RNA 10:779-786 Nishiyama T, Nakashima N internal ribosome entry site 39 elements in dicistroviruses and computational search for their counterparts Hatsugai N, Kuroyanagi M, 2004 A plant vacuolar protease, VPE, Science 305:855-858 Yamada K, Meshi T, Tsuda S, mediates virus-induced 40 Kondo M, Nishimura M, Hara- hypersensitive cell death Nishimura I Hayakawa T, Shitomi Y, 2004 GalNAc pretreatment inhibits FEBS Letters 576:331-335 Miyamoto K, Hori H trapping of Bacillus thuringiensis 41 Cry1Ac on the peritrophic membrane of Bombyx mori Hayashi M, Yonezawa N, 2004 Activity of exoglycosidases in Zygote 12:105-109 Katsumata T, Ikeda K, Imai ejaculated spermatozoa of boar and 42 FL, Kikuchi K, Hamano S, bull Nakano M Hayashi T, Awata T 2004 Efficient method for analysis of QTL Genetica 122:173-183 43 using F1 progenies in an outcrossing species He C, Sayed-Tabatabaei BE, 2004 AFLP targeting of the 1-cM region Genome 47:1122- Komatsuda T conferring the vrs1 gene for six- 1129 44 rowed spike in barley, Hordeum vulgare L. He C, Komatsuda T 2004 PCR-based screening BAC library and Acta Genetica 31(11):1262 45 direct end sequencing of BAC clones Sinica -1267 Henmi T, Miyao M, Yamamoto 2004 Release and reactive-oxygen-mediated Plant & Cell 45:243-250 Y damage of the oxygen-evolving Physiology 46 complex subunits of PSII during photoinhibition

- 160 - Higuchi M, Miyashita N, 2003 The complementary DNA sequence and Journal of 120:322-330 Nagamine Y, Watanabe A, polymorphisms of bovine Animal 47 Awata T procathepsin-D (CTSD) Breeding and Genetics Hinomoto N, Takafuji A 2004 Evaluation of mitochondrial Journal of the 13(1):47-55 cytochrome oxidase subunit I Acarological 48 sequences in Tetranychus kanzawai Society of Kishida (Acari: Tetranychidae) for Japan phylogeographic studies Hinomoto N, Muraji M, Noda 2004 Identification of five Orius species Biological 31(3):276- 49 T, Shimizu T, Kawasaki K in Japan by multiplex polymerase Control 279 chain reaction Hirokawa M, Tatematsu K, 2004 Genetic analysis of the new mutation Journal of 73(3):135- Kosegawa E, Meguro Y “Dark-black ursa” in the silkworm, Insect 139 50 Bombyx mori Biotechnology and Sericology

Ichikawa A, Ono H, Harada N 2004 Stereochemical Studies of Chiral Chirality 16(9):559- 51 Resolving Agents, M9PP and H9PP 567 acids Ieiri S, Nomura T, Hirooka 2004 A comparison of restricted selection Journal of 121:90-100 H, Satoh M procedures to control genetic gains Animal 52 Breeding and Genetics Iiyama k, Chieda Y, 2004 Analyses of the Ribosomal DNA region The Journal of 51(6):598- 53 Yasunaga-Aoki C, Hayasaka in Nosema bombycis NIS 001 Eukaryotic 604 S, Shimizu S Microbiology Ijiro T, Urakawa H, 2004 cDNA cloning, gene structure, and Insect 34:963-969 Yasukochi Y, Takeda M, expression of Broad-Complex (BR-C) Biochemistry 54 Fujiwara Y genes in the silkworm, Bombyx mori and Molecular Biology Imanishi S, Inoue H, Hara 2003 Establishment and characterization In Vitro 39: 1-3 K, Funakoshi M,Yasunaga- of a continuous cell line from pupal Cellular & 55 Aoki C, Mitsuda K ovaries of Japanese oak silkworm Developmental Antheraea Yamamai guerin-meneville Biology. Animal Imanishi T, Itoh T et al. 2004 Integrative annotation of 21,037 PLoS Biology 2(6):856- 56 (158 co-authors) human genes validated by full-length 875 cDNA clones Inoue S, Kanda T, Imamura 2005 A fibroin secretion-deficient Insect 35:51－59 M, Quan G-X, Kojima K, silkworm mutant, Nd-sD, provides an Biochemistry 57 Tanaka H, Tomita M, Hino R, efficient system for producing and Molecular Yoshizato K, Mizuno M, recombinant proteins Biology Tamura T Ishikawa M, Ide H, Price 2004 NMR Micro-imaging for visualizing Cryobiology and 50(1):21-31 58 William S, Arata Y freezing behavior in plant tissues. Cryotechnology Ishimaru K, Kashiwagi T 2005 Identification of a locus for Euphytica 139:141-145 59 asynchronous heading in rice, Oryza sativa L. . Ishimaru K, Kosone M, 2005 Leaf contents differ depending on Plant 42:855-860 60 Sasaki H, Kashiwagi T the position in a rice leaf sheath Physiology and during sink-source transition. Biochemistry Isobe R, Kojima K, 2004 Use of RNAi technology to confer Arch Virol 149:1931- Matsuyama T, Quan GX, Kanda enhanced resistance in BmNPV on 1940 61 T, Tamura T, Sahara K, transgenic silkworm. Asano SI, Bando, H Ito F, Hashim R, Yek Sze 2004 Spectacular Batesian mimicry in ants Naturwissensha 91(10):481- 62 H, Kaufmann E, Akino T, ften 484 Billen J Iwamoto M, Onishi A, 2005 Low oxygen tension during in vitro Theriogenology 63:1277- Fuchimoto D, Somfai T, maturation of porcine follicular 1289 63 Takeda K, Tagami T, Hanada oocytes improves parthenogenetic H, Noguchi J, Kaneko H, activation and subsequent Nagai T, Kikuchi K development to the blastocyst stage Iwamoto M, Higo H, Higo K 2004 Strong expression of the rice Plant 42:241-249 catalase gene CatB promoter in Physiology and 64 protoplasts and roots of both a Biochemistry monocot and dicots

- 161 - Iwata K, Noguchi H, Usami 2004 Crystallization and preliminary Acta 60(12):2340 Y, Nam J, Fujimoto Z, crystallographic analysis of the 2'- Crystallographic -2342 Mizuno H, Habe H, Yamane H, aminobiphenyl-2,3-diol 1,2- a section D 65 Omori T, Nojiri H dioxygenase from the carbazole- Biological Crystallography degrader *Pseudomonas resinovorans* strain CA10 Jan A, Yang G, Nakamura H, 2004 Characterization of Xyloglucan Plant 136:3640- Ichikawa H, Kitano H, endotransglucosylase gene that is Physiology 3681 66 Matsuoka M, Matsumoto H, up-regulated by gibberellin in rice Komatsu S Kusano H, Asano T, Shimada 2005 Molecular characterization of Molecular 272:616-626 H, Kadowaki K ONAC300, a novel NAC gene Genetics and 67 specifically expressed at early Genomics stages in various developing tissues of rice Kaku H 2004 Histopathology of red stripe of rice Plant Disease 88(12):1304 68 -1309

Kameda T, McGeorge G, 2004 13C NMR chemical shifts of the Journal of 29:281-288 69 Orendt MA, Grant MD triclinic and monoclinic crystal Biomolecular forms of valinomycin NMR Kameda T, Miyazawa M, 2005 Conformation of drawn Magnetic 42:21-26 70 Murase S poly(trimethylene terephthalate) Resonance in studied by solid-state 13C NMR Chemistry Kameda T, Miyazawa M, Ono 2005 Hydrogen bonding structure and Macromolecular 5(2):103- 71 H, Yoshida M stability of alpha-chitin studied by Bioscience 106 13C solid-state NMR Kameda T 2004 Molecular structure of crude beeswax Journal of 4(29):1-5 72 studied by solid-state 13C NMR Insect Science

Kameshita I, Nishida T, 2005 Expression cloning of a variety of Journal of 137(1):33- Nakamura S, Sugiyama Y, novel protein kinases in Lotus Biochemistry 39 73 Sueyoshi N, Umehara Y, japonicus Nomura M, Tajima S Kaneko S, Ichinose H, 2004 Structure and function of a family Journal of 279(25):266 Fujimoto Z, Kuno A, Yura K, 10 β-xylanase chimera of Biological 19-26626 74 Go M, Mizuno H, Kusakabe I, Streptomyces olivaceoviridis E-86 Chemistry Kobayashi H FXYN and Cellulomonas fimi Cex Karita E, Yamakawa H, 2004 Three types tobacco calmodulins Plant and Cell 45(10):1371 Mitsuhara I, Kuchitsu K, characteristically activate plant Physiology -1379 75 Ohashi Y NAD kinase at different Ca2+ concentrations and pHs Kasai Y, Taji H, Fujita T, 2004 MαNP acid, a powerful chiral Chirality 16(9):569- Yamamoto Y, Akagi M, Sugio molecular tool for preparation of 585 A, Kuwahara S, Watanabe M, enantiopure alcohols by resolution 76 Harada N, Ichikawa A, and determination of their absolute Schurig V configurations by the 1H NMR anisotropy method Kasai Y, Naito J, Kuwahara 2004 Novel chiral molecular tools for Journal of 62(11):1114 S, Watanabe M, Ichikawa A, preparation of enantiopure alcohols Synthetic -1127 Harada N by resolution and simultaneous Organic 77 determination of their absolute Chemistry, configurations by the 1H NMR Japan anisotropy method Kasuya E, Sakumoto R, Saito 2005 A novel stereotaxic approach to the Journal of 141:115-124 78 T, Ishikawa H, Sengoku H, hypothalamus for the use of push- Neuroscience Nemoto T, Hodate K pull perfusion cannula in Holstein Methods Katagiri S, Wu J, Ito Y, 2004 End sequencing and chromosomal in Breeding 54(3):273- Karasawa W, Shibata M, silico mapping of BAC clones derived Science 279 79 Kanamori H, Katayose Y, from an indica rice cultivar, Namiki N, Matsumoto T, Kasalath Sasaki T Kato H, Hata T, Tsukada M 2004 Antifeedant of Natural Dyestuffs International 9:39-46 against Anthrenus verbasci . (IV) Journal of 80 Effects of tannic acid and catechin Wild Silkmoth on the feeding inhibition in the & Silk larvae

- 162 - Kato H, Hata T, Tsukada M 2004 Potentialities of National Dyestuffs Japan 38(4):241- as Antifeedants against varied Agricultural 251 81 carpet beetle, Anthrenus verbasci . Research Quarterly Katoh E, Takegoshi K, Terao 2004 13C nuclear overhauser polarization- Journal of 126(11):365 T magic-angle spinning nuclear American 3-3657 82 magnetic resonance spectroscopy in Chemical uniformly 13C-labeled solid proteins Society Kawagoe Y, Kubo A, Satoh H, 2005 Roles of isoamylase and ADP-glucose Plant Journal 42:164-174 83 Takaiwa F, Nakamura Y pyrophosphorylase in starch granule synthesis in rice endosperm Kawagoe Y, Suzuki K, Tasaki 2005 The critical role of disulfide bond Plant Cell 17:1141- M, Yasuda H, Akagi K, Katoh formation in protein sorting in the 1153 84 E, Nishizawa N, Ogawa M, endosperm of rice Takaiwa F Kawahigashi H, Hirose S, 2005 Enhanced herbicide cross-tolerance Plant Science 168(3):772 85 Inui H, Ohkawa H, Ohkawa Y in transgenic rice plants co- －781 expressing human CYP1A1, CYP2B6, and Kawai Y, Takeno Y, 2004 Linkage mapping of the locus Experimental 53(4):379- 86 Kobayashi E, Tachibana M, responsible for male Animals 382 Wakafuji Y, Noguchi J, pseudohermaphoroditism ( mp ) on rat Kezuka Y, Kitazaki K, Itoh 2004 Crystallization and preliminary X- Protein and 11(4):401- Y, Watanabe J, Takaha O, ray analysis of plant class I Peptide 405 87 Watanabe T, Nishizawa Y, chitinase from rice Letters Nonaka T Khan Md, Khan A, Ishimoto 2003 Proteome analysis of the Plant Genetic 1:115-123 M, Kitamura K, Komatsu S relationship between bruchid- Resources 88 resistant and -susceptible mungbean genotypes Khoa Le Van, Hatai K, Aoki 2004 Fusarium incarnatum isolated from Journal of 27(9):507- T black tiger shrimp, Penaeus monodon Fish Diseases 515 89 Fabricius, with black gill disease cultured in Vietnam Kimura S, Tahira Y, 2004 DNA repair in higher plants; Nucleic Acids 32(9):2760- Ishibashi T, Mori Y, Mori photoreactivation is the major DNA Research 2767 T, Hashimoto J, Sakaguchi K repair pathway in non-proliferating 90 cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells Kindiger B, Russo V, 2004 Performance and persistence of Grassland 50(3):271- Nakagawa H Japanese cool-season forage grasses Science 279 91 in the central Great Plains of Oklahoma, USA Kiribuchi K, Sugimori M, 2004 RERJ1 , a jasmonic acid-responsive Biochemical 325:857-863 Takeda M, Otani T, Okada K, gene from rice, encodes a basic and Onodera H, Ugaki M, Tanaka helix-loop-helix protein Biophysical 92 Y, Tomiyama-Akimoto C, Research Yamaguchi T, Minami E, Communications Shibuya N, Omori T, Nishiyama M, Nojiri H, Yamane H Kishimoto K, Nishizawa Y, 2004 Transgenic cucumber expressing an Journal of 70(6):314- Tabei Y, Nakajima M, Hibi endogenous Class III chitinase gene General Plant 320 93 T, Akutsu K has reduced symptoms from Botrytis Pathology cinerea Kitani H, Yagi Y, Naessens 2004 The secretion of acute phase Acta Tropica 92(1):35-42 J, Sekikawa K, Iraqi F proteins and inflammatory cytokines 94 during Trypanosoma congolense infection is not affected by the absence of the TNF-alpha gene Kobayashi S, Gotoh-Yamamoto 2004 Retrotransposon-induced mutations in Science 304:982 95 N, Hirochika H grape skin color Koga-Ban Y, Tabei Y, 2004 Biosafety assessment of transgenic Japan 38(3):167 Ishimoto M, Nishizawa Y, plants in the greenhouse and the Agricultural 96 Tsuchiya K, Imaizumi N, field-a case study of transgenic Research Nakamura H, Kayano T, cucumber- Quarterly Tanaka H Kojima M, Takeya M 2004 Cloning of six full-length cDNAs Journal of 50(5):518- encoding pig cytochrome P450 enzymes Health Science 529 97 and gene expression of these enzymes in the liver and kidney

- 163 - Kojima M, Masui T, Nemoto 2004 Lead nitrate-induced development of Toxicology 154:35-44 K, Degawa M hypercholesterolemia in rats: Letters 98 sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis Komatsu S, Yang G, Hayashi 2004 Alterations by a defect in a rice G Plant, Cell 27:947-957 99 N, Kaku H, Umemura K, protein α subunit in probenazole and Iwasaki Y and pathogen-induced responses Environment Komatsuda T, Tanno K 2004 Comparative high resolution map of Hereditas 141:68-73 the six-rowed spike locus 1 ( vrs1 ) 100 in several populations of barley, Hordeum vulgare L. Komatsuda T, Maxim P, 2004 High-density AFLP map of non-brittle Theoretical 109:986-995 101 Senthil N, Mano Y rachis 1 (btr1 ) and 2 (btr2 ) genes and Applied in barley (Hordeum vulgare L.) Genetics Komoto N 2004 The silkworm BmXDH2 gene encodes an Journal of 73(3):129- active xanthine dehydrogenase Insect 133 102 Biotechnology and Sericology Kondo A, Kaikawa J, 2004 Clumping and dispersal of Planta 219:500-506 103 Funaguma T, Ueno O chloroplasts in succulent plants Konishi H, Kitano H, 2005 Identification of rice root proteins Plant, Cell 28:328-339 104 Komatsu S regilated by gibberellin using and proteome analysis Environment Kuji N, Tanaka Y, Komatsu 2005 Protein kinase activity and protein Archives of 51:55-64 105 S, Yoshimura Y phosphorykation during the mouse Andrology sperm acrosomal reaction Kumarevel T, Fujimoto Z, 2004 Crystal structure of activated HutP: Structure 12(7):1269- Karthe P, Oda M, Mizuno H, an RNA binding protein that 1280 106 Kumar PKR regulates transcription of the hut operon in Bacillus subtilis Kumarevel T, Fujimoto Z, 2004 Crystallization and preliminary X- Biochemica et 1702(1):125 Mizuno H, Kumar PKR ray diffraction studies of the Biophysica -128 107 metal-ion-mediated ternary complex Acta of the HutP protein with L-histidine and its cognate RNA Kurusu T, Sakurai Y, Miyao 2004 Identification of a putative Plant and Cell 45(6):693- A, Hirochika H, Kuchitsu K voltage-gated Ca2+ -permeable Physiology 702 108 channel (OsTPC1) involved in Ca2+ influx and regulation of growth and development in rice Kusama T, Nomura T, 2004 Development of primers for detection Journal of 67:1289- Kadowaki K of meat and bone meal in ruminant Food 1292 109 feed and identification of the Protection animal of origin Lee JH, Ma Y, Wako T, Li 2004 Flow karyotypes and chromosomal DNA Chromosome 12(1):93- 110 LC, Kim Ky, Park SW, contents of genus Triticum species Research (洋雑 102 Uchiyama S, Fukui K and rye (Secale cereale ) 誌） Li L, Xue C, Bruno K, 2004 Two PAK kinase genes, CHM1 and Molecular 17:547-556 111 Nishimura M, Xu Jin-Rong MST20 , have distinct functions in Plant-Microbe Magnaporthe grisea Interactions Lin HX, Zhu MZ, Yano M, Gao 2004 QTLs for Na+ and K+ uptake of shoot Theoretical 108:253-260 112 JP, Liang ZW, Su WA, Hu XH, and root controlling rice salt and Applied Ren ZH, Cha DY tolerance Genetics Ma JF, Mitani N, Nagao S, 2004 Characterization of the silicon Plant 136:3284- Konishi S, Tamai K, uptake system and molecular mapping Physiology 3289 113 Iwashita T, Yano M of the silicon transporter gene in rice Maeno K, Tanaka S 2004 Hormonal cotnrol of phase-related Journal of 50(855):865 changes in the number of antennal Insect 114 sensilla in the desert locust, Physiology Schistocerca gregaria: possible involvement of [His 7]-corazonin Maeno K, Gotoh T, Tanaka S 2004 Phase-related morphological changes Bulletin of 94: 349-357 induced by [His 7]-corazonin in two Entomological 115 species of locusts, Schistocerca Research gregaria and Locusta migratoria (Orthoptera: Acrididae)

- 164 - Masood MS, Nishikawa T, 2004 The complete nucleotide sequence of Gene 340:133-139 Fukuoka S, Njenga P, wild rice (Oryza nivara) chloroplast 116 Tsudzuki T, Kadowaki K genome : first genome wide comparative sequence analysis of wild and cultivated rice Matsui K, Kiryu Y, 2004 Identification of AFLP markers Genome 47:469-474 Komatsuda T, Kurauchi N, linked to non-seed shattering locus 117 Ohtani T, Tetsuka T (sht1 ) in buckwheat and conversion yo STS markers for marker-assisted selection Matsumoto T, Niino T, 2004 Long-term conservation of Diospyros Plant 21(3):229- Shirata K, Kurahashi T, germplasm using dormant buds by a Biotechnology 232 118 Matsumoto S, Maki S, prefreezing method Itamura H Matsuyama S, Ohkura S, 2004 Simultaneous observation of the GnRH Journal of 50(6):697- Ichimaru T, Sakurai K, pulse generator activity and plasma Reproduction 704 Tsukamura H, Maeda K, concentrations of metabolites and and 119 Okamura H insulin during fasting and Development subsequent refeeding periods in Shiba goats Medan M, Akagi S, Kaneko H, 2004 Effects of re-immunization of Reproduction 128:475-482 120 Watanabe G, Tsonis C, Taya heifers against inhibin on hormonal K profiles and ovulation rate Medvedev S, Onishi A, 2004 Advanced in vitro production of pig Journal of 50:71-76 Fuchimoto D, Iwamoto M, blastocysts obtained through Reproduction 121 Nagai T determining the time for glucose and supplementation Development Mikawa A, Suzuki H, Suzuki 2004 Characterization of 298 ESTs from Mammalian 15: 315-322 K, Toki D, Uenishi H, Awata porcine back fat tissue and their Genome 122 T, Hamasima N assignment to the SSRH radiation hybrid map Mikawa S, Shimanuki S, 2004 Comparative analysis and development Animal 35(6):445 Morozumi T, Domukai M, of microsatellite markers on swine Genetics Shinkai H, Uchida Y, Mikawa (Sus scrofa) chromosome 1qter 123 A, Miyake M, Miyake Y, Hayashi N, Kusumoto H, Uenishi H, Hayashi T, Awata T Mita K, Morimyo M, Okano K, 2003 The construction of an EST database Proceeding of 100:14121- Koike Y, Nohata J, Kawasaki for Bombyx mori and its application the National 14126 H, Kadono-Okuda K, Yamamoto Academy of 124 K, Suzuki GM, Shimada T, Sciences of Goldsmith MR., Maeda S the USA

Mita K, Kasahara M, Sasaki 2004 The genome sequence of silkworm, DNA Researchy 11:27-35 S, Nagayasu Y, Yamada T, Bombyx mori Kanamori H, Namiki N, Kitagawa M, Yamashita H, Yasukochi Y, Kadono-Okuda 125 K, Yamamoto K, Ajimura M, Ravikumar G, Shimomura M, Nagamura Y, Shin-i T, Abe H, Shimada T, Morishita S, Sasaki T Mitsuhashi W, Fukuda H, 2004 Male-killing Wolbachia in the Entomologia 112:57-64 Nicho K, Murakami R butterfly Hypolimnas bolina Experimentalis 126 et Applicata

Miyashita N, Fulka J Jr, 2004 Do cloned mammals skip a Nature 22:25-26 127 Nagai T, Ogura A reprogramming steps? Biotechnology Miyashita N, Shiga K, 2003 Normal telomere lengths of Theriogenology 59:1557- 128 Fujita T, Umeki H, Sato W, spermatozoa in somatic cell-cloned 1565 Suzuki T, Nagai T bulls Momma M, Fujimoto Z 2004 Expression, crystallization and Acta 60(12):2352 preliminary X-ray crystallographic Crystallographic -2354 129 studies of Klebsiella pneumoniae a section D maltohexaose-producing α-amylase Biological Crystallography

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Nakamura Y, Itoh T, Matsuda 2004 Biased biological functions of Nature 36(7):760- 148 H, Gojobori T horizontally transferred genes in Genetics 766 prokaryotic genomes Nakayama S 2004 Species-specific accumulation of Genes & 79(6):361- 149 interspersed sequences in genus Genetic 365 Saccharum Systems Nakazawa H, Tsuneishi E, 2004 Antiviral activity of a serine Virology 321: 154- Ponnuvel KM, Furukawa S, protease from the digestive juice of 162 150 Asaoka A, Tanaka H, Bombyx mori larvae against Ishibashi J Yamakawa M nucleopolyhedrovirus Nam JW, Noguchi H, Fujimoto 2005 Crystal structure of the ferredoxin PROTEINS: 58(4):779- Z, Mizuno H, Ashikawa Y, component of carbazole 1,9a- Structure, 789 Abo M, Fushinobu S, Kobashi dioxygenase of Pseudomonas Function, and 151 N, Wakagi T, Iwata K, resinovorans strain CA10, a novel Bioinfomatics Yoshida T, Habe H, Yamane rieske non-heme iron oxygenase H, Omori T, Nojiri H system

Kishimoto N, Higo H, Higo 2003 RFLP mapping of nuclear DNA Rice Genetics 20:107-110 152 K, Saito A sequences homologous to organellar Newsletter DNAs Nemoto T, Cho Eun-Min, 2004 Stemar-13-ene synthase, a diterpene FEBS Letters 571:182-186 Okada A, Okada K, Otomo K, cyclase involved in the biosynthesis Kanno Y, Toyomasu T, of the phytoalexin oryzalaxin S in 153 Mitsuhashi W, Sassa T, rice Minami E, Shibuya N, Nishiyama M, Nojiri H, Yamane H Karja NWK, Medvedev S, 2004 Effect of replacement of Journal of 50(5):587- Onishi A, Fuchimoto D, pyruvate/lactate in culture medium Reproduction 592 154 Iwamoto M, Otoi T, Nagai T with Glucose on preimplantation and development of porcine embryos in Development vitro Nii M, Hayashi T, Mikawa S, 2005 Quantitative trait loci mapping for Journal of 83(2):308- Tani F, Niki A, Mori N, meat quality and muscle fiber traits Animal Science 315 155 Uchida Y, Fujishima-Kanaya in a Japanese wild boar x Large N, Komatsu M, Awata T White intercross Nishibori M, Hayashi T, 2004 Complete Nucleotide Sequence of Journal of 41(4):259- 156 Yasue H Numida meleagris (Helmeted Poultry 268 Guineafowl) Mitochondrial Genome Science Nishikawa T, Vaughan DA, 2005 Phylogenetic analysis of Oryza Theoretical 110(4):696- Kadowaki K species, based on simple sequence and Applied 705 repeats and their flanking Genetics 157 nucleotide sequences from the mitochondrial and chloroplast genomes Nishimura S, Hinomoto N, 2005 Gene flow and spatio-temporal Experimental 35(1-2):59- Takafuji A genetic variation among sympatric and Applied 71 populations of Tetranychus kanzawai Acarology 158 (Acari: Tetranychidae) occurring on different host plants, as estimated by microsatellite gene diversity Niwa R, Matsuda T, 2004 CYP306A1, a Cytochrome P450 enzyme, Journal of 279(34):359 Yoshiyama T, Namiki T, Mita is essential for ecdysteroid Biological 42-35949 159 K, Fujimoto Y, Kataoka H biosynthesis in the prothoracic Chemistry glands of Bombyx and Drosophila Noguchi J, Kobayashi E, 2004 Fine mapping of a region of rat Experimental 53(5):429- Akiyama K, kawai Y, Ozawa chromosome 12 close to the aspermia Animals 435 160 M, Ohnuma K, Kikuchi K, (as ) locus and comparison with the Kaneko H, Kunieda T human orthologous regions Nonomura K, Nakano M, 2004 The novel gene HOMOLOGOUS PAIRING Plant Cell 16:1008- Fukuda T, Eiguchi M, Miyao ABERRATION IN RICE MEIOSIS1 of rice 1020 161 A, Hirochika H, Kurata N encodes a putative coiled-coil protein required for homologous chromosome pairing in meiosis

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Sasaki K, Asaoka K 2005 Different physiological properties Neuroscience 374:166-170 in a pool of mandibular closer motor Letters 193 neurons in a caterpillar, Bombyx mori

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Gottwald S, Stein N, Borner 2004 The gibberellic-acid insensitive Molecular 271: 426- A, Sasaki T Graner A dwarfing gene sdw3 of barley Is Genetics and 436 195 located on chromosome 2HS in a Genomics region that shows high colinearity with rice chromosome 7L Sato H, Saito C, Handa H 2004 Mitochondrial DNA is decreased Protoplasma 224(3- during the pollen development of 4):179-185 rapeseed (Brassica napus L..), but 196 RNA polymerase encoded by the mitochondrial linear plasmid still exists in the mature pollen Nagao H, Sato T, Kakishima 2004 Three species of Exobasidium causing Mycoscience 45:85-95 M Exobasidium leaf blight on subgenus 197 Hymenanthes , Rhododendron spp., in Japan Satoh M 2004 A method of computing restricted Journal of 82(8):2253- best linear unbiased prediction of Animal Science 2258 198 breeding values for some animals in a population Satoh M 2004 Improving environmental management Animal Science 75(6):499- for higher reproduction accelerates Journal 502 199 genetic improvement in closed herd of swine Satoh M 2004 Including an additional systematic Animal Science 75:97-102 environmental effect within a Journal generation in evaluation model 200 improves accuracy of prediction of breeding values in a closed herd of pigs Sawada H, Nakagoshi M, 2002 Purification and characterization of Journal of 71:103-108 Yamamoto T, Kato T, Mase K, an ommin-binding protein from an Insect 201 Yamamoto T, Izumi S acid-methanol extract of diapause Biotechnology eggs of the silkworm, Bombyx mori and Sericology

Sawada H, Nakato H, Togawa 2003 Molecular cloning and Comparative 134:519-527 T, Nakagoshi M, Takikawa S, characterization of a cDNA encoding Biochemistry 202 Dohke K, Iino T, Mase K, a novel cuticle protein in the and Physiology Yamamoto T, Izumi S silkworm, Bombyx mori Part B Scandiani M, Ruberti D, 2004 Recent outbreak of soybean sudden Plant Disease 88(9):1044 O'Donnell K, Aoki T, Pioli death syndrome caused by Fusarium 203 R, Giorda L, Luque A, virguliforme and F. tucumaniae in Biasoli M Argentina Seo ST, Tsuchiya K, 2004 Characterization and differentiation Journal of 70(2):120- 204 Murakami R of Erwinia carotovora strains from General Plant 123 mulberry trees Pathology Sharma R, Komatsu S, Noda H 2004 Proteomic analysis of brown Insect 34:425-435 planthopper: application to the Biochemistry 205 study of carbamate toxicity and Molecular Biology Sharma A, Isogai M, 2004 A novel interaction between Plant and Cell 45:684-692 206 Yamamoto T, Sakaguchi K, calreticulin and ubiquitin-like Physiology Hashimoto J, Komatsu S nuclear protein in rice Shibuya N, Nishiyama T, 2004 Cell-free Synthesis of polypeptides Journal of 136(5):601- Nakashima N lacking an amino-terminal methionine Biochemistry 606 207 by using a dicistroviral intergenic internal ribosome entry site Shiotsuki T, Kuwano E 2004 Identification of proteins Journal of 29(2):121- possessing a high affinity with Pesticide 123 208 imidazole insect growth regulator, Science KK-42

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Sumitani M, Yamamoto SD, 2005 Isolation of white gene orthologue Insect 35(3):231- Lee JM, Hatakeyama M of the sawfly, Athalia rosae Biochemistry 240 212 (Hymenoptera) and its functional and Molecular analysis using RNA interference Biology Sun QY, Fuchimoto D, Nagai 2004 Regulatory roles of ubiquitin- Theriogenology 62(1- 213 T proteasome pathway in pig oocyte 2):245-255 meiotic maturation and fertilization Suto J, Sekikawa K 2004 Confirmation and characterization of Journal of 66(9):1039 murine body weight QTLs, Bwq1 and Veterinary 214 Bwq2, identified in C57BL/6J x KK- Medical Ay/a F2-Ay/a mice Science Suto J, Sekikawa K 2004 Further mapping and characterization Journal of 66(9):1033 of Naq1, a quantitative trait locus Veterinary 215 responsible for maternal inferior Medical nurturing ability in RR mice Science Suto J, Sekikawa K 2004 Quantitative trait locus analysis of Biochemical 42(9&10):34 216 plasma cholesterol and triglyceride Genetics 7 levels in C57BL/6J x RR F2 mice Suzuki M, Funaguma S, Kanda 2005 Role of the male BmDSX protein in Evolution and 7:58-68 217 T, Tamura T, Shimada T the sexual differentiation of Bombyx Development mori Suzuki N, Shikamoto Y, 2005 Crystallization and preliminary X- Acta 61(1):147- Fujimoto Z, Morita T, ray analyses of coagulation factor Crystallographic 149 Mizuno H IX-binding protein from Habu snake a section F 218 venom at pH 6.5 and 4.6 Structural Biology and Crystallization Communications Suzuki R, Fujimoto Z, Kuno 2004 Crystallization and preliminary X- Acta 60(10):1895 A, Hirabayashi J, Kasai K, ray crystallographic studies of the Crystallographic -1896 219 Hasegawa T C-terminal domain of galactose- a section D binding lectin EW29 from the Biological Crystallography earthworm Lumbricus terrestris Takabe E, Sentoku N, Nagato 2003 Morphological classification of Rice Genetics 20(1):27-28 Y, Matsuoka M, Kitano H twelve rice mutants that cease Newsletter 220 embryogenesis at early globular stages Takahashi T, Imai K, 2004 A direct time-resolved The Journal of 66(3):225- Hamanaka S, Hashizume K fluoroimmunoassay (TR-FIA) for Veterinary 229 221 measuring plasma estradiol-17 beta Medical concentrations in cattle Science Takahashi T, Imai K, 2004 Generation and characterization of Journal of 50(6):717- Hashizume K anti-leptin antisera against Reproduction 724 222 synthetic peptides and recombinant and protein Development Takase T, Yanagawa Y, 2004 Overexpression of a gene for 26S Plant 21(3):233- Mitsuhara I, Ohashi Y, proteasome subunit RPN10 confers Biotechnology 236 223 Nakagawa H, Hashimoto J enhanced resistance to canavanine,an analog of arginine,in transgenic rice (Oryza sativa L..) Takeda K, Satoh M, Neopane 2004 Mitochondrial DNA analysis of Animal Science 75:103-110 SP, Kuwar BS, Joshi HD, Nepalese domestic dwarf cattle Journal 224 Shrestha NP, Fujise H, 'Lulu' Tasai, Tagami T, Hanada H

- 171 - Takehisa H, Shimodate T, 2004 Identification of quantitative trait Field Crops 89(1):85-95 225 Fukuta Y, Ueda T, Yano M, loci for plant growth of rice in Research Yamaya T, Kameya T, Sato T paddy field flooded with salt water Takenouchi T, Takayama Y, 2004 Co-treatment with dexamethasone and Cell Biology 28:209-216 226 Takezawa T octanoate induces adipogenesis in International 3T3-L1 cells Takenouchi T, Miyashita N, 2004 Role of caveolin-1 and cytoskeletal Cell Biology 28(8- Ozutsumi K, Michael TR, Aso proteins, actin and vimentin, in International 9):615-623 227 H adipogenesis of bovine intramuscular preadipocyte cells Takezawa T, Ozaki K, Nitani 2004 Collagen vitrigel: A novel scaffold Cell 13:463-473 228 A, Takabayashi C, Simo-oka that can facilitate a three- Transplantatio T dimensional culture for n Takigami S, Wakabayashi Y, 2004 Fetal development of vomeronasal Developmental 149:113-120 Ohsako S, Ohkura S, Okamura system in the goat Brain Research 229 H, Ikai A, Ichikawa M, Osada T Tamura Y, Hattori M, Konno 2004 Triterpenoid and caffeic acid Chemoecology 14:113-118 K, Kono Y, Honda H, Ono H, derivatives in the leaves of Yoshida M ragweed, Ambrosia artemisiifolia L. 230 (Asterales: Asteraceae), as feeding stimulants of Ophraella communa LeSage (Coleoptera: Chrysomelidae) Tanaka D, Niino T, 2004 Cryopreservation of shoot apices of CryoLetters 25:167-176 Isuzugawa K, Hikage T, in vitro grown Gentian plants: 231 Uemura M Comparison of vitrification and encapsulation-vitrification protocols Tanaka N, Fujita M, Handa 2004 Proteomics of the rice cell: Molecular 271:566-576 H, Murayama S, Uemura M, systematic identification of the Genetics and 232 Kawamura Y, Mitsui T, protein populations in subcellular Genomics Mikami S, Yozawa Y, compartments Yoshinaga T, Komatsu S Tanaka S 2003 Effects of temperature and [His 7]- Physiological 28: 290-297 233 corazonin on the body darkening in Entomology Locusta migratoria . Tanaka S 2004 Environmental control of body color Annals of 97: 293-301 polyphenism in the American Entomological 234 grasshopper, Schistocerca americana Socisty of Ameriaca Tanaka S 2004 Hormonal control of body-color Annals of 97: 302-309 polyphenism in the American Entomological 235 grasshopper, Schistocerca americana : Socisty of a physiological function of [His 7]- Ameriaca corazonin Tanaka S, Zhu Dao-Hong 2003 Phase-related differences in mating Annals of 96: 498-502 strategy of a locust(Orthoptera: Entomological 236 Acrididae) Society of America Tanaka S, Zhu Dao-Hong 2003 Presence of three diapauses in a Pysiological 28: 323-330 237 subtropical cockroach: control Entomology mechanisms and adaptive significance Tang WR, Shioya N, Eguchi 2005 Characterization of new Veterinary 103:113-127 T, Ebata T, Matsui J, monoclonalantibodies against porcine Immunology and Takenouchi H, Honma D, lymphocytes: molecular Immunopatholog 238 Yasue H, Takagaki Y, characterization of clon 7G3,an y Enosawa S, Itagaki M, antibody reactive with the constant Taguchi T, Kiyokawa N, region of the T-cell receptor δ- Amemiya H, Fujimoto J chains Taniai K, .Zhou CLE, Lee 2005 Expression of a non-secreted form of Japan 39(1):11-18 DG, Maeda S, Hammock BD juvenile hormone esterase in a Agricultural 239 baculovirus Research Quarterly Terami F, Fukumoto F, 2004 Cucumber mosaic virus isolated from Journal of 70(3):192- 240 Hanada K Amazon lily (Eucharis grandiflora) General Plant 193 Pathology Teramoto H, Nakajima K, 2004 Chemical modification of silk Biomacromolecu 5(4):1392- Takabayashi C sericin in lithium chloride/dimethyl les 1398 241 sulfoxide solvent with 4-cyanophenyl isocyanate

- 172 - Tewary P, Oka S 2003 Isolation and electrofusion of Sericologia 43(4):535- 242 interspecific mulberry protoplasts 541 Todoroki J, Noguchi J, 2004 Plasma concentrations of inhibin A Domestic 27(11):333- Kikuchi K, Ohnuma K, Ozawa in cattle with follicular cysts: Animal 344 243 M, Kaneko H relationships with turnover of Endocrinology follicular waves and plasma levels of gonadotropins and steroid Todoroki J, Noguchi J, 2004 Retrospective analysis of the Journal of 50: 369-373 Kikuchi K, Kaneko H efficacy of controlled internal drug Reproduction 244 relaese in follicular cysts in an and embryo donor beef herd Development Tokuda G, Lo N, Watanabe H, 2004 Major alteration of the expression Molecular 13:3219- 245 Arakawa G, Matsumoto T, site of endogenous cellulases in Ecology 3228 Noda H members of an apicaltermite lineage Akino T, Nakamura K, 2004 Diet-induced chemical phytomimesis Chemoecology 14:165-174 Wakamura S by twig-like caterpillars of Biston 246 robustum Butler (Lepidoptera: Geometridae) Akino T, Yamamura K, 2004 Direct behavioral evidence for Applied 39(3):381- Wakamura S, Yamaoka R hydrocarbons as nestmate recognition Entomology and 387 247 cues in Formica japonica Zoology (Hymenoptera: Formicidae) Toyoshima S, Hinomoto N 2004 Intraspecific Variation of Applied 39(3):351- Reproductive Characteristics of Entomology and 355 248 Amblyseius californicus (McGregor) Zoology (Acari:Phytoseiidae) Tsubouchi K, Igarashi Y, 2005 Sericin enhances attachment of Bioscience 69(2):403- Takasu Y, Yamada H cultured human skin fibroblasts Biotechnology 405 249 and Biochemistry Tsuge S, Ochiai H, Inoue Y, 2004 Involvement of phosphoglucose Phytopathology 94:478-483 250 Oku T, Tsuno K, Kaku H, isomerase in pathogenicity of Kubo Y Xanthomonas oryzae pv. oryzae Turuspekov Y, Mano Y, Honda 2004 Identification and mapping of Theoretical 109:480-487 251 I, Kawada N, Watanabe Y, cleistogamy genes in barley and Applied Komatsuda T Genetics Ueda S, Kimura T, Onuki M, 2004 Three distinct groups of isolates of Journal of 70(4):232- Hanada K, Iwanami T Tomato yellow leaf curl virus in General Plant 238 252 Japan and construction of an Pathology infectious clone Sato T, Numa H, Yano M 2003 Delimitation of the chromosomal Theoretical (108):385- region for a quantitative trait and Applied 391 253 locus, qUVR-10 , conferring Genetics resistance to ultraviolet-B radiation in rice ( Oryza sativa L.) Ueno O, Wakayama M 2004 Cellular expression of C 3 and C4 Journal of 117:433-441 photosynthetic enzymes in the Plant Research 254 amphibious sedge Eleocharis retroflexa ssp. chaetaria Ueno O 2004 Environmental regulation of Plant, Cell 27:627-639 photosynthetic metabolism in the and 255 amphibioussedge Eleocharis baldwinii Environment and comparisons with related species

Ui-Tei K, Ueda R, Zenno S, 2004 RNA interference induced by trangent Molecular 38(2):228- Takahashi F, Doi N, Naito or stable expression of heparin Biology 238 256 Y, Yamamoto M, Hashimoto N, structures of double-stranded RNA in Takahashi K, Hamada T, dirosophila and mammalian cells Tokunaga T, Saigo K Umemoto T, Aoki N, Lin H, 2004 Natural variation in rice starch Functional 31:671-684 Nakamura Y, Inouchi N, Sato synthase IIa affects enzyme and Plant Biology 257 Y, Yano M, Hirabayashi H, starch properties Maruyama S Ushizawa K, Herath BC, 2004 cDNA microarray analysis of bovine Reproductive 2(1):77 Kaneyama K, Shiojima S, embryo gene expression profiles Biology & Hirasawa A, Takahashi T, during the pre-implantation period. Endocrinology 258 Imai K, Ochiai K, Tokunaga T, Tsunoda Y, Tsujimoto G, Hashizume K

- 173 - Ushizawa K, Kaneyama K, 2005 Cloning and and expression of a new Biochemical and 326(2):435- Takahashi T, Tokunaga T, member of prolactin-related protein Biophysical 441 259 Tsunoda Y, Hashizume K in bovine placenta: bovine Research prolactin-related protein-VII Communications Volkoff AN, Rocher J, 2003 Characterization and transcriptional Gene 319:43-53 d'Alencon E, Bouton M, profiles of three Spodoptera Landais I, Quesada-Moraga frugiperda genes encoding cysteine- 260 E, Vey A, Fournier P, Mita rich peptides. A new class of K, Devauchelle G defensin-like genes from lepidopteran insects? Wakamura S, Arakaki N 2004 Sex pheromone components of pyralid Chemoecology 14(3- moths Terastia subjectalis and 4):181-185 Agathodes ostentalis feeding on the 261 coral tree, Erithrina variegata : Two sympatric species share common components in different ratios Wako T, Fukui K 2003 Quantitative analysis of nuclear Bioimages (洋 11(3-4):97- 262 chromocenter in Spiranthes sinensis 雑誌） 103 (Pers.) Ames Wang Pi-Chao, Okada N, 2004 Co-culture of glomerular epithelial Biochemical 20:149-154 263 Takezawa T cells and mesangial cells on Engineering collagen-gauze-fiber gel Journal Wang XW, Kaga A, Tomooka N, 2004 The development of SSR markers by a Theoretical and 10 Mar., Vaughan DA new method in plants and their Applied 2004 application to gene flow studies in Genetics, 264 azuki bean [Vigna angularis (Willd.) International Journal of Plant Ohwi & Ohashi] Breeding Research Warren J, Petryk A, Marques 2004 Phantom encodes the 25-hydroxylase Insect 34(9):991- G, Parvy J-P, Shinoda T, of Drosophila melanogaster and Biochemistry 1010 Itoyama K, Kobayashi J, Bombyx mori : a P450 enzyme critical and Molecular 265 Jarcho M, Li Y, O'Connor in ecdysone biosynthesis Biology BM, Dauphin-Villemant C, Gilbert L Watanabe M, Hirai Y 2004 Host-use pattern of the ragweed Applied 39: 249-254 beetle Ophraella communa LeSage Entomology and 266 (Coleoptera : Chrysomelidae) for Zoology overweintering and reproduction in Tsukuba Watanabe M, Nakano T, 2004 Developmental stage-specific Comparative 139:125-131 Shiotani B, Matsushima- expression and tissue distribution Biochemistry Hibiya Y, Kiuchi M, of pierisin-1, a guanine-specific and 267 Yukuhiro F, Kanazawa T, ADP-ribosylationg toxin, in Pieris Physiology, Koyama K, Sugimura T, rapae Part A Wakabayashi K Watanabe M, Kikawada T, 2004 Physiological traits of European 101:439-444 268 Fujita A, Forczek E, Adati invertebrates entering cryptobiosis Journal of T, Okuda T in a post-embryonic stage Entomology Watanabe S, Iwamoto M, 2005 A novel method for the production of Biology of 72:309-315 Suzuki S, Fuchimoto D, transgenic cloned pigs: Reproduction 269 Honma D, Nagai T, Hashimoto electroporation-mediated gene M, Yazaki S, Sato M, Onishi transfer to non-cultured cells and A subsequent selection with puromycin Wei-Ran Tng, Kiyokawa N, 2004 Development of Novel Monoclonal Hybridoma and 23(3):187- Eguchi T, Matsui J, Antibody 4G8 against Swine Leukocyte Hybridomics 191 Takenouchi H, Honma D, Antigen Class I α chain Enosawa S, Mimori K, 270 Itagaki M, Taguchi T, Katagiri U Y, Okita H, Amemiya H, Yasue H, Fujimoto J Wu J, Yamagata H, Hayashi- 2004 Composition and structure of the The Plant Cell 16:967-976 Tsugane M, Hijishita S, centromeric region of rice Fujisawa M, Shibata M, Ito chromosome 8 Y, Nakamura M, Sakaguchi M, Yoshihara R, Kobayashi H, 271 Ito K, Karasawa W, Yamamoto M, Saji S, Katagiri S, Kanamori H, Namiki N, Katayose Y, Matsumoto T, Sasaki T

- 174 - Liu X, Tanaka Y, Song Q, Xu 2004 Bombyx mori prothoracicostatic Archives of 56(4):155- B, Hua Y peptide inhibits ecdysteroidogenesis Insect 161 272 in vivo . Biochemistry and Physiology

Yagi Y, Shiono H, Chikayama 2004 Transport stress increases somatic Journal of 66(4):381- Y, Ohnuma A, Nakamura I, cell counts in milk, and enhances Veterinary 387 273 Yayou K the migration capacity of peripheral Medical neutrophils of dairy cows Science Yamada M, Nakamura K, 2004 Effect of modified oligopeptides Journal of 66:137-142 Saido-Sakanaka H, Asaoka A, from the beetle Allomyrina dichotoma Veterinary 274 Yamakawa M, Samejima T, on Escherichia coli in mice Medical Motobu M, Hirota Y Science Yamaguchi T, Tanabe S, 2004 Activation of phospholipase D Plant and Cell 45(9):1261 275 Minami E, Shibuya N induced by hydrogen peroxide in Physiology suspension-cultured rice cells Yamamoto DS, Sumitani M, 2004 Cloning of a decapentaplegic Development 214: 128- Tojo K, Lee JM, Hatakeyama orthologue from the sawfly, Athalia Genes and 133 276 M rosae (Hymenoptera), and its Evolution expression in the embryonic appendages Yamamoto M, Yamao M, 2004 New and highly efficient method for Biotechnol 88:849-853 Nishiyama H, Sugihara S, silkworm transgenesis using Bioeng 277 Nagaoka S, Tomita M, Autographa californica Yoshizato K, Tamura T, Mori nucleopolyhedrovirus and piggyBac H transposable elements Yamamoto T, Mori Y, 2004 Characterization of Rad6 from a Biochemical 314:434-439 Ishibashi T, Uchiyama Y, higher plant, rice ( Oryza sativa and 278 Sakaguchi N, Furukawa T, L..) and its interaction with Sgt1, Biophysical Hashimoto J, Kimura S, a subunit of the SCF ubiquitin Research Sakaguchi K ligase complex Communications Yanagawa Y, Sullivan J, 2004 Arabidopsis COP10 form a complex Genes & 18:2172- Komatsu S, Gusmaroli G, with DDB1 and DDB1 and DET1 in vivo Development 2181 Suzuki G, Yin J, Ishibashi and enhances the activity of 279 T, Saijo Y, Rubio V, Kimura ubiquitin conjugating enzymes S, Wang J, Deng Xing Wang

Yang G, Jan A, Shen S, 2004 Microarray analysis of Molecular 271:468-478 Yazaki J, Ishikawa M, brassinosteroids- and gibberellin- Genetics and 280 Shimatani Z, Kishimoto N, regulated gene expression in rice Genomics Kikuchi S, Matsumoto H, seedlings Komatsu S Yasukochi Y, Ashakumary L, 2004 Organization of the Hox gene cluster Development 214:606-614 Wu C, Yoshido A, Nohata J, of the silkworm, Bombyx mori : A Genes and 281 Mita K, Sahara K split of the Hox cluster in a non- Evolution Drosophila insect Yasunaga-Aoki C, Imanishi 2004 Establishment of phagocytic cell In Vitro Cell. 40:183-186 282 S, Iiyama K, Kawarabata T lines from larval hemocytes of the Dev. Biol.- Beet Armyworm, Spodoptera Exigua Animal. Yazaki J, Shimatani Z, 2004 Transcriptional profiling of genes Physiological 17:87-100 Hashimoto A, Nagata Y, Fujii responsive to abscisic acid and Genomics F, Kojima K, Suzuki K, Taya T, gibeberellin in rice: phenotyping 283 Tonouchi M, Nelson C, Nakagawa and comparative analysis between A, Otomo Y, Murakami K, Matsubara K, Kawai J, Carninci rice and Arabidopsis P, Hayashizaki Y, Kikuchi S Yokomizo T, Nakasako M, 2005 Hydrogen-bond patterns in the Chemical 401(4- 284 Yamazaki T, Shindo H, Higo hydration structure of a protein Physics 6):332-336 J Letters Yonezawa N, Amari S, 2005 Participation of the nonreducing Molecular 70:222-227 Takahashi K, Ikeda K, Imai terminal β-galactosyl residues of Reproduction 285 FL, Kanai S, Kikuchi K, the neutral N-linked carbohydrate and Nakano M chains of porcine zona pellucida Development glycoproteins in sperm-egg binding Yoshida H, Akimoto H, 2004 Alteration of methylation profiles Euphytica 135:247-253 Yamaguchi M, Shibata M, in distinct cell lineages of the 286 Habu Y, Iida S, Ozeki Y layers during vegetative propagation in carnations (Dianthus caryophyllus)

- 175 - Yoshii M, Nishikiori M, 2004 The Arabidopsis CUCUMOVIRUS Journal of 78(12):6102 Tomita K, Yoshioka N, MULTIPLICATION 1 and 2 loci encode Virology -6111 287 Kozuka R, Naito S, Ishikawa translation initiation factors 4E M and 4G Yoshimura Y, Kubota F, Ueno 2004 Structural and biochemical bases of Planta 220:307-317 O photorespiration in C4 plants: 288 quantification of organelles and glycine decarboxylase Yoshioka K, Suzuki C, Ito 2004 Production of piglets derived from Biology of 69:2092- S, Kikuchi K, Iwamura S, in vitro -produced blastocysts Reproduction 2099 Rodriguez-Martinez H fertilized and cultured in 289 chemically defined media: effects of theophylline,adenosine, and cysteine during in vitro fertilization Yotushimma K, Sakaguchi M, 2004 Effect of fatty-free bovine serum Journal of 50(4):471- Shimizu M, Okimura T, alubumin and fetal calf serum Reproduction 476 Izaike Y supplementing repair cultures on and 290 pre- and post-warm viability of Development biopsied bovine embryos produced in vitro Yuhashi N, Tomiyama M, 2005 Development of a novel glucose Biosensors and 20:2145- Okuda J, Igarashi S, enzyme fuel cell system employing Bioelectronics 2150 291 Ikebukuro K, Sode K protein engineered PQQ glucose dehydrogenase Ishikawa Y, Endo M, Abe K, 2004 Isolation of four Rad23 genes from Plant 21(1):65-71 Osakabe K,Nakajima N, Saji Arabidopsis thaliana and detection Biotechnology 292 S, Ito Y, Ichikawa H, of alternative splicing variants Kameya T, Toki S Zhu Dao-Hong, Tanaka S 2004 Photoperiod and temperature affect Physiological 29: 16-25 the life cycle of a subtropical Entomology 293 cockroach, Opisoplatia orientalis : seasonal pattern shaped by winter Zhu DH, Tanaka S 2004 Summer diapause and nymphal growth Physiological 29: 78-83 294 in a subtropicalcockroach: response Entomology to changing photoperiod. Zhuang T, Kashiwabara S, 2004 Transgenic expression of testis- Journal of 50:207-213 295 Noguchi J, Baba T specific poly(A) polymerase TPAP in Reproduction wild-type and TPAP-deficient mice. and

Reviews Benjamin R, Blackwell M, 2004 Insect- and other arthropod- Biodiversity 395-433 Chapela I, Humber R, Jones associated fungi of fungi : K, Klepzig K, Lichtwardt R, inventory and 1 Mallock D, Noda H, Roeper monitoring R, Spatafora J, Weir A methods

Fukaya M, Arakaki N, Yasui 2004 Effect of colour on male orientation Chemoecology 14(3- H, Wakamura S to female pheromone in the black 4):225-228 2 chafer Holotrichia loochooana loochooana Gill BS, Apples R, Botha- 2004 A workshop report on wheat genome Genetics 168(2):1087 Oberholster Anna-Maria, sequencing: International Genome Buell CR, Bennetzen JL., Research on Wheat Consortium Chalhoub B, Chumley F, 3 Dvorak J, Iwanaga M, Keller B, Li W, McCombie WR, Ogihara Y, Quetier F, Sasaki T Hirochika H 2003 Insertional mutagenesis in rice Rice science: 205-212 using the endogenous retrotransposon Innovations 4 and impact for livelihood Hirohiko H, Guiderdoni E, 2004 Rice mutant resources for gene Plant 54:325-334 An G, Hsing Y , Young ME, discovery Molecular 5 Han Cd, Upadhyaya N, Biology Ramachandran S, Zhang Q, Pereira1A, Sundaresan V,

- 176 - Inagaki N, Satoh K 2004 C-terminal processing peptidase of HANDBOOK OF 2028-2031 chloroplasts PROTEOLYTIC 6 ENZYMES, 2nd Edition Ishikawa M, Okada Y 2004 Replication of tobamovirus RNA Proceedings of 80(5):215- the Japan 224 7 Academy series B Kaga A, Vaughan DA, Tomooka 2004 Molecular markers in Vigna Biotechnology 55:171-187 N improvement: understanding and using in Agriculture 8 gene pools and Forestry

Khan Md Monowar Karim, 2004 Phosphoproteomics: recent Recent 2:73-83 Komatsu S advancements and fiture prospect in Research 9 rice phosphoproteome research Development Plant Science Khan M, Monowar Karim, 2004 Rice proteomics: recent developments Phytochemistry 65:1671- 10 Komatsu S and analysis of nuclear proteins 1681 Komatsu S, Tanaka N 2004 Rice proteome analysis: A step Proteomics 4:938-949 11 toward functional analysis of the rice genome Kusaba M 2004 RNA interference in crop plants Current 15:139-143 12 Opinion in Biotechnology Miura K, Lin S Y, Araki H, 2004 Genetical studies on germination of Japan 38(1):1-5 Nagamine T, Kuroki M, seed and seedling establishment for Agricultural 13 Shimizu H, Ando I, Yano M breeding of improved varieties Research suitable for direct seeding culture Quarterly Miyao A, Hirochika H 2004 Transposon-insertion lines of rice Rice Blast: 107-112 for analysis of gene function Interaction 14 with Rice and Control Naito M, Kuwana T 2004 Production of chimeric chickens Methods in 245-253 Molecular Biology, Vol.256: Germ Cell Protocoles, 15 Volume 2: Molecuar Embryo Analysis, Live Imaging, Transgenesis, and Cloning Nakayama S, Fujishita M, 2004 FISH shows structural Plant Genome: 235-246 Ohyama K differentiation between liverwort Biodiversity 16 sex chromosomes and Evolution, Volume 2 Part A: Lower Nisiguchi M, Shimono M, 2004 Microarray analysis of gene Rice Blast: 145-154 Eguchi Y, Okuizumki H, expression in rice treated with Interaction Yazaki J, Nakamura K, Fujii probenazole, a resistance inducer, with Rice and F, Shimbo K, Shimatani Z, in special reference to blast Control 17 Nagata Y, Hashimoto A, Ohta disease T, Sato Y, Honda S, Iwano M, Yamamoto K, Sakata K, Sasaki T, Kishimoto N, Nishizawa Y, Minami E, 2004 Analysis of rice/blast interactions Genomic and 174-184 Akimoto-Tomiyama C, focused on chitinolytic enzyme and Genetic Mizobuchi R, Tagiri A, Itoh chitin elicitor Analysis of 18 Y, Watanabe T Plant Parasitism and Defense Ohashi Y, Seo S, Mitsuhara 2004 Signal transduction in TMV-infected Genomic and 249-257 I, Yamakawa H, Takabatake R and wounded tobacco plants Genetic Analysis of 19 Plant Parasitism and Defense

- 177 - Okamura H, Mori Y 2005 Characterization of the primer Chemical 30(suppl pheromone molecules responsible for Senses 1):i140- 20 the 'male effect' in ruminant Supplement i141 species Okuda T, Watanabe M, 2004 Cryptobiosis in the African Proceedings of 39: 1-7 Kikawada T, Fujita A, chironomid: physiological mechanism Arthropod 21 Forczek E to survive complete dehydration Embryology Society of Japan Okuizumi H, Matsuyama T, 2004 Genome Scanning Method; Ristriction Encyclopedia 5:413-439 Hayashizaki Y Landmark Genomic Scanning (RLGS) of Molecular 22 Cell Biology and Molecular Medicine Okuno K 2004 Molecular mechanisms of cold Japanese 20(2):51-60 tolerance in rice and wheat Journal of 23 Hyperthermic Oncology Saito T, Hirai K, Way OM 2005 The rice water weevil, Lissorhoptrus Applied 40(1):31-39 24 oryzophilus Kuschel Entomology and (Coleoptera:Curculionidae) Zoology Sasaki T 2004 Rice genome sequence analysis and Farming Japan 38: 18-24 25 the development of rice science Sasaki T 2004 Chapter 11. Rice functional Plant 261-278 26 genomics: from nucleotide sequence Functional to gene function Genomics Sasaki T, Antonio BA. 2004 Chapter 18 : Rice genome as a model Cereal 535-557 system for cereals Genomics, ISBN 27 : 1-4020-2358- 8 Sasaki T, Matsumoto T, 2005 From mapping to sequencing, post- Plant and Cell 46(1):3-13 28 Antonio Baltazar A., sequencing and beyond Physiology Nagamura Y Sasaki T, Christou P 2004 Plant biotechnology: editorial Current 15: 117-119 29 overview Opinion in Biotechnology Fukuoka S, Shimizu T, Yano 2004 Genetic dissection and mapping of Rice Blast: 131-136 M, Okuno K, Nagamine T genes conferring field resistance to Interaction 30 rice blast in Japanese upland rice with Rice and Control Takenouchi T, Takezawa T 2004 Isolation and characterization of Animal Cell 13: 219-223 fetal bovine cells derived from Technology: 31 liver and bone marrow Basic & Applied Aspects Takezawa T 2004 A novel technology for conversion of Animal Cell 13:19-23 in vivo tissue architecture into a Technology: 32 three-dimensional in vitro culture Basic & mode Applied Aspects Vaughan DA, Miyazaki S, 2004 The rice genepool and human Biological 1-13 33 Miyashita K migrations Resources and Migration Wako T, Fukui K 2004 Histone modifications in plant Recent 1:313-322 chromosomes Research Developments 34 in Plant Molecular Biology

- 178 - Yamazaki T, Katoh E, Katoh 2004 Deformation of one α-helix leads to PEPTIDES, 559-561 S, Tsunoda Y, Mizuno T S-S bond formation of YhhP Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings of the Eighteen 35 American Peptids Symposium, Michael Chorev and Tomi K. Sawyer, Eds.

Yang G, Komatsu S 2004 Microarray and proteomic analysis of Genomics 2(2):77-83 brassinosterid- and gibberellin- Proteomics & 36 regulated gene and protein Bioinformatics expression in rice Yano M, Takeuchi Y, Nonoue 2003 Marker-assisted dissection and Rice Science: 257-263 Y, Ando T, Shomura A, pyramiding of complex traits in rice Innovations Shimizu T, Kono I, and Impact for 37 Takahashi Y, Yamanouchi U, Livelihood Konishi S, Liang ZW, Ueda T, Yamamoto S, Izawa T, Sasaki T Yasuda N, Hirayae K, 2004 Relationship between two avirulence Rice Blast: 65-69 Hayashi N, Fujita Y, genes: Avr-Hattan 3 and Avr-Piks Interaction 38 Tsujimoto M, Nakajima T with Rice and Control

- 179 - Author Index Author Department Paper No.

Physiology and Genetic Regulation Akino Toshiharu 3, 4, 21, 22, 23, 62, 246, 247 Department Aoki Takayuki Genetic Diversity Department 89, 163, 203 Asano Takayuki Molecular Genetics Department 5, 6, 67 Physiology and Genetic Regulation Asaoka Kiyoshi 193 Department Awata Takashi Genome Research Department 19, 43, 47, 122, 123, 155 Fuchimoto Daiichiro Developmental Biology Department 63, 121, 154, 209, 213, 269 16, 17, 65, 74, 106, 107, 129, 151, Fujimoto Zui Biochemistry Department 184, 218, 219 Insect Biotechnology and Sericology Fukui Kuniaki 24 Department Fukuoka Shuuichi Genebank 116 Furusawa Tadashi Developmental Biology Department 26 Molecular Biology and Immunology Goto Hideo 29 Department Habu Yoshiki Plant Biotechnology Department 286 Hamasima Noriyuki Genome Research Department 122, 131 Department of Research Planning Hanada Kaoru 240, 252 and Coordination Handa Hirokazu Plant Biotechnology Department 196, 232 Harumi Takashi Developmental Biology Department 33, 143 Insect Biomaterial and Technology Hata Tamako 80, 81 Department Hatakeyama Masatsugu Developmental Biology Department 38, 212, 276 Insect Genetics and Evolution Hattori Makoto 230 Department Insect Biotechnology and Sericology Hayasaka Shoji 53 Department Hayashi Nagao Genetic Diversity Department 6, 99, 123 Hayashi Takeshi Genome Research Department 11, 43, 155, 156 Higo Ken-ichi Vice President 64, 152 Insect Genetics and Evolution Hinomoto Norihide 48, 49, 158, 248 Department Hirochika Hirohiko Molecular Genetics Department 7, 95, 108, 161, 186 Insect Genetics and Evolution Hirokawa Masahiko 50 Department Physiology and Genetic Regulation Ichikawa Akio 51, 76, 77 Department Ichikawa Hiroaki Plant Biotechnology Department 66, 292 Insect Biotechnology and Sericology Imanishi Shigeo 55, 282 Department Molecular Biology and Immunology Ishibashi Jun 150, 183 Department Ishikawa Masaya Genetic Diversity Department 58 Ishikawa Masayuki Plant Physiology Department 287 Ishimaru Ken Plant Physiology Department 59, 60 Physiology and Genetic Regulation Ito Yuuji 19 Department Itoh Takeshi Genome Research Department 56, 148 Iwabuchi Masaki President 185

- 180 - Iwamoto Masao Plant Physiology Department 63, 64, 209 Izaike Yoshiaki Developmental Biology Department 174, 290 Izawa Takeshi Molecular Genetics Department 15 Jiang Chang-Jie Plant Physiology Department 144 Kadono-Okuda Keiko Genome Research Department 124, 125 Kadowaki Kouichi Genetic Diversity Department 5, 67, 109, 116, 157, 171 Haga Atsunobu Genetic Diversity Department 10, 190, 264 Kaku Hanae Biochemistry Department 14, 147, 169 Kaku Hisatoshi Genetic Diversity Department 27, 68, 99, 250 Insect Biomaterial and Technology Kameda Tsunenori 69, 70, 71, 72 Department Kaneko Hiroyuki Genetic Diversity Department 63, 120, 160, 243, 244 Kanayama Kanako Developmental Biology Department 258, 259 Physiology and Genetic Regulation Kasuya Etsuko 37, 78, 187 Department Katayose Yuuichi Genome Research Department 271 Insect Biotechnology and Sericology Kato Hiroshi 80, 81 Department Katoh Etsuko Biochemistry Department 82, 84 Kawagoe Yasushi Plant Biotechnology Department 83, 84 Kawahigashi Hiroyuki Plant Biotechnology Department 85 Physiology and Genetic Regulation Kawasaki Kenjiro 49, 132 Department Department of Research Planning Kayano Toshiaki 34, 96 and Coordination Physiology and Genetic Regulation Kikawada Takahiro 268 Department 42, 63, 160, 209, 243, 244, 285, Kikuchi Kazuhiro Genetic Diversity Department 289 Kikuchi Shoushi Molecular Genetics Department 140, 141, 142, 210, 280, 283 Insect Biotechnology and Sericology Kinoshita Haruo Department Kishimoto Naoki Molecular Genetics Department 152, 210, 280 Molecular Biology and Immunology Kitani Hiroshi 94, 135, 136 Department Department of Research Planning Kiuchi Makoto 267 and Coordination Koga-Ban Yasunori Plant Biotechnology Department 96 Insect Biomaterial and Technology Kojima Katura 57 Department Physiology and Genetic Regulation Kojima Misaki 97, 98 Department 1, 2, 6, 35, 66, 88, 99, 104, 105, Komatsu Setsuko Molecular Genetics Department 164, 165, 179, 180, 205, 206, 232, 279, 280 Komatsuda Takao Genetic Diversity Department 44, 45, 100, 101, 117, 189, 251 Insect Genetics and Evolution 102 Komoto Natsuo Department Insect Genetics and Evolution Konno Koutaro 230 Department Insect Genetics and Evolution Kosegawa Eiichi 50 Department Insect Biotechnology and Sericology Mase Keisuke 201, 202 Department Matsubara Yuko Developmental Biology Department 33, 143 Matsumoto Takashi Genome Research Department 79, 271

- 181 - Insect Genetics and Evolution matsumoto yukiko 29 Department Meshi Tetsuo Plant Physiology Department 40, 185 Mikawa Satoshi Genome Research Department 123, 155 Minami Eiichi Biochemistry Department 14, 92, 153, 181, 184, 275 Minezawa Mitsuru Genebank 211

Mita Kazuei Genome Research Department 13, 124, 125, 159, 172, 260, 281

Mitsuhara Ichiro Plant Physiology Department 75, 166, 192, 223 Physiology and Genetic Regulation Mitsuhashi Tadayoshi 191 Department Insect Genetics and Evolution Mitsuhaｓhi Wataru 126, 133 Department Insect Genetics and Evolution Miyamoto Kazuhisa 41, 133 Department Department of Research Planning Miyao Akio 108, 161, 186 and Coordination Miyao Mitsue Plant Physiology Department 46 Department of Research Planning Miyashita Kiyotaka 184 and Coordination Physiology and Genetic Regulation Miyashita Norikazu 47, 127, 128, 227 Department Insect Biomaterial and Technology Miyazawa Mitsuhiro 70, 71 Department Momma Mitsuru Biochemistry Department 129, 184 Insect Genetics and Evolution Muraji Masahiko 28, 49, 132 Department Insect Genetics and Evolution Murakami Ritsuko 126, 133, 204 Department Myohara Maroko Developmental Biology Department 134 Nagai Toshirou Genebank 137, 138 Nagamura Yoshiaki Genome Research Department 125 Naito Mitsuru Developmental Biology Department 33, 143 Nakagawa Hitoshi Institute of Radiation Breeding 91 Insect Genetics and Evolution Nakashima Nobuhiko 39 Department Insect Biotechnology and Sericology Nakajima Kenichi 241 Department Niino Takao Genebank 118, 231 Nishikawa Tomotarou Genetic Diversity Department 116, 157 Nishimura Marie Genetic Diversity Department 111 Nishizawa Yoko Plant Biotechnology Department 87, 93, 96 Insect Genetics and Evolution Noda Hiroaki 9, 30, 205, 245 Department Insect Genetics and Evolution Noda Takashi 49, 132 Department Noguchi Junko Genetic Diversity Department 63, 86, 160, 243, 244, 295 Numa Hisataka Genome Research Department 253 Ochiai Hirokazu Genetic Diversity Department 27, 250 Ohkoshi Katsuhiro Developmental Biology Department 26 Physiology and Genetic Regulation Ohkura Satoshi 32, 119, 167, 188 Department Oka Seibi Plant Biotechnology Department 242 Physiology and Genetic Regulation Okamura Hiroaki 119, 167 Department

- 182 - Physiology and Genetic Regulation Okuda Takashi 268 Department

Onishi Akira Developmental Biology Department 63, 121, 154, 209, 269

Physiology and Genetic Regulation Saito Toshiyuki 12, 78, 187 Department Physiology and Genetic Regulation Sakumoto Ryousuke 78, 187 Department Sasaki Takuji Genome Research Department 79, 125, 195, 271 Sato Mamoru Genetic Diversity Department 52, 198, 199, 200, 224 Sato Toyozou Genebank 138, 139, 197 Sentoku Noki Plant Physiology Department 220 Seo Shigemi Plant Physiology Department 166, 192 Insect Genetics and Evolution shinoda tetsuro 265 Department Shiotsuki Takahiro Developmental Biology Department 146, 208 Shirata Akira Genebank 118 Sugimoto Kazuhiko Molecular Genetics Department 7 Molecular Biology and Immunology Suto Jun-ichi 214, 215, 216 Department Suzuki Shun-ichi Developmental Biology Department 269 Tabei Yutaka Plant Biotechnology Department 93, 96 Insect Biotechnology and Sericology Takabayashi Chiyuki 8, 228, 241 Department Takahashi Hideaki Genebank 194 Takahashi Sakiko Genetic Diversity Department 5 173, 174, 175, 176, 221, 222, 258, Takahashi Toru Developmental Biology Department 259 Takaiwa Fumio Plant Biotechnology Department 83, 84 Insect Biomaterial and Technology Takasu Youko 249 Department Takatsuji Hiroshi Plant Physiology Department 144, 145 Molecular Biology and Immunology Takenouchi Takato 36, 226, 227 Department Takeuchi Kasumi Genebank 138 Physiology and Genetic Regulation Takezawa Toshiaki 174, 226, 228, 263 Department Takyu Toshio Institute of Radiation Breeding 170 Insect Biotechnology and Sericology Tamura Toshiki 57, 61, 217, 277 Department Insect Genetics and Evolution Tamura Yasumori 230 Department Molecular Biology and Immunology Tanaka Hiromitsu 150, 182 Department Physiology and Genetic Regulation 114, 115, 178, 233, 234, 235, 236, Tanaka Seiji Department 237, 293, 294 Tanaka Yoshiaki Developmental Biology Department 272 Insect Biotechnology and Sericology Taniai Kiyoko 239 Department Insect Genetics and Evolution Tatematsu Kenichiro 50 Department Insect Biotechnology and Sericology Teramoto Hidetoshi 241 Department Toki Seiichi Plant Biotechnology Department 1, 170, 292 Tokunaga Tomoyuki Developmental Biology Department 26, 256, 259

- 183 - Tomioka Keisuke Genebank 138 Tomiyama Masamitsu Genetic Diversity Department 291 Tomiyama-Akimoto Chiharu Biochemistry Department 92 Tomooka Norihiko Genetic Diversity Department 190, 264 Tsukada Masuhiro 80, 81 Ueda Tadamasa Molecular Genetics Department 225 Uenishi Hirohide Genome Research Department 19, 122, 123, 131, 168 Ueno Osamu Plant Physiology Department 103, 254, 255, 288 Umehara Yousuke Plant Physiology Department 73

Vaughan Duncan Alexander Genetic Diversity Department 157, 190, 264

Insect Genetics and Evolution Wada Sanae 133 Department Physiology and Genetic Regulation Wakamura Sadao 3, 4, 21, 22, 23, 246, 247, 261 Department Wako Toshiyuki Biochemistry Department 110, 262 Insect Genetics and Evolution Watanabe Hirofumi 245 Department Physiology and Genetic Regulation Watanabe Masahiko 266, 267 Department Watanabe Satoshi Developmental Biology Department 269 Wu Jianzhong Genome Research Department 79, 271 Molecular Biology and Immunology Yamakawa Minoru 130, 150, 182, 183, 274 Department Yamamoto Kimiko Genome Research Department 124, 125 Yamanouchi Hiroaki Institute of Radiation Breeding 170 Yamazaki Toshimasa Biochemistry Department 284 15, 18, 112, 113, 162, 225, 253, Yano Masahiro Molecular Genetics Department 257 Physiology and Genetic Regulation Yasuda Tetsuya 21, 22, 23 Department Yasue Hiroshi Genome Research Department 11, 156, 168, 191, 238, 270 Physiology and Genetic Regulation Yasui Hiroe 4, 21, 22, 23 Department Yasukochi Yuji Genome Research Department 54, 125, 281 Physiology and Genetic Regulation Yayou Kenichi 273 Department Yoshikawa Manabu Molecular Genetics Department 177 Yoshioka Terutaka Institute of Radiation Breeding 170

- 184 - Reviews Benjamin R, Blackwell M, 2004 Insect- and other arthropod- Biodiversity 395-433 Chapela I, Humber R, Jones associated fungi of fungi : K, Klepzig K, Lichtwardt R, inventory and 1 Mallock D, Noda H, Roeper R, monitoring Spatafora J, Weir A methods

Fukaya M, Arakaki N, Yasui 2004 Effect of colour on male orientation Chemoecology 14(3-4):225- H, Wakamura S to female pheromone in the black 228 2 chafer Holotrichia loochooana loochooana Gill BS, Apples R, Botha- 2004 A workshop report on wheat genome Genetics 168(2):1087 Oberholster Anna-Maria, sequencing: International Genome Buell CR, Bennetzen JL., Research on Wheat Consortium Chalhoub B, Chumley F, 3 Dvorak J, Iwanaga M, Keller B, Li W, McCombie WR, Ogihara Y, Quetier F, Sasaki T Hirochika H 2003 Insertional mutagenesis in rice using Rice science: 205-212 the endogenous retrotransposon Innovations 4 and impact for livelihood Hirohiko H, Guiderdoni E, An 2004 Rice mutant resources for gene Plant 54:325-334 G, Hsing Y , Young ME, Han discovery Molecular 5 Cd, Upadhyaya N, Biology Ramachandran S, Zhang Q, Pereira1A, Sundaresan V, Inagaki N, Satoh K 2004 C-terminal processing peptidase of HANDBOOK OF 2028-2031 chloroplasts PROTEOLYTIC 6 ENZYMES, 2nd Edition Ishikawa M, Okada Y 2004 Replication of tobamovirus RNA Proceedings of 80(5):215- the Japan 224 7 Academy series B Kaga A, Vaughan DA, Tomooka 2004 Molecular markers in Vigna Biotechnology 55:171-187 N improvement: understanding and using in Agriculture 8 gene pools and Forestry

Khan Md Monowar Karim, 2004 Phosphoproteomics: recent Recent 2:73-83 Komatsu S advancements and fiture prospect in Research 9 rice phosphoproteome research Development Plant Science Khan M, Monowar Karim, 2004 Rice proteomics: recent developments Phytochemistry 65:1671-1681 10 Komatsu S and analysis of nuclear proteins Komatsu S, Tanaka N 2004 Rice proteome analysis: A step toward Proteomics 4:938-949 11 functional analysis of the rice genome Kusaba M 2004 RNA interference in crop plants Current 15:139-143 12 Opinion in Biotechnology Miura K, Lin S Y, Araki H, 2004 Genetical studies on germination of Japan 38(1):1-5 Nagamine T, Kuroki M, seed and seedling establishment for Agricultural 13 Shimizu H, Ando I, Yano M breeding of improved varieties Research suitable for direct seeding culture Quarterly Miyao A, Hirochika H 2004 Transposon-insertion lines of rice Rice Blast: 107-112 for analysis of gene function Interaction 14 with Rice and Control

- 185 - Naito M, Kuwana T 2004 Production of chimeric chickens Methods in 245-253 Molecular Biology, Vol.256: Germ Cell Protocoles, 15 Volume 2: Molecuar Embryo Analysis, Live Imaging, Transgenesis, and Cloning Nakayama S, Fujishita M, 2004 FISH shows structural differentiation Plant Genome: 235-246 Ohyama K between liverwort sex chromosomes Biodiversity 16 and Evolution, Volume 2 Part A: Lower Nisiguchi M, Shimono M, 2004 Microarray analysis of gene Rice Blast: 145-154 Eguchi Y, Okuizumki H, expression in rice treated with Interaction Yazaki J, Nakamura K, Fujii probenazole, a resistance inducer, in with Rice and F, Shimbo K, Shimatani Z, special reference to blast disease Control 17 Nagata Y, Hashimoto A, Ohta T, Sato Y, Honda S, Iwano M, Yamamoto K, Sakata K, Sasaki T, Kishimoto N, Kikuchi S Nishizawa Y, Minami E, 2004 Analysis of rice/blast interactions Genomic and 174-184 Akimoto-Tomiyama C, focused on chitinolytic enzyme and Genetic Mizobuchi R, Tagiri A, Itoh chitin elicitor Analysis of 18 Y, Watanabe T Plant Parasitism and Defense Ohashi Y, Seo S, Mitsuhara 2004 Signal transduction in TMV-infected Genomic and 249-257 I, Yamakawa H, Takabatake R and wounded tobacco plants Genetic Analysis of 19 Plant Parasitism and Defense Okamura H, Mori Y 2005 Characterization of the primer Chemical 30(suppl pheromone molecules responsible for Senses 1):i140-i141 20 the 'male effect' in ruminant species Supplement

Okuda T, Watanabe M, 2004 Cryptobiosis in the African Proceedings of 39: 1-7 Kikawada T, Fujita A, chironomid: physiological mechanism Arthropod 21 Forczek E to survive complete dehydration Embryology Society of Japan Okuizumi H, Matsuyama T, 2004 Genome Scanning Method; Ristriction Encyclopedia 5:413-439 Hayashizaki Y Landmark Genomic Scanning (RLGS) of Molecular 22 Cell Biology and Molecular Medicine Okuno K 2004 Molecular mechanisms of cold Japanese 20(2):51-60 tolerance in rice and wheat Journal of 23 Hyperthermic Oncology Saito T, Hirai K, Way OM 2005 The rice water weevil, Lissorhoptrus Applied 40(1):31-39 24 oryzophilus Kuschel Entomology and (Coleoptera:Curculionidae) Zoology Sasaki T 2004 Rice genome sequence analysis and the Farming Japan 38: 18-24 25 development of rice science Sasaki T 2004 Chapter 11. Rice functional genomics: Plant 261-278 26 from nucleotide sequence to gene Functional function Genomics Sasaki T, Antonio BA. 2004 Chapter 18 : Rice genome as a model Cereal 535-557 system for cereals Genomics, ISBN 27 : 1-4020-2358- 8 Sasaki T, Matsumoto T, 2005 From mapping to sequencing, post- Plant and Cell 46(1):3-13 28 Antonio Baltazar A., sequencing and beyond Physiology Nagamura Y

- 186 - Sasaki T, Christou P 2004 Plant biotechnology: editorial Current 15: 117-119 29 overview Opinion in Biotechnology Fukuoka S, Shimizu T, Yano 2004 Genetic dissection and mapping of Rice Blast: 131-136 M, Okuno K, Nagamine T genes conferring field resistance to Interaction 30 rice blast in Japanese upland rice with Rice and Control Takenouchi T, Takezawa T 2004 Isolation and characterization of Animal Cell 13: 219-223 fetal bovine cells derived from liver Technology: 31 and bone marrow Basic & Applied Aspects Takezawa T 2004 A novel technology for conversion of Animal Cell 13:19-23 in vivo tissue architecture into a Technology: 32 three-dimensional in vitro culture Basic & mode Applied Aspects Vaughan DA, Miyazaki S, 2004 The rice genepool and human Biological 1-13 33 Miyashita K migrations Resources and Migration Wako T, Fukui K 2004 Histone modifications in plant Recent 1:313-322 chromosomes Research Developments 34 in Plant Molecular Biology Yamazaki T, Katoh E, Katoh 2004 Deformation of one α-helix leads to PEPTIDES, 559-561 S, Tsunoda Y, Mizuno T S-S bond formation of YhhP Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings of the Eighteen 35 American Peptids Symposium, Michael Chorev and Tomi K. Sawyer, Eds.

Yang G, Komatsu S 2004 Microarray and proteomic analysis of Genomics 2(2):77-83 brassinosterid- and gibberellin- Proteomics & 36 regulated gene and protein expression Bioinformatics in rice Yano M, Takeuchi Y, Nonoue 2003 Marker-assisted dissection and Rice Science: 257-263 Y, Ando T, Shomura A, pyramiding of complex traits in rice Innovations Shimizu T, Kono I, Takahashi and Impact for 37 Y, Yamanouchi U, Konishi S, Livelihood Liang ZW, Ueda T, Yamamoto S, Izawa T, Sasaki T

Yasuda N, Hirayae K, Hayashi 2004 Relationship between two avirulence Rice Blast: 65-69 N, Fujita Y, Tsujimoto M, genes: Avr-Hattan 3 and Avr-Piks Interaction 38 Nakajima T with Rice and Control

- 187 - International Meetings and Foreign Visitors

International Meetings

■ NIAS/COE International Symposium: “Frontiers of Arthropod Protein Peptide Science” The symposium was held at Tsukuba International Conference Hall on September 6-7, 2004. The Participants were 80 people, including domestic and international. In this symposium, we dealt with specific protein in arthropod such as silkworms, spiders, and horseshoe crab and set up 3 sessions and discussed on the latest research results and topics. At the session, “Structural chemistry of protein peptide derived from arthropod”, lectures on elucidation of molecular structure and functions of the antibacterial protein nuclear receptor, derived from insect and horseshoe crab by analyses of X-ray and NMR were given. At the session, “fibroin protein of spiders and silkworms”, lectures on transformation of silk protein using genetically modified technology and on its materialization were given. At the session, “protein science of insects”, lectures of research on synthesis of insect chitin and on gene functions of hormone receptor protein were given. With the rapid advancement of analysis of genomics of silkworms, the participants were aware of the importance of protein science as post genomes and every lecture was heated with discussions.

■ The 12th Animal Genome International Workshop The 12th animal genome international workshop was held as the joint symposium entitled "Toward the advance of animal breeding utilizing genome information" under the joint auspices of NIAS, Japanese Society of Animal Breeding and Genetics, the Society for Techno-innovation of Agriculture, Forestry and Fisheries (STAFF), and the Livestock Technology Association in Japan (JLTA) on September 16-17, 2004 at Josui Kaikan in Tokyo. Four invited speakers from overseas presented lectures on genome research achievements and their application to animal breeding on the first day. Among the topics presented included a strategy for sequencing the pig genome, prospects for positional cloning using the cattle whole genome sequence, strategies for marker-assisted selection in cattle and full-length sequencing and expression analysis of the pig major histocompatibility complex as a useful information for disease resistance. On the second day, four speakers from Japan gave presentation on the present status and future of marker-assisted selection in Japan, for dairy and beef cattle, pig and fish, respectively. The subject for beef cattle especially was followed by a panel discussion with field researchers. About 300 participants attended the symposium.

- 188 - ■ The 12th NIAS International Workshop on Genetic Resources entitled “Genetic and Functional Diversity of Agricultural Microorganisms” NIAS sponsored the 12th NIAS International Workshop on Genetic Resources as one of the symposia during the 10th International Congress for Culture Collection to be held 11-16 October 2004 in Tsukuba. About 400 scientists involving more than 200 from abroad participated in the workshop focusing on genetic and functional diversity of agricultural microorganisms. Drs. Kerry O’Donnell, USA and Marie-Anne van Sluys, Brazil discussed the developments of molecular-based phylogeny and taxonomy and genomics of major plant pathogenic microorganisms, Fusarium and Xanthomonas, respectively. Innovative roles of culture collections in near future were also discussed at other symposium on “New Paradigms of Biological Resource Centers”. Poster presentations contributed to the information exchange of recent research progress in agricultural microorganisms. A total of 10 researchers from 5 Asian countries, China, Myanmar, Pakistan, Thailand and Vietnam took part in the training course on identification and preservation of plant pathogens which was held on 16 October at NIAS Genebank.

■ “World Rice Research Conference 2004” World Rice Research Conference with a title “Rice is Life” was held at Tsukuba International Congress Center on 5-7 November 2004 in collaboration with International Rice Research Institute (IRRI). The conference had 20 concurrent sessions, 6 workshops and poster sessions. NIAS co-sponsored WRRC 2004 and organized 3 concurrent sessions focusing on “The genus Oryza, its diversity, evolution and utility”, “Structure and function of rice genome” and “Opportunities and challenges of transgenic rice”. We invited 19 speakers worldwide and discussed recent research highlights on biodiversity, genome and transgenic plants in cultivated rice and its wild relatives. A total number of participants in the conference was 1,235 from 42 countries and areas involving 274 from abroad. A part of participants visited the Genebank at NIAS as a post conference study tour in the afternoon on 7 November.

■ World Rice Research Congress (WRRC), FAO/IAEA/RCA Workshop on "Nuclear techniques for rice improvement in Asia" Recently, mutation breeding has been promoted and succeeded in developing various kinds of new rice varieties in RCA (Regional Cooperative Agreement for Research, Development and Training Related to Nuclear Science and Technology for Asia and the Pacific) member countries. The workshop was sponsored by FAO/IAEA and NIAS, and held as a session of WRRC on 4 - 7 November, 2004. The expected outputs of the Workshop are to (1) Increase the public awareness of and advocate mutation techniques in rice research and development; (2) Update knowledge and information for the participants of rice science and technology; (3) Formulate a consensus of future directions in mutation induction for rice, and a possible consortium of mutation techniques for rice improvement/functional genomics in Asia. Twenty Invited speakers from Vietnam, Thailand, Suri Lanka, the Philippines, Korea, China,

- 189 - Indonesia, Pakistan, Malaysia, Indonesia, Japan, and FAO/IAEA introduced their mutation breeding activities and induced varieties of rice developed in their countries, and discussed the future directions in 3 sessions at JICA Guesthouse, Ecopal Tsukuba Internationl Congress Center, and NIAS Conference Room, respectively.

■ “The First Rice Annotation Project Meeting（RAP1)“ With the completion of the sequencing of the rice genome (Oryza sativa ssp. japonica,cv. Nipponbare), the First Rice Annotation Project Meeting (RAP1) was held in Tsukuba, Japan on December 13-18, 2004. The meeting was co-organized by the National Institute of Genetics, National Institute of Advanced Industrial Science and Technology, and National Institute of Agrobiological Sciences in collaboration with the International Rice Genome Sequencing Project (IRGSP). About 90 researchers of rice and bioinformatics participated in RAP1. The meeting focused on manual curation of predicted genes in the rice genome. An automated annotation pipeline facilitated the prediction of 29,757 loci on the genome by using the IRGSP Build 3 genome sequence, full-length cDNAs, ESTs, and ab initio gene-finding methods. Curation was subsequently conducted in 11,148 loci for which protein functions were electronically inferred. The results of annotations were discussed from the view points of functional genomics, comparative genomics and biological databases. A WWW-based database of the annotations will be released in 2005.

■ NIAS/COE International Symposium: “Genetic resources and functional genomics in insects” The symposium was held at Tsukuba International Conference Hall on March 8-9, 2005. One hundred and eighteen persons including 20 foreigner participated to the symposium,. In this symposium, we dealt with the recent progress of Bombyx genomic analysis research and present status of genetic resources of Bombyx mori At the first session, “Genomics resources and genome analyses”, 9 lectures on genomic resources and genome analysis were given by 7 Japanese and by 2 foreign researchers. At the second session “Gene function studies, 15 lectures were given by 8 Japanese and by 7 foreign researchers. NIAS has been promoted genome sequence analysis of Bombyx mori by WGS method as the main research organization since 2004. The draft of Bombyx genome was revealed in February, 2005. Consequently about 80% of silkworm genes were verified in the sequence of Bombyx genome. Hence, the analysis of functional genes specific to insects will advance remarkably by the use of information of Bombyx genome. This symposium was quite successful in this respect.

- 190 - Main Foreign Visitors to NIAS (April 2004-March 2005)

13 Maｙ Dr. Henglu Guan President, Nanjin Agricultural University (China)

4June Dr.S.B.Dandin Director, Central Sericultural Research and Training Institute (India)

8Jul. Dr. Hyoug-Chin Kim Chief, Bioevaluation Center, Korean Research Institue of Bioscience and Biotecnology (Korea)

13 Jul. Dr.Lai Po-Yung Director, Taiwan National Pingtong Science and Tecnology University (Taiwan)

13 Jul. Dr. Udin S. Nugraha Director, BALAI PENELITIAN TANAMAN SAYURAN (BALITSA) (Indonesia)

3Sep. Dr. Deng Wei Director, Northeast Institute of Geography and Agricultural Ecology (China)

9Sep. Dr.Dra.AnaMariaSadir Director, Reseatch Center of Veterinary and Agricultural Science (Argentina)

7Oct. Haji Abdul Ghani bin Othman Chief Minister, Johor, Malaysia (Malaysia)

1Feb. Juan Carlos Tafur Ribera Chief editor, Correo Co.Ltd. (Peru)

22 Feb. Anuar Mohamad Head, Sabah Biodiversity Center (Malaysia)

- 191 - Executive Members and Research Staff Members

(as of March 31,2005) Executive Members President Iwabuchi Masaki Vice President Kitamura Chikayoshi Vice President Higo Kenichi Auditor Motoi Yoshiko Auditor Matsui Takehisa Research Staff Department of Research Plannin Director Shinbo Hiroshi and Coordination Senior Research Planner Miyashita Kiyotaka Research Planner Hirai Kazuo Research Planner Awata Takashi Research Planner Kadowaki Kouichi Research Planning Section Head Kiuchi Makoto Watanabe Shinichirou Research Evaluation Section Head Hanada Kaoru Research Coordination Section Head Shirata Akira Imai Tsuneo Office for GMO Research and Head Tabei Yutaka Development Miyao Akio Technology Transfer Section Head Ogawa Masafumi Kayano Toshiaki Hirogari Yasuhiro Field Management Section Head Obo Masahiro Head Kawauchi Ikuo Head Kobayashi Toru Genome and Biodiversity Research Director Obata Taro Center Genome Research Department Director Sasaki Takuji Research Leader Yasue Hiroshi Plant Genome Laboratory Head Matsumoto Takashi Katayose Yuuichi Wu Jianzhong Mizuno Hiroshi

- 192 - Animal Genome Laboratory Head Awata Takashi Hamasima Noriyuki Hayashi Takeshi Mikawa Satoshi Uenishi Hirohide Insect Genome Laboratory Head Mita Kazuei Kadono Keiko Yamamoto Kimiko Yasukochi Yuji Bioinfomatics Laboratory Maeda Miki Ito Takeshi Numa Hisataka DNA Bank Head Nagamura Yoshiaki Baltazar Alcaraz Antonio Rice Genome Resource Center Nagamura Yoshiaki Miyao Akio Genetic Diversity Department Director Kurisaki Jun-ichi Research Leader Kaku Hisatoshi Research Leader Umehara Masamichi Molecular Biodiversity Laboratory Head Kadowaki Kouichi Nakayama Shigeki Nishikawa Tomotaro Takahashi Sakiko Biosystematics Laboratory Aoki Takayuki Ochiai Hirokazu Evolutionary Dynamics Laboratory Head Vaughan Duncan Alexander Tomooka Norihiko Kaga Akito Germ Cell Conservation Laboratory Head Kaneko Hiroyuki Noguchi Junko Kikuchi Kazuhiro Applied Microbiology Laboratory Head Hayashi Nagao Tomiyama Masamitsu Kitamoto Hiroko Nishimura Marie Adaptation Systems Laboratory Ishikawa Masaya Komatsuda Takao Biometrics Laboratory Sato Masahiro Takeya Masaru

- 193 - Genebank Director Okuno Kazutoshi Research Leader Shirata Kazuto Niino Takao Plant Genetic Resources Laboratory Kawase Makoto Ebana Kaoru Fukuoka Shuuichi Uga Yuusaku Microorganism Genetic Resources Head Sato Toyozou Laboratory Nagai Toshirou Tomioka Keisuke Takeuchi Kasumi Animal Genetic Resources Laboratory Head Minezawa Mitsuru Takahashi Hideaki Kawada Masae Developmental Biology Department Director Izaike Yoshiaki Miyashita Norikazu Research Leader Furutachi Hiroshi Developmental Mechanisms Laboratory Head Myohara Maroko Nakao Hajime Kato Yusuke Hatakeyama Masatsugu Development and Differentiation Head Tokunaga Tomoyuki Laboratory Furusawa Tadashi Ohkoshi Katsuhiro Animal Genetic Engineering Laboratory Head Naito Mitsuru Matsubara Yuko Harumi Takashi Embryonic Technology Laboratory Head Onishi Akira Watanabe Satoshi Fuchimoto Daiichiro Suzuki Shunichi Insect Growth Regulation Laboratory Head Shiotsuki Takahiro Tanaka Yoshiaki Tateishi Ken Kamimura Manabu Shimura Sachiko Reproductive Biology and Technology Takahashi Toru Laboratory Hosoe Misa Kanayama Kanako

- 194 - Molecular Biology and Immunology Research Leader Sakurai Michiharu Department Molecular Immunology Laboratory Head Kitani Hiroshi Takenouchi Takato Sato Mitsuru Innate Immunity Laboratory Head Yamakawa Minoru Ishibashi Jun Tanaka Hiromitsu Experimental Animals Laboratory Head Goto Hideo Suto Jun-ichi Physiology and Genetic Regulation Director Kawasaki Kenjiro Department Research Leader Hirai Yoshio Research Leader Inouchi Jun Insect Life-Cycles and Physiology Head Tanaka Seiji Laboratory Okuda Takashi Kotaki Toyomi Watanabe Masahiko Insect Nutrition and Metabolism Head Nakamura Masatoshi Laboratory Hirayama Chikara Kikawada Takahiro Insect Neurobiology Laboratory Head Asaoka Kiyoshi Inoue Hisashi Ichikawa Akio Kihara Mami Insect Behavior Laboratory Head Wakamura Sadao Yasuda Tetsuya Akino Toshiharu Yasui Hiroe Animal Gene Function Laboratory Head Mitsuhashi Tadayoshi Kojima Misaki Ito Yoshiyasu Animal Cell Biology Laboratory Takezawa Toshiaki Animal Neurophysiology Laboratory Head Saito Toshiyuki Kasuya Etsuko Sakumoto Ryousuke Animal Neuroendocrinology Laboratory Head Okamura Hiroaki Ohkura Satoshi Yayou Kenichi

- 195 - Insect Genetics and Evolution Director Sato Mamoru Department Kato Masao Insect-Plant Interactions Laboratory Head Hattori Makoto Konno Koutaro Tamura Yasumori Natural Enemies Laboratory Head Noda Takashi Hinomoto Norihide Maeda Taro Symbiosis Laboratory Head Noda Hiroaki Shinoda Tetsuro Watanabe Hirofumi Nakashima Nobuhiko Watanabe Kenji Matsumoto Yukiko Insect Pathology Laboratory Head Miyamoto Kazuhisa Mitsuhashi Wataru Wada Sanae Murakami Ritsuko Insect Genetics Laboratory Head Kosegawa Eiichi Hirokawa Masahiko Tatematsu Kenichirou Insect Molecular Evolution Laboratory Head Nagayasu Kenichi Yukuhiro Kenji Muraji Masahiko Tomita Shuuichiro Hasegawa Tsuyoshi Komoto Natsuo Insect Biomaterial and Technology Director Takeda Satoshi Department Research Leader Tsukada Masuhiro Biopolymer Characterization Laboratory Toshima Yoshiyuki Hata Tamako Takasu Youko Biomaterial Development Laboratory Miyazawa Mitsuhiro Kameda Tsunenori Biomimetic Laboratory Head Tamada Yasushi Goto Yoko Kuwana Yoshihiko Kojima Katsura Insect Products Utilization Laboratory Haga Atsunobu

- 196 - Insect Biotechnology and Sericology Director Machii Hiroaki Department Research Leader Kato Hiroshi Research Leader Hayasaka Shoji Research Leader Hara Wajiro Research Leader Kinoshita Haruo Research Leader Ichihashi Takahisa Insect Cell Engineering Laboratory Head Imanishi Shigeo Taniai Kiyoko Akizuki Gaku Insect Gene Engineering Laboratory Head Tamura Toshiki Yonemura Naoyuki Shimoda Masami Sezutsu Hideki Mass Production System Laboratory Head Ohura Masanobu Koyama Akio Arakawa Toru New Silk Materials Laboratory Head Takabayashi Chiyuki Nakajima Ken-ichi Teramoto Hidetoshi Sericultural Science Laboratory Head Mase Keisuke Okada Eiji Fukui Kuniaki Iizuka Tetsuya Molecular Genetics Department Director Hirochika Hirohiko Functional Genomics Laboratory Hagiwara Kiyoshi Miyao Akio Yamazaki Muneo Takahashi Akira Applied Genomics Laboratory Head Yano Masahiro Izawa Takeshi Yamanouchi Utako Yamamoto Shin-ichi Ueda Tadamasa Epigenetics Laboratory Okuizumi Hisato Mochizuki Atsuko Gene Expression Laboratory Head Kikuchi Shoushi Mori Masaki Kishimoto Naoki Gene Regulation Laboratory Head Komatsu Setsuko Yoshikawa Manabu

- 197 - Asano Takayuki Biochemistry Department Director Kobayashi Mikihiko Crystallography Laboratory Takase Kenji Momma Mitsuru Fujimoto Zui Wako Toshiyuki Biophysics Laboratory Yamazaki Toshimasa Katoh Etsuko Suzuki Rintaro Glycobiology Laboratory Head Minami Eiichi Kaku Hanae Akimoto Chiharu Membrane Biology Laboratory Head Shimomura Shouji Kajiwara Hideyuki Taguchi Fumio Plant Physiology Department Director Meshi Tetsuo Research Leader Tanaka Yoshiyuki Research Leader Kawasaki Shinji Photosynthesis Laboratory Head Tokutomi Mitsue Inagaki Noritoshi Fukayama Hiroshi Carbon Metabolism Laboratory Head Ueno Osamu Ishimaru Ken Sentoku Noki Developmental Biology Laboratory Head Takatsuji Hiroshi Sugano Shouji Chang-Jie Jiang Baba Akiko Environmental Physiology Laboratory Head Takano Makoto Takeichi Tetsuo Yazaki Yoshiaki Kiyota Seiichirou Iwamoto Masao Disease Physiology Laboratory Head Ishikawa Masayuki Mitsuhara Ichiro Fukuda Atsunori Seo Shigemi Nitrogen Fixation Laboratory Head Kouchi Hiroshi Nakayama Yasuji Umehara Yousuke

- 198 - Plant Biotechnology Department Director Oka Seibi Gene Design Laboratory Head Miyahara Kenzo Ichikawa Hiroaki Nishizawa Yoko Plant Gene Engineering Laboratory Head Takaiwa Fumio Toki Seiichi Kawagoe Yasushi Plant Cell Engineering Laboratory Head Tabei Yutaka Hagio Takashi Ogawa Taiichi Habu Yoshiki Molecular Breeding Laboratory Koga-Ban Yasunori Otake Yuko Biosystems Laboratory Head Handa Hirokazu Kawahigashi Hiroyuki Institute of Radiation Breeding Director Nakagawa Hitoshi Mutation Genetics Laboratory Head Nishimura Minoru Radiation Technology Laboratory Head Morishita Toshikazu Yamaguchi Hiroyasu Mutation Breeding Laboratory Head Yoshioka Terutaka Takyu Toshio Yamanouchi Hiroaki

- 199 - Members of NIAS Evaluation Comittee

(as of March 31,2005)

Ueda Ryu : National Institute of Genetics

Kiguchi Kenji : Shinshu University

Kouno Tomohiro : Tokyo University of Agriculture

Takeda Kazuyoshi : Okayama University

Nishimura Ikuko : Kyoto University Graduate School of Science

Hirai Atsushi : Meijyou University

Sakaki Yoshiyuki : Riken Genomics Sciences Center

Sano Hiroshi : Nara Institute of Science and Technology

- 200 - FINANCIAL OVERVIEW Fiscal Year 2004 (April 2004 - March 2005) thousands of yen

TOTAL BUDGET 16,976,816

OPERATING COSTS 9,453,544

Personnel(419)＊ 4,356,330

President(1) Vice President(2) Auditor(2)

Administrators Ⅰ(93) ＊＊ Administrators Ⅱ(44) ＊＊＊ Researchers(277) ＊ Number of persons shown in () ＊＊ General administration ＊＊＊ Field management and transportations

Administrative costs 5,097,214

RESEARCH PROMOTION COSTS 7,523,272

Research Grant from MAFF 3,027,086 Entrusted Research Expenses from MAFF 3,474,653 Entrusted Research Expenses from MEXT 366,496 Entrusted Research Expenses from others 655,037

*MAFF : Ministry of Agriculture, Forestry and Fishries *MEXT : Ministry of Education, Culture, Sports, Science and technology

Entrusted Research Expenses Entrusted Research Expenses from MEXT from others 366,496(2.2%) 655,037(3.9%)

Personnel Entrusted Research Expenses from 4,356,330 (25.7%) MAFF 3,474,653(20.5%)

Administrative costs 5,097,214(30.0%)

Research Grant from MAFF 3,027,086(17.8%)

- 201 - - 202 - Annual Report 2005

（Apr. 2004 － Mar.2005） No.4

Published by National Institute of Agrobiological Science 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan TEL : 029(838)7272 FAX : 029(838)7408 URL : http://www.nias.affrc.go.jp/index_e.html