2006 Annual Report 2006 (Apr.2005 Mar.2006) (In the fiscal year 2005) !$--"&$ %,*( */, +,$-'#$).

Teruo Ishige, President

The National Institute of Agrobiological Sciences (NIAS), which is the largest agricultural research institute of basic life science in , was established on 1 April 2001 as an independent administrative institution of the Ministry of Agriculture, Forestry, and Fisheries and a center for basic studies to develop innovative agricultural biotechnologies and new bioindustries. The Second Five-year Plan started on 1 April 2006. During the next five years, the Institute will advance the technologies developed in the First Five-year Plan. These include sequencing of the entire rice genome; sequencing of the silkworm genome and recombination of livestock and silkworm genes; and bioscience research that will help improve human nutrition and promote the creation of new bioindustries. In the Second Five-year Plan, the Institute will focus on: (1) improvement, diversity and utility of agrobiological resources - we will utilize resources that we have built up in the course of research on the genes of rice, silkworms, and pigs and will secure other genetic resources; (2) research on, and development of, innovative agricultural technologies using biological and genome information - we will study organisms with a view to their ability to adapt to their environments, differentiate, and interact with other organisms; and (3) research and development aimed at creating new biotechnology-based industries - we will develop biotechnologies to produce useful materials, such as silk-based products that can be used in everyday items and in medical practice. There is an urgent need to develop technologies in these three subject areas, because each of them is important and is expected to contribute greatly to society. The Institute will make further efforts to maximize the benefits of biotechnology. One of the major biotechnologies in which the Institute is involved is the recombination of genes in crops, (silkworms), and . This technology is based on fundamental principles related to the building blocks of life in which DNA provides the templates for building amino acids into proteins. The principles are essentially the same as those on which conventional breeding technologies and successes are based. Gene recombination and other biotechnologies are developing rapidly; however, their high level of sophistication may make them difficult for the general public to understand. In addition, the media is now reporting on the potential risks of using biotechnology. Since the development of basic gene recombination technologies in the 1970s, these technologies have been used to develop a number of drugs to help patients with diseases such as diabetes, and to improve human health. Agricultural plants developed using these new technologies are now widely cultivated in many countries and are consumed in Japan and elsewhere in foods for humans and in feeds. Because due consideration has been given to securing safety in both the research and development of gene-recombination technology and its application, few concerns have arisen in regard to its influence on the environment and the safety of its use in food production. Biotechnologies have great potential, and there is fierce global competition to lead in these fields. Gene-recombination technology is spreading widely because it provides opportunities to make use of scientific discoveries in many areas of biology. Advanced biotechnologies are indispensable in helping to solve problems in the areas of global food supply, the environment, and medicine. We believe that biotechnology research will contribute to the well-being of the human race. Thus there is a need for the public to gain an understanding of the importance of the wise use of biotechnology for the future good of humankind. As part of the Second Five-year Plan, all the staff at NIAS are determined to make a concerted effort to achieve more great results like the sequencing of the entire rice genome achieved under the First Five-year Plan. We thank all of those involved for their cooperation, and we look forward to your continued support, understanding, and collaboration.

The buildings of NIAS in the cherry blossoms season !$#&"#&%

Message from the President Organization

Topics of Research in This Year

Genome and Biodiversity Research Division

" Positional cloning of ,/)"$! a densovirus-resistance gene in %-+(10 +-.* !!!!!!!!!! $ " The development of a genetic linkage map for azuki bean !!!!!!!!!!!!!!!!! % " Allele diversity and evolution of &%'' # gene generated by multiple transposable elements in foxtail millet !!!!!!!!!!!!!!!!!! ' " Development of prectical method to discriminate Nagoya breed from other chicken breeds !! )

Insect and Animal Sciences Division

" Intrabody technology: a novel method for domain-specific knockdown of cytosolic protein in mice !!!!!!!!!!!!!!!!!!!!!!!!!!!!! * " Mulberry latex defense mulberry trees from herbirous insects !!!!!!!!!!!!!!! , " %-+(10 +-.* cultured cell line (NIAS-Bm-Kel) that adjusted to serum-free media !!!!!! $%

Plant Science Division

" Functional analysis of phytochromes in rice!!!!!!!!!!!!!!!!!!!!!!!! $& " Transgenic rice for allergy immunotherapy: Oral feeding of rice seeds expressing T cell epitopes suppresses clinical symptoms in a mouse model of pollen allergy !!!!! $'

Research Activities

Genome and Biodiversity Research Division

" Genome Research Department!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $* " Genetic Diversity Department !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %& " Genebank !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! %,

Insect and Animal Sciences Division

" Developmental Biology Department !!!!!!!!!!!!!!!!!!!!!!!!!!! &' " Molecular Biology and Immunology Department !!!!!!!!!!!!!!!!!!!!! '# " Physiology and Genetic Regulation Department !!!!!!!!!!!!!!!!!!!!!! '' " Insect Genetics and Evolution Department !!!!!!!!!!!!!!!!!!!!!!!! ', " Insect Biomaterial and Technology Department !!!!!!!!!!!!!!!!!!!!!! (% " Insect Biotechnology and Sericology Department !!!!!!!!!!!!!!!!!!!!! ('

Plant Science Division

" Molecular Genetics Department !!!!!!!!!!!!!!!!!!!!!!!!!!!!! )# " Biochemistry Department !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! )* " Plant Physiology Department !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! *# " Plant Biotechnology Department!!!!!!!!!!!!!!!!!!!!!!!!!!!!! *) " Institute of Radiation Breeding !!!!!!!!!!!!!!!!!!!!!!!!!!!!! +# List of Publication

" Original papers 1) Western language !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '% 2) Japanese language with English Summary !!!!!!!!!!!!!!!!!!!!! (&

" Review and Monograph (in English) !!!!!!!!!!!!!!!!!!!!!!!!!!! ('

Main meeting related NIAS !!!!!!!!!!!!!!!!!!!!!!!! $## Executive Members and Research Staff Members !!!!!! $#$ Members of NIAS Evaluation Comittee !!!!!!!!!!!!!!! $#'

Financial overview !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $#( Location and How to access !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! $$# !'#"%$)"($&%

President Physiology and Genetic Regulation Department Vice President(Insect and Animal Sciences) Insect Life-Cycles and Physiology Laboratory Vice President(Plant Sciences) Insect Nutrition and Matabolism Laboratory Auditor Insect Neurobiology Laboratory Department of Research Planning and Coordination Insect Behavior Laboratory Research Planning Division Research Evaluation Division Animal Gene Function Laboratory Research Coordination Section Animal Cell Biology Laboratory Office for GMO Research and Development Animal Neurophysiolory Laboratory Technology Transfer Section Animal Neuroendocrinnolory Laboratory Administration Section Insect Genetics and Evolution Department Information and Public Relation Section Insect-Plant Interactions Laboratory Field Management Section Natural Enemies Laboratory Genome Resource Center Symbiosis Laboratory QTL Genomics Research Center Center for GMO Research and Information Insect Patholory Laboratory Insect Gene Function Research Center Insect Genetics Laboratory Department of Administration Insect Molecular Evolution Laboratory General Affairs Section Insect Biomaterial and Technology Department Accounting Section Biopolymer Characterization Laboratory Facility Management Section Biomaterial Development Laboratory Biomimetic Laboratory Insect Products Utilization Laboratory $,120, )1+ ".2+.6,34.57 Insect Biotechnology and Sericology Department Insect Cell Engineering Laboratory ',4,)3*- #.6.4.21 Insect Gene Engineering Laboratory Director of Genome and Biodiversity Research Division Mass Production System Laboratory Genome Research Department New Silk Materials Laboratory Plant Genome Laboratory Sericultural Science Laboratory Animal Genome Laboratory Insect Genome Laboratory Bioinformatics Laboratry DNA Bank &/)15 (*.,1*, #.6.4.21 Genetic Diversity Department Molecular Genetics Department Molecular Biodiversity Laboratory Functional Genomics Laboratory Biosystematics Laboratory Applied Genomics Laboratory Evolutionary Dynamics Laboratory Epigenetics Laboratory Germ Cell Conservation Laboratory Gene Expression Laboratory Applied Microbiology Laboratory Gene Regulation Laboratory Adaptation Systems Laboratory Biochemistry Department Biometrics Laboratory Crystallography Laboratory Genebank Biophysics Laboratory Plant Genetic Resources Labolatory Glycobiology Laboratory Microorganism Genetic Resources Laboratory Membrane Biology Laboratory Animal Genetic Resources Laboratory Plant Physiology Department Genetic Resources Management Section Photosynthesis Laboratory Carbon Metabolism Laboratory Development Biology Laboratory %14,*5 )1+ !1.0)/ (*.,1*,4 Environmental Physiology Laboratory Disease Physiology Laboratory #.6.4.21 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 Topics of Research in This Year

Positional cloning of ),&"#! a densovirus-resistance gene in $*(%.- (*+'

Keiko KADONO-OKUDA, Katsuhiko ITO, Junko NOHATA, Kimiko YAMAMOTO, Motoe SASANUMA, Shunichi SASANUMA, Ryokitsu EGUCHI, Wajiro HARA and Kazuei MITA Genome Research Department

%-+(10 +-.* densovirus ( BmDNV ) candidate gene for ,/)"$ on the deletion region multiplies in the columnar cell nuclei of the (Fig. 3). The gene was expressed only in the midgut epithelia of the silkworm, %-+(10 +-.*. midgut throughout the larval stage. RT-PCR It is classified into two species, DNV-1 and DNV revealed that mutant strains examined showed -2 based on their symptoms, serological a common shorter transcript compared with characters, genome structure and sequences. that of susceptible strains, which was caused by Some silkworm strains were identified as the common deletion in the gene among resistant against DNV-1 and/or DNV-2. In such resistant strains (Fig. 4). Sequence analysis of strains the response reflects non-susceptibility the cDNA tells that ,/)"$ encodes a rather than resistance because even high dose transmembrane protein (Fig. 5). Mutation of of inoculum does not affect their survival rate. this gene endows the resistance against the So far four non-susceptibility genes have been virus. Identification of ,/)"$ gene strongly reported, namely; ,/)"# (L21-8.3 cM), &*)"# (L17 accelerates the virus research through -31.1 cM), ,/)"$ (L17-24.5 cM) and ,/)"' (L15- establishing transgenic cultured cell lines 50.7 and 30.0 cM from apical and distal ends, hypersensitive to the virus, in addition to the respectively). However, none of them have yet direct marker-assisted breeding of %-+(10 been isolated as responsible genes. Studies on strains resistant to DNV-2. these genes are useful in understanding the mechanism of the viral invasion and multiplication, and introducing those genes into the silkworms or cultured cells will lead us to understand the functions of the genes. We have identified four EST markers closely linked with ,/)"$! By taking advantage of Bombyx genome information, positional cloning, BAC-contig construction and linkage analysis of the virus- selected BC1 (backcross 1) progenies (Fig. 1) enabled to find the resistant strain-specific Fig. 1 deletion within the candidate region of 500 kb Segregation of BC1 with DNV-2 infection. (Fig. 2). We have succeeded in identifying the

%**/') &(,+-. #!!$ " Fig. 2 BAC-contig construction and fine mapping of %&$"#!

Fig. 3 Gene organization of %&$"# in the susceptible and resistant strains.

Fig. 5 Predicted amino acid sequence secondary structure.

Fig. 4 RT-PCR analysis of %&$"# in the midgut of the strains.

The development of a genetic linkage map for azuki bean

Akito KAGA, Takehisa ISEMURA and Norihiko TOMOOKA Genome Research Department

Azuki bean (!$#&" "&#)%"'$( var. "&#)%"'$() oligo-primed second-strand synthesis enrichment belongs to the Asian !$#&" that consists of 21 procedure of the single strand genomic library. species among them 10 are cultivated. Until We found in the azuki bean genome (AG)n now none of the Asian !$#&" had a genetic motifs are a rich source of markers and thus linkage map that resolved the 11 linkage constructed an (AG)n-SSR enriched library. The groups representing the haploid chromosome enriched library enabled primer pairs to be number. In order to develop a saturated linkage designed and these primers had a high map of azuki bean we first constructed an SSR percentage (98%) of successful single locus (microsatellite) library using a new method in amplification. A total of 401 SSR primer pairs plants. The library construction involved an were developed for use in azuki genome

" $)).&( %'+*,- "!!# studies. order was highly conserved among the three To construct a fully saturated genetic maps. To integrate the marker information into linkage map for azuki bean, three mapping a single map a consensus linkage map was populations consisting of a total 592 individuals synthesized based on common SSR markers were developed using azuki bean and three among three maps as a bridge. The consensus closely related taxa (#" $*&/)$-'. var. *',,+*%*.'.! linkage map consists of a total 896 markers and #" *%,$)%*.'. and #" -'/('/%*.'.). The linkage has an over all length of 854 cM with an maps based on the newly developed SSR average distance of 3.1 cM between SSR markers with RFLP and AFLP markers markers (Fig. 1). covered 11 linkage groups and the marker The significance of the SSR primers

Fig. 1 A genetic linkage map for azuki bean Red: SSR markers. Blue: RFLP markers. Black: AFLP markers.

%**/') &(,+-. "!!$ # developed for azuki bean is that it can be used The significance of this azuki bean genetic not only for genetic and QTL mapping in azuki linkage map is that it can act as a standard for bean but also the many other cultivated Asian comparative mapping in the Asian *0.3+.In *0.3+. We have confirmed that approximately addition, since azuki bean belongs to the 70% of the SSR primer pairs amplify a certain temperate group of legumes, that includes DNA fragment even in Asian *0.3+ not closely common bean ('/+7-4197 :91.+607) and soybean related to azuki bean. Thus using the SSR (%1<,03- 2+;), this map will be helpful for primers genome maps for other important comparative genome research across the crops, like mungbean, can now be developed. worlds most important legume crops.

Allele diversity and evolution of #"$$ ! gene generated by multiple transposable elements in foxtail millet

Makoto KAWASE Genebank

Foxtail millet, (-8+60+ 08+10,+ P. $-+9:! 775! insertions, TSI-1 to TSI-11, studied in the 08+10,+ is one of the worlds oldest cultivated present study. crops. It is most likely to have been domesticated Type I is distributed through Asia and in Eurasia and genetically differentiated into Europe, while others have their own various landrace groups. The naturally occurring distribution particularly in East Asia and waxy and low-amylose variants of this millet Southeast Asia (Fig. 2). All of the twelve and other cereals were developed in East Asia accessions of ssp. viridis, the presumed wild and Southeast Asia under human selection for ancestor of ssp. 08+10,+, have the type I allele. sticky foods. Mutations in the %$(( # gene for Type II similarly produces non-waxy starch, granule-bound starch synthase 1 are known to although it has an insertion in an intron, )(&"#. be associated with these traits. Twelve different alleles were found on %$(( # gene in foxtail millet based on the analysis of 871 landraces collected from various regions mainly in Europe and Asia, which are conserved in NIAS Genebank. Twelve %$(( # alleles were identified by Long PCR: types I to X, IVa and IVb (Fig. 1). Types I and II produce non-waxy endosperm starch containing over 22% amylose, types III, VI and IX forms low amylose endosperm (less than 22% amylose), and types IV, IVa, IVb, V, VII, VIII and X cause waxy endosperm (almost no amylose). The Fig. 1 differentiation of those alleles has been Allele diversity of #"$$ ! locus in foxtail millet mediated by eleven transposable elements (Kawase et al., 2005)

# %**/') &(,+-. "!!$ Fig. 2 Geographical distribution of %$&& # alleles of foxtail millet (Kawase (* ')"! 2005)

Types III, IV, IVa, IVb and IX have deleted portion. Type III has an additional insertions of different length at the same site of insertion ()('!$)on)('!# of type IX. Type V the Intron 1, and types VI and VIII have has a retroposon ()('!% ) inserted in intron 3. insertions of different length at the same site of )('!& of type X is its Solo-LTR variant. intron 12. )('!# of type IX is homologous with Intergenic reconbinat of types II and VI were )('!" of types IV, IVa and IVb but have a rarely found.

Fig. 3 Mode of evolution of %$&& # alleles of foxtail millet (Kawase (* ')"! 2005)

%**/') &(,+-. "!!$ # An evolutionay sequence was suggested viewpoint. most likely from type I to type IV, from type IV Allele types I to X can be identified by to type IX, and from type IX to type III (Fig. 3). Long PCR using four sets of primers, and Types IVa and IVb were independently formed additional two sets can distinguish types IVa from type IV by different short insertions. and IVb from type IV. Type VI was also evolved from type I, and type VIII from type VI. Publication Five landraces of type V ('&%!" ) and one of 1) Kawase M., K. Fukunaga, K. Kato (2005) type X ('&%!#) have an completely identical Diverse origins of waxy foxtail millet crops in LTR sequence. The LTRs of '&%!$ were East and Southeast Asia mediated by multiple identical among six landraces of type VII. transposable element insertions. Mol. Gen. These findings indicate that the insertions Genomics, 274:131-140. occurred quite recently from a paleontological

Development of practical method to discriminate Nagoya breed from other chicken breeds

Hideaki TAKAHASHI, Akihiro NAKAMURA* and Mitsuru MINESAWA Genebank *Akichi-ken Agricultural Research Center

Nagoya breed (Fig. 1) is a famous native Livestock and Poultry Breeding Center, which chicken in of Japan, a dual supplies parent stocks to the hatcheries using purpose breed for eggs and meat. The the four strains. Therefore, all commercial purebred (Nagoya breed Nagoya breed) has Nagoya breed chicken are derived from four been commercialized, so the commercial strains. Since the taste, palatability and texture chickens are produced by intra-strain mating. of Nagoya breed meat are well recognized in Nagoya breed has four strains (NG1, NG2, NG3 Japan, the market price of Nagoya breed meat and NG4) established at Aichi-ken Agricultural is much higher than that of broiler meat. Research Center. They are maintained at Aichi However, cut-up meat of Nagoya breed and broiler cannot be easily distinguished by appearance. So, a technology to discriminate between Nagoya breed and broiler is vital for preventing false sales and guaranteeing the quality of meat. The objective of the present study is to develop a method to discriminate between Nagoya breed and all other chicken on the market using microsatellite DNA markers. Four strains of Nagoya breed established at the Aichi-ken Agricultural Research Center were analyzed using twenty-five microsatellite Fig. 1 markers. In these strains, five of the markers Nagoya breed

# $)).&( %'+*,- "!!# (ABR0015, ABR0257, ABR0417, ABR0495 and combination as the Nagoya breed. These five ADL0262) had a single allele. Other 448 chicken microsatellite markers provide a practical samples of various breeds and hybrids were method to accurately discriminate the Nagoya analyzed using the same five markers. None of breed from other chicken and can contribute to these chicken samples had the same allele checking the validity of labeling Nagoya breed.

Intrabody technology: a novel method for domain-specific knockdown of cytosolic protein in mice

Mitsuru SATO Molecular Biology and Immunology Department

Intracellularly expressed antibodies affinity of the parental antibody. However, (intrabodies) have been considered useful for technical problems, such as stability and not only clinical applications such as viral functional expression of intrabodies in the neutralization and cancer therapy but also cytosol remain to be overcome. functional analysis of proteins inside the cell. A In this study, we constructed the scFv variety of intrabody formats have been intrabodies derived from hybridoma cells designed. Single-chain variable fragments producing the monoclonal antibodies (Fig. 1A) (scFvs) consist of one heavy chain variable that specifically bind to an Ena/VASP region (VH) linked through a flexible peptide homology 1 (EVH1) domain of Wiskott-Aldrich spacer, a repeated motif of 3 GGGGS, to one syndrome protein (WASP). WASP resides in light chain variable (VL). They are able to fold the cytosol as a multifunctional adaptor and retain the antigen-binding specificity and molecule and mediates actin polymerization

Fig. 1 Antibody structure (A) The basic structure of a conventional antibody consists of four polypeptide, two identical heavy (H) chains and two identical light (L) chains, held together by disulfide bonds. Each chain has both a variable (V) and a constant (C) region. (B) Schematic representation of the two scFv DNA constructions for microinjection. Shown are the leader signal sequence, VH region, polypeptide linker (Gly4Ser)3,VL region, light chain constant (CL) region and Myc tag sequence. The scFv DNA fragments were cloned into the pCAGGS-MCS expression vector.

%**/') &(,+-. "!!# $ and interleukin (IL)-2 synthesis in the T-cell production induced by TCR stimulation was receptor (TCR) signaling pathway. The anti- strongly inhibited in the scFv transgenic mice WASP-EVH1 scFvs were constructed with a T cells (Fig. 2A). Similar T-cell defects were

VH leader signal sequence at the N-terminus observed in WASP-EVH1 domain overexpressing and with a light chain constant (CL) region transgenic mice (Fig. 2A). So, the WASP-EVH1 behind the VL region (Fig. 1B), and injected into scFv intrabodies inhibit only the function of fertilized mouse eggs. In T cells from EVH1 domain that regulates IL-2 synthesis transgenic mice overexpressing the anti-WASP signaling, but do not affect the rest of the -EVH1 scFvs, binding of anti-WASP-EVH1 domain functions of WASP (Fig. 2B). The novel scFvs to native WASP was confirmed by procedure presented here can be applicable to immunoprecipitation. Furthermore, IL-2 functional analysis of other cytosolic proteins.

Fig. 2 IL-2 production induced by TCR stimulation was inhibited by anti-WASP-EVH1 intrabodies (A) Splenic T cells from anti-WASP scFv 21SHL and 21SHL-CL Tg mice, WASP-EVH1 Tg and wild-type mice were cultured in medium alone or in the presence of anti-CD3εAb. Each cell culture supernatant was collected at 24 h. IL-2 in the supernatant was quantified by ELISA. (B) Schematic representation of mode of intrabody action against WASP-EVH1 domain in T cells. WASP contains multiple domains that enable it to interact with different proteins. Shown are the EVH1 domain, GTPase-binding domain (GBD), proline rich region (PRR), verprolin homology (VH) and cofilin homology (CH) domain. Anti-WASP scFvs can specifically bind to the EVH1 domain of WASP in the cytosol of T cells, blocking the collect interaction between WASP and signaling molecules involved in IL-2 synthesis.

$ %**/') &(,+-. "!!# Mulberry latex defense mulberry trees from herbivorous insects

Kotaro KONNO Insect Genetics and Evolution Department

Latex is widely found among plant species; indicated a need to investigate latex ingredients 12,000-35,000 species have been reported to and their biological functions. exude it and many kinds of chemicals and Mulberry trees (&-/20 0.." Moraceae) proteins have been reported from plant latex. grow in Asia and their leaves are used for The biological functions of plant latex and its rearinganeconomicallyveryimportantinsect, ingredients, however, remained obscure. Recently, the silkworm, #-,(54 ,-/+, for thousands of we found that several latex-producing plants years. Moraceae plants are characterized by with no reported toxicities are strongly toxic to the presence of latex, and mulberry trees also insects due to the ingredients of latex. For exude latex (Fig. 1, lower left photo) when their example, papaya ($'/+)' .'.'5'! Moraceae) and leaves are damaged by caterpillars. In wild fig (%+)20 3+/*'1'! Moraceae) leaves are strongly condition, mulberry leaves are not often toxic to insects because of the cysteine damaged by herbivorous insects, although the proteases in their latex. Our recent findings leaves are soft and contains a lot of nutrients

Fig. 1 Defense activity of mulberry latex and three sugar-mimic alkaloids, the active compounds contained in the latex in very high concentrations Upper left photo, left part: the cabbage larvae fed intact (excised) mulberry leaves; Upper left photo, right part: the cabbage moth larvae fed mulberry leaves from which latex was washed of by cutting into narrow leaf strips and washing with water; Lower left photo: latex exuded from mulberry leaves(arrows), Right: three sugar-mimic alkaloids contained in mulberry latex in high concentrations, which are glycosidase inhibitors and were reported to have anti- diabetic activities.

%**/') &(,+-. "!!# $ such as proteins. However, there have been no the silkworm, $" 134/ (Fig. 2 D). Our results detailed studies about the defense activities of suggest that mulberry latex and sugar- mulberry trees against insect herbivory or the mimicking alkaloids in it play key roles in involvement of mulberry latex in the plant defense of mulberry against insect herbivory, defense. In this study, we addressed these and also suggest the existence of some adaptive subjects. mechanisms in the silkworm, $" 134/. We found that mulberry leaves are highly This study shows, for the first time in long toxic to lepidopteran larvae other than the history of sericulture, the existence of strong silkworm, $" 134/! (such as the Eri silkworm, latex-borne defense mechanisms in mulberry, (*1/* 4/,/2/ and notorius pest species, the and gives practical answer to mulberry- cabbage moth larvae, %*1.564* +4*55/,*.), due silkworm interactions. This study, together to the ingredients of the latex. When Eri with our previous studies on latex-borne silkworm and the cabbage moth larvae were defense mechanisms of papaya and fig trees fed mulberry leaves, they bit in the leaves but carried out by cysteine proteases in latex, they didnt grow, and died at last (Fig. 1, upper shows that medically applicable chemicals (i. e. left photo, left part). The toxicity of mulberry anti-diabetic sugar-mimic alkaloids) exist in leaves was lost when latex was washed off (Fig. mulberry latex in surprisingly high 1, upper left photo, right part). Also, latex-added concentrations (18% of dried latex), and they artificial diets showed toxicity and growth are easily purified from latex, and shows the inhibitory effects to the Eri silkworm (Fig. 2A). possibility of mulberry latex as a source of Mulberry (%" *7564*0/5) latex contained very interesting and applicable chemicals in medical high concentrations of alkaloidal sugar-mimic and agricultural field. Also, this study will glycosidase inhibitors (sugar-mimic alkaloids) contribute to researches on pest resistance of reported to have anti-diabetic activities, such as mulberry varieties. 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1), 1- deoxy nojirimycin (DNJ), and 1,4-dideoxy-1,4- Reference imino-D-ribitol (Fig 1, right). Their concentrations, 1) Konno K., Ono H., Nakamura M., Tateishi K., altogether, in latex reached 1.5-2.5% (8-18% to Hirayama H., Tamura Y., Hattori M., Koyama dry weight) in several mulberry varieties A., Kohno K. (2006) Mulberry latex rich in anti- (Table 1), which were 100 times the concentrations diabetic sugar-mimic alkaloids forces dieting on previously reported from whole mulberry caterpillars. '43," &*60" #,*-" (,/" )(# 103, leaves. These sugar-mimicking alkaloids showed 1337-1341. toxicities to caterpillars (Fig. 2 B,C), but not to

Table 1 Concentrations of sugar-mimic alkaloids in mulberry latex Concentrations in latex (%) Species, populations, and cultivars D-AB1 DNJ 1,4-dideoxy-1,4-imino-D-ribitol "! #.,-+#'&, (or "! $*($/%&,) Wild, Ishigaki, Okinawa, Japan $!)& " #!%$#!&) " #!#,#!'+ " #!#& Wild, Tsukuba, Honshu, Japan $!%+ " #!')#!'( " #!%&# Cultivar Yukishirazu $!'$ " #!%)#!&( " #!#'# Cultivar Ichibei $!#' " #!#%#!'* " #!#*# "! #'$# Cultivar Shinichinose ##!&% " #!### Values indicate means SD () = 4-5 for wild populations, ) = 2-3 for cultivars)

"! %**/') &(,+-. #!!$ Fig. 2 Growth inhibitory effects of mulberry latex and sugar mimic alkaloids on the Eri silkworm, $%('% +'&')'" Mulberry latex (A) and two sugar-mimic alkaloids, D-AB1 (B) and DNJ (C), show remarkable growth inhibitory effects on the Eri silkworm, $%('% +'&')'" Thelarvaeofthesilkworm,#*(-, (*+'! are not at all affected by either of sugar-mimic alkaloids even at the high concentrations, suggesting that this species has developed some adaptive mechanisms to these compounds(D). Both sugar-mimic alkaloids and unidentified high molecular weight components contribute to the defense activities of mulberry latex (E). Percentages in graphs indicate mortalities of larvae (A-D).

%**/') &(,+-. #!!$ "" !%$"(' $%&# cultured cell line (NIAS-Bm-Ke1) that adjusted to serum-free media

Shigeo IMANISHI1), Gaku AKIDUKI1), Yoshiya YOSHIDA2) and Chun Ying YANG2) 1) Insect Biotechnology and Sericology Development 2) Kohjin Bio Co.Ltd. Biology and Immunology Department

Both Sf9 cell line derived from $32)236*4& recombinant BmNPV when added to Ke1 cells +47,.3*4)& and High 5 cell line derive from cultured in serum-free medium. In comparison %4.(-23/75.& 1.! are utilized actually as a gene of luciferase gene expression of Ke1 cells and Sf expression system by baculovirus .1 8.642" 9 cells, Ke1 cells cultured in KBM700 serum- However, there is not #20':9 024. cell line free medium containing heat-treated silkworm that is suitable for serum-free culture and high hemolymph, showed higher luciferase activity level gene expression. This time, we established than Sf9 cells cultured in SF900! serum-free #20':9 024. cell line that is almost equal to medium containing 10% of FBS. A large scale $32)236*4& +47,.3*4)& cell line and leaded to luciferase expression in spinner culture was develop a new .1 8.642 gene expression system possible when Ke1 cells were cultured in KBM that can actualize a large scale production of 700 serum-free medium containing heat-treated recombinant protein. silkworm hemolymph, because Ke1 cells were NIAS-Bm-Ke1 cell line (Ke1 cells) that floatage ( Fig. 3 ) . Heat-treated silkworm adjusted to KBM700 of serum-free media was hemolymph was eliminated from KBM700 established. Ke1 cells showed high cell proliferation serum-free medium at 24 hours after the virus and high susceptibility to BmPTLNPV, a inoculation and cultured in fresh KBM700 recombinant BmNPV expressing the luciferase serum-free medium. As a conclusion, it was gene (Figs. 1, 2). shown that NIAS-Bm-Ke1 cell line was able to The heat-treated silkworm hemolymph be used as baculovirus gene expression system. showed a role of promoter of susceptibility of

Fig. 1 Fig. 2 NIAS-Bm-Kel cell line derived from !%$"(' $%&# Formation of BmNPV polyhedra in ke1 cells embryo (serum-free) (serum-free media+heat-treated silkworm hemolymph 1 % volume)

"# %**/') &(,+-. #!!$ Fig. 3 Change of the levels of luciferase activities in Kel cells infected with a recombinant BmNPV in spinner culture Kel cells of 5107 cells were added to spinner flask containing 250 ml of KBM700 serum-free medium. After 1% or 3% of silkworm heat-treated hemolymph was added, BmPTLNPV expressing the luciferase gene was infected with Kel cells. The stir speed of the medium was adjusted to be 100rpm. At indicated hours after culture started, cells were harvested and luciferase activities in the cells were measured. Date shows luciferase activity per one cell.

Functional analysis of phytochromes in rice

Makoto TAKANO Plant Physiology Department

We have isolated phytochrome A (phyA), significant residual phytochrome responses, phytochrome B (phyB) and phytochrome C indicating that not only phyA but also phyC is (phyC) mutants from rice ($)-.% *%+',%)and involved in the photoperception of FRc in rice. have produced all combinations of double Interestingly, the (&-"(&-# double mutant mutants. Seedlings of (&-" and (&-"(&-# displayed clear R/FR reversibility in the pulse- mutants exhibited a partial loss of sensitivity to irradiation experiments, indicating that both continuous red light (Rc) but still showed phyA and phyB can mediate the low-fluence significant de-etiolation responses. The responses response for gene expression (Fig. 1). Rice is a to Rc were completely canceled in (&-!(&-" short-day plant and we found that mutation in double mutants. These results indicate that either phyB or phyC caused moderate early phyA and phyB act in a highly redundant flowering under the long-day photoperiod, while manner to control de-etiolation under Rc. Under monogenic (&-! mutation had little effect on continuous far-red light (FRc), (&-! mutants the flowering time. The (&-! mutation, however, showed partially impaired de-etiolation and in combination with (&-" or (&-# mutation (&-!(&-# double mutants showed no caused dramatic early flowering (Fig. 2).

&++0(* ')-,./ #!!% "$ Fig. 1 Comparison of photoperception modes of phytochromes between in rice and in ",%&)'*+-)- In ",%&)'*+-)-! phyA is mainly responsible for the VLFR, whereas phyA is involved in both VLFR and R /FR reversible LFR in rice. FR-HIR: Far Red-High Irradiance Response; VLFR: Very Low Fluence Response; LFR: Low Fluence Responce. Fig. 2 Flowering times of various phytochrome mutants under long day conditions Monogenic +(." mutation had little effect on the flowering time. The +(." mutation, however, in combination with +(.# or +(.$ mutation caused dramatic early flowering.

Transgenic rice for allergy immunotherapy: Oral feeding of rice seeds expressing T cell epitopes suppresses clinical symptoms in a mouse model of pollen allergy

Lijun YANG, Fumio TAKAIWA and Hidenori TAKAGI Plant Biotechnology Department

Immunotherapy using allergen-specific T Japanese cedar pollen as a fusion protein with cell epitopes is a safe and effective treatment the soybean glycinin (Fig. 1). Under the control for the control of IgE-mediated allergic of the rice seed storage protein glutelin GluB-1 diseases, such as Japanese cedar pollinosis. We promoter, the fusion protein was specifically developed transgenic rice plants expressing T expressed and accumulated in seeds at a level cell epitopes of Cry j I and Cry j II allergens of of 0.5 % of the total seed protein.

Fig. 1 A schematic illustration of the transformation plasmid

"$ &++0(* ')-,./ #!!% Next, we examined the effect of transgenic IL-5, IL-13, and histamine release in serum rice seeds on allergic immune responses. Mice were also significantly suppressed. In were orally fed with rice seeds, and then addition, the development of pollen-induced systemically challenged with pollen clinical symptoms was inhibited in our allergens (Fig. 2). Compared to the control experimental sneezing mouse model (Fig. 4). group of mice, the development of allergen- These results provide a positive first step specific IgE and CD4+ T cell proliferative toward developing rice seed-based edible responses was inhibited in the group of mice vaccines for the prevention and treatment of fed with transgenic seeds (Fig. 3). The levels of allergic diseases. allergy-associated cytokine production of IL-4,

Fig. 2 Experimental schedule

Fig. 3 Fig. 4 Allergen-specific IgE Sneezing

&++0(* ')-,./ #!!% "$ Research Activities

Genome and Biodiversity Research Division

The National Institute of Agrobiological these model organisms could be very useful for Sciences (NIAS) is composed of three divisions: analysis of related species by comparative (1) the Genome and Biodiversity Research genomic approaches. Division, (2) the Insect and Animal Sciences Genetic erosion caused by various Division, and (3) the Plant Science Division. The environmental, socio-economical and ecological research activities of the Genome and changes necessitates research on genetic Biodiversity Division are interconnected with resources to maintain the genetic diversity of the Insect and Animal Science Division and the biological world. The Genetic Diversity Plant Science Division. Therefore, research Department focuses on basic research on plant, data and biological resources are shared among microbe and animal diversity. Extensive studies these three divisions to maximize research on various germplasm have contributed to the efficiency of the institute. development of rational and efficient methods The Genome and Biodiversity Research for classification, characterization, preservation Division aims to elucidate major biological and discovery of new biological resources. phenomena involved in agricultural production. To accelerate research on genetic It consists of three departments: (1) the Genome resources, the NIAS is also the center of the Research Department, (2) the Genetic Diversity MAFF Genebank System for agricultural Department and (3) the Genebank. The overall genetic resources in Japan. The Genebank has research output of these three departments is the responsibility for conservation and transmitted to the basic and applied research maintenance of plant, microorganism and community. As a result, the Genome and animal genetic resources and their DNA. A Biodiversity Research Division contributes to close linkage between NIAS and other sub- science, agriculture and social life by genebanks across Japan facilitates efficient distributing useful bio-resources and scientific collection, management and distribution of a results to the local as well as international wide range of genetic resources. All genetic research community. resources available through the Genebank can The Genome Research Department be used for research and breeding purposes in focuses on genome analyses of three major accordance with the regulations of the Convention organisms, which serve as the backbone of on Biological Diversity and International Japanese agriculture, namely, rice, pig and Treaty. Information related to genetic resources silkworm. Large-scale analysis of the genome and DNA materials available at Genebank can by genetic mapping, physical mapping and be accessed at http://www.gene.affrc.go.jp/ genome sequencing is now considered as the and http://www.dna.affrc.go.jp. The research most efficient strategy for understanding the activities of the Genome Research Department, structure and function of many genes the Genetic Diversity Department and controlling the expression of economically Genebank are described below. important traits. The genome information from

"$ %**/') &(,+-. #!!$ G enome Research Department

Introduction projects, as well as other genome-related Extensive analysis of the genomes are projects, is provided by the DNA Bank and the being undertaken through the three genome Rice Genome Resource Center (RGRC). The research projects of the institute, namely, the following reports summarize the major Rice Genome Research Program (RGP), research topics and achievements of the six Animal Genome Research Program (AGP) laboratories of this department. and Silkworm Genome Research Program (SGP). In the case of rice, the RGP in Assignment of 205 genes collaboration with the International Rice localized in HSA17 to a swine RH Genome Sequencing Project (IRGSP) has (IMpRH) map to generate a dense successfully completed the sequencing of the comparative map between swine entire genome in 2004. The accurate map-based and human/mouse sequence of the rice genome is now considered Bi/uni-directional chromosomal painting as the most important resource for cereal (Zoo-FISH) and gene mapping have earlier genomics and provides the link to several indicated the correspondence of swine important projects on rice functional genomics, chromosome (SSC) 12 harboring economically comparative genomics and applied genomics. important quantitative trait loci (QTLs) to Immediately after completion of the sequence, human chromosome (HSA) 17. In the present the Rice Annotation Project (RAP) was initiated study, we have attempted to assign the genes with the aim of manually curating the annotation localized in HSA17 to SSC12 to generate a of the genome on a regular basis. The first comprehensive and comparative map between jamboree-style annotation meeting (RAP1) HSA17 and swine chromosomes. A total of 255 paved the way for the establishment of the Rice primer-pairs were designed using porcine Annotation Project Database (RAP-DB), a sequences indicated to be orthologous to comprehensive database of manually curated human genes. Of the 255 primer pairs, 208 rice genes. Analysis of the genome structure of (81.6%) were found to be mappable to a swine pig through the Animal Genome Research chromosome using the INRA-Minnesota 7000- Program (AGP) led to the development of DNA rad porcine Chinese hamster whole genome markers for QTL analysis, construction of a radiation hybrid (IMpRH) panel. The mapping catalog of porcine genes and facilitating results revealed the following: 205 genes were traceability in the Japanese pork market. The integrated into an SSC12 RH linkage map with Silkworm Genome Research Program (SGP), logarithm of odds (lod) scores greater than 6. which aims to characterize the silkworm Onegene(&(,") was suggested to localize on genome, has also made significant progress in SSC12, while the remaining two (',!!% and EST analysis, genetic and physical mapping, +*)#$) were not linked to any markers/ whole genome shotgun sequencing and expressed sequence tags (ESTs)/genes expression profiling. Informatics support for registered, including those in the present study. these projects facilitate large-scale analysis of A comparison of the gene orders among SSC12, genome sequence data, development of analysis HSA17, and mouse chromosome (MMU) 11 tools, construction of genome databases and corresponding to HSA17 indicated that intra- release of information to the public domain. chromosomal rearrangements occurred Access to genome information and biological frequently in HSA17 of human ancestral materials generated from these genome species after its speciation.

&++0(* ')-,./ #!!$ "% Analysis of Diversity in the variation among wild rice species using 900 Genus !#$%" by Comparative ESTs mapped at intervals of approximately 500 Genomics Approach kb throughout the genome. Primers were The high-quality sequence of the rice designed mainly from the 3-UTR region of the genome based on the japonica cultivar EST sequences and used for PCR amplification Nipponbare was revealed in 2004. Since then, of DNA from 45 accessions belonging to 21 the information derived from the genome Oryza species from the germplasm collection of sequence has been widely used in NIAS and the National Institute of Genetics understanding the biology of rice including the (NIG). The overall efficiency of amplification for identification and functional characterization of each species is shown in Fig. 1. Most of the novel genes for agricultural traits. As a model regions were amplified in the AA genome ("! cereal crop, the rice genome sequence could +#,&-# complex, 94% in average for 15 accessions), also be used in clarifying how the genus "*./# suggesting that the genome sequence is originated from the ancestral cereal genome, as conserved. On the other hand, the "! )%%&$&(#'&+ well as how it evolved and diversified. This complex, which includes BB, BBCC, CC, CCDD would not only elucidate the lineage between and EE genomes, has 50-70% amplification cultivated rice varieties from their wild efficiency. The other remote genomes (FF, GG relatives but would also be useful in searching and HHJJ) showed only 20-30% amplification. for new beneficial alleles of the genes in the We have clearly shown here that the rice wild rice species that had been lost during the genome sequence has gradually changed in the establishment of modern varieties. A comparative course of evolution from the wild species. These genomics approach is becoming more and more results also indicate the utility of the standard important because the wild rice has been genome sequence from the japonica cultivar recognized as one of the major source for novel Nipponbare for analysis of genome diversity as gene alleles. Many agriculturally important well as the evolutionary relationships between genes such as disease resistance genes have modern rice varieties and wild rice species. been found in wild rice and introduced into the Moreover, the conserved regions across species modern rice varieties. could be good markers for comparative We investigated the nucleotide sequence genomics among "*./# species.

Fig. 1 Amplification of "! *#+%,# ssp. &#)('%$# cv. Nipponbare EST markers in wild rice accessions The height of the bars represents the efficiency (%) of marker amplification.

"% &++0(* ')-,./ #!!$ The Rice Annotation Project representative plant species, rice and %0'(+)./1+1 Database (RAP-DB) and 2*',+'-'" First, our analysis revealed that the Comparative Genome Analysis number and average length of exons in rice were quite similar to those in %" 2*',+'-'! while Completion of the rice genome sequencing the lengths of introns and intergenic regions by the International Rice Genome Sequencing varied between the two species. This may be Project (IRGSP) enabled us to annotate all the because transposable elements had been rice genes. Automated annotations of the gene inserted extensively in non-exonic regions of functions were manually curated in a jamboree- rice. Second, we found that 22% of the rice style annotation meeting of the Rice Annotation genes had no homologs in the %0'(+)./1+1 gene Project (RAP). The annotation data is available set. Although this observation is consistent in the RAP Database (RAP-DB, http://rapdb. with the previous view that the rice genome lab.nig.ac.jp/) which was constructed in possesses a number of unique genes, the collaboration with the DNA Data Bank of Japan majority of the rice-specific genes might be due (DDBJ), National Institute of Genetics (Fig. 2). to gene loss in the %0'(+)./1+1 lineage or The RAP-DB provides two types of viewers mispredictions of the protein coding regions. (GBrowse and G-integra), BLAST/BLAT Lastly, despite independent gene duplication similarity searches, and keyword searches. events after the speciation between the Information about TIGR and NCBI annotations monocot and dicot plant species, the distribution on the IRGSP genome assembly is also of the paralogs and protein functions had been available. Additionally, the RAP genes are retained in a similar manner in both of rice and linked to other rice genomics data, such as full- %" 2*',+'-'" These data suggest that the rice length cDNAs and &.1#$ insertion mutant and %0'(+)./1+1 gene sets have shared a lines. We expect that the RAP-DB will serve as common feature for over 100 million years. The a hub for rice genomics. phenotypic difference between the two species The RAP annotation data was envisaged to may have been derived from changes of a small elucidate the evolutionary process of flowering number of genes or the divergence of gene plants. We compared the genomes of two regulation.

Fig. 2 The Rice Annotation Project Database (RAP-DB) with an overview of the two genome viewers, G-integra and GBrowse

&++0(* ')-,./ #!!$ "% Construction of a silkworm SNP domesticated for an estimated 5,000 years and linkage map based on BAC used extensively for silk production. In addition, end-sequences it is a key model of the , the second largest group of holometabolous insects, which The silkworm, !%$"(' $%&#,isan include many beneficial insects as well as the agriculturally important insect that has been most destructive agricultural pests. Due to

Fig. 3 A !%$"(' $%&# SNP linkage map The map is based on 534 SNP markers segregated into 28 linkage groups, represented by vertical lines. The BAC clones corresponding to the mapped SNP markers are shown on the right and the corresponding recombination distances between the markers are indicated on the left.

"! $)).&( %'+*,- "!!# industrial and agricultural concerns, genome industrial and agricultural interest. analysis has become an urgent necessity for a comprehensive characterization of silkworm. So Single nucleotide polymorphisms far, we have been carrying out silkworm whole- in Toll-like receptor (TLR) genes genome-shotgun sequencing, large-scale EST revealed in pigs collection, BAC-contig construction by fingerprinting methoods, etc. The integration of Opportunistic infections have become major those results based on BAC clones will provide problems for modern pork production. Diseases a useful tool for post-genome investigations of like pneumonia and diarrhea, which result from silkworm and other Lepidopteran species. We intensive breeding of swine, lead to extensive have developed a linkage map for silkworm economic losses. The causative pathogens are based on single nucleotide polymorphisms resident organisms in pig farms and are (SNPs) initially found on regions corresponding difficult to eradicate. Hence, the genetic to the end sequences of bacterial artificial improvement of swine, based on resistance to chromosome (BAC) clones. Using 190 segregants pathogens may prove to be an effective control from a backcross of a p50T female an F1 (p50 measure against these infections. Toll-like T C108T) male, we analyzed the segregation receptors (TLRs) play a crucial role in the patterns of 534 SNPs between p50T and C108T recognition of pathogen-associated molecular detected among 3,840 PCR amplicons, each patterns (PAMPs) derived from the pathogenic associated with a p50T BAC end-sequence. microbes. Polymorphisms in TLRs may This enabled us to construct a linkage map influence their recognition of pathogen-derived composed of 534 SNP markers spanning 1,305 molecules; swine TLRs are predicted to be cM in total length distributed over the associated with responses to infectious diseases expected 28 linkage groups (Fig. 3). Among 534 such as pneumonia. We cloned and determined BACs with end sequences harboring the SNPs the complete sequence of porcine TLR genes used to construct the linkage map, 89 were ()'("! )'(#! )'($! )'(% and )'(&)that associated with 107 different ESTs. Each of the are expressed on the cell surface and closely SNP markers is directly linked to a specific related to the microbial infection. We searched genomic BAC clone and to whole genome for single nucleotide polymorphisms (SNPs) in sequence data. In addition, some are also linked the coding sequences of porcine )'("! )'(#! to the EST data. Therefore, the SNP linkage )'($! )'(%! and )'(& genes in 96 pigs map will be a powerful tool for investigating from 11 breeds and elucidated 21, 11, 7, 13, and silkworm genome properties, mutation 11 SNPs, respectively, which caused amino acid mapping, and map-based cloning of genes of substitutions in the respective TLRs (Fig. 4).

Fig. 4 Heterozygosity of the SNPs in porcine %#$! and %#$" genes investigated in 96 individuals Synonymous and non-synonymous SNPs are indicated by closed and open circles, respectively. The green area in each gene corresponds to LRRs, which are important for pattern recognition of molecules, and the yellow area corresponds to TIR (Toll/IL-1 receptor) domains, which are necessary for intracellular signaling. Heterozygosity of the SNPs is calculated using the following formula: 2 2 Heterozygosity = 1 - (p1 +p2 ) where p1 and p2 are the observed frequencies of the first and second alleles at each SNP locus, respectively. Biases in the heterozygosity between non-synonymous and synonymous SNPs are indicated by red lines and were calculated as the differences of the sums of non-synonymous SNPs (SN) and synonymous SNPs (SS)withinevery 100 bp window sliding 1 bp. %**/') &(,+-. #!!$ #" The distribution of these non-synonymous researchers to simultaneously characterize SNPs was biased; many were located in the thousands of rice genes associated with leucine-rich repeats (LRRs), particularly in %#$ biological functions, growth processes, and "! These data demonstrated that the biotic and abiotic stress response. The open heterogeneity of TLR genes has been laboratory for microarray analysis is equipped preserved in various porcine breeds despite with facilities for hybridization, scanning and intensive breeding that was carried out for data analysis. A 2-day protocol for microarray livestock improvement. This suggests that the analysis was developed to allow researchers to heterogeneity in TLR genes is advantageous in conduct expression analysis of samples from increasing the possibility of survival in porcine hybridization to data analysis. Prior to populations. hybridization, the RNA samples are checked for quality using the BioAnalyzer and Optimization of expression concentration using the NanoDrop in order to profiling output from microarray assure that only high-quality RNA samples are analysis used for linear amplification and labeling. Sample labeling using Cy3 and Cy5, and Much of the researches in the post-genome subsequent hybridization can be performed on era will depend on high-throughput technologies. the first day. Hybridization is carried out for 17 The Rice Genome Resource Center (RGRC, hours at 60!. Subsequently, washing and data http://www.rgrc.dna.affrc.go.jp/) is providing spot analysis using the Agilent Feature access to microarray technology to allow a Extraction and Genespring analysis software large number of investigators to apply global can be performed on the second day. It is a expression profiling into their specific research highly reliable protocol eliminating most of the programs on rice. A rice oligonucleotide microarray limiting factors that affect microarray with 22,000 non-redundant oligonucleotide expression analysis such as sensitivity of the probes based on the full-length cDNA sequence quantity of RNA and background intensity. information was constructed in collaboration This suggests that hybridization of target with Agilent Technologies (http://www.agilent. genes can be performed with high reproducibility co.jp). This rice microarray system enables and enhanced sensitivity.

Fig. 5 Optimization of conditions for microarray analysis facilitates analysis of very small amount of RNA derived from various tissues or cells therefore expanding the utility of gene expression profiling in functional genomics.

"" $)).&( %'+*,- "!!# One of the main problems involving reduced to 10 ng of total RNA with almost expression profiling is the high level of similar expression intensity for highly and variability in microarray data. Although some medium expressed genes. Reducing the amount of this variability is relevant because it of samples to 2 ng also gave a clear expression corresponds to the differential expression of pattern. This indicates that the microarray genes, a large portion of variability usually could be used for analysis of small samples of results from undesirable biases introduced RNA derived from tissues or organs, specific during the many technical steps of the tissues isolated by laser dissection or transient experimental procedure. The so-called assay in cell culture (Fig. 5). experimental noise has been addressed to The microarray open laboratory has been normalize data according to the related effects. used by almost 247 research groups from Color swaps are now routinely included in different institutes and universities all over microarray experimental designs to correct for Japan since it became operational in August 1, labeling biases. The amount of samples for 2003. Currently, an average of two users/ successful hybridization has also been groups per week use the facilities. In addition to investigated. Although it is normally suggested the rice microarray, the RGRC open laboratory that about 400 ng of total RNA should be used also supports microarray analysis in #6)*0,45707, for labelling, the amount of sample could be silkworm and cow.

G enome Diversity Department

The objectives of the Genetic Diversity 2005 are described below. Department are to conduct basic research into plant, animal and microorganism diversity from Very close relationship of the the molecular to the population level. Such chloroplast genomes among research contributes to the development of !"##$"&'% species new and improved methods for classification, characterization and preservation of germplasm We recently determined the complete and discovery of new biological resources for sequence of the sugarcane chloroplast genome. use in agriculture and other industries. Here, we have used the information for a Current plant research includes molecular comprehensive phylogenetic analysis of the and biological characterization and evaluation genus &)++/)692! usingallsixspecies(13 of the genera Hordeum, %6:;)! &)++/)692! accessions). The polymorphisms between '6080+92! $1:+03- and (0.3)" Mechanisms in sugarcane and maize in 26 chloroplast genome plants that confer cold hardiness are also being regions were used for the analysis. In 18 of the investigated. Microbiological research includes 26 regions (a total of 5,381 bp), we found 41 functional genomics, biosystematics and mutations involving 17 substitutions, three proteomics of such organisms as fungi, yeasts inversions, six insertion/deletion mutations, and and bacteria. In particular, plant pathogens 15 simple sequence repeat length polymorphisms. have been focused on. Animal research includes Based on these results, we calculated a the development of the methods to utilize phylogenetic tree of the genus Saccharum, in various types of germplasm and to improve the which all six species are clearly separated (Fig. genetic performance of domestic animals. 1). By the analysis, (1) &" 703-37- and &" *)6*-60! Major research outputs of the department which have identical sequences, belong to the except for Topics of Research during fiscal same clade, whereas the other four species, &"

%**/') &(,+-. "!!$ "# Fig. 1 Phylogenetic relationships among !"##$"&'% species A strict consensus tree was calculated using the maximum parsimony method based on the sequence data of 18 chloroplast DNA regions. Tree length = 264, confidence interval =0.8659, retention index =0.9299, rescaled consistency index =0.8912. Species names and abbreviations of each accession (in parentheses) are shown. If two accessions of the same species have identical sequences, they are shown as 2 acc. The numbers above the nodes represent bootstrap values expressed as the percentage of 1,000 bootstrap replications.

2++.(.1&470! $" 42'75670! $" *)7/*! and $" 53216&1*70! form an independent clade; (2) $" 53216&1*70 has a paraphyletic relationship with the other five species; and (3) no or very Fig. 2 low intraspecific variation was observed in $" Variation in (a) seeds and (b) leaves of mutant, 2++.(.1&470! $" 42'75670! $" 5.1*15*! $" '&4'*4.! cultivated and wild black gram and $" *)7/*! whereas higher intraspecific variation was observed in $" 53216&1*70" wild black gram. In a BC1F1 population of 180 Based on the number of nucleotide individuals from this cross 148 marker loci substitutions, the divergence time between $" could be assigned to the 11 linkage groups that 2++.(.1&470 and $" 53216&1*70! and between $" corresponds to the haploid chromosome 2++.(.1&470 and maize were calculated to be number of black gram. This is the first genetic about 730-780 thousand years ago and about 5.9 linkage map for black gram and it was million years ago, respectively. These results compared with that of a close relative, azuki suggest that the cytoplasm of $&((-&470 bean. Inversions, insertions, deletions / species are very closely related. duplications and a translocation were detected between the black gram and azuki bean linkage Analysis of a large seeded maps. Domestication trait QTL, including seed mutant in black gram size, locations have been identified on the !#&%($ '*(%)" genome map of black gram.

The Asian %.,1& consists of domesticated Identification and mapping of species among them is black gram. A mutant of QTL for rachis internode length this species has a seed size about double that of associated with cleistogamy in traditional cultivated black gram and six times barley the size of wild black gram (Fig. 2a). This mutant is also larger in other organs such as General knowledge of the closed flowering leaves (Fig. 2b). In order to determine the trait, or cleistogamy, of barley, #24)*70 location of this mutation(s) on the black gram 87/,&4* L., is still limited. We studied the genome, a genome map was developed based relationship between cleistogamy and characters on a cross between the mutant black gram and of spike morphology and detected a linkage of

"# %**/') &(,+-. "!!$ Fig. 3 Mapping of QTL for rachis internode length ($'%&!"#) on the long arm of chromosome 2H of barley A: Genetic map of 126 DH lines of Mikamo Golden x Harrington; B: Genetic map of 150 F2 plants of Misato Golden x Satsuki Nijo. The genetic distance between markers is given in cM. Arrows represent positions; shaded bars represent 90% confidence interval of $'%&!"#! cleistogamy genes with a highly significant people. Crop research within the GCP has many quantitative trait locus (QTL) for rachis interrelated data types, focused on germplasm. internode length on the long arm of The scope of the GCP scientific domain model is chromosome 2H(Fig.3). The mapping to cover this range of data types spanning populations consisted of 129 doubled haploid germplasm, phenotype, genotype, mapping, lines of Mikamo Golden Harrington and 150 functional genomics and location environment

F2 plants of Misato Golden Satsuki Nijo. The data. NIAS has a role to develop the functional phenotypic variance explained by this QTL genomics sub-domain model. Software accounted for 77.5% and 82.6% of the variance implementations of the GCP domain model will in rachis internode length in these two form the middleware of the GCP platform and populations. The peaks of the QTL coincided network that will link and user tools and with the positions of the cleistogamy gene loci. interfaces to local and remote (web service connected) data source (Fig. 4). The model is Development of GCP domain model documented in Unified Modelling Language (UML). Computable versions of the UML model The Generation Challenge Programme are published in the Demeter package of the (GCP) aims to assist farmers in the developing GCP Middleware project in CropForge (http:// world by using advances in molecular biology cropforge.irri.org/ projects/ gcpmiddleware / ) . to harness the rich global heritage of plant The alpha release of the domain model is also genetic resources and create a new generation published to http://www.generationcp.org/ of crops that meet the needs of resource-poor model.

Fig. 4 Relationship of GCP domain model to platform

%**/') &(,+-. "!!$ "# Taxonomic analysis of !-+#*&-' Canada) and Japan is caused by two closely species, as the causal pathogens related species, #" 2,&4)1.- and #" (60)-314536/" of soybean sudden death The SDS species do not form an exclusive syndrome and dry bean root-rot group within the molecular phylogeny, indicating they may not have a monophyletic origin. The etiological agents of soybean sudden Artificial inoculation tests on a susceptible death syndrome (SDS) have been reported as #" variety of soybean, using the isolates from 41.&0- or its forma specialis, f. sp. +.8(-0)4" On soybeans, dry beans and mung beans, revealed the other hand, the causal pathogens of dry that all six species can induce typical SDS bean or mung bean root-rot have been known symptoms on the inoculated soybean plants. as #" 41.&0- f. sp. 2,&4)1.-" Soybean SDS pathogens isolated from the US, Argentina and Genome-wide gene expression Brazil, dry bean root-rot pathogens from the US analysis of "#(,%)')(#+ )*./#$ and Japan, and mung bean root-rot pathogen pv. )*./#$ from Canada were investigated. Detailed phenotypic comparisons of macro- and To survey genes regulated by HrpG or microscopic features, and phylogenetic analyses HrpX, we constructed a DNA macroarray of multilocus DNA sequence data indicated that system consisting of 2,384 of genomic DNA the soybean SDS and dry bean root-rot fragments of strain T7174 (MAFF311018) of pathogens comprised six morphologically and %11" It comprised about 95.5% of the whole phylogenetically distinct species (Fig. 5). genome DNA. Using this macroarray system, it Soybean SDS in North and South America (US, was confirmed that the ,32 gene cluster (about Argentina and Brazil) was found to be caused 87k to 120k region of the Xoo genome) was up by four species: #64&3-6/ 7-3+6.-*13/)! #" regulated in wild type strain under ,32- 56(6/&0-&)! #" '3&4-.-)04) and an undescribed inducing medium, while it was not up regulated species of #64&3-6/" By way of contrast, dry or in the ∆,32% and ∆,32$ mutants (Fig. 6). mung bean root-rot in North America (US and Moreover, it was revealed that thirteen genomic regions were regulated by HrpG or HrpX. To check that expression of genes within these regions were really controlled by the HrpG or HrpX, a real-time quantitative RT-PCR system was used. Finally, six genes (XOO0037, XOO0078, XOO1388, XOO2263, XOO4042 and XOO4134) were newly identified. In addition,

Fig. 5 Four soybean SDS pathogens (A-C, F) and two dry bean root-rot pathogens (D, E) A: #64%3-6/ 56'6/%0-%)! B: #" 7-3+6.-*13/)! Fig. 6 C: #" &3%4-.-)04)! Schematic representation of regions with up- and down D: #" 2,%4)1.-! -regulated gene expression in ,32 inducing condition E: #" '60)-314536/ %0( (!,32$ / wild type strain) F: #64%3-6/ 42" (undescribed species).

"# $)).&( %'+*,- "!!# two genes, XOO2263 and XOO4134 contained a several antifungal proteins, isolated from plants, plant-inducible- promoter (PIP) box, which was are also reported as mediated by sphingolipid. a consensus sequence of HrpX regulons, Thus the structure of sphingolipid in the target upstream region of putative initiation codon. cell is a key of proteinaceous toxin spectrum. These obtained results showed that this macroarray system was useful tool for genome- Sex chromosome inactivation in wide gene expression analysis in '11" male FKBP6 deficient animals

!&,.-$)('+.#$* &"#+%* killer FK506 binding protein 6 (FKBP6) is a protein entry to sensitive yeast component of the synaptonemal complex (SC). cell mediated by plasma In the male mice and rats, lack of this protein membrane sphingolipid defects chromosome pairing, resulting in meiotic arrest in spermatogenesis. The female Although a number of microorganisms deficient animals, however, enable to complete surround us, very few succeed in causing meiosis and shows normal fertility, although the infection. Recently, innate immune system in protein has been detected in the SC. The eukaryotes, through the action of antimicrobial discrepancy brought us an idea that FKBP6 peptides and proteins, is recognized as playing might have a role in inactivation of XY an important role in survival and defense. A chromosomes at pachytene stage, which is a killer strain of the yeast %/687+3108*+4 /(*5-4 male specific meiotic event. Therefore we secretes a protein toxin (zymocin) that prevents examined co-localization of phosphorylated the proliferation of &(**,(3108*+4 *+3+7-4-(+ histone H2AX (γH2AX) with paired sex (baker yeast) cells. We analyzed one of the chromosomes, as a marker of inactivation of mutants, .5-#! which resists exogenous zymocin XY. but is sensitive to intracellularly expressed Germ cells obtained from $.)2# +/- toxic subunit of zymocin (Fig.7). KTI6 is allelic (phenotypic normal) and -/- male (meiotic to IPT1, and codes for mannosyl- arrest) mice and rats were processed by diinositolphospho-ceramide [M(IP)2C] synthase, surface spread method. Nuclei spread on slide the enzyme which catalyzes the reaction to glass were reacted with anti SCP3 (for produce M(IP)2C, the major plasma membrane chromosome staining) and γH2AX antibodies, sphingolipid. Mutants defective in the synthesis followed with fluorescent labeled 2nd antibodies, of M(IP)2C intermediates were also found then examined under the razor scanning resistant to zymocin. In addition, kti6/ipt1 cells microscope (Fig. 8). In $.)2# +/- mice, most prevented the import of toxic subunit of zymocin. In summary, our results indicates that

M(IP)2C plays a role in the accumulation of zymocin in target cells. Interestingly, actions of

Fig. 8 Pachytene nuclei of "$#%! +/- (upper panels) and -/- (lower panels) mice Fig. 7 SCP3 (red) and γH2AX (green) staining. Nucleus of +/ Response to zymocin of heterozygous diploids (kti6/ - showed localization of γH2AX in the restricted region EUROSCARF gene disruptants) surrounding XY pair. In the -/- nucleus γH2AX kti6∆ipt1 was resistant to zymocin. diffusively localized to the region surrounding abnormally paired chromosomes including XY (arrows).

%**/') &(,+-. "!!# "$ pachytene nuclei (96.3%) displayed γH2AX co- computer simulation. A large number of localizing to XY pair. In #'$(" -/- mice, 71.9% of animals did not cause slow convergence if the pachytene nuclei showed restricted staining of data structure (%!&! number of generations and surrounding paired XY. Other nuclei showed mating ratio) was fixed. The numbers of diffused γH2AX staining possibly caused by breeding animals and their progeny affected #'$(" A A non-homologous pairing of XY. The the structure of the !!-inverse matrix ( : deficient rats showed similar tendency to mice. additive genetic relationship matrix), but The results evidenced that sex chromosome selection methods did not. An increasing inactivation could occur, unless XY paired number of iterations seemed to be due to an A normally, in FKBP6 deficiency. imbalance of nonzero elements in the !-inverse matrix, which resulted in the imbalance of Effects of data structures on the nonzero elements in the MME. The number of solving of animal model equations iterations until convergence was roughly by preconditioned conjugate constant for two-trait animal model when the gradient iteration heritability of a trait was constant regardless of the heritability of another trait, and it was the To predict breeding values in dairy cattle, smallest when the heritabilities of both traits preconditioned conjugate gradient (PCG) were about 0.5 to 0.6 (Fig. 9). Increasing algorithm has become of interest lately as a absolute values of genetic and environmental means of solving large sets of mixed model correlations resulted in slow convergence. In equations (MME). Thereby, the effects of data particular, genetic correlation affected structure on the solving of MME by PCG convergence strongly. algorithms were investigated by using data in a

Fig. 9 Number of iterations until convergence for different heritabilities (a) and different genetic and environmental correlations (b)

"$ %**/') &(,+-. "!!# G enebank

Genebank Project GRs, and 878 accessions of animal GRs as of The NIAS Genebank Project consisting of December 31, 2005. In FY2005, 4524 accessions plant, microorganism, animal, and DNA of plant GRs, 799 accessions of microorganism sections has been implemented in national and GRs and 28 accessions of animal GRs were international collaboration with a large number distributed to internal and external users for of organizations in public and private sectors. basic and applied researches. Passport and The Genebank, NIAS coordinates the Project characteristics data of freely distributable as a center of the project activities including accessions are uploaded on the website (http:// the exploration, collection, characterization, www.gene.affrc.go.jp/). evaluation, preservation, multiplication, and International Genetic Resources Workshop information management of genetic resources was held on August 22nd and 23rd, 2005 at (GRs). Tsukuba on two topics, Rice Genome: In the fiscal year 2005, international Challenges and Opportunities and Global and collaborative field studies were carried out in Regional Strategies for Conservation and cooperation with relevant research institutes in Sustainable Use of Plant Genetic Resources. It the partner countries. The Project dispatched was held jointly by the SABRAO and the NIAS scientists to explore tartary buckwheat GRs in as a part of the 10th International Congress of Sakhalin, wild rice and vegetables GRs in the Society of Breeding Researches in Asia and Myanmar, and yam GRs in Vietnam, to do Oceania (SABRAO). NIAS Genebank supported feasibility study on apples, pears, and stone the Congress as a member of the JSCC and held fruits GRs in Xinjiang Uygur Autonomous a training course for instruction of techniques Region of China, to survey wild rice and wild to preserve microorganisms and revive them, and cultivated legumes GRs in Papua New as ICCC10 training course at NIAS Genebank. Guinea, to collect !4.&%0*(*2, GRs in Taiwan and Malaysia. The #)/*++% GRs collected in the Discovery of a Chromosomal collaborative field study program in Korea Region Associated with Root were studied using AFLP analysis for possible Structure Using a Set of and effective conservation on farm. Five Representative Cultivars exploration missions were organized by the Derived from Rice Germplasm Project to explore different GRs inside Japan: Collection pear GRs in Aomori, Akita, and Iwate Asian cultivated rice ("/56% 0%1*3% L.) holds prefectures, plum and pear GRs in Yamagata diversified root systems depending on different prefecture, $%''*-*2, GRs in water-stress conditions such as rainfed-lowland prefecture and Yakushima Island of Kagoshima and upland. Although root traits should play a prefecture, Cruciferous vegetables in Kagoshima key role in drought avoidance, limited analyses and Miyazaki prefectures, and tea GRs in of QTL loci on a few traits such as length and . thickness of root have been reported. We did Additionally, several sub-banks did microscopic observation and comparative explorations by their own efforts, from which analysis of root structures in the Rice Diversity collected materials are also registered in the Research Set (RDRS) of rice germplasm NIAS Genebank system. The total numbers of developed in the NIAS Genebank. Nodal roots GRs preserved at the NIAS Genebank and of the main stem were collected from rice partner institutions are 235,946 accessions of plants grown in upland condition for six weeks plant GRs, 22,231 accessions of microorganism of summer season, 2004 at Tsukuba. We took

%**/') &(,+-. "!!# "$ digital images of stained cross-sections using and 14 Japonica varieties were divided into two digital microscope and measured root area, groups corresponding to small (n=9) and large stele area, area and number of xylem vessel. (n=5) stele/root area ratios (Fig. 1). We analyzed Association between alleles at 147 RFLP loci genotype frequency at RFLP loci between two and phenotypic data obtained from 61 varieties groups divided based on variation in stele/root (47 of Indica type and 14 of Japonica type) were area ratio and found one marker (orange box) calculated. We discovered one region on associated with stele/root area ratio in long chromosome 4 associated with the stele/root arm of chromosome 4 (Fig. 2). This region is area ratio. suggested to harbor a gene or genes Microscopic observation showed the responsible for size factors of root organs. similarity of frequency distribution for root area and number of xylem vessels between Indica Development of Cryopreservation type and Japonica type cultivars. Japonica rice Protocol for Long-term Storage was more variable than Indica rice in root traits of Plant Germplasm such as areas of stele and xylem vessels, xylem Cryopreservation is becoming a very vessel area/no. ratio, and stele/root area ratio, important tool for long-term storage of plant germplasm, especially vegetatively propagated crops and recalcitrant seed species, with a minimum of space and maintenance. Cryopreservation should be considered as a backup to the collections to insure against loss. In this, we mean the priority of collections to be cryopreserved should be given to the at risk crops that have an increased chance of being lost from a collection. In our genebank, the !"#%$ germplasm collection at Tsukuba, which include about 1,400 accessions with several Fig. 1 species, are processed for long-term preservation. Relationship between root area and stele area in 61 To date, about 750 accessions of them have rice cultivars (Uga #% "$! 2005) been cryopreserved in liquid nitrogen tanks

Fig. 2 Comparison of graphical genotypes between the two different groups on stele/root area ratios (Uga #% "$! 2005) Light blue: Nipponbare (Japonica) type allele; Pink: Kasalath (Indica) type allele; White: other type allele; and Yellow: heterozygous allele. In each chromosome, each line from left to right indicates genotype of each variety, and each column from top to bottom indicates a genotype of RFLP markers from a short to a long arm.

#! %**/') &(,+-. "!!$ using dormant winter buds. Also, we started the dark are flat with little aerial mycelium, three cryopreservation projects such as smooth and cream to salmon or pale brown in practical storage of (+ 2(0.,-grown potato color. Conidiophores are unbranched or shoots, development of cryopreservation protocol occasionally branched, with conidiogenous cells of mat rush and sugarcane. In these, practical often arising at right angles from vegetative cryopreservation method of (+ 2(0.,-grown hyphae. Conidiogenous cells are monophialides potato shoot tips by encapsulation-vitrification or rarely polyphialides formed at the apices, or was improved. Mass propagation system of (+ as short (adelophialides) or long blanches from 2(0.,-grown mat rush shoots was established vegetative hyphae, hyaline, smooth, cylindrical using induction of multiple shoots. to obclavate, sometimes crooked or sinuous at the tips, often with single conidiogenous Morphological, molecular and apertures, and occasionally with second phytopathological variations of apertures. Conidia are produced in colorless !'%$.*-+*,&/( ."#"$&)/( slime masses at the tips of the phialides, hyaline, #)'&0,/-,.(1* 0$%$&(+1* (van Beyma) M.E. smooth, oblong-ellipsoidal, usually asymmetrical Palm, W. Gams & Nirenberg, which was validly to slightly curved, multiguttlate and most are 1- described in 1995 (Palm et al. 1995), had been septate and with a few aseptate. known as a common soil fungus at rhizospheres, Growth speed of mycelia and appearance and was isolated from lake sediment for the rates of aseptate conidia on PDA at 20!in the first time in Japan (Tubaki and Ito, 1975). We dark varied from 2.7-4.7 mm/day and 0-28.6%, identified several fungal isolates pathogenic to respectively, in 6-8 isolates. Sizes of septate and plants, ("'" pumpkin, garden ranunculus, anemone, aseptate conidia that had formed on synthetic sweet pepper, lotus ginger and Japanese radish low nutrient agar (SNA) at 20!in the dark (Fig. 3), as #" 0$%$&(+1*! and found that it had ranged from 4.0-12.01.0-5.5 µm and 2.5-8.51.0- been differentiated morphologically, molecularly 3.5 µm, respectively, depending on the isolates. and phytopathologically. The isolates used are The 10 isolates were classified into 2 groups shown in Table 1. Their morphological based on their sequence data of rDNA ITS characters are as follows (Fig. 4). Colonies in culture on potato dextrose agar (PDA) at 20!in

Fig. 3 Fig. 4 Diseases of pumpkin (A), garden ranunculus (B), Colonies of "! )#$#%&(*' on potato dextrose agar anemone (C), sweet pepper (D) , lotus ginger (E) and plates (upper left: surface view, upper right: reverse Japanese radish (F) caused by "! )#$#%&(*' view), conidiogenous cells and conidia of "! )#$#%&(*' (lower left: an isolate from pumpkin, lower right: an isolate from ranunculus)

&++0(* ')-,./ #!!% $" regions, though their homologies were highest original host plants, whereas the others caused (>94%) with those of "! )#$#%&(*' registered in no disease in the plants tested (Table 1). There the DNA Data Bank of Japan (Fig. 5). When is the possibility that "! )#$#%&(*' containing pumpkin, garden ranunculus and lotus ginger various strains morphologically and molecularly were inoculated with the isolates, the has been also differentiating strains with high respective isolates were virulent only to the host-specificity.

Fig. 5 Grouping of $! 7'(')/281 isolates based on neighbor-joining analysis for sequence of rDNA ITS regions ITS1F(5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) were used as primers for PCR. Numbers above branches are bootstrap values (%).

Table 1 Isolates used and their pathogenicity to pumpkin, garden ranunculus and lotus ginger. Pathogenicity to Collection Fig. Isolation source garden lotus locality 1 pumpkin ranunculus ginger Kagoshima, Japan pumpkin [#8)85(/7' sp.] A + Ibaraki, Japan pumpkin [#8)85(/7' sp.] + Kagawa, Japan garden ranunculus [%'282)8086 '6/'7/)86]B + Chiba, Japan garden ranunculus [%'282)8086 '6/'7/)86]+ Chiba, Japan anemone ["2+132+ )3532'5/']C Ehime, Japan sweet pepper [#'4)/)81 '22881 var. -536681] D , Japan lotus ginger [#58)81' '0/61'7/,30/']E+ Tokyo, Japan lotus ginger [#58)81' '0/61'7/,30/']+ Tokyo, Japan lotus ginger [#58)81' '0/61'7/,30/']+ Tokyo, Japan lotus ginger [#58)81' '0/61'7/,30/']+ Chiba, Japan lotus ginger [#58)81' '0/61'7/,30/']+ Miyazaki, Japan radish [%'4.'286 6'7/986]F Ezypt sweet violet [&/30' 3*35'7'] Hyogo, Japan lake sediment

+: Virulent. : Virulence was not detected.

#" %**/') &(,+-. "!!$ Insect and Animal Sciences Division

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

%**/') &(,+-. "!!$ ## D evelopmental Biology Department

Expression of a nanos homologue mechanism(s) of germ cell formation during the in "*)$/. eggs evolution. The silkworm $/-'54 -/1+ has no recognizable germ plasm, a morphologically Application of the tetracyclin distinguishable egg cytoplasm that contains controlled binary gene expression germ cell determinant(s), and its germ cells system (tTA/TRE system) to the appear first on the ventral side of the embryo, sawfly, !-&#('# +*,#% not on the posterior pole as in %1/2/0*+,& One of the powerful tools to analyze gene -(,&./)&23(1" During the search of germ cell function in non-model organisms is the specific genes in $/-'54! we found that technique to regulate gene expression. We transcripts of a .&./2 homologue were dispersed demonstrated that a transgenesis-based binary along the ventral midline of the egg, the region gene expression system worked in #3*&,+& where germ cell formation occurs in the future 1/2&( (Fig. 2). Embryos bearing both tetracycline- (Fig. 1a). In later stages of embryogenesis, the controlled transactivator (tTA) and a marker transcript was observed specifically in the EGFP gene driven by the tTA response germ cells (Fig. 1b). The localization of the element (TRE-EGFP) expressed EGFP, while .&./2 transcripts may be interpreted as the the embryos bearing either the tTA or the presence of $/-'54 germ plasm in the ventral TRE-EGFP did not express EGFP. The EGFP midline of the egg. However, its composition expression in embryos bearing both tTA and and localization mechanisms seem to be TRE-EGFP was suppressed when tetracycline different from those of %1/2/0*+,&! suggesting was orally applied to the parents at a that $/-'54 has developed a specific concentration of 100 µg/ml.

Fig. 2 The tTA/TRE binary expression system works in the sawfly, !.&#('# ,+-#% The embryo bearing tTA/TRE -EGFP expresses the marker EGFP (left panel, middle embryo). The expression was suppressed by application of tetracycline (right panel, middle embryo).

Fig. 1 Expression pattern of a "+)$0/ *#*+- homologue during the early development (a) An egg at cleavage stage. Ventral view. Anterior is to the left. (b) An embryo after blastokinesis. The *#*+- transcript is specifically observed in the gonad (arrow).

#$ &++0(* ')-,./ "!!% Expression patterns of two novel pluripotency of ES cell subpopulations. In order genes isolated from regenerating to identify novel regulatory factors in ES cell individuals of an oligochaete differentiation, we have performed comparison annelid, !(#%/-+"$., '"*)($(,&, of gene expression profiles by oligo-DNA array analysis between three subpopulations. In this The spatiotemporal expression profiles of fiscal year, we focused on uncharacterized 23 two novel genes, $*.1-" and $*.1-#! which genes (Ppet: PECAM-1 Positive ES cell-derived were isolated from regenerating $+&(20.%'1/ Transcripts) and attempt to elucidate the *%-,+'+/)/ were examined by RT-PCR and functions of them in ES cells and early embryos whole mount )+ /)01 hybridization (WISH). For by small interfering RNA (siRNA) mediated both genes, the expression was scarcely gene knockdown. detectable in intact worms, but was dramatically When fluorescein-labeled Ppet siRNAs activated following amputation. The expression were transfected into ES cells, plating efficiency levels were highest at the blastema formation of these ES cells showed no significant stage (around 12-24 hours after amputation). difference between fluorescein negative and The results suggest that these genes play positive cells in all groups. Akaline phosphatase important roles in annelid regeneration (Fig. 3). (AL-P, a marker for undifferentiated ES cells) positive colonies were decreased by Ppet genes have a function on knockdown of Ppet 002, 005, 010, 015, 021 and differentiation / proliferation of 023 gene as well as Oct3/4 (Fig. 4). In contrast, ES cells and also early AL-P positive colonies were increased by development in the mouse knockdown of Ppet 001, 003, 004 and 019 genes. Next, we performed knockdown of Ppet gene in We have previously reported that mouse early mouse embryos. The embryos that ES cells were divided into three subpopulations injected Ppet015, 019 and 021 siRNA exhibited according to the expression levels of platelet developmental retardation and degradation. In endothelial cell adhesion molecule 1 (PECAM-1) cases of Ppet 019 and 021 gene knockdown, and stage-specific embryonic antigen (SSEA)-1. total cell numbers of blastocysts were reduced Quantitative RT-PCR and chimera formation without morphological abnormality. Conversely, revealed that the expression level of PECAM-1 Ppet 010, 022 and 023 gene knockdown caused and SSEA-1 were positively correlated with developmental facilitation. In cases of Ppet 022

Fig. 3 Expression patterns of $(,.+" and $(,.+# RT-PCR was performed using the total RNAs from intact and regenerating $! (%+*)&)-'- at 3-96 hours after amputation (A). The number of PCR cycles used are indicated at the right of each panel. WISH analysis was performed using DIG-labeled antisense riboprobes against regenerating fragments at 24 (B-D) or 36 hours after amputation (E). Lateral views, the anterior is to the left. Arrowheads indicate the base of the blastema. e, esophagus; g, gut. Scale bar = 200 µminB.

&++0(* ')-,./ "!!% #$ Fig. 4 Undifferentiated state of the ES cell was influenced by the Ppet gene knockdown and 023 gene total cell numbers of blastocyst expected that the nuclear transferred PGCs are were incresed without morphological abnormality. produced and the manipulated PGCs migrate to This study showed that some Ppet genes the germinal ridges after being transferred to probably play an important role in the specific the bloodstream of recipient embryos and mechanism of differentiation / proliferation of successfully differentiate into germ cells in the ES cells and early embryo. gonads of the chimaeric chickens. By mating This work was supported by the Program these chimaeric chickens, somatic cell-derived for Promotion of Basic Research Activities for offspring are expected to be produced. In order Innovative Biosciences (PROBRAIN). to apply this strategy for nuclear transfer in chickens, the techniques for each step involved Fusion of primordial germ cell in the nuclear transfer in PGCs should be with embryonic blood cell in developed. The present study was conducted to chickens produce nuclear transferred PGCs, and here we Manipulation of primordial germ cells report the preliminary results of fusing PGC (PGCs) provides a useful method for nuclear with embryonic blood cell (EBC) using transfer in chickens. The system for producing inactivated Sendai virus (Hemagglutinating germline chimaeric chickens by the transfer of Virus of Japan; HVJ) or electrical stimulation. PGCs has already been established. If a nucleus Fused cells of PGC and EBC were produced of PGC can be replaced by a somatic cell using inactivated HVJ as shown in Fig. 5 (A-I). nucleus, the nuclear transferred PGC could give PGC-EBC fused cells were identified by their rise to viable offspring via germline chimaeric morphology and the difference in size of the chicken after being transferred to recipient two nuclei after staining with Hoechst 33342. embryo. Enucleation of PGCs could be done by PGC has a large nucleus while EBC has a UV irradiation or some other methods, and relatively small nucleus. The fused cell has thus somatic cell nuclear transfer could be achieved both large and small nuclei. The fused cells by fusing the enucleated PGC with a somatic were PGC-EBC, PGC-PGC, and EBC-EBC. More cell. Through these manipulations, it is than three cells were occasionally fused. The

#$ %**/') &(,+-. "!!$ Fig. 5 PGC and EBC fused cells PGCs and EBCs were were fused using inactivated HVJ (A-I) or electrical stimulation (J and K). Cells were stained with Hoechst 33342 to visualise their nuclei (B, D, F, I), and incubated for 2 hours (E, J, K) or 16 hours (A, C, H). Photographs E and F are merged and shown in photograph G. PGC and EBC fused cells were identified by their morphology and difference in size of the two nuclei. Scale bar represents 10 µm. mean fusion rate of PGC with EBC using and the complement system. It is considered inactivated HVJ was 0.96%. When cell fusion that natural antibodies against αGal epitope on was carried out using electrical stimulation, pig cells are the main cause of hyperacute pearl chains were formed within 20 seconds by rejection. Recently, the overseas companies exposing cells to the AC field. After applying succeeded to produce α1,3GT knocked out pigs DC pulses, adjacent cells in a pearl chain were by somatic cell cloning for elimination of αGal fused in some places. Fused cells of more than epitope. three cells were also occasionally observed. The The endo-β-galactosidase C (EndoGalC) fused cells of PGC and EBC were shown in Fig. secreted from !'*-.,&"&/( +#,$,&)%#)- cleaved 5 (J and K). The mean fusion rate of PGC with αGal epitope from pig cells under physiological EBC using electrical stimulation was 5.2%. The pH conditions. In addition, transfection with system for producing viable offspring derived EndoGalC gene effectively reduced αGal from nuclear transferred PGCs makes it expression in cultured porcine aortic possible to manipulate the germline of chickens endothelial cells. Thus, the cleavage of αGal through somatic cells. epitope by EndoGalC would be the alternative to knock out α1,3GT gene for elimination of Lack of αGal epitope in αGal epitope in whole pigs. transgenic cloned pigs by The EndoGalC expression vector was expression of EndoGalC transfected in Meishan fetal fibroblasts by electroporation. After selection under G418 for Because organs of pigs are biologically and 10-14 days, the cells lacking αGal epitope were anatomically similar to human organs, porcine separated and collected by FACS (fluorescent- organs are expected to use as replacement for activated cell sorting). The separated cells were human organs, which are chronically in short cultured and expanded sufficient for nuclear supply. The major problem with transfer to produce live piglets. 1144 of nuclear xenotransplantation of porcine organs into transferred embryos at 2-8 cell stage were humans is the hyperacute rejection caused by transferred to 7 synchronized recipient pigs. the reactions involving natural human antibodies Two recipients were pregnant but one

&++0(* ')-,./ "!!$ #% recipient aborted two fetuses on day 61 and the from follicle cells of the silkworm at late pupal other recipient delivered one live piglet (Fig. 6). state with high reactivity with JH was Genomic PCR of DNA extracted from the piglet phospholylated by incubation with methoprene and the aborted fetuses confirmed the presence for 5 min. (2) New prothoracicostatic factors, of EndoGalC gene. The FACS analysis showed !%$"(' FMRFamide (BRFa) were identified in that the levels of elimination of αGal epitope in !%$"(' $%&#. BRFa is predominantly expressed the fibroblasts collected from the piglet and the in neurosecretory cells of thoracic ganglia, and aborted two fetuses were 98.3%, 99.0% and the neurons in prothoracic ganglia innervate 99.2% respectively. The cloned piglet had no the prothoracic glands to supply the peptides to physical abnormalities and reached normal the gland surface (Fig. 7). These results suggest maturity. that BRFa is controlling the activity of These results clearly showed that the prothoracic gland by direct innervation. (3) We combination of FACS with nuclear transfer has analyzed transactivation abilities of three much superiority to produce transgenic pigs isoforms of a metamorphosis-specific like our previous success of cloned pig with transcriptional factor Broad-complex (BR-C) highly expression of human decay accelerating isolated from !%$"(' $%&# by luciferase gene factor (hDAF). Moreover, we are planning to assay using a !%$"(' cell line. BR-C Z2 isoform produce transgenic pigs with both hDAF and that processes zinc finger DNA binding EndoGalC expression by mating with each motives induced luciferase expression via transgenic cloned pig for xenotransplantation. either of three metamorphosis-specific gene promoters, but BR-C NZ1 and NZ4 isoforms without zinc finger motif did not, suggesting that BR-C isoforms with zinc finger motives play an important role for induction of multiple metamorphosis-specific genes. (4) We cultured pieces of larval integument in MGM-450 medium with 10 % fetal bovine serum (FBS). Reduced form glutathione was added in the medium to inhibit melanization of medium and integument. The pieces of integument were put on the supports of polyester membrane in order to expose air on surface of cuticle. At 48 hours after initiation of the culture we observed Fig. 6 malformed cells at surround of the wound, but Cloned transgenic pig expressing EndoGalC for elimination of αGal epitope recognized only a few melanizing or damaged

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) A protein of cultured cells derived from silkworm ovary, which was phospholylated by methoprene, a juvenile homone (JH) analog, was MAP kinase, Fig. 7 and it was also observed that the MAP kinase BRFa-immunoreactive axons on the surface of prothoracic glands

#% &++0(* ')-,./ "!!$ cells. The epidermal cells maintained apparently were detected only in the placenta by RT-PCR. cell growth activity on the culture condition for mRNA was primarily expressed in the cotyledon the immunofluorecent staining of incorporated and intercotyledonary tissues throughout bromodeoxyuridine (BrdU). These observations gestation. An !" #!$% hybridization analysis suggested that the integument culture was revealed the presence of bPRP-VIII and -IX available method for study of growth and mRNA in the trophoblastic binucleate and /or development on epidermal cells. trinucleate cells. bPRP-VIII mRNA was observed in the extra-embryonic membrane on Identification of new molecules in Day 27 of gestation, however, no bPRP-IX bovine placenta: Prolactin-related mRNA was observed in the extra-embryonic proteins and BCL2A1 membrane in the same stage of pregnancy by Prolactin-related proteins (PRPs) are quantitative real-time RT-PCR analysis. Both specific proteins of the growth hormone/ new bPRP genes were possible to translate a prolactin (GH/PRL) family in bovine placenta. A mature protein in a mammalian cell expression full-length cDNA for two new members of system with approximately 28 kDa in bPRP- bovine PRPs, bPRP-VIII and -IX were VIII and 38 kDa in bPRP-IX. identified, and their placental localization and Their different temporal and spatial quantitative expression were examined. expressions suggest a different role for these New bPRP-VIII and -IX were identified genes in bovine placenta during gestation. from bovine placentome. Localization and A full-length cDNA for the bovine BCL2 quantitative gene expression in the placenta antiapoptotic family member, BCL2-related were respectively investigated by !" #!$% protein A1 (BCL2A1) was identified. hybridization and real-time RT-PCR methods. Spaciotemporal expression profiles of BCL2A1 Recombinant proteins of these genes were suggested the implication to trophoblast cell produced by a mammalian HEK293 cell proliferation and differentiation during expression system. Full-length bPRP-VIII and - pregnancy. We cloned a full-length bovine BCL IX cDNA were respectively cloned with 909 2A1 cDNA with 725 nucleotides and an open- and 910 nucleotide open-reading-frames reading frame corresponding to a protein of 175 corresponding to proteins of 236 and 238 amino amino acids. The predicted amino acid acids. The predicted bPRP-VIII amino acid sequence shared 78% homology with human sequence shared about 40 to 70% homology BCL2A1. All BCL2 homology domains (BH1, BH with other bPRPs, and bPRP-IX had about 50 to 2, BH3, and BH4) in bovine BCL2A1 were 80% homology of others. The two new bPRPs conserved as in other mammalian BCL2A1. In

Fig. 8 !# $"%& hybridization for BCL2A1, BAX, CASP3 and PL in bovine placenta

&++0(* ')-,./ "!!$ #% the placentomes, !" #!$% hybridization revealed with progression of gestation and remained that the BCL2A1 expression was localized in elevated in postpartum. Caspase-3 protein binucleate cells expressing various pregnancy- (CASP3) and mRNA (CASP3) were detected specific molecules like placental lactogen. BCL2- from late gestation to postpartum in placenta as associated X protein (BAX) was also expressed well as in the results of TUNEL detection. It is in binucleate cells (Fig. 8). Quantitative real- likely that apoptosis of binucleate cells is time RT-PCR detection showed a high-level regulated by the balance of the BCL2A1 and expression of BCL2A1 in the conceptus at Day BAX. This molecule is a new candidate for anti- 21 of gestation, and it was expressed and apoptotic maintenance of the binucleate cells increased in the extra-embryonic membrane, that support placental functions throughout cotyledon, and intercotyledon from implantation gestation in bovine. to term. BAX expression intensity increased

M olecular Biology and Immunology Department

Production and characterization which represented one of the observed alleles of allospecific anti-bovine CD34 were produced in HeLa cells by DNA monoclonal antibody transfection, and the reactivity of mAb N21 to CD34 is a cell-surface glycoprotein that is these mutants was assessed; mAb N21 reacted specifically produced in hematopoietic and to W261 allele, but not to R261 allele. This nonhematopoietic stem/progenitor cells. In assignment of immunodominant amino-acid order to facilitate the study on bovine residue well explained the correlation between hematopoiesis, we produced a monoclonal genotype and reactivity to mAb N21 of the antibody (mAb) against bovine CD34 (boCD34) calves examined. Taken together, mAb N21 designated as N21 by using mouse cells was shown to recognize boCD34 in an producing recombinant boCD34 as antigens. allospecific manner, discriminating a single The mAb N21 stained relatively high amino-acid change. This mAb would not only percentages (23% in average) of bone marrow facilitate the identification and characterization mononuclear cells (BMMNCs) from 4 neonatal of bovine hematopoietic progenitor cells, but be Holstein calves, but did not stain BMMNCs used as an allelic cell-surface marker in from the other 6 calves. Cell sorting experiments allogeneic transplantation studies using the showed that hematopoietic progenitor cells cattle. such as colony-forming unit-granulocyte macrophage (GM-CFU) and burst-forming unit- erythroid (BFU-E) (Fig. 1) were enriched in N 21+ (thus boCD34+) cell fraction, suggesting that boCD34 was produced specifically in hematopoietic progenitor cells. To examine the cause of the difference in reactivity to mAb N21 among the calves, the sequence in the coding region for boCD34 of each calf was determined. As a result, 4 single-nucleotide polymorphisms within the coding region were found; 3 of them lead to amino-acid changes, S159F, W261R, and

A276V, respectively. The CD34 mutants each of Fig. 1 A bovine BFU-E (day 12) grown in methylcellulose Colony was unstained. (magnification 40 x)

#! %**/') &(,+-. "!!$ Animal and cell culture models transduction pathway in LPS-activated for the study of mammalian microglia. In addition, we have established immune system immortalized microglial cell lines from the Microglia have critical roles in transgenic mice overexpressing prion-protein, development, homeostasis and pathogenesis in to study the mechanisms of neuropathogenesis the brain. As immune surveillant cells, and possible involvement of microglial cells in microglia respond to various extracellular prion diseases. These cell lines are highly signals such as neuronal injury or infection, and susceptible to various strains of mouse-adapted secrete several proteases, neurotrophic factors scrapie and BSE prions. We demonstrated that and various cytokines. Wiskott-Aldrich syndrome both expression and activation of P2X7, one of protein (WASP), the gene product responsible the ionotropic receptor channels for ATP, were for the Wiskott-Aldrich immunodeficiency increased in microglial cells after persistent syndrome, acts as an important adaptor infection with scrapie ME-7 strain. Further molecule in T cell receptor signaling. However, studies will be needed to determine whether little attention was paid on the roles of WASP in the changes in P2X7 properties are observed in microglial activation. So, we have established a prion-infected animal brain. microglial cell line from WASP-dominant negative transgenic mice. These microglial cells Innate Immune System in Insects produced lower levels of inflammatory cytokines, A short antibacterial peptide was designed such as tumor necrosis factor alpha and and synthesized on the basis of the active sites interleukin 6 than the wild-type microglia, after of defensins. The 9-mer peptide, ALYLAIRRR- stimulation with bacterial lipopolysaccharide NH2, was tested to determine whether it can (LPS). Furthermore, LPS-stimulated WASP- suppress the proliferation of methicillin- microglia showed less neuronal killing in resistant "1#.(4*-%-%%20 #2/'20 (MRSA) ), 3)3- coculture system. Concomitantly, release of and ), 3)1/- evaluation of the peptide against nitric oxide was reduced in WASP-microglia MRSA was carried out histopathologically. Silk (Fig. 2). These results strongly suggest that sutures pretreated or non-treated with the WASP has important roles in the signal peptide were embedded in the back skin of mice for 24h. Sutures treated with the peptide showed strong inhibition of the proliferation of MRSA judging from examination by light microscopy of biopsy samples. On the contrary, non-treated sutures showed numerous Gram- positive loci. For ), 3)1/- experiments, silk fibroin films containing different concentration of the 9-mer peptides were prepared. MRSA seeded on the culture plates was covered with a transparent fibroin film and incubated at 37! for 24h. No MRSA colonies were detected under the films containing the 9-mer peptide, whereas many colonies appeared under the control film without the peptide (Fig. 3). These results suggest this synthetic antibacterial peptide is a useful lead peptide for development of novel therapeutic agents against infection with antibiotic-resistant bacterial pathogens. Antibacterial peptide defensin isoform A

Fig. 2 was previously isolated from the midgut Possible involvement of WASP in LPS-TLR4 mediated contents of !/,)1(-&-/-0 +-2$#1# blood-fed signal transduction pathway in microglia

&++0(* ')-,./ #!!% $" Fig. 3 Inhibition of MRSA proliferation by fibroin films containing the 9-mer peptide, ALYLAIRRR-NH2 MRSA seeded on the culture plates was covered with the fibroin films containing 0 (a) and 300 mg/cm2 (b)ofthe peptide. The plates were incubated at 37!for 24h. The transparent moist films were gradually dried in an a incubator and formed creases. Note that many MRSA colonies are detected under the film containing no peptides (a), whereas no MRSA colonies are seen under the films containing the peptides (b). females. However, not only defensin A, but also showed a broad antibacterial spectrum against three other defensin isoforms showed gene Gram-positive and negative bacteria. Sl moricin expression in the midgut, suggesting the gene was inducible by bacterial injection and possibility that these antibacterial peptides are expressed tissue specifically in the fat body and secreted into the midgut lumen. To further hemocytes. Furthermore, the solution structure understand tick immune mechanisms, the of Sl moricin was determined by two-dimensional involvement of antibacterial peptides in midgut (2D) 1H-nuclear magnetic resonance (NMR) defense was investigated. Three antibacterial spectroscopy and hybrid distance geometry- peptides with molecular masses near defensin simulated annealing calculation. The tertiary isoforms B, C and D were detected in the structure revealed a long a-helix containing midgut contents of blood-fed females. Enzyme- eight turns along nearly the full length of the linked immunosorbent assay analysis revealed peptide like that of moricin, confirming that Sl that the antibacterial peptides in the midgut moricin is a new moricin-like antibacterial contents cross-reacted with defensin A peptide. These results suggest that moricin is antibodies and increased as a response to blood present not only in "-+%54 +-/) but also in feeding. Simultaneously, the antibacterial other lepidopteran insects forming a gene activity of the midgut contents was enhanced family. by blood feeding. Secretion of antibacterial peptides into the midgut lumen and an increase Development of mammalian in the peptide concentration following blood selection markers feeding was also confirmed. These findings We have developed a series of new further support the hypothesis that mammalian cell surface marker fusion genes antibacterial peptides play an important role in using a 01/(.1$3)'), gene from #1/(.1-+5&(0 the midgut defense of ticks. $3)'),)) as an antigen. The fusion genes are An antibacterial peptide was isolated from intended to use as selection markers to a lepidopteran insect, #.-'-.1(/$ *)12/$! The separate transformed mammalian cells rapidly molecular mass of this peptide was determined without any toxic effect on cell growth. Two to be 4489.55 by matrix assisted laser different length of the 01/(.1$3)'), gene was desorption/ionization-time of flight mass used: a longer fragment contains the native (MALDI-TOF-MS) spectrometry. The peptide bacterial signal sequence which the shorter consists of 42 amino acids and the sequence has fragment lacks. To express the streptavidin 69-98% identity to those of moricin-related antigenonmammaliancellsurface,the peptides, antibacterial peptides from streptavidin gene was sandwiched by a lepidopteran insects. Thus, the peptide was mammalian signal sequence and a trans- designated #! *)12/$ (Sl) moricin. Sl moricin membrane sequence at N-terminus and C-

#" %**/') &(,+-. "!!$ terminus, respectively. A signal sequence and a against streptavidin, and the antibody bound trans-membrane sequence from either the cells were pulled out using a paramagnetic mouse &"!' gene or the mouse ',1 gene were beads coupled with the corresponding used. Some constructs contains the #%$( gene secondary antibody. Highly pure population of sequence next to the 01/+.1)2,*,- gene transformed cells was separated (Fig. 4). Then, sequence. Totally eight kinds of fusion genes effects of fusion proteins on cell growth were were constructed in this study. To examine the assayed. Cell proliferation rate of transformed expression of the fusion genes on mammalian HeLa cells were compared with that of the cell surface, a series of plasmids encoding the untransformed HeLa cells. No significant fusion protein were transfected into the HeLa difference of cell growth was observed in any of cells. Expression of the fusion protein on cell the fusion genes. The property of eight fusion surface was observed for any of the constructs. genes developed in this study was found to be Then, an antibody-mediated immunomagnetic similar. These results suggest that the fusion separation methodology was applied to genes and the immunomagnetic separation separate transformed cells. Transfected cells protocol are useful for various transformation were incubated with a polyclonal antibody applications.

Fig. 4 Expression of fusion protein on HeLa cells transfected with a plasmid encoding the FSAH2K-EGFP fusion protein (A) Localization of streptavidin antigen on the surface of a transformed cell. Serial images captured at 5-µm intervals on the z-axis. (B) Transfected HeLa cells. Red staining indicates transformed cells. (C-E) Immunomagnetically separated cells, same field. (C) Light-interference view. (D) Cells immunostained with anti-streptavidin antibody. (E) EGFP-positive cells. Scale bar: 10 mm (A) and 100 mm (B-E).

&++0(* ')-,./ "!!% $# P hysiology and Genetic Regulation Department

Odor coding by group of neurons depending on odor quality. A group of PNs may in insect brain encode information of specific odor by sending It has been thought that olfactory their synchronized spikes from AL to MB. The information processing in the insect brain is results support the view that odor representation mediated by dynamic modulation of groups of canbeaccomplishedbyensemblenetworks. neurons firing synchronously. We characterized correlations of firing among several single unit Induction of anhydrobiosis in activities by simultaneous recordings from the isolated fat body tissue from an antennal lobe (AL) and mushroom body (MB) to insect odor stimuli in the male cockroach (!%-&,("*%." Some organisms can stand almost complete ")%-&#"*"). We found odor-dependent dehydration. Such a biological state without synchronous activity correlations between the water is referred to as anhydrobiosis. Since AL and the MB. Cross-correlograms of anhydrobiotic organisms are in zero metabolism, activities of AL neuron pairs indicated that they can be in dormant eternally unless water they had common excitatory synaptic input, is given. The Sleeping Chironomid, !+(1,%$&(/) shared reciprocal synaptic input and 0"*$%-,("*'& (Diptera, Chironomidae) is only monosynaptic inhibitory input in the AL circuit. insect species enables to enter anhydrobiosis. A Cross-correlograms of activities of neuron pair question is whether or not the central nervous obtained between the AL and the MB revealed system is involved in the induction mechanism. direct monosynaptic connection from AL We excised tissues from the larvae followed by neuron (presumptive projection neuron: PN) to complete dehydration &* 0&.-+ in different MB neuron (presumptive intrinsic Kenyon cell: media to determine the ability for trehalose KC). Each PN formed several different neural synthesis and the viability of these tissues after circuits functionally connected to KCs. Cross- rehydration. Only fat-body tissues produced a correlograms of the KC pairs suggested that large amount of trehalose in certain medium the cells received common excitatory synaptic upon desiccation (Fig. 2). We also found that the input from the PNs. These results indicate that fat body tissues could be preserved in a dry some functional synaptic connections in a group state at room temperature for an extended of neighboring AL neurons are formed period of more than 18 months in viable form. Thus we have confirmed that the central nervous system is not involved in the induction of anhydrobiosis, even in this complex multicellular organism. This is an important novel finding because insect diapause is strictly under regulation of the central nervous system. This gives us a great encouragement for developing dry preservation technology in tissues and cells.

Novel flavonoids isolated from Fig. 1 the green cocoon shell of the The neural connection between the PNs in the AL and silkworm, !%$"(' $%&# the KCs in the MB Two flavonoids containing the L-proline Activities of KCs in MB are controlled by activities of a PN or combination of PNs and these cells form moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin functional connection.

## %**/') &(,+-. "!!$ Fig. 2 Trehalose content of body parts, mainly including fat body (A) and alimentary canals (B) desiccated over 2 days or 0.5 day (QD) with various kinds of media BmHem is the supernatant of heat-treated hemolymph of !%$"(' $%&# larvae.

(prolinalin A) and 6-C-[(2S,5R)-proline-5-yl] the flavoniods are metabolites of the insect. qurcetin (prolinalin B), were isolated from the This is the first time that flavonoids with an green cocoon shell of the silkworm, $31):9 amino acid moiety have been found as naturally 135.. Extract of the green cocoon shell (160 g) occurring compounds. was applied to a solid-phase extraction cartridge. The elute was applied to Toyoperl Morphology of foretarsal ventral HW-40F column chromatography and further surfaces of Japanese !"&#$#% purified by reversed-phase HPLC. Prolinarin A butterflies (3.2 mg) and prolinalin B (1.5 mg) were obtained. Comparison of ventral surface of foretarsa Their structural elucidation was achieved by among Japanese '(4.0.3 butterflies showed application of acid hydrosis and spectroscopic that the shapes of fifth foretarsi, numbers and methods (Fig. 3). These compounds were not localization of chemosensilla for contact found in the leaves of mulberry (&3586 (0)( L.), chemicals, and spines in these areas were the host plant of the silkworm, suggesting that closely related to both phylogeny and behavior of these species. The results basically supported the classification that Japanese '(4.0.3 species are divided into five subgenera -- '(4.0.3 ('" 1(*-(32), '5.2*,46 ('" 987-86 and '" +,130,86), #*-.00.+,6 ('" 1((*/.. and '" ).(235), &,2,0(.+,6 ('" -,0,286! '" 430:7,6! '" 4537,235 and '" 1(*.0,2786)and%0.(+,6 ('" 1,1232). Moreover, foretarsal morphology of female also corresponded to the physical features of their preferable host plant leaves. The specific character of female '" 1(*-(32! '" 1(*.0,2786 and '" 1((*/.. seems to relate to the area of Fig. 3 distribution the species and their hostplants. Structure of novel flavonoids isolated from the green cocoon shell of the silkworm 1: 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A), 2: 6-C-[(2S,5R)-proline -5-yl] qurcetin (prolinalin B).

&++0(* ')-,./ "!!% #$ Fig. 4 Fig. 5 The ventral surfaces of female 5th foretarsal segment Amale#" -" -00'*00%/% landing on an untreated black of $%1+-+0 31" 1: $" .%'*%0/! 2: $" 654*53! 3: $" lure (-) paired with a white lure (+) treated with ().0-)53! 4: $" .%%',++! 5: $" &+%/02,6:$" *)-)/53! 7: pheromone (10 mg of anthranilic acid) and set on a $" 10-74)3! 8: $" 1204)/02! 9: $" .%'+-)/453! 0: $" lawn with a stick and bar .)./0/".D:$" ().0(0'53! P: $" 1%2+3" The interval between lures is 5 cm.

Visual pinpoint location Relationship between imprinting associated with pheromonal cue disorder and overweight at birth in males of the black chafer in bovine somatic cell nuclear !(&(*)%#$%" &((#$(("'" &((#$(("'" transfer clones (Coleoptera: Scarabaeidae) Genomic imprinting, which is the parental- Females of the black chafer !(&(*)%#$%" origin-specific gene regulation mechanism in &((#$(("'" &((#$(("'" (Sawada) (Coleoptera: mammals, plays essential roles in development Scarabaeidae) release anthranilic acid, which and growth. The mechanisms discriminating functions as sex attractant pheromone for between paternal and maternal genes are not males and aggregation pheromone for females. clear yet but DNA methylation as epigenetic When a white lure treated with anthranilic acid marker is thought to be a major clue for their was placed next to an untreated black lure in recognition. DNA methylation for parental the field, males were observed to make pinpoint imprinted markers persists in somatic cells landings significantly more frequently on the after fertilization, and is erased and re- latter (Fig. 5). When the distance between the established in germ cells according to sex. two lures was increased from 0 cm to 20 cm, Recent remarkable technique of somatic cell frequency of pinpoint landing onto the nuclear transfer (SNT) cloning made us to untreated black lures significantly decreased reproduce animals without germ cell while that onto the treated white lures slightly transmission and fertilization. But survival rate increased. When the lures were further of SNT clone at birth is still miserable. One separated to 2-m intervals, males approached possibility of this inefficiency has been thought only to the treated lures regardless of the color to be epigenetic disorder. Weight of bovine but significantly more frequently landed on the SNT clones at birth are usually heavier than black ones than on the white. These average and the neonatal survival rate is low. observations demonstrated that males locate We had determined bovine imprinting and land on a female by visual cues after genes and identified several genes expressed reaching the vicinity by olfaction. paternally monoallelic in Holstein Japanese black families. Based on the information, we successively investigated allelic expressions for polymorphic imprinting genes in adult

#$ %**/') &(,+-. "!!$ Fig. 6 Birth weight of 11 adult SNT clones Red characters indicate individuals with bi-allelic expression of imprinting gene(s). Blue characters indicate those with mono-allelic ones. Green circle and yellow square indicate average birth weight of female and male artificially inseminated(AI).

Japanese black SNT clones. Surprisingly, andturbiddiskofcollagengelintoastrongand almost half of healthy and well-grown adult transparent gel-membrane by utilizing a concept bovine SNT clones had imprinting disorder, i.e. for the vitrification of heat-denatured proteins bi-allelic expressions. Some of them were also and named the novel gel in a stable condition confirmed fertile. These results were clearly for vitrigel. The collagen vitrigel membrane indicated that imprinting disorder was common involving a nylon frame can be easily handled for bovine SNT clones with no apparent with tweezers, consequently it functions as a abnormality in adulthood. But our further scaffold excellent for three-dimensionally analysis revealed the relationship between culturing cells on double surfaces of it. Here, we overweight at birth and bi-allelic disorder (Fig. investigated the molecular permeability of the 6). Our results indicate that the management of collagen vitrigel membrane in time-course imprinting will crucial for effective SNT clone using glucose and serum proteins. The glucose reproduction. added to one compartment penetrated into another compartment via the collagen vitrigel Protein-permeable scaffold of a membrane and the glucose concentration of collagen vitrigel membrane each compartment came nearly up to a plateau useful for reconstructing level within 24 hours. Serum proteins with not crosstalk models between two only low molecular weight but also high one different cells more than 100 kDa passed gradually through We recently succeeded in converting a soft the collagen vitrigel membrane (Fig. 7). These

Fig. 7 Permeability tests in time-course for the collagen vitrigel membrane using glucose and serum proteins Change of glucose concentration (A), quantitative clearance of FBS (B), and qualitative clearance of FBS (C) were estimated using the samples at the indicated times.

&++0(* ')-,./ "!!$ #% Fig. 8 EEG recording in the absence of eyelid movement obtained from a piglet while lying at rest Relative power of each activity (delta, theta, alpha and beta) is shown at the bottom.

Fig. 9 EEG recording in the presence of eyelid movement obtained from a piglet while lying at rest Other explanations are as described in Fig. 8. (Saito et al. , 2005)

data suggest the protein-permeable scaffold of absence of eyelid movement, slow waves with collagen vitrigel membrane is useful for the large amplitude appeared in the EEG. The reconstruction of crosstalk models between two power of the delta (1-3.9 Hz) and theta (4-7.9 Hz) different cells. activities were larger than that of the alpha (8- 12.9 Hz) and beta (14.1-25 Hz) (Fig. 8). While Electroencephalogram (EEG) eyelid movement was present, faster waves changes with eyelid movements with small amplitude were recorded in the EEG in piglets trace. With the eyelid movement, the power of It is known that the amplitude and rhythm the alpha and beta activities was stronger than of the EEG is largely altered in response to that of the delta and theta (Fig. 9). According to opening and closing eyes. In this study, a the power spectral analysis of the EEG, the wireless recording system was applied to delta and theta activities, which appeared in the examine EEG activity with or without opening absence of eyelid movement, were replaced eyes in unrestrained, male Landrace piglets. with faster alpha and beta activity once eyelid Electrodes and telemetry devices were implanted movement appeared. These findings strongly under halothane anesthesia. Recordings were suggest arousal in the piglets while lying at performed while lying at rest, following rest. recovery from the surgical operation. In the

#% &++0(* ')-,./ "!!$ The role of glucose as a metabolic (MUA volleys) were examined. Infusion of the regulator of hypothalamic highest dose of 2DG increased the mean gonadotropin-releasing hormone interval between MUA volleys, whereas the pulse generator activity in goats lower doses of 2DG had no effect on volley We examined the relative importance of interval (Fig. 10). The MUA volley intervals blood glucose &%! free fatty acids as a metabolic lengthened as insulin-induced hypoglycemia signal regulating gonadotropin-releasing became profound. There was a negative hormone ( GnRH ) release as measured correlation between MUA volley intervals and electrophysiologically by multiple-unit activity blood glucose concentrations during insulin (MUA) in the arcuate nucleus/ median infusion, and coinfusion of glucose with insulin eminence region in ovariectomized, estradiol- returned the MUA volley interval to a normal treated goats. MUA was recorded before, frequency. Infusion of MA alone or MA with 2 during, and after: 1) cellular glucoprivation by DG did not increase MUA volley intervals. intravenous infusion of 2-deoxy-D-glucose (2 These findings demonstrate that glucose DG); 2) peripheral hypoglycemia in response to availability, but not fatty acids, regulates the intravenous infusion of insulin; and 3) cellular GnRH pulse generator activity in the ruminant. lipoprivation induced by intravenous infusion of Glucose is considered a key metabolic regulator sodium mercaptoacetate (MA), and effects on that fine-tunes pulsatile GnRH release. the interval of characteristic increases in MUA

Fig."! Intravenous infusion of 2DG increased MUA volley intervals in a dose-dependent manner in ovariectomized, estradiol-treated (OVX+E2) goats A, A representative rofile of MUA and volley intervals (closed circle) in an OVX+E2 goat during 2DG infusion (0- 2.5 h; shaded periods). B, Mean ( SEM) volley intervals (percent of OVX+E2 value) during xylose or 2DG infusion (n = 5 goats). *, "<0.05 vs. xylose infusion. (Adapted from Ohkura $& #%! (2004), Endocrinology 145(7): 3239-3246)

I nsect Genetics and Evolution Department

Molecular and evolutional skin RNAs identified some genes whose analyses of insects expression is repressed in a translucent skin Oligo array analyses of silkworm larval mutant, $" or $#! We estimated a magnitude of

&++0(* ')-,./ "!!$ #% nucleotide diversity of molybdenum cofactor laccases have been identified in cuticle, we sulfrase (;5) gene using 15 &;91CB 9;=7 know only an example of laccase of salivary regional races. We also estimated that of gland origin. To characterize the enzymological mitochondrial 2;B 1 gene and comparatively properties, we expressed a salivary laccase as a analyzed these results. To detect transposition recombinant protein. ("2;87 cells harboring the event of &;91CB MITE-like transposon, expression plasmid produced laccase as a -=50:3C! we carry out excision assay on soluble protein in the cytoplasms. Laccase specific insertion sites. acitvity was detected when copper chloride was added to (" 2;87 culture medium, suggesting Genetics and evaluation of the presence of a cupper center essential for silkworm stocks the oxidase activity. Bisexually reproductive tetraploid strains We have started to isolate brown were established with high fertility in the planthopper resistant genes from rice using &;91CB 9;=7. It was reconfirmed that the map-based cloning method. Norin PL10 has a female sex was determined by the presence of resistance gene (&<6 %) which is originated W chromosome in the tetraploid and triploid from a Sri Lanka variety Rathu Heenati. The offspring of the strains. Our database search of chromosome segment of Rathu Heenati was the deleted sequence found in one of the found in the chromosome 4 of Norin PL10, intersex strain, *>B#$! showed no significant between the two SSR (Simple Sequence Repeat) homology to the previously registered genes. In makers, RM6487 and RM3735. normal cells we detected fragments of mRNA We found that the latex of a wild fig transcribed from the deleted region, but it was species, )72@> >4B gene was silkworms were fed the artificial diet containing increased in the cultured male cells transfected 2% of mulberry latex, growth inhibition was with one of these BAC clones. Mapping of observed, and when the diet containing more homeotic mutant genes was examined by a than 10% of mulberry latex was fed, high three-point test. Three homeotic mutant genes mortalities were observed. We also discovered belonging to (-pseudoalleles and ,2! (+@!(,2! that the mulberry leaves are toxic to insect ('A and ,2 were arranged in order from the except the silkworm, &;91CB 9;=7,andthat proximal side of the sixth linkage chromosome. the latex ingredients, three sugar mimic The new mutant gene, maternal brown egg of alkaloids, which are well known as glycosidase Shimizu (1#$ >) was confirmed to be a novel inhibitors, and unknown high-molecular-weigh mutant as a member of recessive pseudoalleles factor(s) are responsible for the defense of in the &;91CB 9;=7. mulberry leaves against herbivorous insects. As a trial in order to develop longterm preservation of silkworm eggs, ovary Natural enemies transplantation using fourth instar larva of ,4;>47@8@> A;94=>84C7 is one of the most silkworm was technically too difficult to keep important predators of spider mites of the alive. A correlation between total amount of genus /4?=0:C26@> in Japan. We found that ," sugar alcohols contained in eggs and viability in A;94=>84C7 learns a specific blend of several two-years preservation of silkworm eggs was volatiles emitted from prey-infested plants and low score. is attracted to the volatile complex. The predatory mite has intra-specific variation in its Insect-plant interactions olfactory response. The predator showing We have cloned a cDNA for laccase from strong olfactory response could reach prey- the salivary glands of the green rice leafhopper, infested plants more efficiently than the mites ,4<6;?4??7B 27:2?724<>" While many insect with weak olfactory responses did. Genetic

#! %**/') &(,+-. "!!$ analysis using microsatellite markers showed virus binds with 80S ribosomes in the absence that a population of 40-60 adult females per of eukaryotic initiation factors. This indicates generation would be sufficient to conserve the that the IRES-mediated protein synthesis genetic diversity of the wild populations. would be possible in a complete reconstituted Minute pirate bug, '5/86 675/-/+300/6! is one of system, containing 80S ribosome, mRNA, the most effective predators of small insect tRNAs, aminoacyl tRNA synthetases (AARSs), pests, such as thrips. We evaluated genetic elongation factors, ATP, and GTP. We prepared differentiations among wild populations of this translation apparatus from eggs of brine species using microsatellite DNA markers and shrimp and reconstituted translation was found that no difference among populations 100 examined. Our AARSs were inactivated during km apart, suggesting that this species has large the purification procedure. If stable AARSs are gene pools in the field. We also analyzed purified, eukaryotic reconstituted translation polymorphic DNA regions of mitochondrial and system would be available with the IRES. nuclear DNAs amplified from Japanese '5/86 We found the infectivity against rice species. Phylogenetic analyses based on yellow drawf-phytoplasma was different between nucleotide sequences showed that these DNA regional strains of green rice leafhopper, regions can be used to examine genetic &,4.37,77/: +/2+7/+,46! and thereby were able to interrelationships among species and strains. build the experiment system of virulence Based on the results, we developed PCR mechanism of phytoplasma insect pathogens. primers to amplify polymorphic DNAs from It was examined by measuring the initial many other insects. quantity of #231)0) +845,) entomopoxvirus (AcEPV) fusolin gene in the ectoperitrophic Symbiotes area of #" +845,) larvae with a method of real- About 70,000 expressed sequence tags time quantitative PCR of the gene whether or were generated from the brown planthopper, not a greater number of the AcEPV virions had &/0)4)59)7) 08-,26! and new oligo-microarray passed the peritrophic membrane (PM) after was designed based on a sequence clustering the feeding of spindles mixed with spheroids analysis of the planthopper ESTs. A microarray compared with that after the feeding of -analysis-room was newly set up for silkworm- spheroids only. The experimental results and planthopper-arrays. The silkworm-array is indicated that a greater number of AcEPV used by more than 12 domestic research virions had passed through the disintegrated groups and planthopper-array by three groups. sites generated in the PM by the spindles, and RNAi protocol was successfully applied to the that they had reached the ectoperitrophic area planthoppers and is useful for the functional and then had entered the midgut epithelium analyses of the planthopper genes. Nucleotide within 6 hr after the administration of the sequence analyses of ribosomal RNA and spindles. mitochondria were performed in culicoides Resistance of the silkworm to Cry1Ac species in Japan, and the species were identified toxin of $)+/0086 7.85/2-/,26/6 was related to based on the nucleotide sequences. recessive major gene. We selected two We found two key amino-acid positions silkworm lines on the susceptibility against affecting adaptability for heterologous over- entomogenous fungus, $,)89,5/) *532-2/)57//! expression of termite cellulases (432 amino- infection. One line was resistant and the other acids, a member of the glycoside-hydrolase was susceptible. First filial generation (F1) of family 9) which generally resist against two lines showed resistance and half of recombinant production. By introducing amino- backcrosses between F1 and susceptible line acid mutations at the two positions, the termite showed resistance. These results suggested cellulases were over-expressed in %" +30/". that the resistance of the silkworm against $" The internal ribosome entry site (IRES) in *532-2/)57// infection was related to dominant the intergenic region of (0)87/) 67)0/ intestine major gene.

&++0(* ')-,./ #!!% $" I nsect Biomaterial and Technology Department

Development of biosensors and transfect plasmid DNA transiently into related materials focusing on the silkgland cells of silkworm larva, and we immolization of chemical optimized the experimental conditions. recognition molecules We have been reported that liposome Development of measurement containing insect sensory organ extracts and and recording methods for the membrane potential sensitive dye is a new obtaining bio-physical information type biosensor. We immobilized the liposome on on insects a photodiode and measured membrane Multichannel microelectrodes are being potential changes triggered by salt stimulation developed in order to record action biopotentials (Fig. 1 Left). The photodiode output was in of insects. Silicon was conventionally used as proportional to the concentration of salt the electrode material and microelectrodes solution. We also constructed the other type of were fabricated by anisotropic etching and biosensor that uses a planar lipid bilayer reactive ion etching. Because electrode membrane which is used for immobilization of microprobe shape and size were very difficult receptor proteins from insect sensory organs. to control, we proposed a novel pin-shaped This biosensor is fabricated by combining multichannel microelectrode that used epoxy- receptors with a field effect transistor (FET). based UV sensitive photoresist as the electrode Our FET device could detect the membrane material. Analysis of the electrical properties of potential changes stimulated by salt solution this type electrode showed that it has (Fig. 1 Under). Lactose immobilized silk fibroin properties excellent enough to record insect was indicated a better substrate for fibroblast biopotentials. We could record muscle action culture than no-treated silk fibroin by the potentials from a flapping silk moth using our results of mice fibroblast cultivation study. We epoxy-based multichannel microelectrodes (Fig. established the lipofection method which 2).

Fig. 1 Recorded membrane potential changes (Left: Photodiode system; Under: FET system)

#" %**/') &(,+-. "!!$ Fig. 2 Recorded Electromyograms (left: at rest; right: flapping)

Development of functional !&*$#)"#" (#)&,% silk fibroin films were materials such as fine chemical incubated with Protease Type XXI at 37!,to using chitin and fibroin etc. investigate the degradation behaviour in an %& The porous microsphere was prepared by +%*)' model system. The enzyme-resistant the addition of the lithium bromide. The fractions of films were collected after dissolution rate from the microsphere of the incubation time of 17 days, and analyzed by FT- theophylline was quickly finished further than Raman spectroscopy. The intensity ratio (I850/ 1 non-pore microsphere. The dissolution rate was I830) between the bands at 850 and 830 cm is rapider as particle size was smaller. Oleic acid, sensitive to the hydrogen-bonding state of the palmitic acid and ricinoleic acid were extracted Y phenoxy group. The I850/I830 intensity ratio from the beetle larva cuticula by the decreased from 1.55 to 1.30 upon supercritical carbon dioxide (Fig. 3). biodegradation of silk films, suggesting a more buried state of Tyrosine (Tyr) residues in the biodegraded film. This feature can be explained assuming that a certain amount of exposed Tyr residues, initially present in the amorphous film domains more accessible to protease, were lost with the peptide fragments removed by proteolytic cleavage.

Fig. 3 Chitin microsphere containing theophylline

&++0(* ')-,./ "!!% $# Characterization silk fibiroin and fibroin after autoclaving penetrates phospholipids other biopolymers from chemical membrane as well. and physical respect and New process for forming the film of hornet development of their applications silk using the harmless solvent system has been to wound covering materials and developed. This hornet silk film was found to others exhibit the quite low cell adhesion activity The primary structure of sericin A was when the film coexists with serum. This finding determined by RT-PCR; it consists of 1,271 indicates that the hornet silk film is useful for amino acid residues with a high serine content the biomaterials. The sequence of a silk protein up to 44% in molar ratio, and additionally, the from Japanese spider #&,'()$ %)$/$.$ was presence of repetitive sequences with 86 and 8 determined from a partial cDNA clone. The amino acids per repeat was revealed. The repeating unit was composed of several sequences were predicted less preferable to segments such as a polyalanine, glycine-glicine- form β-sheet structure compared to the 38- Xaa with Xaa being alanine, tyrosine, glutamine amino acid-repeats that constitute sericin M. or leucine. Circular dichiroism (CD) studies This is consistent with the experimental result showed the stable secondary structures of using cast films; sericin M tends to favorably spider silk in silk gland. form β-sheet structure through humidity The compressive modulus of silk fibroin compared to sericin A. Electron microscopic sponge influenced growth rate of fibroblasts observations of liposomes showed that newly cultured in the sponge, and the higher cell constructed silk fibroin recovered from "! *+-( growth rate was observed in the softer silk posterior silk gland quickly penetrates sponge. The secondary structure changes of phospholipids membranes without bursting silk fibroin in the sponge construction during them. Observations of the planar lipid bilayer storage were observed by 13C solid-state NMR membrane showed that the regenerated silk measurement.

I nsect Biotechnology and Sericology Department

Building of the data retrieval softwares for providing security of the private system for the genetic resources server. The program function was added on of silkworm the retrieval system configured with HTML by The database on the preserved silkworm using JavaScript. Still more, the retrieval races that have been maintained as the genetic system was composed of the preserved resources by National Institute of silkworm races and the commercial races. The Agrobiological Sciences is presently disclosed system made it possible to gain access to the on the following ; http://ss.nises.affrc.go.jp/ preserved silkworm races and to the nisesDB/bombygen/tablemaster-eng.html in commercial silkworm races either in Japanese English (Table 1 and Fig. 1). This database is or English for many users. configured with a hierarchical tree structure. It is troublesome to find the target characteristic Improvement and application of races. So, the retrieval function was added to mapping systems in the silkworm, supplement the faults and to correspond !%$"(' $%&# quickly. Some database softwares which have Molecular linkage map has been improved retrieval function on the Web are commercially and finally 330 EST-cDNA clones have been available. But we did not use these database mapped on 28 linkage groups. The methods for

$# &++0(* ')-,./ "!!% Table 1 Menu of Commercial silkworm races

Fig. 1 Selection of quantitative characteristics (Commercial)

%**/') &(,+-. "!!$ ## Fig. 2 p/+ and +/+ tagged by RFLP linkage analysis and mapping that were very IBMX. Particularly, Dexamethason promoted efficient for the organisms without crossing to accumulate the fat in the cells. We analyzed over in one sex were developed. Scanning the gene related to form the fat. The gene was linkage analysis (SLA) is the method for linkage designated to the BmFABP1. This genes analysis using the same backcross segregants promoter region was analyzed in detail. We to know where genes are linked. BCMAP is the discovered the suppression-region of mapping method for the same individuals of the expression of the gene. By a method of gel sift backcross segregants. These methods were assay, we clarified the existence of protein that introduced for making the molecular genetic combined to origo DNA that contained the map of EST-cDNA clones in the silkworm, partial sequences of the BmFABP1 gene in the !%$"(' $%&#. The newly developed EST- extracts of BmN4 cells. We established the cDNA clones were examined by Southern blot insect cell culture system of Baculovirus gene hybridization to clarify whether those showed expression by using !%$"(' $%&# serum-free effective RFLP or not. The clones showing cultured cell line, NIAS-Bm-Ke1. To produce a effective RFLP were used for SLA and high amount of the gene product of luciferase, BCMAP. Finally new 70 clones were added to !%$"(' $%&# heat-treated serum has to be the map this year. contained in culture medium (see p12, Figs. 1 The markers on the map and the methods and 2; p13, Fig. 3). As an experimental system, were introduced to analyze many kinds of clone cell lines from !%$"(' $%&# cell line characters like resistant genes against BT- (NISES-BoMo-Cam1) was selected. toxin, polyphagous gene and etc. The molecular markers on the map was also used to make Utilization of transposon for the homozygote of p and +p genes on the second construction of transgenic insects chromosome. As shown on Fig. 1, p/+ and +/+ and application for the analysis were identified by using very closely linked of insect genes RFLP marker. The technologies that developed a transgenic insect for these five years has been Induction of cell-differentiation joined and the standard methods for making of insect cultured cell by drags, the transgenic silkworm, including the injection and its application method for the preblastodermal embryos, Characterization of new insect cultured marker genes for the screening of the transgenic cell lines was analyzed. BmN4 cells that was silkworm and non-diapausing strains for the derived from ovary tissues was induced injection, have been authorized. The method strongly to fat cells by treatment of the three development has been shown to be very useful complex drags, Insulin, Dexamethason and and gave very high efficiency of transformation

#$ %**/') &(,+-. "!!$ Fig. 3 Expression of GFP gene in the various organs of the transformed silkworm rate compared to the previous methods, and Newly developed mulberry many different transgenic silkworms that cultivar Ayanobori with high introduced many different foreign genes were leaf quality and productivity produced. Especially, the $#%" ! '#& strains Recently, the sericulture is on the decline using yeast to control the gene expression of in Japan. However, the improvement of the the introduced foreign gene was very effective productivity in mulberry field is still of for the study of gene function and for the importance for the development of sericulture. production of useful materials. The efficient We have developed a new mulberry cultivar production of recombinant proteins in the Ayanobori (Fig. 4), which was selected out of transgenic silkworm has been shown to be seedlings obtained by crossing the mother possible and to be useful as the new developed Wasemidori and the father Hayatesakari system for it. In addition, jump starter strains Tetraploid. Its resistance to dwarf disease has have been constructed for the enhancer trap in the silkworm. The strains have been shown to be used to transpose the mutator gene in the silkworm and can be used for the production of useful proteins. Furthermore, the method for the analysis of brain and nervous system using GAL4-RNAi has been confirmed and the transgenic insects that introduced human FMR 1 gene was constructed.

Fig. 4 The shoot (left) and leaf (right) of a new mulberry cultivar Ayanobori

&++0(* ')-,./ "!!$ #% been studied at two experiment stations, in improve the artificial diet suitable for Tokushima and Kagoshima prefectures, for five production of useful substances using years since 1996. Local adaptability has been transgenic silkworm . tested at two experiment stations, in Gunma and Miyazaki prefectures, for four years since Breeding and utilization of a new 1998. Ayanobori was found to be superior in silkworm race having special leaf yield and lodging resistance to the control features cultivar Shinichinose. In order to officially The sericin produced by the sericin cocoon register Ayanobori as a new variety, an strain SERICIN HOPE was experimentally application has been made to the Ministry of used to food and cosmetic items. Furthermore, Agriculture, Forestry and Fisheries of Japan, we are developing another two sericin cocoon and the application has been announced strains, which simultaneously secrete functional officially since June, 2005. Ayanobori has the colored ingredients with sericin (Fig. 5). following characteristics: SERICIN FLAVO, whose flavonol ingredients Ayanobori is a triploidy cultivar in the green cocoon strongly inhibit oxidation, belonging to !%&(' "$#" L. Its form is was released as a commercial race. A yellow characterized by moderate expansion resulting sericin cocoon strain (provisional name: in tolerance to lodging. The shoots are longer in SERICIN CAROTENE) produced 50mg of length and nearly the same in the number of sericin cocoon containing 2.5mg of carotenoids shoots, respectively, as compared to per larva. The yellow cocoon shows the Shinichinose. The sprouting time in spring is a stronger inhibition of tyrosinase activity and few days earlier than in the case of less anti-oxidation than the SERICIN FLAVO Shinichinose. The young shoots grow cocoon. These ingredients also appeared to vigorously and uniformly after sprouting. suppress some bacterial growth. These Resprouting after intermediate pruning in additional activities of the SERICIN HOPE summer and autumn is also vigorous. The yield cocoon were effective even in gel and emulsion is high in both normal planting and dense states for hours, because the ingredients were planting. In the silkworm rearing both with slowly released into the water solution. Based mulberry shoots and by the artificial diet added on these findings, the colored sericin produced with the Ayanobori leaf powder, the leaf by SERICIN FLAVO and SERICIN quality is almost the same in Ayamobori as in CAROTENE is expected to be widely used as a Shinichinose whose leaf quality is said to be new functional cosmetic material. pretty high. A strain having twice as much sericin Ayanobori is resistant to dwarf disease, solubility in 60!water as ordinary races was but rather sensitive to bacterial blight and found in the silkworm genetic resources. powdery mildew as compared to Shinichinose. Differences in cocoon filament intensity were The injury of shoot tips to cold is less pronounced than in Shinichinose. Ayanobori has a good rooting ability; production of sapling by hard-wood cutting is easy. Taken together, Ayanobori can be easily cultivated in wide areas from Kyushu district to Tohoku district of Japan and is adaptable to both normal and densely planted cultivations in summer and spring pruning. Introduction of Fig. 5 Ayanobori would lead to improve the Three kinds of cocoons produced by sericin cocoon productivity in mulberry field. Since race series Ayanobori has a high quality of leaves, left, SERICIN HOPE; center, SERICIN CAROTENE; Ayanobori might be able to contribute to right, SERICIN FLAVO.

#% &++0(* ')-,./ "!!$ also surveyed in the genetic stocks. The cotton and wool were treated with high strongest cocoon filament showed an intensity temperature. The activated value of the of over 4.5 g/d, or 1 g/d higher than ordinary antibacterial activity was investigated. As a races. These strains might be useful resources result, the value of silk was the biggest among for breeding new silkworm races. them. The silk thread characteristics of the Chemical and physical modifications silkworm races, Koishimaru, Ohkusa and of silk and the development of Shohkoh were investigated. As a result, it was characteristic silk products recognized that they were very similar to the 13C solution and solid-state NMR analyses silk characteristics of Edo period (1603-1867). were employed to investigate native structure The flexible type silk reeling machine was and degradation pattern of silk sericin. Sericin developed to produce five kinds of thick silk samples were prepared from Sericin Hope fibers, that is, the high bulk silk, the special flat silkworm, which secrets almost exclusively silk, the combination silk yarn of high bulk silk sericin. The 13C solution NMR spectra of native and special flat silk, the super thick silk and the sericin solution obtained from Sericin Hope covered silk yarn. Those thick silk fibers are larvae exhibited that sericin was largely produced by reeling from large number of random coil structure before spinning. The cocoons, for example, from around 200 cocoons structural changes by thermal degradation to 300 hundred cocoons per one thread. Using were followed by 13C solution NMR measurements. these fibers, jackets, sweaters and shawls were The 13C NMR spectrum after thermal produced; those products show each specific degradation exhibited obvious peak splitting at characteristic on the aspects of bulkiness, luster Asp Cα,Cβ and Cγ peaks. This observation and texture. indicated that Asp residues might be a weak point toward hydrolysis. It was examined to form the artificial skein and blood tube using cocoon filaments. The basement of artificial skein was weaved by using single cocoon filament which reeled up from a cocoon (Fig. 6), the artificial blood tube basement was made by the formation of knitting and winding with cocoon filaments. In order to produce the silk tube except sericin, it was degummed (dissolving sericin), the fibroin solution was put on the tube, and the fibroin was fixed with alcohol treatment. As the changing treatment of the fibers Fig. 6 Artificial skein basement weaved by using single characteristics, the knit materials made of silk, cocoon filament for the warp and weft filaments

&++0(* ')-,./ "!!$ #% Plant Science Division

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

M olecular Genetics Department

The research activities of this department scanning (RLGS) is a two-dimensional are mainly focused on the analysis of the electrophoresis of genomic DNA, which structure and function of rice genome, genes visualizes thousands of loci. We improved RLGS and their products, the development of tools method to detect methylated sites directly. The and resources for functional analysis of rice isoschizomers, #.,Iand",$II that recognize genes, and the mechanisms regulating gene the same sequence (CCGG) but have different expression. Major topics in the fiscal year 2005 methylation sensitivity were employed. We are described as follows. detected 22 spots on both RLGS patterns (#.,I and ",$II) in !-$%(&+,.(. /'$)($*$ ecotype DNA methylation analysis Columbia. In comparison of them, 18% of the DNA methylation is an epigenetic spots were polymorphic, which indicated the modification that a methyl group is added to methylation of C5mCGG sites. And, 52 and 54 the 5th carbon of pyrimidine ring of cytosine. It restriction enzyme sites were also analyzed in is informative to systematically scan genome- two other ecotypes, Wassilewskija and wide changes of methylation through each Landsberg erecta, respectively. Consequently, developmental stage, and to precisely analyze 15% of the 52 common sites showed methylation interactions between methylation status and polymorphism among the three ecotypes. gene expressions. Restriction landmark genome Almost all the restriction sites analyzed in this

#! $)).&( %'+*,- "!!# research were located in or near genes. This prokaryotic and eukaryotic cells. On the other improved RLGS method is readily applicable to hand, environmental situations within cells practical analyses of methylation dynamics in differ among organisms: unicellular organisms an un-sequenced species and even in a cloned cannot control the ion concentrations outside animal/plant. the cell, but multicellular eukaryotes (especially animals) can precisely regulate the ion Gene family-oriented rice gene concentrations of their environments within annotation micro molar ranges. Therefore, we can expect After the completion of rice genome organisms to differ the gene numbers, sequence by the International Rice Genome structure, and functions according to their Sequence Project (IRGSP) and the collection of biological abilities and/ or environmental over 32,000 rice full-length cDNA clones and situations. Because transport activities are their complete sequence analysis by the Rice necessary in most tissues at distinct levels, we full-length cDNA Consortium, next challenging expected that the transcripts of most target is the comprehensive annotation of rice transmembrane transporters would be genes and their functional analyses. For the contained within the standard materials establishment of bioinformatics platform for (including various developmental stages, rice gene annotation, Rice genome annotation tissues, and plants stimulated with various program (RAP) has been initiated in December treatments) held in full-length cDNA libraries. 2004. RAP activity is the human curated We searched for ortholog with known annotation of the gene structure generated by membrane transport genes by using the 32,127 the mapping and alignment of full-length cDNA full-length cDNA data for rice and also clones, individual EST clones and combiner ",#$(%*+-(- and rice genomic sequence data. EST sequences to the rice genome sequence. We used the BLASTX program to search for Currently about 25K locus have been assigned sequence homologies at the amino-acid level. on the IRGSP built 3 Pseudomolecules (See Because membrane transport proteins have RAP-DB: http://rapdb.lab.nig.ac.jp/ ). In RAP-2 specific structural features, the identification of annotation meeting held in February 2006 in orthologs is clear from computer calculations. Tsukuba, 580K single pass sequences from 380 There have been many precise reports of K full-length cDNA clones (FL-ESTs) were individual transporter protein families (e.g. Pao incorporated for the mapping and alignment. et al. 1998; Sanchez-Fernandez et al. 2001; Eng As pointed out in RAP-2 discussion meeting et al. 1998; Mäser &. #)! 2001; Wipf &. #)! 2002), held in November 2005 in Manila, next direction but we have little overall information about of rice gene annotation is toward the gene whole transport systems. We tried to examine family specific- and much deeper gene function- the topics and selection of total membrane related annotation. transport systems, as indicated by the overall Along with the future direction of rice outline of gene diversity in organisms. gene annotation, our research team has also Comparison of membrane transport genes focused some gene families, such as calcium or indicated that these genes are examples of the Ca2+ related proteins in signal transduction evolutionary diversity of homeostasis systems pathways (Nagata &. #)! 2004, 2005), and the in organisms. The increase in the ratio of membrane protein. In this year we have mainly membrane transport genes was smaller than in focused to the membrane transport protein other gene categories (&!'! transcription factors, families. metabolism) in higher eukaryotes (350-850). Cells maintain their biological activities by Usually, according to the complexity of the importing and exporting various materials. organisms, the gene numbers increases drastically Supplementation with energy, materials, and by divergence, evolution and duplication of the substrates and efflux of salts, drugs, and ions genes. Therefore, the indispensable number to are necessary to maintain biological activity in retain the cell membrane transport homeostasis

%**/') &(,+-. #!!$ $" seems 300-350 and the increases of the genes sterility, and seed. The categories were divided are strongly limited by selection pressures. into 53 sub category with phenotype ID Especially the numbers of the pump system numbers for description more detailed genes are 70-170. This indicates that, the active phenotypes. The phenotype ID system enables transport systems (= the system that operates to input phenotype data into barcode handy structure material concentration against a terminal in the field. Even using the physicochemical situations ) are strongly standardized phenotyping system, personal controlled its working situations. Many of the bias for observation is still remaining, '"("! newly diverged genes of higher animals and determination between dwarf and semi-dwarf plants are channels and secondary transporters phenotypes, yellow green and pale green works by adapting already existing phenotypes. To solve the personal bias concentration gradients. problem, photo images were also obtained in the field. All phenotype data with phenotype ID Phenotype catalog of &')"# and photo images have been stored into web- insertion mutants based relational database with PostgreSQL and Functional identification of rice genes is an CGI script written in perl language. important target after whole genome sequencing Descriptions of special phenotypes were saved of rice. Gene disruption is one of the most into free style comment box. Phenotyping of powerful methods for this purpose. The 50,000 insertion lines is almost finished. Nearly endogenous retrotransposon in rice, %)*#$ ,is half of insertion lines showed at lease one only active in cultured cell, and is transposed phenotype. Most frequently appeared with copy and past manner. This phenotype was dwarf and sterile (including characteristic is suitable for gene disruption partial sterile). Pigmentation mutant also system. Because the copy number of original relatively frequently appeared. For the low %)*#$ in japonica rice Nipponbare is two, frequency but characteristic phenotype, detection of newly transposed %)*#$ by narrow leaf, brittle culm, viviparous, abnormal hybridization and isolation of flanking region of shoot, abnormal tillering, abnormal flower, '+&! %)*#$ are relatively easier than other high- were observed. Isolation and characterization copy number of transposons. Furthermore, of the responsible gene of these phenotypes will because %)*#$ is an endogenous retrotransposon, be helpful to design new shape of rice for gene disruption line with %)*#$ is same as improvement of photosynthesis, yield, resistance spontaneous mutation lines for handling mutant of plant pathogens, etc.. Currently, more than plants and is not under regulation of the 172,000 phenotype descriptions and 58,000 genetically modified organism (GMO). This photo images are maintained with the relational enables large scale phenotyping in the field. database and stored data is still growing. We Currently, more than 50,000 insertion lines have are also constructing the database for been produced and stocked. disruption site by %)*#$ on the rice genome. In the project of MAFF Isolation and Mutant lines can be searched by BLAST Functional Analysis of Useful Rice Genes and search (http://tos.nias.affrc.go.jp). When the Development of Techniques for Their Utilization, candidate of the knock-out line is detected, large scale phenotyping proceeded with phenotype description with photo images will collaboration of 7 laboratories for 3 years be listed. Seed of mutant lines is distributed by project. From 1000 to 3000 lines were observed rice genome resource center. in each laboratory for a year. It was difficult to keep the quality of data without standard. We A major QTL, (%$"! chose standardized vocabularies for phenotyping. for shattering habit in rice Their consist 12 categories, germination, Loss of seed shattering habit is one of the growth, leaf color, leaf shape, culm shape, mimic critical events during domestication of rice response, tillering, heading date, flower, panicle, plant, because the easy-to-shatter trait in wild

#" $)).&( %'+*,- "!!# relatives results in severe reduction in yield. pedicel and spikelet at the base of the rice seed Therefore, seed shattering habit is one of the (Fig. 1C). while no abscission layer was most important agronomic traits in rice observed in Nipponbare at all (Fig. 1C). This breeding. To reveal a molecular basis of seed indicated that Kasalath allele of <+%" was shattering habit in rice, we genetically analysed involved in the formation of abscission layer. naturally occurring variations in seed A large-scale linkage analysis of 10,388 shattering among cultivars. We first performed plants segregating at the <+%" region was a QTL (Quantitative Trait Locus) analysis performed for the fine mapping of <+%"! We between a shattering-type 69160. variety, finally defined functional natural variation in Kasalath, and a non-shattering type 7.;:960. 612 bp between the flanking markers qSH1-F cultivar, Nipponbare (Fig.1A). Five QTLs were and qSH1-H and found only one single detected on five chromosomes of rice in an F2 nucleotide polymorphism (SNP) within this population of the cross between Kasalath and region (Fig. 2A). Gene prediction using the Nipponbare (Fig. 1B). The QTL with the largest Nipponbare rice genome and the Kasalath effect, ),& :3 >221 >5.??2=694 69 05=:8:>:82 " genome at the <+%" locus predicted no distinct (<+%"), explained about 68.6% of the total open-reading frame (ORF) in the candidate FNP phenotypic variation in the population (Fig. 1B). region. However, we found one ORF that was We made a near-isogenic line (NIL) that an ortholog of the #=./61:;>6> *$(&-'&$++ contained a chromosome segment from (*(&) gene encoding a BEL1-type homeobox Kasalath at the <+%" locus in a Nipponbare (Fig. 2B), located 12 kb away from the SNP. The background. The NIL exhibited the formation *(& gene is required for pod shatter and is of a complete abscission layer between the involved in the formation of a dehiscence zone

Fig. 1 qSH1 is required for formation of the abscission layer at the base of the rice grain (A) Seed shattering habits of rice panicles. Left: non-shattering-type cultivar, Nipponbare. Right: shattering-type cultivar, Kasalath. (B) Chromosomal locations of QTLs for seed shattering degree, based on an F2 population from a cross between Nipponbare and Kasalath. qSH1 is marked on chromosome 1 with the nearest DNA marker (C434). (C) Photo of a rice grain. White box indicates position of abscission layer formation.

%**/') &(,+-. "!!$ $# Fig. 2 Map-based cloning of qSH1 and qSH1 expression (A) Delimitation of qSH1 by a large scale linkage analysis. (B) Neighbor-joining phylogenetic tree of BEL1-type homeobox genes found in !)"#%$'(*%* and rice genomes. (C) Complementation test for qSH1 gene. A 26-kb Kasalath fragment (TAC9) in TAC vector, pYLTAC7, was transformed into Nipponbare. Top: Non-shattering degrees of T0 progeny were measured. Bottom: Lines 203 and 204 were partly transformed, because these lines lost the ORF region upon transformation. (D) and (E) %& *%+, analysis of qSH1 expression at the abscission layer Nipponbare and NIL, respectively.

(or abscission layer) alongside the valve in the formation of the provisional abscission layer at fruit (silique), which originates from the carpels. the base of the spikelet (Fig. 2E), and the stage To verify the function of candidate gene, we of rapid elongation of rachis and branches (Fig. introduced ten of 10- to 25-kb Kasalath genomic 2E). We therefore concluded that this )(' fragments scanning the predicted ORF and the ortholog was the 2*%# gene and that the SNP into the non-shattering Nipponbare identified SNP affected only the spatial mRNA cultivar (Fig. 2C). Only transgenic lines that expression pattern of 2*%#, which was lost at contained the Kasalath fragment with both the the abscission layer in Nipponbare. ORF and the SNP exhibited complete seed shattering, although one fragment, which Flowering-time control of rice contained a full ORF region but not the SNP, We have previously identified that a novel partly complemented the phenotype (Fig. 2C). B-type response regulator, $.,#! promote &1 3/45 hybridization analysis revealed that, flowering mainly under short-day conditions in Nipponbare, the ORF was expressed in the (Doi -4 +0"! 2004). $.,# encodes a B type rachis meristem (Fig. 2D), but it was not response regulator, which consists of one expressed in the provisional abscission layer receiver and one GARP DNA binding domain. (Fig. 2D). On the other hand, in the NIL the $.,# mRNA was induced by short-day ORF was expressed at the stage of treatments and exhibited two peaks a day; one establishment of the rachis meristem (Fig. 2E), before dawn and the other after dawn. This the stage of differentiation of glumes and indicates that both photo signal transduction

$# %**/') &(,+-. "!!$ and circadian clocks may control the &.-" Under the same conditions, (-#, mRNA, a mRNA. To reveal the molecular mechanism downstream gene of &.-", was not affected by further, we here over expressed several &.-" the application regardless of the functional &.- cDNA derivatives driven by an maize +%* ". This suggests that &.-" may not receive promoter. The results have clearly shown that cytokinin signaling in rice. Therefore, what over- expressing a full length &.-" cDNA kind of chemical signals are received by &.-" induce flowering of rice under short-day receiver domain is an open question. conditions (SD). Interestingly, an &.-" derivative, termed IGR, which lost only the A novel gene that is up regulated receiver domain of &.- ", significantly by gibberellin is involved in promoted flowering of rice under SD. In rice growth contrast, other two &.-" derivatives, which The plant hormone gibberellin (GA) plays lost C terminal region after the GARP domain an important role in regulating many physiological and which lost the N terminal region before the processes in the growth and development of GARP did not promote and repress flowering at plants, including seed germination, shoot and all. These indicate that several functional stem elongation, and flower development. To domains may exist in the &.-" gene product. understand how GA stimulates leaf sheath In addition, over expression of the full length elongation, a cDNAs microarray containing &.-" and IGR did not flowered early under 4000 clones randomly selected from a rice long-day conditions. Therefore, post cDNA library prepared from seedlings treated transcriptional regulation of &.-" mRNA and/ with GA3 was analyzed to identify new or the modification of &.-" gene product would members involved in cell elongation. A novel be the target for long-day repression of GA enhanced gene, designated as )/'$&"! flowering in rice. Exogenous applied cytokinin was identified using microarray analysis of GA induced A type response regulators in rice. regulated genes. )/'$&" expressed in a dose

Fig. 3 Histochemical localization of GUS activities in transgenic rice plants expressing GUS gene driven by the %&$"#! promoter region Leaf sheath longitudinal sections (a), young seedlings (b) and panicle (c) of transgenic rice plants were incubated with GUS staining solution for 2-12 h. SAM; shoot apex meristem. PL; primary leaf.

%**/') &(,+-. "!!$ $# and time course-dependent manner with and root. %- 0+12 hybridization and &0$"#! minimum expression at 1 µMGA3 and maximum promoter analysis revealed that &0$"#! expression at 50 µMGA3 starting from 1 h and expressed in shoot apex meristem and young peaked at 24 h after GA3 treatment. &0$"#! primary leaves (Fig. 3). Northern blot, Western expression was up regulated by GA3 at blot and GUS activities revealed that OsGAE1 transcript level while no significant effect was transcript and translation are up regulated by observed for other hormones. OsGAE1 was GA3. Transgenic rice expressing &0$"#! in expressed in #0(*)/+(*+' (.,+ with N-terminal antisense orientation exhibited severely affected

His6 tag and the recombinant protein migrated vegetative and reproductive growth (Fig. 4). at 38 kDa, slightly larger than the predicted 29 The transgenic plants were 55 to 70 % short kDa, during SDS-polyacrylamide gel compared to control. These results suggest that electrophoresis. Anti-OsGAE 1 antibodies OsGAE1 is differentially expressed in rice leaf immunoreacted with a protein of 40 kDa in rice sheath in relation to GA3 and it encodes a leaf sheath. &0$"#! expressed mainly in functional protein which is involved in GA growing leaf sheath and callus compared to leaf regulated growth and development of rice.

Fig. 4 Phenotype of transgenic rice constitutively expressing %&$"#! in antisense direction a) The transgenic plants were grown in an isolated greenhouse and photographed two months after transfer to soil. b) Antisense transgenic plants exhibited stem bifurcation usually at 2nd or 3rd node. c) Spikes of antisense transgenic plants remained in the leaf sheath of flag leaf.

## $)).&( %'+*,- "!!# B iochemistry Department

Structural biology from the snake venom and target cyclic nucleotide-gated ion channels. The structure X-ray crystallographic analysis consisted of an N-terminal domain that has a of proteins fold similar to the group 1 plant pathogenesis- Crystal structure studies of several related proteins and a cysteine-rich C-terminal biologically important proteins have been domain. The multidomain strucutre seemed to carried out. α-Galactosidases catalyze the play an important role to recognize the target hydrolysis of α-linked galactosyl residues from proteins. galacto-oligosaccharides and polymeric galacto- (gluco)mannans. The crystal structure of 3D-structure of barnacle cement !()*%$)$&&" +%'"#$" α-galactosidase I was protein, !$"#-20k determined at 1.6 ! resolution (Fig. 1). The Structure determination by X-ray structure consisted of a catalytic domain crystallography and/or NMR spectroscopy is comprising a (β/α)8-barrel structure and a C- the powerful tool for research of proteins with terminal domain made up of eight β-strands unknown functions because protein function is containing a Greek key motif. Owing to the high strictly regulated by three-dimensional (3D) resolution X-ray data, four carbohydrate chains structure. Barnacle cement proteins, which are were observed in one α-galactosidase I secreted for underwater adhesion, were molecule and their structures were identified to recently isolated and cloned. The molecular be high mannose type. α-Galactosidase I seemed functions of these proteins in adhesion are not to form a tetramer around the crystallographic to be established. We have determined the 3D- four-fold axis. structure of one of these proteins, !)cp-20k, in The crystal structure of elapid snake solution by NMR spectroscopy in order to toxins pseudechetoxin (PsTx) and pseudecin obtain insight into its biological function. (Pdc) have been determined at around 2 ! !)cp-20k contains 32 cysteine residues. resolution. These proteins belong to the They are assembled in regular repetitive cysteine-rich secretory protein family isolated positions in the primary structure, leading to

Fig.2 Structure of !*#)-20k Fig. 1 Six homologous units are numbered, and the boundaries of The ribbon model of the crystal structure of each repeats are marked by dotted lines. Cystines are shown !(*+%$*$&&" ,%'"#$" α-galactosidase I in yellow sticks. Two catalytic residues, disulfide bridges and the sugar chains were shown as black ball- and-stick drawings in red, yellow and gray color, respectively.

%**/') &(,+-. "!!# #$ the prediction of six repeated sequences with defense related genes. However, the overall low homology. The NMR analyses of the understanding of the biological significance of protein revealed the repetitive structure with ROS is not sufficient. For example, ROS six homologous units corresponding to the six produced by the host plants has been repeats. The calculated structure revealed that postulated to be toxic to the invading microbes, the conformation of the N-terminal part in each however, some pathogenic bacteria and fungi unit is supported by two disulfide bonds, while show strong viability in the presence of ROS. the C-terminal part forms a β-hairpin structure "),*-+'+ %'(&*&$! a necrotic pathogen in a (Fig. 2). The C-terminal part of each unit has variety of crops, secretes several kinds of one more disulfide bond with the exception of catalases which likely play important roles to the fifth and sixth units. The second unit has an avoid toxic activity of ROS. However, in the additional β-hairpin. In the whole structure of infection process of hemi-autotrophic fungi the protein, the C-terminal three units formed a including rice blast fungus, no conclusive novel triangular domain (Fig. 2). The central evidence on the role of ROS has been shown. To part of the domain was supported by disulfide address the question, we examined ROS- bonds and the three β-hairpins were exposed degrading activities in rice blast fungus and into solvent. Because several atoms in the N- found a stable catalase activity in the culture terminal domain, especially in the third unit, filtrate of the fungus. The activity was detected were not observable, we modeled the structure in the 6 different field isolates, which was likely of the N-terminal domain based on the to be carried in a single polypeptide because similarity between the C-terminal and N- the in gel activity was detected in the presence terminal halves of r#*cp-20k. The resulted of sodium dodecylsulfate, in contrast to catalase whole structure consists of two triangular from mammals and higher plants that is domains which were connected by a linker homotetramer of 60kDa subunit. As shown in sequence. The unique structure determined by the figure (Fig. 3), antibody against bovine us should accelerate further biochemical catalase did not react to any secreted studies which lead to identification of the polypeptide from rice blast fungus, supporting precise molecular function of #*cp-20k. the idea that the catalase is phylogenetically distinct from that of higher organisms. We are going to identify the gene(s) encoding the Molecular analysis of signal catalase and analyze the role of the catalase by perception and transduction reverse genetics approach.

Interaction between rice and rice blast fungus: Identification of a secretory protein from rice blast fungus as catalase Rice blast is one of the most important diseases for rice production in Japan as well as in the world. Recently, the whole genome Fig. 3 sequence of the fungus was reported, providing Western blot analysis of the total an excellent model system for the analysis of secreted proteins from rice blast pathogenic interaction between fungi and fungus Ten mg of total protein secreted higher plants, in combinationwithavarietyof into the culture media of P91-15B the genome resources of rice. A common (lane 3) and INA86-137 (lane 4), together with 50 ng of purified feature in the defense responses by the host bovine calatase (lane 1) and 10 mg plant is the rapid production of reactive oxygen of protein of culture media (lane 2). As shown here, no bands were species (ROS), which is believed to be a signal to detected by anti-bovine catalase the following events such as expression of antibody.

#$ %**/') &(,+-. "!!# Proteomic analysis Analysis of auxin-signaling Based on the proteomic approach, mainly pathway silkworm have been analyzed using matrix- Synthetic peptides corresponding to the C- assisted laser desorption/ ionization time-of- terminus of auxin-binding protein 1 (ABP1) flight mass spectrometry or ion-trap mass have been shown to function as auxin agonists. spectrometry coupled with high-performance The identification of a C-terminal receptor liquid chromatography after the separation by would be key importance in analyzing the two-dimensional polyacrylamide gel electrophoresis auxin-signaling pathways. To define the or two-dimensional high-performance liquid receptor, photoaffinity crosslinking studies chromatography. The results obtained by were performed and isolated two types of proteomic analysis, the silkworm proteome maize proteins as candidates for the receptor. database has been constructed using Make 2 One was a GPI-anchored plasma membrane ddb II software linking with silkworm genome protein (termed C-terminal peptide-binding data. In addition to these conventional protein 1, CBP1). CBP1 was found to be a approaches, new methods and apparatus have copper-binding protein, and is highly been developed for protein analysis, native two- homologous to the proteins functioning on cell dimensional electrophoresis to identify the elongation processes relating to plant cell protein interactions on gels, on-target polarity such as "+%&(')*,(, SKU5, SKS6 and sequential biochemical reactions for de novo tobacco NTP303. Furthermore, a null mutation sequences or identification of amino acid of the "#$! gene has been shown to disturb modifications after the measurements of the directional cell elongation at the early proteins in a high-mass range, and the direct embryogenesis. The present results indicate protein analysis which were extracted from a that an ABP1-CBP1 pathway may contribute to small tissue section using a mass spectrometry. directional cell growth processes relating to cell Evidences of differential expression of proteins polarity among the auxin-mediated cell in the small tissues were showed by the help of expansion processes. hierarchical cluster analysis. Proteins were not only distributed with a gradient, but also they were specifically expressed in the part of tissue.

%**/') &(,+-. "!!# #$ P lant Physiology Department

The research activities of our department approach molecular basis of the high specificity, are mainly focused on the elucidation of we examined each residue of the spinach CtpA molecular mechanisms of important physiological by several concepts that tendency of processes in plants. The major topics in fiscal conservation in the CtpA family, distance from 2005 are as follows. the catalytic center and accessibility of substrates. I400, V416 and Y419 were extracted as candidate residues involved in substrate Photosynthesis and carbon metabolism recognition of the protease (Fig. 1A). Identification of critical residues We introduced a series of site-directed for substrate recognition of mutations about these residues of the spinach CtpA, an integral protease for ",*! gene and transformed the mutagenized assembly of oxygen-evolving genes into $,*! deleted #-(%$&)$-+,'+ sp. PCC complex of photosystem II. 6803 cells, which helps rapid evaluation of Carboxyl-terminal processing protease A effects of each mutation onto the CtpA activity. (CtpA) is an indispensable protease for These analyses revealed that V416 and Y419 assembling of the photosynthetic oxygen are not so important for the CtpA action, evolving machinery, which performs because these positions could be accepted proteolytic processing of the precursor form of various substitutions without deactivation of the D1 protein, a pivotal subunit of photosystem the CtpA. In contrast, I400 is likely to be critical II. The CtpA is well known to have very for CtpA activity. It is noteworthy that I400T, a narrow substrate specificity, which is one of the relatively mild substitution, significantly common properties of proteases involved in abolishes the activity and then #-(%$&)$-+,'+ regulation of cellular processes. In order to cells with the mutant gene could not grow on

Fig. 1 (A) Three dimensional structure of the CtpA and positions of our target residues Catalytic center residues, S372 and K397 were Fig. 1 (B) colored with red and blue, respectively. CtpA has two Photoautotrophic growth test of the several mutants grooves close to the catalytic center. I400, V416 and #-(%$&)$-+,'+ cells carrying the I400T mutant ",*! Y419 (colored with orange) emerged on surface of the gene could not grow on photoautotrophic medium (- narrow groove. glucose). In contrast, V416T cells did not abolish the CtpA activity and then the mutant as well as WT cells grew well on the same medium.

$! %**/') &(,+-. "!!# photoautotrophic medium (Fig. 1B). It clearly characterize these genes more in detail to suggested that hydrophobic environment elucidate possible involvements in C4 leaf supplied from this aliphatic residue is required development. for maintenance of the CtpA activity. It is most probable that the I400 is a key residue for Identification and physiological substrate recognition of the CtpA and analyses of a locus for rice yield contributes the high specificity of the protease. potential We analyzed quantitative trait loci (QTLs)

Genetic analysis of C3-C4 for the ratio of filled grains, a yield component, intermediate photosynthesis in backcrossed inbred lines of Nipponbare and Kasalath. Only one QTL (5-"), with a positive

C3-C4 intermediate plants have lower Kasalath allele, was detected across environments. activities of photorespiration than C3 plants. We In a near-isogenic line (NILrg5) with a Kasalath investigated structural and photosynthetic chromosome segment containing 5-"! characteristics in leaves of reciprocal hybrids carbohydrate storage capacity before heading differing in genome constitution produced or sink activity was significantly higher than in between &35/*)2+/) )58,26/6 (C3-C4; MaMa) and Nipponbare (control). The ratio of filled grains

$5)66/*) 30,5)*,) (C3; CC). We found that the and yield per plant were significantly higher in hybrids had intermediate features between the NILrg5 than in Nipponbare, by 5% (( < 0.01) parents and expressed more strongly the C3-C4 and 15% (( < 0.05), respectively. These results intermediate characteristics with an increase in suggest that 5-" improves carbohydrate storage the constitution ratio of the Ma:C genome. The capacityandkeepssinkactivityhigherinthe same pattern of expression of the C3-C4 reproductive stage, and consequently increases intermediacy was found between the hybrids yield potential. that the parents were reciprocally exchanged.

We conclude that the C3-C4 intermediate Light response characteristics are inherited in the hybrids depending on the constitution ratio of the Distinct functions of different parent genomes and there is no evidence of phytochrome species in rice maternal inheritance in these characteristics. We have isolated phytochrome B (4.9$) and 4.9% mutants from rice ('59:) 6)7/8))and Isolation of genes that are have produced all combinations of double differentially expressed during mutants. Seedlings of 4.9$ and 4.9$ 4.9%

C3 and C4 leaf development mutants exhibited a partial loss of sensitivity to

C4 leaves have a specialized structural continuous red light (Rc) but still showed feature, Kranz anatomy, that differs from C3 significant deetiolation responses. The responses leaves. #0037,5346/6 6,1/)0)7) is a unique grass to Rc were completely canceled in 4.9# 4.9$ including both Kranz (C4) and non-Kranz (C3) double mutants. These results indicate that forms within this one species. We used phyA and phyB act in a highly redundant subtractive hybridization to isolate genes that manner to control deetiolation under Rc. Under are differentially expressed during leaf continuous far-red light (FRc), 4.9# mutants development of the two forms. Northern blot showed partially impaired deetiolation, and analyses of the subtracted clones revealed that 4.9# 4.9% double mutants showed no six and 13 genes were C3-andC4-abundant, significant residual phytochrome responses, respectively, most of which showed high indicating that not only phyA but also phyC is similarities to genes involved in mechanisms of involved in the photoperception of FRc in rice. regulation, signaling and transport, carbon Interestingly, the 4.9$ 4.9% double mutant metabolism, and protection from abiotic or displayed clear R/FR reversibility in the pulse biotic stress. We are now attempting to irradiation experiments, indicating that both

&++0(* ')-,./ #!!$ %" phyA and phyB can mediate the low-fluence phenotype of yeast strain G19 that lacks a response for $)'& gene expression (Fig. 2). Rice major component of Na+ efflux (Fig. 3). The is a short-day plant, and we found that mutation intracellular K+ contents under salt stress in either ,)/" or ,)/# caused moderate early conditions increased in OsKAT1-expressing flowering under the long-day photoperiod, yeast during the logarithmic growth phase, while monogenic ,)/! mutation had little effect suggesting the enhancement of K+ uptake by on the flowering time. The ,)/! mutation, OsKAT1. At the late linear phase, however, the however, in combination with ,)/" or ,)/# transformants accumulated lesser amounts of mutation caused dramatic early flowering. Na+ than mutant cells whereas the K+ amounts of these cells were almost the same. Intracellular Na+/K+ ratio in the OsKAT1 expressing cells was maintained at a steady level throughout the culture, whereas that in the mutant cells changed dramatically. These results suggest that OsKAT1 may participate in maintenance of cytosolic ion homeostasis during salt stress and protect the cells from Na +. The function of OsKAT1 observed in yeast is expected to take place also in rice.

Fig. 2 Induction of (-+* gene expression by R and/or FR pulses in etiolated rice seedlings Rice seedlings from Nipponbare ( WT ) and phytochrome single (/-0%! /-0&! and /-0') and double (/-0% /-0'! /-0& /-0'! and /-0% /-0&) mutants were grown in complete darkness for 7 d (D) and then treated with a single Rp (R), a FRp immediately after an Fig. 3 Rp (R/FR), a train of Rp-FRp-Rp (R/FR/R), or a single Suppression of salt sensitivity of the ,.)#"$ yeast FRp (FR). mutant (G19) by K+ channel of rice (OsKAT1) Serial tenfold dilution of salt sensitive yeast mutant expressing empty plasmid (top row) and OsKAT1 Salt tolerance (bottom row) were spotted onto YPG plate containing 0.3MNaCl. Isolation of cation transporter genes functioning in salt tolerance Disease resistance Some cation transporters are known to mediate ion homeostasis in cytoplasm and BTH-inducible WRKY transcription contribute to the salt tolerance in plants. factor plays a critical role in blast Finding a novel cation transporters involved in resistance of rice the salt tolerance is important to unravel and The signaling pathway mediated by improve the salt tolerance in crops. In this salicylic acid (SA) plays a critical role in purpose, we carried out the screening of a rice activating defense responses in some dicot full-length cDNA expression library through plants. Benzothiadiazole (BTH) is one of the functional complementation in yeast. We found chemicals known as plant activator that a cDNA clone encoding the rice homologue of protect plants from diseases by activating the the !-%&*(+,.*. potassium channel protein SA signaling pathway in a manner termed KAT1 (OsKAT1) suppressed the salt-sensitive priming. BTH is effective also in rice, however,

$" %**/') &(,+-. "!!# Fig. 4 Blast resistance of OsWRKY45-KD and OsWRKY45-ox rice plants (A) BTH-induced blast resistance is compromised in OsWRKY45-knockdown rice plant. (B) Overexpression of $,&%#'!" under .()+.)-)* promoter conferred blast resistance to rice plants. (C) Disease symptoms on the fourth leaves of OsWRKY45-ox and control rice plants. BTH (0.5 mM, 1 mL/plant) was sprayed onto leaves of 3.5 4-leaf stage and conidia of rice blast fungus were sprayed onto rice plants 5 days after BTH application, followed by estimation of symptoms 10 days after inoculation. little is known about the roles of SA and BTH in of these genes was observed when grown in a the defense system in rice. To investigate the greenhouse, suggesting some environmental molecular mechanisms underlying the BTH- factor(s) that influences the PR-gene expression. induced defense program in rice, we first Only small adverse effects of the transgene analyzed gene expression profiles by microarray expression on the growth were observed in following BTH application to rice plants and these transformants. These results suggest that revealed a number of BTH-responsive genes the overexpression of '0*)&+$% mimicked including WRKY transcription factor genes. the priming effect of BTH in activating the The WRKY genes were induced within 3 h defense program in rice. after BTH application, preceding some pathogenesis-related ( PR ) genes. RNAi- Assessing real time variability in mediated silencing of one of them, '0*)&+$%! various genome regions compromised BTH-induced resistance to a All organisms need a system to protect compatible race of rice blast fungus (Fig. 4A), them from various pathogens, by differentiating indicating that this transcription factor plays a them from their self-components. Also each critical role in blast resistance of rice. Moreover, plant line has several disease resistance (R) overexpression of '0*)&+$%! driven by the genes to recognize infection of specific pathogens 2,-/2-1-. promoter, markedly enhanced the through their corresponding avirulence gene blast resistance (Fig. 4 B and C). The '0*)&+ products, directly or indirectly, and induce $%-overexpressing rice plants showed various defense reactions, including upregulation of ()", and ()# genes when hypersensitive death of the infected cells. This grown in a growth chamber, but no upregulation recognition system of the pathogen molecules is

&++0(* ')-,./ "!!$ %# analogous to vertebrates immunity system Catalytic activation of plant based on the recognition of pathogens specific MAPK phosphatase NtMKP1 by molecule (antigen) with the highly variable its physiological substrate SIPK antibody: immunoglobulins. However, although In plants, two MAPKs, wound-induced plants lack special diversifying system of the R- protein kinase (WIPK) and salicylic acid- genes as the case of vertebrate globulin induced protein kinase (SIPK), are key molecules, we have shown that the genome molecules of signal transduction upon wound regions around the R-genes are high in the and disease responses. The activity of MAPKs mutation frequencies (2004 Annual Report), is strictly regulated via phosphorylation by an especially in the base substitution rate. In order upstream MAPK kinase. Conversely, MAPKs to clarify whether this accumulation of are dephosphorylated and inactivated by variability around the R-genes are due to the protein phosphatases. Previously, we identified simple selective accumulation, or reflecting the NtMKP1 (MAPK phosphatase) as a novel higher mutation rate in this region, we have calmodulin (CaM)-binding protein. Here, we assessed the real-time mutation (base- investigated the interaction between NtMKP1 substitution) rate in the !("#%$&')%) genome and substrate MAPKs or CaMs. NtMKP1 and checked the correlation of this with the inactivated SIPK through dephosphorylation. distance from the R-genes. β-glucuronidase CaM interacted with NtMKP1, but did not (GUS) genes with a stop codon mutation was activate its phosphatase activity. NtMKP1 is transformed into !("#%$&')%),andthe composed of four domains: a dual-specificity transformants were checked for their insertion phosphatase catalytic domain, a gelsolin sites in the genome. As shown in the Fig. 5, the homology domain, a CaM-binding domain, and somatic mutation during the individual C-terminal domain. Deletion analysis revealed development was assessed by the development that the N-terminal non-catalytic region of of revertant of the GUS genes as the blue spot NtMKP1 interacted with SIPK and was of digested X-Gluc. Some correlation of higher essential for inactivating SIPK, whereas the substitution rate with the R-gene was CaM-binding and C-terminal domains were suggested, and more confirmation is now being dispensable. Moreover, the phosphatase activity done. If this was confirmed, it will become the of NtMKP1 was increased strongly by the first case of verification of the plant R-gene binding of SIPK, but weakly by WIPK, another mutation development system. MAPK. The strong activation of NtMKP1

Fig. 5 Fig 6 Development of GUS blue spots revealing mutations in Transient expression of NtMKP1 suppresses MEKDD- the "/$%*'-.0*0 genome induced cell death This spots frequency correlated with the GUS Expression of constitutively active MAPK kinase localization in the genome suggests the presence of (MEKDD)in#*&-1*$,$ %(,1)$+*$,$ induced activation hot spots of mutation in the plant genome, related to of MAPK (WIPK and SIPK) and cell death. the plant disease resistance genes to recognize the Simultaneous expression of NtMKP1 compromised the corresponding pathogens avirulence molecules. constitutively active MAPK kinase-induced responses. NtMKP1 and MEKDD were transiently expressed in #! %(,1)$+*$,$ by agroinfiltration. The photograph was taken at 3 days after agroinfiltration.

%# &++0(* ')-,./ "!!$ phosphatase activity by SIPK depended that the $.580"!# nodule infected cells were partially on the putative common docking significantly smaller than those of wild type domain of SIPK. On the other hand, conversion plants, contained enlarged symbiosomes with of Lys41 and Arg43 of NtMKP1 to Ala (K41A/R multiple bacteroids, and underwent deterioration 43A) abolished the interaction with SIPK. of the symbiosomes prematurely as well as Expression of constitutively active MAPK disintegration of the whole infected cell kinase in &-*26-(1( )+16,(0-(1( induced cytoplasm (Fig. 7C and D). These results activation of SIPK and cell death. Simultaneous indicate that the ineffectiveness of the $.580 expression of either NtMKP1 (Fig. 6) or NtMKP "!# nodules is primarily due to impaired 1 L443R, which was unable to bind CaM, growth of infected cells accompanied with the compromised the constitutively active MAPK premature senescence induced at early stages kinase-induced responses, whereas that of of nodule development. We delimited the NtMKP1K41A/R43A did not. These results $.'80"!# locus in a few hundred kb region on indicate that the regulation of NtMKP1 activity the upper portion of chromosome 4, and are in by SIPK binding, but not by CaM binding, is progress towards molecular identification of the important for the function of NtMKP1. $.'80"!# gene.

Symbiotic nitrogen fixation A novel Fix- symbiotic mutant of %,/0. )&-,+('0.! %).1*#"$! shows impaired development and premature deterioration of nodule infected cells and symbiosomes Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant $.580"!# from a model legume $2675 .(321-*75 MG-20 by somaclonal mutation through extensive culture of suspension cells followed by regeneration of the plant. The $.580"!# mutants displayed nitrogen deficiency symptoms after inoculation with %+524,-92)-70 /26- under nitrogen free conditions, but their growth recovered when supplied with nitrogen-rich nutrients (Fig. 7A). Fig. 7 The mutant $.580"!# formed an increased (A, B) Growth and nodulation phenotypes of wild type MG-20 (left) and %(,.*#"$ (right) plants inoculated number of small and pale-pink nodules (Fig. 7B). with &! )+-'! Bars=10mm(A)and4mm(B). Nitrogenase (acetylene reduction) activity per (C, D) Micrographs of nodule infected cells of wild type MG-20 (C) and %(,.*#"$ (D). Wild-type nodule nodule fresh weight was low but retained more infected cells are densely packed with bacteroids, than 50% of that of the wild type nodules. Light whereas %(,.*#"$ infected cells show less-dense and and electron microscopic observations revealed aggregated bacteroids (arrow). U, uninfected cells; V, vacuoles. Bars = 10 µm.

&++0(* ')-,./ "!!$ %# 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. The major topics in fiscal 2005 are as follows.

Characterization of transcription factors responisible for endosperm-specific expression of seed proteins Fig.1 Cis-regulatory elements involved in the Expression pattern of )($% endosperm-specific regulation of cereal storage Total RNA from roots, seedlings and seeds during seed development (5, 10, 15, 20 and 30 Days After protein genes have been mainly characterized Flowering (DAF)) was analyzed by Northern blot using by producing stable transgenic plants or by gene-specific sequences of )($%! )'*$+# and the rice storage protein glutelin (&,-$"#) genes. 25S transient expression assays using particle rRNAs are shown as a loading control. bombardment. A number of consensus sequences such as the prolamin box (P-box), GCN4, AACA callus protoplasts that the isolated RPBF trans- and ACGT motifs, among others, have been activated several storage protein genes via an identified as elements that are critical in AAAG target sequence located within their determining the endosperm specificity of cereal promoters, and with methylation interference seed storage protein genes. A conserved experiments that additional AAAG-like element, referred to as the endosperm box, sequences in promoters of genes expressed in located 300 basepairs upstream of the maturing seeds were recognized by the RPBF transcriptional start site, has been found in protein. Binding was sequence-specific, since many cereal prolamin storage protein genes. mutation of the AAAG motif or its derivatives The endosperm box has a bifactorial motif that decreased both binding and trans-activation by is composed of the P-box (TGTAAAG) and the RPBF. Synergism between RPBF and RISBZ1 GCN4 motif (TGA(G/C)TCA), which are recognizing the GCN4 motif (TGA(G/C)TCA) separated by less than 10 nucleotides. was observed in the expression of many We characterized here the transcription storage protein genes. Over-expression of both factor which is involved in binding to the P-box. transcription factors gave rise to much higher This Dof zinc finger protein called RPBF (rice levels of expression than the sum of individual prolamin box binding factor) has been isolated activities elicited by either RPBF or RISBZ1 from rice cDNA EST clones containing the alone(Fig. 2). Furthermore, mutation of conserved Dof domain by examining their trans recognition sites suppressed reciprocal trans- -activation abilities with storage protein activation ability, indicating that there are promoters (Fig. 1). RPBF is found as a single mutual interactions between RISBZ1 and gene per haploid genome. Comparison of RPBF RPBF. The RPBF gene is predominantly genomic and cDNA sequences revealed that expressed in maturing endosperm and the genomic copy is interrupted by one long coordinately expressed with seed storage intron of 1892 bp in the 5 noncoding region. We protein genes, and is involved in the demonstrated by transient expression in rice quantitative regulation of genes expressed in

$# %**/') &(,+-. "!!# the endosperm in co-operation with RISBZ1. products, chitin elicitors, in rice / blast interactions. Previous studies revealed that chitin elicitor causes various defense-related cellular responses and induces blast resistance, and overexpression of chitinase results in enhanced fungal disease resistance in many species. Clarification of the signal transduction pathwaystartingwithchitin elicitor will result in a better understanding of basal resistance lied in rice/blast interactions. This year we evaluated involvement of the chitin-elicitor binding protein (CEBiP) in the basal resistance. Expression of CEBiP was repressed by RNAi and using leaf sheaths of the stable trasformants we scored blast disease resistance. As a result, we observed that growth of blast fungus was promoted in the Fig. 2 CEBiP RNAi lines 48 hr after inoculation. This Functional analysis of RISBZ1 and RPBF binding sites result indicates that recognition of chitin elicitor using a transient expression assay Schematic diagrams of the proximal %)*$!" (-199 to + contributes to the expression of basal 23) and 13kDa prolamin &('## (-386 to -13) are resistance in rice/blast interactions. shown. The mutagenized promoters, each carrying one mutated site (X), were designed for loss-of-function assays. These promoters were linked to the GUS Transgenic crops expressing reporter gene and electroporated into protoplasts in the presence of RISBZ1, RPBF or RISBZ1+RPBF. P450 monooxygenases (CYP) for Expression of a GUS reporter gene in the presence of phytoremediation the RISBZ1 was used as a control (100%). RISBZ1 The increased use of pesticides and and RPBF binding sites are the GCN4 motif and the P- box, respectively. herbicides on the agricultural scene has caused serious problems for plants, fish, insects, mammals, and sometimes to humans. These Molecular analysis of rice/blast chemicals are usually removed from the interactions environment by natural degradation and Plants activate self-defense systems to degradation by bacteria and plants. To decrease resist pathogens upon sensing them. There are the load on the environment, the enhancement several different classes of resistance: non-host of degradation of these chemicals by plants resistance, variety-specific resistance mediated should be as effective as the reduction in their by " (resistance) genes, and basal (or general) usage. resistance. Among these, basal resistance is not Phytoremediation is the use of living plants strong enough to avoid infection completely but to remove and or detoxify organic and inorganic understanding it at the molecular level is compounds. It is a possible method for reducing important to manipulate the durability of the risks of exposure of people and the disease resistance. environment to pesticides. Current physical It is believed that basal resistance is clean-up methods such as removing established after plants perceive invading contaminated soil from a site and chemical microbes through non-specific elicitors generated remediation treatments are very costly and during infection process. One of them is sometimes environmentally destructive. Plants fragmented chitin (! -acetylchitooligosaccharide), can remove organic pollutants, including which derives from a common component of pesticides, by root and leaf uptake of fungal cell walls. We have focused on the contaminants, biochemical degradation and contribution of chitinolytic enzymes and their subsequent accumulation of non-phytotoxic

%**/') &(,+-. "!!# $$ metabolites in plant tissue. been confirmed food and feed safety and Cytochrome P450 monooxygenase plays an influence on biodiversity by authorities. important role in the oxidative metabolism of However, gene confinement technology must xenobiotics in higher plants as well as in be important to achieve co-existence and mammals. The enzyme system on microsomes utilization of future generations of transgenic consists of many P450 species and a few crops. NADPH-cytochrome P450 oxidoreductase We have been developing to develop male molecules. Agrochemicals including herbicides sterile transgenic crops using several molecular were metabolized by P450 species, conjugated approaches for gene confinements. We cloned with glutathione or sugars, and tapetum or anther specific expressed genes compartmentalized into vacuoles, or cell walls, from "*#++&$# ('%*#$%# and "! *#)# and isolated in plant body. The oxidation by P450 species is promoter region by 5-Race. Then tissue and considered to be the limiting step of metabolism time specificity of isolated promoters were of foreign chemicals. confirmed by GUS expression. We could not Rice genome contained 246 P450 genes, but detect GUS expression except in anther using the P450 species involved in xenobiotic several promoters such as BoA3, BoMS2, BrA6, metabolism have not been studied well yet. BrMS2 (Fig. 3 A). But other promoters induced However, mammalian P450 species involved in GUS expression not only tapetum and anther the xenobiotic metabolism have a high activity but in petal, ovule and other tissues. We have to metabolize various herbicides with different also isolated nine genes, which have the modes of action and in different chemical potentiality of inducing male sterility, including functional groups. ribonuclease, protease, apoptosis related genes We produced three types of transgenic rice and phytohormone biosynthesis related genes plants expressing human P450 species, CYP1A from "*#++&$# genus. And we constructed 1 or CYP2B6 or three P450s, CYP1A1, CYP2B6 vectors to combine the promoters and those and CYP2C19 simultaneously. The transgenic isolated gene. Subsequently we demonstrated rice plants had an enhanced ability to effects of combination of promoters and those metabolize herbicides with different chemical structures owing to the introduced P450s. The chemicals were supposed to be absorbed by the transgenic plants in the fields and metabolized rapidly into non-phytotoxic compounds. Therefore, they exhibited a remarkable cross- tolerance toward various herbicides. The transgenic rice plants expressing P450 s involved in xenobiotic metabolism are quite useful for reducing the residual herbicides in plants. I addition, they can be used for phytoremediation of various chemicals that are widespread in agricultural environments.

Development of male sterile transgenic crops for the gene confinement in transgenic crops The possibility of gene transfer from transgenic corps to wild relatives or non- Fig. 3 transgenic crops has often been cited as an GUS gene expression in tapetum and induction of male environmental and consumers concern. sterility Tapetum specific GUS expression by the control of Commercialized transgenic crops have already promoter BoA3 (A) and male sterile flower of transgenic !*"#%()+%+ ,$"&%"'" (B).

$% &++0(* ')-,./ "!!# isolated genes in transgenic "2'(,*013,3 Construction of macroarray 4+'-,'/'! Analysis of transgenic "2'(,*013,3 system for survey genes 4+'-,'/' revealed that expression of protease in regurated by HrpG or HrpX tapetum effectively induced male sterility (Fig. To survey genes regulated by HrpG or 3B). We will introduce those constructions to HrpX, we constructed a DNA macroarray #2'33,)' crops and investigate the stability of system consisting of 2,384 of genomic DNA male sterility by the cultivation in semi-closed fragments of strain T7174 (MAFF311018) of green house and in natural condition such as an &00! It comprised about 95.5% of the whole isolated field. genome DNA. Using this macroarray system, it was confirmed that the +21 gene cluster (about Comparative genomic approach 87k to 120k region of the &00 genome) was to agronomically important genes upregulated in wild type strain under +21- in wheat and barley inducing medium, while it was not up regulated Colinearity in gene content and order in the ∆+21& and ∆+21% mutants (Fig. 4). between rice and closely related cereal crops Moreover, it was revealed that thirteen has been as a powerful tool for gene genomic regions were regulated by HrpG or identification. Using a comparative genomic HrpX. To check that expression of genes within approach, we have identified the rice genomic these regions were really controlled by the region syntenous to the region of the short arm HrpG or HrpX, a real-time quantitative RT-PCR of wheat chromosome 2D, on which a QTL system was used. Finally, six genes (XOO0037, locus for $53'2,5. head blight (FHB) resistance XOO0078, XOO1388, XOO2263, XOO4042 and is located. Utilizing markers known to reside XOO4134) were newly identified. In addition, near the FHB QTL and data from wheat two genes, XOO2263 and XOO4134 contained a genetic maps, we have identified the syntenous plant-inducible-promoter (PIP) box, which was a region of the short arm of rice chromosme 4 to consensus sequence of HrpX regulons, the FHB QTL locus on wheat 2DS. Fifteen upstream region of putative initiation codon. predicted rice genes with similarity to known These obtained results showed that this disease-related genes have been identified in an macroarray system was useful tool for genome- approximately 6-Mb rice sequence spanning wide gene expression analysis in &00! the syntenous region. Rice sequences of these putative genes were used in BLAST searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. RT-PCR analysis and gene mapping of wheat homologues to rice disease-related genes in this region revealed a possible candidate for the FHB QTL on wheat chromosome 2DS. We also applied this approach to explore the genes for heading of wheat. Wheat and rice are closely related species to each other, but rice plant shows a heading under short-day condition, while long-day condition stimulates the heading of wheat. We just cloned several genes orthologous to rice heading-related genes, and analyzed the expression profile of them. Fig.4 Schematic representation of regions with up- and down- regulated gene expression in "$# inducing condition (∆"$#! / wild type strain)

&++0(* ')-,./ "!!# $% I nstitute of Radiation Breeding

The research activities of Institute of of Chl ), to Chlide *. The CAO has a RadiationBreedingarefocusedonthe mononuclear iron-binding motif and a Rieske development of new strains of seed-propagated, binding motif, which are thought to be vegetatively propagated and woody crops important for its activity. Null mutants of '&( through mutation by the application of various in &2)*-+013-3 4,).-)/) lack Chl * and have pale forms of irradiation. Mutations are induced by green leaves. the following radiation sources: Gamma Field We isolated two pale green rice mutants, and the Gamma Green house for γ-ray chronic YM-15 and G-52. These mutants were irradiations to the growing plants, and Gamma induced from cultivars Hitomebore and Room for γ-ray acute irradiations to seed, bulbs, Reimei by gamma ray-irradiation. We tubers, scions and -/ 5-420 materials. The extracted chlorophyll from mature leaves of institute is also involved in the development of these mutants with methanol and analyzed the new technologies for plant breeding mainly chlorophyll composition. The analysis revealed utilizing γ-ray and ion beam irradiation and that both mutants lack Chl *, suggesting that chemical mutagens, including the elucidation of they have defects in Chl * synthesis. The most gene expression mechanisms in mutants. The likely candidate for the mutated gene in these institute provides irradiation service and mutants is '&(! cooperative research at the request of To examine the '&( sequence in these universities, private industries, prefectural mutants, we searched for '&( homologs in the experiment stations, and incorporated agencies rice genome database by using the &2)*-+013-3 of Ministry of Agriculture, Forestry and '&( sequence as a template in a BLAST Fisheries. The major topics in fiscal 2005 are as search. Unexpectedly, we found two '&( follows. homologs, which are tandemly repeated with a 6.6-kbp interval in a BAC clone, OSJNBa0057L Characterization of "(*,.,-(1**)%& 21. The predicted amino acid sequences of $ #01'&+$/& ("!#)inRice these genes, OSJNBa0057L21 Predgene09 Chlorophyll (Chl) plays a central role in ('&(-09) and Predgene11 ('&(-11), show 86.7% capturing light energy for photosynthesis. Land sequence similarity to each other, and 74.2% plants contain two types of chlorophyll, Chl ) and 70.0% similarity, respectively, to and Chl *.TheChl) is a component of the core &2)*-+013-3 '&(! The full-length clones and the peripheral antenna complexes. The Chl corresponding to '&("#% and '&("$$ (J * is contained only in peripheral antenna 013001P04 and 001-114-D11, respectively) have complexes, the light-harvesting chlorophyll a/b- been registered in the rice full-length cDNA protein complexes (LHCPs). The LHCPs are database, suggesting that both genes are classified into light-harvesting complex of expressed. Sequence analysis of '&("#% in the photosystem I (LHCI) and the light-harvesting mutants revealed that YM-15 has a 1-bp complex of photosystem II (LHCII). The only deletion in the 2nd exon, which results in a structural difference between Chl ) and Chl * is frame-shift and a premature stop codon. In G- a methyl group on a C7 of the tetrapyrrole in 52, a 3-bp deletion was found in the 7th exon of Chl ) and a formyl group at the corresponding '&("#%. On the other hand, no mutation was site in Chl *. The key enzyme involved in Chl * observed in the coding region of '&("$$ in synthesis is chlorophyllide ) oxygenase (CAO). either mutant. These observations suggest that It catalyzes a 2-step oxygenase reaction that '&("#% encodes '&(! converts chlorophyllide ) (Chlide )), a precursor To verify this hypothesis, we searched for

$! %**/') &(,+-. "!!# Fig. 1 Gene structures and mutations of the $#%-like genes in rice (A) Boxes and horizontal lines indicate exons and introns. Black triangles show the positions of deleted nucleotides in YM-15 and G-52. White triangles indicate the insertion positions of &'(!". Hatched and crossed boxes indicate the location of the putative Rieske center binding motif and mononuclear iron-binding motifs. (B) Rieske binding and mononuclear iron-binding motifs in CAO. Residues in the binding domain are boxed. A substitution form Y to F in CAO-11 is shaded. The deleted residue in CAO-09 of G-52 is underlined. retrotransposon *,-$% -induced mutants that residue is not necessarily highly conserved in disrupt these two CAO-like genes within the proteins containing mononuclear-iron-binding *,-$% insertion mutant database. We found sites, but is limited to arginine or lysine, both of three mutants (ND0017, NE1005, ND3004) of which are positively charged amino acids, (')"#& and one (ND1064) of (')"$$! We among CAOs of distantly related species. selected plants homozygous for each allele and Complete loss of Chl + in G-52 suggests that this analyzed their phenotypes. Consistent with the positively charged residue is important for analysis of YM-15 and G-52, homozygotes of CAO activity or stability. all three mutations for (')"#& showed pale The rice genome is estimated to have green phenotype and lacked Chl +. On the other many duplicated genes, which are thought to hand, the homozygotes of the (')"$$ mutation have redundant functions. However, the did not show a pale green phenotype and example described here suggests that not contained a normal level of Chl + content. (')" many of such genes are functionally redundant, $$ has an incomplete Rieske binding motif, in even if they are expressed. which the highly conserved tyrosine residue (Y) among proteins containing Reiske binding sites Comparison of the Mutation is replaced with phenylalanine (F), suggesting Inducing Effect between Ion that (')"$$ is a pseudogene. The 3-bp deletion Beams and Gamma Rays in (')"#& of G-52 results in a deletion of the Mutation induction is a useful method for lysine residue (K), which is located in the plant breeding and the type of mutagenic mononuclear iron-binding site in CAO. This treatment is an important factor for successful

&++0(* ')-,./ #!!$ %" results. this experiment, the mutation frequency per M1 One unique characteristic of ion beam spike using 100 MeV helium-ion beams was the irradiation is their high level of linear energy highest at 12.4 %. The frequency was 10.4 % transfer (LET) and the potential to focus that using 320 MeV carbon-ion beams, 9.0 % using high energy on a target site. As a consequence, carbon-ion beams, and 8.4 % using gamma rays. ion beam irradiation can induce a high level of The relative frequency and type of mutagenic effect. For this reason, there is an chlorophyll mutations (albino, xantha, viridis expectation for a higher degree of effect on and others) generated by each treatment, are DNA and a higher level of mutation induction shown in Table 2. Among the four irradiation when compared to gamma rays having a low treatments, the spectrum of chlorophyll level of LET. However, the use of ion beam mutations also did not indicate significant irradiation as a mutagen for mutation breeding differences in treatments. From these results, has not been examined. Therefore, we have the relative frequency of each type of investigated the nature of ion beams as a chlorophyll mutation induced by radiation was treatment for mutation breeding and compared the same without regard to the type of it with gamma ray irradiation treatments. radiation. Seeds of the rice variety, cv. Hitomebore were placed on 6 cm diameter petri dishes and Tabel 2 The relative frequency and type of irradiated with 220 MeV carbon-ion beams (12 chlorophyll mutant 5+ C ,LET;121keV/µm); 320 MeV carbon-ion (%) beams (12C6+,LET;86keV/µm); and 100 MeV albina xantha viridis others helium-ion beams (4He2+,LET;9keV/µm) 220MeV carbon-ion '%!(*!)$'!"#$!* generated by an AVF-cyclotron. Gamma rays beams '"!&##!)$'!##$!* were applied to dry seed at dosage rate of 10 320MeV carbon-ion beams Gy per hour in a gamma-room. 100MeV helium-ion &)!)#'!"$&!(#$!( The effect of mutation induction was beams different among irradiation treatments (Table gamma ray '$!'*!$%"!(*!) 1). Within the irradiation dose range utilized in

Table 1 The effect of ion beam and gamma ray !# '"&%$ culture of a periclinal irradiation on mutation induction chimera for obtaining non-chimera Chlorophyll plantlets at a high frequency Dose mutation frequency (Gy) The generation and development of plant per M1 spike(%) chimeras, following irradiation, is a commonly 220MeV carbon-ion &"+!" encountered obstacle for obtaining a complete %"*!' beams mutant in the mutation breeding especially for $")!* #"(!" vegetatively propagated crops. Among the three types of chimera, sectorial, periclinal and 320MeV carbon-ion #""+!$ beams *"#"!& mericlinal chimeras, a periclinal chimera is most ("*!) stable. The induced mutant comprising the &"'!& periclinal chimera is not easily separated during 100MeV helium-ion $'"#$!& the natural development, even if simple cutting- beams $""#"!+ back techniques are employed. !# '"&%$ culture #'"*!& techniques are believed to be able to isolate a #""&!' '"$!# complete mutant from a chimera. In our study, we demonstrated that only one genotype was gamma rays %""*!& $'"(!* separated from the other genotype in the $"")!) chimera. #'"'!& In 2001, we identified a periclinal #""&!%

$" %**/') &(,+-. "!!# cytochimera among the mulberries (#+-0. %)&% L.) consecutively irradiated since 1979 in the Gamma Field. The chimera has both tetraploid cells and original diploid cells. The tetraploid cells were localized in the L1 layer of the shoot apical meristem (SAM) and diploid ones were identified in the L2 and L3 layers. Generally, Fig. 2. Longitudinal sections of SAMs of a diploid wild type angiosperm, such as mulberry, has a SAM (left) and cytochimera with tetraploid cells in L1 layer constructed with three cell layers, commonly called L1, L2 and L3 from the epidermal cells to mutant derived from cv. Nijisseikiin which self central cell layer. In addition, the L1 cells compatibility is caused by the complete deletion develop into the epidermal cells of a plant body of S4 gene. However, PCR analysis of cv. Osa while L2 and L3 develop into the internal plant Nijisseikireported that the original wild-type cells. The macro-morphological phenotypes of (self-incompatible) cells have been retained and the cytochimera were found to be similar to the are maintained in a chimera state (Sassa, et al. original diploid cultivars. The sizes of the 1997). In our studies, we found that cv. Osa stomata guard cells, which are derived from the Goldis also a chimera similar to cv. Osa L1, were larger than that of the original diploid. Nijisseikibecause a weak amplified fragment of

The longitudinal section of the SAM indicated S4 gene was identified by PCR analysis (Fig. 3). tetraploid L1 and diploid L2 and L3 cell layers Using (*!1(/-+ culture methods, we successfully (Fig. 2). We induced the regenerated plantlets obtained regenerated plantlets via adventitious via (*!1(/-+ direct adventitious bud formation buds that were induced on the leaves of (*!1(/-+ by culturing immature leaves of the cytochimera. -cultured winter buds. The PCR analysis By applying a flow cytometric analysis, we indicated that most of the plantlets were wild recognized that most of the regenerated plants type (Fig. 3). The other regenerated plantlets were tetraploid suggesting that they tended to were chimera similar to the cv. Osa Nijisseiki, be derived from the L1 cells. Other regenerated judging from the PCR fragment pattern. In this plantlets retained the original chimerism. There case, we were not successful in obtaining the was no plantlet derived from only the L2 or L3 non-chimera cell plantlets by the (* 1(/-+ cells among the regenerated plantlets. In this culture method. case, we successfully obtained the complete These results indicate that by (*!1(/-+ tetraploid from the cytochimera by the (*!1(/-+ culture of a periclinal chimera, we can obtain culture method. non-chimera plantlets at a high frequency or Similar results were obtained on a mutant notatall."* 1(/-+ culture techniques are not cultivar of Japanese pear ($2-0. ,2-('+)(% always a veritable panacea. We need to Nakai), cv. Osa Gold.The cultivar is a black- develop new and more robust techniques spot-disease-resistant mutant derived from cv. having application to any types of chimeras. Osa Nijisseikithat is known as a self-compatible

Fig. 3. PCR analysis of the original chimeric pear, cv. Osa Gold, and regenerated plantlets (1-4) after !" &!%$# culture Regenerated plant let 1: same as original cv. Osa Gold; 2-4: same as Nijisseiki, from which Osa Gold is derived S2, 4, 5: Self incompatible gene alleles 2, 4, 5

&++0(* ')-,./ "!!$ %# List of Publication

!)%$%'"& ("(#)*

1) Western language

1 Abe Kiyomi, Nakayama Shigeki, Ichikawa Hiroaki, Toki Seiichi, (2005), Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis., +7/9> +5@=6:7:4@, 139:896

2 Akino Toshiharu, (2005), Chemical and behavioral study on the phytomimetic giant geometer Biston robustum Butler (Lepidoptera: Geometridae), !;;7621 $9>:8:7:4@ /91 .::7:4@, 40(3):497-505

3 Ali Ghulam Muhammad, Komatsu Setsuko, (2006), Proteomic analysis of rice leaf sheath during drought stress, ':?<9/7 :3 +<:>2:82 ,2=2/<05, 5(2):396-403

4 Aoki Takayuki, ODonnell Kerry, Scandiani Maria Mercedes, (2005), Sudden Death Syndrome of soybean in South America is caused by four species of Fusarium: Fusarium brasiliense sp. nov., F. cuneirostrum sp. nov., F. tucumaniae and F. virguliforme., )@0:=062902, 46(3):162-183

5 Arai Shigeki, Chatake Toshiyuki, Ohhara Takashi, Kurihara Kazuo, Tanaka Ichiro, Suzuki Nobuhiro, Fujimoto Zui, Mizuno Hiroshi, Niimura Nobuo, (2005), Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements, *?07260 !061= ,2=2/<05, 33(9):3017-3024

6 Asano Momoko, Atsuko Mochizuki, Tetsuo Meshi, Masayuki Ishikawa, (2005), Tobamovirus-resistant tobacco generated by RNA interference directed against host genes., %$"- (2>>2<=, 579(20):4479-4484

7 Ashikawa Ikuo, Numa Hisataka, Sakata Katsumi, (2006), Segmental distribution of genes harboring a CpG island- like region on rice chromosomes, ):720?7/< &292>60= /91 &29:860=, 275(1):18-25

8 Ashikawa Yuji, Fujimoto Zui, Noguchi Haruko, Habe Hiroshi, Omori Toshio, Yamane Hisakazu, Nojiri Hideaki, (2005), Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a- dioxygenase, !0>/ #<@=>/77:46:9 % ->?/776A/>6:9 #:88?960/>6:9=, 61(6): 577-580

9 Chia Hsiung Cheng, Wu Jianzhong, Matsumoto Takashi, Sasaki Takuji, (2005), A fine physical map of the rice chromosome 5, ):720?7/< &292>60= /91 &29:860=, 274(4):337-345

10 Debi Bakul Rani, Chhun Tory, Taketa Shin, Tsurumi Seiji, Xia Kai, Miyao Akio, Hirochika Hirohiko, Ichii Masahiko, (2005), Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux, ':?<9/7 :3 ;7/9> ;5@=6:7:4@, 162(6):678-685

11 Fujii Yasuyuki, Itoh Takeshi, Sakate Ryuichi, Koyanagi O Kanako, Matsuya Akihiro, Habara Takuya, Yamaguchi Kaori, Kaneko Yayoi, Gojobori Takashi, Imanishi Tadashi, (2005), A web tool for comparative genomics: G- compass, &292, 364:45-52

12 Fukaya Midori,Akino Toshiharu, Yasui Hiroe, Yasuda Tetsuya, Wakamura Sadao, Yamamura Kohji, (2005), Effects of size and color of female models for male mate orientation in the white-spotted longicorn beetle Anoplophora malasiaca (Coleoptera: Cerambycidae), !;;7621 $9>:8:7:4@ /91 .::7:4@, 40(3):513-519

13 Fukui Kuniaki, (2005), Modeling of mulberry shoot elongation and leaf appearance in field conditions, +7/9> +<:1?0>6:9 -062902, 8(2):115-121

14 G. Hariraj, Kinoshita Haruo, T. H. Somashekar, (2005), Studies on bivoltine cocoon cooking : Part2. Influence of permeation treatments in cooking process on reeling characteristics of Japanese bivoltine cocoons, -2<60:7:46/, 45(2):189-199

%# &++0(* ')-,./ "!!$ 15 G. Hariraj, Kinoshita Haruo, T. H. Somashekar, (2005), Studies on bivoltine cocoon cooking : Part1. Influence of permeation treatments in cooking process on water absorption characteristics of Japanese bivoltine cocoons, -3=71;8;57/, 45(2):173-181

16 G. Hariraj, Kinoshita Haruo, T. H. Somashekar, (2005), Studies on bivoltine cocoon cooking : Part3. Influence of permeation treatments in cooking process on quality characteristics of Japanese bivoltine cocoons, -3=71;8;57/, 45(2):209-216

17 Gomi Kenji, Ogawa Daisuke, Katou Shinpei, Kamada Hiroshi, Nakajima Nobuyoshi, Saji Hikaru, Soyano Takashi, Sasabe Michiko, Machida Yasunori, Mitsuhara Ichiro, Ohashi Yuko, Seo Shigemi, (2005), A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco, +8/:? /:2 #388 +6B>7;8;5B,46

18 Gotoh Hideo, Kondo Takashi, (2005), Dynamic rearrangement of telomeres during spermatogenesis in mice., $3A38;<93:?/8 "7;8;5B, 281(2):196-207

19 Gotoh Tetsuo, Akizawa Takashi, Watanabe Masahiko, Tsuchiya Akiko, Shimazaki Sayaka, (2005), Cold hardiness of Neoseiulus californicus and N. womersleyi (Acari: Phytoseiidae), );@=:/8 ;4 ?63 !1/=;8;571/8 -;173?B ;4 )/

20 Gotoh Yohko, Tamada Yasushi, (2005), In vitro study of initial cell attachment to the surfaces coated with conjugates consisting of silk fibroin and chitooligosaccharides, );@=:/8 ;4 (:>31? "7;?316:;8;5B /:2 -3=71;8;5B, 74(2):39-43

21 Han Ouk Kyu, Kaga Akito, Isemura Takehisa, Wang Xing Wang, Tomooka Norihiko, Vaughan Duncan Alexander, (2005), A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi], .63;=3?71/8 /:2 !<<8732 &3:3?71>, III:1278-1287

22 Hanada Hirofumi, Takeda Kumiko, Tagami Takahiro, Nirasawa Keijiro, Akagi Satoshi, Adachi Noritaka, Takahashi Seiya, Izaike Yoshitaka, Iwamoto Masaki, Fuchimoto Daiichiro, Miyashita Norikazu, Kubo Masanori, Onishi Akira, W. Allan King, (2005), Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer, *;831@8/= ,3<=;2@1?7;: /:2 $3A38;<93:?, 71:36-44

23 Hasebe Hiroyuki, Sato Shin-ichi, Asahi Yoshimi, Hayashi Takeshi, Kobayashi Eiji, Sugimoto Yasukazu, (2005), High-resolution physical mapping and construction of a porcine contig spanning the intramuscular fat content QTL, !:79/8 &3:3?71>, 37:113-120

24 Hattori Makoto, Konishi Hirosato, Tamura Yasumori, Konno Koutaro, Sogawa Kazushige, (2005), Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps, );@=:/8 ;4 (:>31? +6B>7;8;5B, 51(12):1359-1365

25 Hayashi Takeshi, Awata Takashi, (2006), Interval mapping for loci affecting unordered categorical traits, '3=327?B, 96:185-194

26 He Fang, Morita Hirotsugu, Kubota Akira, Ouwehand Arthur C., Hosoda Masataka, Hiramatsu Masaru, Kurisaki Jun- ichi, (2005), Effect of orally administered non-viable Lactobacillus cells on murine humoral immune responses, *71=;07;8;5B /:2 (99@:;8;5B, 49(11):993-997

27 Hidema Jun, Teranishi Mika, Iwamatsu Yutaka, Hirouchi Tokuhisa, Ueda Tadamasa, Sato Tadashi, Benjamin Burr, Betsy M Sutherland, Yamamoto Kazuo, Kumagai Tadashi, (2005), Spontaneously occuring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice, +8/:? );@=:/8, 43(1):57-67

28 Hirokawa Masahiko, Tatematsu Kenichiro, Kosegawa Eiichi, (2005), Genetic analysis of a new mutation Extra- abdominal legs and extra crescents belonging to the *E* pseudoalleles in the silkworm, *Bombyx mori*, );@=:/8 ;4 (:>31? "7;?316:;8;5B /:2 -3=71;8;5B, 74(3):111-116

29 Hirose Sakiko, Kawahigashi Hiroyuki, Inoue Tomomi, Inui Hideyuki, Ookawa Hidero, Ookawa Yasunobu, (2005), Enhanced expression of CYP2C9 and tolerance to sulfonylurea herbicides in transgenic rice plants, +8/:? "7;?316:;8;55B, 22(2):89-96

30 Hirose Sakiko, Kawahigashi Hiroyuki, Ozawa Kenjirou, Shiota Noriaki, Inui Hideyuki, Ookawa Hidero, Ookawa Yasunobu, (2005), Transgenic rice containing human CYP2B6 detoxifies various classes of herbicides., );@=:/8 ;4 !5=71@8?@=/8 /:2 %;;2 #6397>?=B, 53(9):3461-3467

31 Honda Ichiro, Turuspekov Yerlan, Komatsuda Takao, Watanabe Yoshiaki, (2005), Morphological and physiological analysis of cleistogamy in barley (Hordeum vulgare)., +6B>7;8;57/ +8/:?/=@9, 124:524-531

&++0(* ')-,./ "!!$ %# 32 Ichikawa Akio, Ono Hiroshi, (2005), Preparation of single-enantiomer semiochemicals using 2-methoxy-2-(1- naphthyl)propionic acid and 2-methoxy-2-(9-phenanthryl)propionic acid, -2>

33 Ikeo Kazuho, Gojobori Takashi, (2006), Evolution of metabolic networks by gain and loss of enzymatic reaction in eukaryotes, (292, 365:88-94

34 Ishiga Yasuhiro, Takeuchi Kasumi, Taguchi Humiko, Inagaki Yoshishige, Toyota Kazuhiro, Shiraishi Tomonori, Ichinose Yuuki, (2005), Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tabaci wild type and defective mutants for flagellin (∆fliC) and flagellin-glycosylation (∆orf1), ):?<9/7 :3 (292 +/>5:7:4C, 71(4):302-307

35 Ishikawa Masaya, Kitashima Tomomi, Hemechandra PV, Yamaguchi Eiji, Toyomasu Takayuki, (2005), Constant relative humidity chambers using phosphoric acid for controlled desiccation of recalcitrant biological samples., ,221 ,062902 /91 -2059:7:4C, 33:741-752

36 Ishikawa Masaya, Suzuki Mitsuteru, Nakamura Toshihide, Kishimoto Tadashi, Robertson Albert J, Gusta Lawrence V, (2006), Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses., "99/7= :3 #:>/9C, 97:453-459

37 Ishikawa Ryo, Tamaki Shojiro, Yokoi Shuji, Inagaki Noritoshi, Shinomura Tomoko, Takano Makoto, Shimamoto Ko, (2005), Suppression of the floral activator gene Hd3a is the principal cause of the night-break effect in rice, +7/9> $277, 17(12):3326-3336

38 Ishikawa Satoru, Noriharu Ae, Masahiro Yano, (2005), Chromosomal regions with quantitative trait loci controlling cadmium concentration in brown rice (Oryza sativa)., *2A +5C>:7:46=>, 168:345-350

39 Ishimaru Ken, (2005), Factors responsible for decreasing sturdiness of the lower part in lodging rice (Oryza sativa L.)., +7/9> +<:1?0>6:9 ,062902, 8(2):166-172

40 Ishimaru Ken, Kashiwagi Takayuki, Hirotsu Naoki, Madoka Yuka, (2005), Identification and physiological analyses of a locus for rice yield potential across the genetic background., ):?<9/7 :3 &B;2<6829>/7 #:>/9C, 56:2745-2753

41 Ishizuka Toru, Tanabata Takanari, Takano Makoto, Shinomura Tomoko, (2005), Kinetic measuring method of rice growth in tillering stage using automatic digital imaging system, &9@6<:9829> $:9><:7 69 #6:7:4C, 43(2):83-96

42 Itoh Hironori, Sasaki Akie, Ueguchi-Tanaka Miyako, Ishiyama Kanako, Kobayashi Masatomo, Hasegawa Yasuko, Minami Eiichi, Ashikari Motoyuki, Matsuoka Makoto, (2005), Dissection of the phosphorylation of rice DELLA protein, SLENDER RICE1, +7/9> /91 $277 +5C=6:7:4C, 46(8):1392

43 Iwaki Jun, Suzuki Ryuichiro, Fujimoto Zui, Momma Mitsuru, Kuno Atsushi, Hasegawa Tsunemi, (2005), Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1, "0>/ $/77:46:9 ' ,>?/776D/>6:9 $:88?960/>6:9=, 61(11):1003-1005

44 Iwamoto Masaki, Onishi Akira, Fuchimoto Dai-ichiro, Somfai Tamas, Suzuki Shun-ichi, Yazaki Satoko, Hashimoto Michiko, Takeda Kumiko, Tagami Takahiro, Hanada Hirofumi, Noguchi Junko, Kaneko Hiroyuki, Nagai Takashi, Kikuchi Kazuhiro, (2005), Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer, .C4:>2, 13:335-345

45 Jan Asad, Nakamura Hidemitsu, Handa Hirokazu, Ichikawa Hiroaki, Matsumoto Hiroshi, Komatsu Setsuko, (2006), Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice, +7/9> /91 $277 +5C=6:7:4C, 47(2):244- 253

46 Jetty S S Ammiraju, Katayose Yuuichi, Matsumoto Takashi, Wu Jianzhong, Sasaki Takuji, (2005), Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome, -52:<2>60/7 /91 ";;7621 (292>60=, 111(8):1596-1607

47 Kameda Tsunenori, (2005), 13C solid-state NMR analysis of heterogeneous structure of beeswax in native state, ):?<9/7 :3 +5C=60= %! ";;7621 +5C=60=, 38:4313-4320

48 Kameda Tsunenori, Ishii Tadashi, Matsunaga Toshiro, Ashida Jun, (2006), 11B solid-state NMR investigation of the rhamnogalacturonan II-borate complex in plant cell walls, "9/7C>60/7 ,062902=, 22:321-323

$# %**/') &(,+-. "!!# 49 Kameda Tsunenori, Teramoto Hidetoshi, (2006), Phase transition of L-Ser monohydrate crystal studied by 13C solid-state NMR, *28=6B:4 -6A>=2=46 := $96<:AB@E, 44:318-324

50 Kameda Tsunenori,Kojima Katsura, Miyazawa Mitsuhiro, Fujiwara Seita, (2005), Film formation and structural characterization of silk of the hornet Vespa simillima xanthoptera Cameron., 16:BA49@:7B 7C@ +2BC@7>@A49 $, 60(11- 12):906-914

51 Kanako O. Koyanagi, Masato Hagiwara, Takeshi Itoh, Takashi Gojobori, Tadashi Imanishi, (2005), Comparative genomics of bidirectional gene pairs and its implications for the evolution of a transcriptional regulation system, '6=6, 353(2):169-176

52 Kanegae Hiromi, Miyoshi Kazumaru, Hirose Tetsuro, Tsuchimoto Suguru, Mori Masaki, Nagato Yasuo, Takano Makoto, (2005), Expression of rice sucrose non-fermenting-1 related protein kinase 1 genes are differently regulated during the caryopsis development, ,;2=B ,9EA:>;>8E 2=5 #:>496<:AB@E, 43:669-679

53 Kaneko Hiroyuki, Kazuhiro Kikuchi, Noguchi Junko, Ozawa Manabu, Ohnuma Katsuhiko, Maedomari Naoki, Kashiwazaki Naomi, (2006), Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice, -6?@>5C4B:>=, 131:279-288

54 Kapoor Meenu, Baba Akiko, Kubo Kenichi, Shibuya Kenichi, Matsui Keisuke, Tanaka Yoshikazu, Takatsuji Hiroshi, (2005), Transgene-triggered, epigenetically regulated ectopic expression of a flower homeotic gene pMADS3 in Petunia., ,;2=B )>C@=2;, 43:659-661

55 Katoh Shizue, Murata Katsuyoshi, Kubota Yoshiki, Kumeta Hiroyuki, Ogura Kenji, Inagaki Fuyuhiko, Asayama Munehiko, Katoh Etsuko, (2005), Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli, ,@>B6:= %D?@6AA:>= 2=5 ,C@:7:42B:>=, 43:149-156

56 Katoh Shizue, Tsunoda Yuki, Murata Katsuyoshi, Minami Eiichi, Katoh Etsuko, (2005), Active-site residues and amino acid specificity of the E2-binding RING-H2 finger domain, /96 )>C@=2; >7 3:>;>8:42; 496<:AB@E,

57 Katou Shinpei, Karita Eri, Yamakawa Hiromoto, Seo Shigemi, Mitsuhara Ichiro, Kuchitsu Kazuyuki, Ohashi Yuko, (2005), Catalytic activation of the plant MAPK phosphatase NtMKP1 by its physiological substrate salicylic acid- induced protein kinase but not by calmodulins, )>C@=2; >7 #:>;>8:42; $96<:AB@E, 280(47):39569-39581

58 Katsuyam Susumu, Tanaka Shinichiro, Omuro Naoko, Takabuchi Lisa, Daimon Takaaki, Imanishi Shigeo, Yamashita Shuichi, Iwanaga Masashi, Mita Kazuei, Maeda Susumu, Kobayashi Masahiko, Shimada Toru, (2005), Novel Macula-Like Virus identified in Bombyx mori Cultured Cells, )>C@=2; >7 0:@>;>8E, 79(9):5577-5584

59 Kawabata Hiroyuki, Niwa Atsuko, Tsuji-Kawahara Sachiyo, Uenishi Hirohide, Iwanami Norimasa, Matsukuma Hideaki, Abe Hiroyuki, Tabata Nobutada, Matsumura Haruo, Miyazawa Masaaki, (2006), Peptide-induced immune protection of CD8+ T cell-deficient mice against Friend retrovirus-induced disease, (=B6@=2B:>=2; (<;>8E,18 (1):183-198

60 Kawahigashi Hiroyuki, Hirose Sakiko, Ookawa Hidero, Ookawa Yasunobu, (2005), Phytoremediation of metolachlor by transgenic rice plants expressing human CYP2B6, )>C@=2; >7 "8@:4C;BC@2; 2=5 &>>5 $96<:AB@E, 53:9155-9160

61 Kawahigashi Hiroyuki, Hirose Sakiko, Ookawa Hidero, Ookawa Yasunobu, (2005), Transgenic rice plants expressing human CYP1A1 remediate the triazine herbicides atrazine and simazine, )>C@=2; >7 "8@:4C;BC@2; 2=5 &>>5 $96<:AB@E, 53:8557-8564

62 Kawahigashi Hiroyuki, Hirose Sakiko, Ozawa Kenjiro, Ido Yoshiko, Kojima Misaki, Ookawa Hidero, Ookawa Yasunobu, (2005), Analysis of substrate specificity of pig CYP2B22 and CYP2C49 towards herbicides by transgenic rice plants., /@2=A86=:4 -6A62@49, 14:907-917

63 Kayukawa Takumi, Chen Bin, Miyazaki Shoichiro, Itoyama Kyo, Shinoda Tetsuro, Ishikwa Yukio, (2005), Expression of mRNA for the t-complex polupeptide-1, a subunit of chaperonin CCT, is upregulated in association with increased cold hardiness in Delia antiqua, $6;; .B@66 ! $92?6@>=6A, 10(3):204-210

64 Khan Md Monowar Karim, Jan Asad, Karibe Hideji, Komatsu Setsuko, (2005), Identification of phosphoproteins regulated by gibberellin in rice leaf sheath, ,;2=B *>;64C;2@ #:>;>8E, 58:27-40

65 Khan Monowar, Takasaki Hironori, Komatsu Setsuko, (2005), Comprehensive phosphoproteome analysis in rice and identification of phosphoproteins responsive to defferent hormone/stresses, )>C@=2; >7 ,@>B6><6 -6A62@49,4 (5):1592-1599

&++0(* ')-,./ "!!# %$ 68 Kikuchi Kazuhiro, Kaneko Hiroyuki, Nakai Michiko, Noguchi Junko, Ozawa Manabu, Ohnuma Katsuhiko, Kashiwazaki Naomi, (2006), In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice, ';@=:/8 ;4 +3<=;2@1?7;: /:2 $3A38;<93:?, 51(6):51-57

67 Kikawada Takahiro, Minakawa Noboru, Watanabe Masahiko, Okuda Takashi, (2005), Factors inducing successful anhydrobiosis in the African chironomid Polypedilium vanderplanki: significance of the larval tubular nest, &:?35=/?7A3 /:2 #;9

69 Kikui Satoshi, Sasaki Takayuki, Maekawa Masahiko, Miyao Akio, Hirochika Hirohiko, Matsumoto Hideaki, Yamamoto Yoko, (2005), Physiological and genetic analyses of aluminium tolerance in rice, focusing on root growth during germination, ';@=:/8 ;4 7:;=5/:71 07;16397>?=B, 99(9):1837-1834

70 Kiribuchi Kyoko, Jikumaru Yusuke, Kaku Hanae, Minami Eiichi, Hasegawa Morifumi, Kodama Osamu, Seto Hideharu, Okada Kazunori, Nojiri Hideaki, Yamane Hisakazu, (2005), Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants, "7;>173:13 "7;?316:;8;5B /:2 "7;16397>?=B, 69(5):1042

71 Kitagawa N Washio T, Kosugi S, Yamashita T, Higashi K, Yanagawa H, Higo K, Satoh K, Ohtomo Y, Sunako T, Murakami K, Matsubara K, Kawai J, Carninci P, Hayashizaki Y, Kikuchi S, Tomita M., (2005), Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice (Oryza sativa).,, 21 no9

72 Kobayashi Toshihiro, Niino Takao, Kobayashi Masatomo, (2005), Simple cryopreservation protocol with an encapsulation technique for tobacco BY-2 suspension cell cultures, *8/:? "7;?316:;8;5B, 22(2):105-112

73 Komatsu Setsuko, (2005), Rice Proteome Database: a step toward functional analysis of the rice genome, *8/:? (;831@8/= "7;8;5B, 59:179-190

74 Komatsu Setsuko, Abbasi Fida, Kobori Eiko, Fujisawa Yukiko, Kato Hisatoshi, Iwasaki Yukimoto, (2005), Proteomic analysis of rice embryo: An approach for investigating G protein-regulated proteins, *=;?3;971>, 5:3932 -3941

75 Komatsu Setsuko, Zang Xin, Tanaka Naoki, (2006), Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice, ';@=:/8 ;4 *=;?3;93 +3>3/=16, 5(2):270-276

76 Konihsi Hirosato, Maeshima Masayoshi, Komatsu Setsuko, (2005), Characterization of vacuolar membrane proteins changed in rice root treated with gibberellin, ';@=:/8 ;4 *=;?3;93 +3>3/=16, 5(5):1775-1780

77 Konno K., Ono H., Nakamura M., Tateishi K., Hirayama H., Tamura Y., Hattori M., Koyama A., Kohno K. (2006) Mulberry latex rich in anti-diabetic sugar-mimic alkaloids forces dieting on caterpillars. *=;13327:5> ;4 ?63 )/?7;:/8 !1/239B ;4 ,173:13> ;4 ?63 .:7?32 ,?/?3> ;4 !93=71/ 103: 1337-1341

78 Kotaki Toyomi, (2005), Oosorption in the stink bug, Plautia crossota stali: follicle cells as the site of protein degradation., &:A3=?30=/?3 +3<=;2@1?7;: /:2 $3A38;<93:?, 47(3):147-153

79 Kurusu Takamitsu, Yagala Toshikazu, Miyao Akio, Hirochika Hirohiko, Kuchitsu Kazuyuki, (2005), Identification of putative voltage-gated Ca2 channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen- activated protein kinase activation in rice, *8/:? ';@=:/8, 42(6):798-809

80 Kuwana Takashi, Kawashima Takaharu, Naito Mitsuru, Yamashita Hiroaki, Matsuzaki Masaharu, Takano Toshinori, (2006), Conservation of a threatened indiginous fowl (Kureko Dori) using the germline chimeras transplanted from primordial germ cells, ';@=:/8 ;4 *;@8?=B ,173:13, 43(1):60-66

81 Maeda Taro, Takabayashi Junji, (2005), Effects of Foraging Experiences on Residence Time of the Predatory Mite Neoseiulus womersleyi in a Prey Patch, ';@=:/8 ;4 &:>31? "36/A7;=, 18(3):323-333

82 Maegawa Kenichi, Itoyama Kyo, Shinoda Tetsuro, Yoshimura Tetsuo, Kobayashi Jun, (2005), Effects of medium compositions on Autographa californica nucleopolyhedrovirus replication and cellular gene expression in an Antheraea pernyi cell line, ';@=:/8 ;4 &:>31? "7;?316:;8;5B /:2 ,3=71;8;5B, 74(2):63-73

83 Matsumoto Takashi, Wu Jianzhong,Katayose Yuuichi, Mizuno Hiroshi, Yamamoto Kimiko, Baltazar A. Antonio, Nagamura Yoshiaki, Yamamoto Sin-ichi, Yano Masahiro, Sasaki Takuji, (2005), The map-based sequence of the rice genome, )/?@=3, 436(7052):793-800

84 Matsumura H, S . Kawasaki, R. Takahashi, (2005), Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean., -63;=3?71/8 /:2 !<<8732 %3:3?71>, 110(7):1203-9

$$ %**/') &(,+-. "!!# 85 Matsuyama Shuichi, Ohkura Satoshi, Sakurai Katsuyasu, Tsukamura Hiroko, Maeda Kei-Ichiro, Okamura Hiroaki, (2005), Activation of melanocortin receptors accelerates the gonadotropin-releasing hormone pulse generator activity in goats, ,5B?=@395<35 +5AA5?@, 383(3):289-294

86 Mikawa Satoshi, Hayashi Takeshi, Nii Masahiko, Shimanuki Shinichi, Morozumi Takeya, Awata Takashi, (2005), Two quantitative trait loci on Sus scrofa chromosomes 1 and 7 affecting the number of vertebrae, *=B?<1: =6 #<9;1: /395<35, 83(10):2247-2254

87 Mikawa Satoshi, Kishi hisashi, Ogawa Hidehiko, Iga Kosuke, Uenishi Hirohide, Yasue Hiroshi, (2005), Analysis of recessive lethality on swine chromosome 6 in a Goettingen miniature resource family, #<9;1: (5<5A93@, 36(5):376 -380

88 Mitsuhara Ichiro, Yatou Osamu, Iwai Takayoshi, Naito Yumi, Nawa Yoshiko, Ohashi Yuko, (2006), Genetic studies of transgenic rice plants overproducing an antibacterial peptide show that a high level of transgene expression did not cause inferior effects on host plants, -:1

89 Miura-Ohnuma Jun, Nonaka Tsuyoshi, Murata Katsuyoshi, Kita Akiko, Miki Kunio, Katoh Etsuko, (2005), Improved expression!purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice(Oryza sativa), #3A1 %?D@A1::=7?1>8931 /53A9=< ', F61:1058-1061

90 Miyata Maiko, Komori Toshiyuki, Yamamoto Toshio, Ueda Tadamasa, Yano Masahiro, Nitta Naoto, (2005), Fine scale and physical mapping of Spk(t) controlling spreading stub in rice., $?5549<7 /395<35, 55(2):237-239

91 Murakami Ritsuko, Kobayashi Takao, Takahashi kokichi, (2005), Myrothecium leaf spot of mulberry caused by Myrothecium verrucaria, *=B?<1: =6 (5<5?1: -:1

92 Murakami Ritsuko, Shirata Akira, (2005), Possible roles of myrotoxin B produced by pathogen of Myrothecium leaf spot of mulberry, /5?93=:=791, 45(1):33-43

93 Murakami S, Matsui Katsuhiro, Komatsuda Takao, Yoshihiko Furuta, (2005), AFLP-based STS markers closely linked to a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley., -:1

94 Murayama Seiji, Handa Hirokazu, (2005), Central region of wheat UCP acts as a major signal for protein transport to mitochondria, but both distal regions can also drive a protein import to mitochondria., $?5549<7 /395<35, 55(4): 447-452

95 Nagata Toshifumi, Todoriki Setsuko, Kikuchi Shoshi, (2005), Levels of control of active oxygen species in Arabidopsis, .5@! #4C 9< #7?93B:AB?1: "'==4 %85; ., 6:1-13

96 Nagata Toshifumi, Iizumi Shigemi, Sato Kouji, Ooka Hisako, Kawai Jun, Piero Carninci, Hayashizaki Yoshihide, Ohtomo Yasuiro, Murakami Kazuo, Matsubara Kenichi, Kikuchi Shoshi, (2005), Comparative molecular biological analysis of plant and animal signal transduction genes, Res. Adv. )< #7?93B:AB?1: "'==4 %85; ., 6:55-78

97 Naito Mitsuru, Harumi Takashi, Matsubara Yuko, Kuwana Takashi, (2005), An attempt at fusing primordial germ cell with embryonic blood cell in chickens using inactivated Sendai virus or electri stimulation, *=B?<1: =6 -=B:A?D /395<35, 42(4):369-374

98 Naito Mitsuru, Sano Akiko, Kawashima Takaharu, Nakamichi Hiroyuki, Harumi Takashi, Matsubara Yuko, Kuwana Takashi, (2005), Culture of chicken embryos obtained from the anterior region of the magnum of the oviduct after removing a thin layer of dense albumen capsule from the ovum, *=B?<1: =6 -=B:A?D /395<35, 42(2):140-144

99 Nakajima Yoshiro, Saido-Sakanaka Hisako, Ogihara Kazuma, Taylor DeMar, Yamakawa Minoru, (2005), Antibacterial peptides are secreted into the midgut lumen to provide antibacterial midgut defense in the soft tick, Ornithodoros moubata (Acari: Argasidae), #>>:954 &

100 Nakashima Nobuhiko, Kawahara Nobuyuki, Omura Toshihiro, Noda Hiroaki, (2006), Characterization of a novel satellite virus and a strain of Himetobi P virus (Dicistroviridae) from the brown planthopper, Nilaparvata lugens., *=B?<1: =6 )

101 Nakayama Hiroki, Nagamine Tsukasa, Hayashi Nagao, (2005), Genetic variation of blast resistance in foxtail millet (Setaria italica (L.) P. Beauv.) and its geographic distribution, (5<5A93 .&@=B?35@ 1<4 %?=> &C=:BA9=<, 52:863- 868

102 Nakayama Shigeki, (2005), Molecular cytological diversity in cultivated rice Oryza sativa subspecies japonica and indica., $?5549<7 /395<35, 55(4):425-430

&++0(* ')-,./ "!!# $% 103 Nathalie Choisne, Wu Jianzhong, Mizuno Hiroshi, Matsumoto Takashi, Sasaki Takuji, (2005), The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications, %+& "%9=+54 35

104 Ni Jinfeng, Takehara Motomi, Watanabe Hirofumi, (2005), Heterologous overexpression of a mutant termite cellulase gene in Esherichia coli by DNA shuffling of four orthologous parental cDNAs, %?9A9@8 .=B:A?C 1395<35,69 (9):1711-1720

105 Nishibori Masahide, Shimogiri Takeshi, Hayashi Takeshi, Yasue Hiroshi, (2005), Molicular evidence For hybridization of species in the genus Gallus except for Gallus varius, $<9;2: )5<5A93@, 36:367-375

106 Nojiri Hideaki, Ashikawa Yuji, Noguchi Haruko, Nam Jeong-Won, Urata Masaaki, Fujimoto Zui, Uchimura Hiromasa, Terada Tohru, Nakamura Shugo, Omori Toshio, (2005), Crystal structure of the terminal oxygenase component of carbazole 1,9a-dioxygenase, a non-heme iron oxygenase system catalyzing the novel angular dioxygenation for carbazole and dioxin, *=B?<2: =6 +=:53B:2? %9=:=7C, 351(2):355-370

107 Nomura Mika, Higuchi Tomonori, Ishida Yuji, Ohta Shozo, Komari Toshihiko, Imaizumi Nobuyuki, Miyao Tokutomi Mitsue, Matsuoka Makoto, Tajima Shigeyuki, (2005), Differential expression pattern of C4 bundle sheath expression genes in rice, a C3 plant, .:2

108 Nomura Mika, Higuchi Tomonori, Katayama Kenichi, Taniguchi Mitsutaka, Miyao Tokutomi Mitsue, Matsuoka Makoto, Tajima Shigeyuki, (2005), The promoter for C4-type mitochondrial aspartate aminotransferase does not direct bundle sheath-specific expression in transgenic rice plants, .:2

109 Nonaka Sumie, Hashizume Tsutomu, Etsuko Kasuya, (2005), Effects of leptin on the release of luteinizing hormone, growth hormone and prolaction from cultured bovine anterior pituitary cells., $<9;2: 1395<35 *=B?<2:, 76: 435-440

110 Nonaka Tsuyoshi, Kita Akiko, Miura Ohnuma Jun, Katoh Etsuko, Inagaki Noritoshi, Yamazaki Toshimasa, Miki Kunio, (2005), Crystal structure of putative N-acety1-gamma-glutamy1-phosphate reductase(AK071544) from rice(Oryza sativa), PROTEINS:Structure, (B<3A9=

111 Ochiai Hrokazu, Inoue Yasuhiro, Takeya Masaru, Sasaki Aeni, Kaku Hisatoshi, (2005), Genome sequence of Xanthomonas oryzae pv. oryzae suggests contribution of large numbers of effector genes and insertion sequences to its race diversity, *2>2< $7?93B:AB?2: 05@52?38 /B2?A5?:C, 39(4):275-287

112 Oizumi Yuki, Hemmi Hikaru, Minami Masayoshi, Asaoka Ai, Yamakawa Minoru, (2005), Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura, %9=385;932 5A %9=>8C@932 $3A2, 1752:83-92

113 Okuizumi Hisato, Takamiya tomoko, Saito Akira, DomonEiji, Iimure Kazuhiko, Tomioka Keisuke, Kawase Makoto, Ohtake Yuhko, Murakami Yasufumi, Okamoto Hiroyuki, (2006), Development of a new cultivar-discrimination method based on DNA polymorphism in a vegetatively propagated crop, *2>2< $7?93B:AB?2: 05@52?38 /B2?A5?:C, 40:65-69

114 Ookawara Kyousuke, Akino Junji, (2005), Seed cleaning behavior by tropical ants and its anti-fungal effect, *-B?<2: =6 'A8=:=7C, 23:93-98

115 Ookura Tetsuya, Komatsu Setsuko, Kawamura Yukio, Kasamo Kunihiro, (2005), A 55-kDa calcium dependent protein kinase phosphorylated Thr residues from the auto-regulatory domein of plasma membrane H+-ATPase in rice, *2>2< $7?93B:AB?2: 05@52?38 /B2?A5?:C, 39(2):99-104

116 Ohyanagi Hajime, Tanaka Tsuyoshi, Sakai Hiroaki, Baltazar A. Antonio, Nagamura Yoshiaki, Itoh Takeshi, Sasaki Takuji, (2006), The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information, ,B3:593 $394@ 05@52?38, 34(DB Issue):D741-D744

117 Osakabe Keishi, Ichikawa Hiroaki, Toki Seichi, (2005), The mutant form of acetolactate synthase genomic DNA from rice is an efficient selectable marker for genetic transformation, +=:53B:2? %?5549<7, 6:313-320

118 Osakabe Keishi, Ichikawa Hiroaki, Toki Seichi, Yamanouchi Hiroaki, Takyuuu Toshio, Yoshioka Terutaka, (2005), Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells., .:2

119 Otsuguro Ken-ichi, Gautam Shree Hari, Ito Shigeo, Habara Yoshiaki, Saito Toshiyuki, (2005), Characterization of forskolin-induced Ca2+ signals in rat olfactory receptor neurons., *=B?<2: =6 .82?;23=:=7932: 1395<35@, 97(4): 510-518

$! %**/') &(,+-. "!!# 120 Ozawa Kenjiro, Kawahigashi Hiroyuki, (2005), Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium- mediated transformation in rice (Oryza sativa L.)., .:1

121 Pillai Ajitha, Ueno Satoshi, Zhang Hong, Lee Jae-Min, Kato Yusuke, (2005), Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum, $9=385;931: ,=B?<1:, 390:207-214

122 Saido-Sakanaka Hisako, Ishibashi Jun, Momotani Eiichi, Yamakawa Minoru, (2005), Protective effects of synthetic antibacterial oligopeptides based on the insect defensins on Methicillin-resistant Staphylococcus aureus in mice, &5C5:=>;51?1A9C5 +;;B<=:=7E, 29:469

123 Saido-Sakanaka Hisako, Momotani Eiichi, Yamada Hiromi, Ishibashi Jun, Tsubouchi kozo, Yamakawa Minoru, (2005), Effect of a synthetic peptide designed on the basis of the active site of insect defensin on the proliferation of Methicillin-resistant Staphilococcus aureus under the conditions for external application, ,=B?<1: =6 9<@53A $9=A538<=:=7E 1<4 /5?93=:=7E, 74:15-20

124 Saito Toshiyuki, Watanabe Yasuko, Nemoto Tetsu, Kasuya Etsuko, Sakumoto Ryosuke, (2005), Radiotelemetry recording of electroencephalogram in piglets during rest., .8E@9=:=7E " $581C9=?, 84(5):725-731

125 Sandal, N., Umehara, Y., Kumagai, H., Murakami, Y., Imaizumi-Anraku, H., Chen, W.L., Shibata, S., Shakhawat- Hossain, M., Kouchi,H., Kawasaki, S., Stougaard, J., (2006), Genetics of symbiosis in Lotus japonicus: Recombinant inbred Lines, comparative genetic maps and map position of 35 symbiotic loci., -=:53B:1? .:1

126 Sano Akiko, Harumi Takashi, Hanzawa Shozo, Kawashima Takaharu, Nakamichi Hiroyuki, Matsubara Yuko, Naito Mitsuru, (2005), New screening test for male germline chimeric chickens by polymerase chain reaction using single nucleotide polymorphism detection primers, ,=B?<1: =6 .=B:A?E /395<35, 42(2):152-157

127 Sasaki Haruto, Aoki Naohiro, Sakai Hidemitsu, Hara Takahiro, Ishimaru Ken, Kobayashi Kazuhiko, (2005), Effect of CO2 enrichment on the translocation and partitioning of carbon at the early grain-filling stage in rice (Oryza sativa L.)., .:1

128 Sasaki Takuji, Baltazar Antonio, (2005), Where does the accurate rice genome sequence lead us?, .:1

129 Sato Masahiro, (2005), Comparison of different solving strategies in multiple-trait mixed model equations with iterative algorithms, ,=B?<1: =6 #<9;1: )5<5A93@, 33(1):11-18

130 Sato Masahiro, (2005), Effects of data structures on the solving of animal model equations by preconditioned conjugate gradient iteration, ,=B?<1: =6 #<9;1: )5<5A93@, 33(1):19-25

131 Sato Mitsuru, Iwaya Ryo, Ogihara Kazumasa, Sawahata Ryoko, Kitani Hiroshi, Chiba Joe, Kurosawa Yoshikazu, (2005), Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells, ('$/ ,=B?<1:, 272:6131-6144

132 Sato Toyozo, Inaba Tadaoki, Mori Mitsutaka, Watanabe Ken, Tomioka Keisuke, Hamaya Etsuji, (2005), Plectosporium blight of pumpkin and ranunculus caused by Plectosporium tabacinum, ,=B?<1: =6 )5<5?1: .:1

133 Sato Toyozo, Muta Taturo, Imamura Yukihisa, Nojima Hidenobu, Moriwaki Jouji, Yaguchi Yukio, (2005), Anthracnose of Japanese radish caused by Colletotrichum dematium, ,=B?<1: =6 )5<5?1: .:1

134 Satoh Masahiro, Ishii Kazuo, (2005), Sex differences in the infertility rate of the syrian hamster, 'D>5?9;5

135 Sekikawa Kenji, Nemoto Kiyomitsu, Degawa Masakuni, (2005), Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate., 0=D93=:=7931: /395<35@, 87(2):537-542

136 Senthil Natesan, Komatsuda Takao, (2005), Inter-subspecific maps of non-brittle rachis genes btr1/btr2 using occidental, oriental and wild barley lines., 'B>8EA931, 145:215-220

137 Shibata, S., Mitsui, H., Kouchi, H., (2005), Acetylation of a fucosyl residue at the reducing end of Mesorhizobium loti nod factors is not essential for nodulation of Lotus japonicus., .:1

&++0(* ')-,./ #!!$ %" 138 Shimanuki Shinichi, Mikawa Ayumi, Miyake Yuko, Hamasima Noriyuki, Mikawa Satoshi, Awata Takashi, (2005), Structure and polymorphism analysis of transforming growth factor beta receptor 1 (TGFBR1) in pigs, "8<274:8219 &4;4@82?, 43(9/10):491-500

139 Shimogiri Takeshi, Kiuchi Sachiko, Hiraiwa Hideki, Hayashi Takeshi, Takano Yuu, Maeda Yoshizane, Rohrer Garry A., Denis Milan, Yasue Hiroshi, (2006), Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse., #C@<64;4@82 1;3 &4;<:4 -4?41>27, 112:114-120

140 Shinkai Hiroki, Morozumi Takeya, Toki Daisuke, Eguchi-Ogawa Tomoko, Muneta Yoshihiro, Awata Takashi, Uenishi Hirohide, (2005), Genomic structure of eight porcine chemokine receptors and intergene sharing of an exon between CCR1 and XCR1, &4;4, 349:55-66

141 Shinkai Hiroki, Muneta Yoshihiro, Suzuki Kohei, Eguchi-Ogawa Tomoko, Awata Takashi, Uenishi Hirohide, (2006), Porcine Toll-like receptor 1, 6, and 10 genes: Complete sequencing of genomic region and expression analysis, )<942A91> '::A;<9<6C, 43(9):1474-1480

142 Shinohara Hiroshi, Matsumoto Mitsuharu, Kurisaki Jun-ichi, Mizumachi Koko, Kusakabe Takahiro, Sugimoto Yasushi, (2005), Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues, "8<274:821 4@ "8<=7C?821 !2@1, 1723:106-113

143 Shiomi Kunihiro, Fujiwara Yoshihiro, Atsumi Tsutomu, Kajiura Zenta, Nakagaki Masao, Tanaka Yoshiaki, Mizoguchi Akira, Yaginuma Toshinobu, Yamashita Okitsugu, (2005), Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori., %$". (;19, 272:3853-3862

144 Shirasawa Seo Naomi, Sano Yoshitaka, Nakamura Shigeo, Murakami Taka, Gotoh Yoko, Naito Yumi, (2005), The promoter of MILK vetch dwarf virus component 8 confers effective gene expression in both dicot and monocot plants, ,91;@ #499 -4=, 24:155-163

145 Shirasawa Seo Naomi, Sano Yoshitaka, Nakamura Shigeo, Murakami Taka, Seo Shigemi, Ohashi Yuko, Hashimoto Yoshifumi, Matsumoto Tsuguo, (2005), Characteristics of the promoters derived from the single-stranded DNA components of Milk vetch dwarf virus in transgenic tobacco, (;19 <5 &4;4>19 08><9<6C, 86:1851-1860

146 Somfai Tamas, Kikuchi Kazuhiro, Medvedev Sergey, Onishi Akira, Iwamoto Masaki, Fuchimoto Dai-ichiro, Ozawa Manabu, Noguc, (2005), Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro, !;8:19 -4=><3A2@8<; .284;24, 90:307-328

147 Somta Prakit, Kaga Akito, Tomooka Norihiko, Kashiwaba Kouichi, Isemura Takehisa, Chaitieng Bubpa, Srinives Peerasak, Vaughan Duncan Alexander, (2006), Development of an interspecific Vigna linkage map between Vigna umbellata (Thunb.) Ohwi & Ohashi and V. Nakashimae (Ohwi) Ohwi & Ohashi and its use in analysis of bruchid resistance and comparative gnomics, ,91;@ ">4438;6, 125:77-84

148 Sudo Jun-ichi, (2005), Apolipoprotein gene polymorphisms as cause of cholesterol QTLs in mice, (;19 <5 04@4>8;1>C )438219 .284;24, 67(6):583-589

149 Suzuki Misae, Misumi Koji, Ozawa Manabu, Noguchi Junko, Kaneko Hiroyuki, Ohnuma Katsuhiko, Fuchimoto Dai- ichiro, Onishi Akira, Iwamoto Masaki, Saito Norio, Nagai Takashi, Kikuchi Kazuhiro, (2006), Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum, /74>8<64;<9<6C, 65:374-386

150 Suzuki Mitsuteru, Akihama Tomoya, Ishikawa Masaya, (2005), Cryopreservation of encapsulated gentian axillary buds following 2 step-preculture with sucrose and desiccation., ,91;@ #499 /8??A4 1;3 +>61; #A9@A>4, 83:115-121

151 Suzuki Nobuhiro, Fujimoto Zui, Morita Takashi, Fukamizu Akiyoshi, Mizuno Hiroshi, (2005), pH-Dependent structural changes at Ca2+-binding sites of coagulation factor IX-binding protein, (;19 <5 )<942A91> "8<9<6C, 353(1):80-87

152 Suzuki Nobuhiro, Yamazaki, Yasuo, Fujimoto Zui, Morita Takashi, Mizuno Hiroshi, (2005), Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels, !2@1 #>C?@199<6>1=7821 .42@8<; % .@>A2@A>19 "8<9<6C 1;3 #>C?@1998D1@8<; #<::A;821@8<;?, 61(8):750-752

153 Suzuki Rintaro, Mori Youichi, Kamino Kei, Yamazaki Toshimasa, (2005), NMR assignment of the barnacle cement protein Mrcp-20k, (;19 <5 "8<:<942A91> *)-, 32:257

154 Taguchi-Shiobara Fumio, Kato Hiroshi, (2005), Alleles in three rice panicle genes, Dn1, Ur1 and Cl, and the effects of genetic backgrounds., -824 &4;4@82? *4B?94@@4>, 22:32-33

$" %**/') &(,+-. "!!# 155 Taguchi-Shiobara Fumio, Yamamoto Toshio, Yano Masahiro, Oka Seibi, (2006), Mapping QTLs that control the performance of rice tissue culture and evaluation of derived near-isogenic lines., 085=?5A932: 2<4 !>>:954 '5<5A93@, 112

156 Takabatake Reona, Seo Shigemi, Mitsuhara Ichiro, Tsuda Shinnya, Ohashi Yuko, (2005), Accumulation of the two transcripts of the N gene,conferring resistance to tobacco Mosaic Virus, is probably important for N Gene- dependent hypersensitive cell death, -:2

157 Takafuji Akio, Hinomoto Norihide, Shih Chain-Ing T., Gotoh Tetsuo, Ho Chyi-Chen, Wang Chien-Chih, (2005), Diapause characteristics of the Taiwanese pupulations of Tetranychus kanzawai Kishida and T. urticae Koch (Acari: Tetranychidae), -:2

158 Takagi Hidenori, Hiroi Takachika, Yang Lijun, Tada Yoshifumi, Yuki Yoshikazu, Takamura Kaoru, Ishimatsu Ryoutaro, Kawauchi Hideyuki, Kiyono Hiroshi, Takaiwa Fumio, (2005) A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responces. -?=35549<7@ =6 A85 ,2A9=<2: !3245;D =6 /395<35@ =6 A85 1<9A54 /A2A5@ =6 !;5?932 102: 17525-17530

159 Takahashi Hideyuki, Hotta Yuji, Hayashi Mitsunori, Kawai Maki, Komatsu Setsuko, Uchimiya Hirofumi, (2005), High throughput metabolome and proteome analysis of transgenic rice plants (Oryza sativa L.), -:2

160 Takahashi S, Furukawa T, Asano T, Terajima Y, Shimada H, Sugimoto A, Kadowaki K, (2005), Very close relationship of the chloroplast genomes among Saccharum species, 085=?5A932: 2<4 !>>:954 '5<5A93@, 110:1523- 1529

161 Takahashi Sakiko, Ishimaru Ken, Sasaki Takuji, Kishimoto Naoki, Kikuchi Shoshi, (2005), Microarray analysis of sink-source transition in rice leaf sheaths, "?5549<7 /395<35, 55:153-162

162 Takahashi Sakiko, Ogiyama Yuki, Kusano Hiroaki, Shimada Hiroaki, Kawamukai Makoto, Kadowaki Koh-ichi, (2006), Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant, &%"/ *5AA5?@, 580:955-959

163 Takano Makoto, Inagaki Noritoshi, Xie Xianzhi, Yuzurihara Natsu, Hihara Fukiko, Ishizuka Toru, Yano Masahiro, Nishimura Minoru, Miyao Akio, Hirochika Hirohiko, Shinomura Tomoko, (2005), Distinct and cooperative function of phytochromes A, B and C in the control of de-etiolation and flowering in rice, -:2

164 Takase Kenji, Hagiwara Kiyoshi, (2005), Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance, "9=385;9@A?D 2<4 #5:: "9=:=7D, 83(2):239-249

165 Takasu Yoko, Yamada Hiromi, Saito Hitoshi, Tsubouchi Kozo, (2005), Characterization of Bombyx mori sericins by the partial amino acid sequences, )=B?<2: =6 (<@53A "9=A538<=:=7D 2<4 /5?93=:=7D, 74:103

166 Takayuki Sakurai, Satoshi Watanabe, Minoru Kimura, Masahiro Sato, (2006), Strain difference in tolerance to low- temperature treatment of fertilized mouse oocytes, .5>?=4B3A9C5 +54939<5 2<4 "9=:=7D, 5:43-50

167 Takeda Kumiko, Tasai Mariko, Iwamoto Masaki, Akita Tomiji, Tagami Takahiro, Nirasawa Keijiro, Hanada Hirohumi, Onishi Akira, (2006), Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells, +=:53B:2? .5>?=4B3A9=< 2<4 $5C5:=>;5

168 Takenouchi Takato, Ogihara Kazumasa, Sato Mitsuru, Kitani Hiroshi, (2005), Inhibitory effects of U73122 and U 73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line., "9=385;932 5A "9=>8D@932 !3A2, 1726(2):177-186

169 Tamada Yasushi, (2005), New process to form a silk fibroin porous 3-D structure, "9=;23?=;=:53B:5@, 60(6): 3100

170 Tanaka Hiromitsu, Furukawa Seiichi, Nakazawa Hiroshi, Sagisaka Aki, Yamakawa Minoru, (2005), Regulation of gene expression of attacin, an antibacterial protein in the silkworm, Bombyx mori, )=B?<2: =6 (<@53A 0538<=:=7D 2<4 /5?93=:=7D, 74:45-56

171 Tanaka Hiromitsu, Yamamoto Masafumi, Moriyama Yuki, Yamao Masafumi, Furukawa Seiichi, Sagisaka Aki, Nakazawa Hiroshi, Mori Hajime, Yamakawa Minoru, (2005), A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori, "9=385;932 5A "9=>8D@932 !3A2, 1730:10-21

172 Tanaka Maiko, Ando Asako, Renard Christine, Chardon Patrick, Domukai Michiko, Okumura Naohiko, Awata Takashi, Uenishi Hirohide, (2005), Development of dense microsatellite markers in the entire SLA region and evaluation of their polymorphisms in porcine breeds, (;;B<=75<5A93@, 57(9):690-696

&++0(* ')-,./ "!!$ %# 173 Tanaka Maiko, Suzuki Kohei, Morozumi Takeya, Kobayashi Eiji, Domukai Michiko, Eguchi-Ogawa Tomoko, Shinkai Hiroki, Awata Takashi, Uenishi Hirohide, (2006), Genomic structure and gene order of swine chromosome 7q1.1-q 1.2, "968.7 &292>60=, 37:10-16

174 Tanaka Naoki, Mitsui Shigeyuki, Nobori Hiroya, Yanagi Koki, Komatsu Setsuko, (2005), Expression and function of proteins during development of the basal region in rice seedling, ):720?7.< ! $277?7.< +<:>2:860=, 4(6):796-808

175 Tanaka Naoki, Takahashi Hideyuki, Kitano Hidemi, Matsuoka Makoto, Akao Shoichiro, Uchimiya Hirofumi, Komatsu Setsuko, (2005), Proteome approach to characterize the methylmalonate-semialdehyde dehidrogenase that is regulated by gibberellin, (:?<9.7 :3 +<:>2:82 ,2=2.<05, 4(5):1575-1582

176 Teramoto Hidetoshi, Kakazu Aya, Asakura Tetsurou, (2005), Native structure and degradation pattern of silk sericin studied by 13C NMR spectroscopy., ).0<:8:720?72=, 39(1):6-8

177 Teramoto Hidetoshi, Miyazawa Mitsuhiro, (2005), Molecular Orientation Behavior of Silk Sericin Film as Revealed by ATR Infrared Spectroscopy, #6:8.0<:8:720?72=, 6(4):2049-2057

178 Teramoto Hidetoshi, Nakajima Ken-ichi, Takabayashi Chiyuki, (2005), Preparation of elastic silk sericin hydrogel, #6:=062902 #6:>2059:7:4A .91 #6:05286=>

179 Thadtha Pattanapong, Yamada Yoshiyuki, Uenishi Hirohide, Wada Yasuhiko, (2005), Molecular cloning of the gene encoding pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3) in pigs, (:?<9.7 :3 "968.7 &292>60=, 33:3-10

180 Toda Kyoko, Kawasaki Shinji, R . Takahashi, (2005), Structure of flavonoid 3-hydroxylase gene for pubescence color in soybean., $<:; -062902, 45(6):2212-2217

181 Tokai Takeshi, Fujimura Makoto, Inoue Hirokazu, Aoki Takayuki, Ohta Kunihiro, Shibata Takehiko, Yamaguchi Isamu, Kimura Makoto, (2005), Concordant evolution of trichothecene 3-O-acetyltransferase and an rDNA species phylogeny of trichothecene-producing and non-producing fusaria and other ascomycetous fungi., )60<:/6:7:4A, 151(2):509-519

182 Tokuda Gaku, Lo Nathan, Watanabe Hirofumi, (2005), Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites, +5A=6:7:460.7 %9>:8:7:4A, 30(4):372-380

183 Toshima Yoshiyuki, (2005), Unusual protein behavior illustrated with silk fibroin, #6:052860. 2> #6:;5A=60. "0>., 1713(1):1-4

184 Tsuge Seiji, Terashima Shinsaku, Furutani Ayako, Ochiai Hirokazu, Oku Takashi, Tsuno Kazunori, Kaku Hisatoshi, Kubo Yasuyuki, (2005), Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv. oryzae, (:?<9.7 :3 #.0>2<6:7:4A, 187(7):2308-2314

185 Tsukamoto Kazumi, Kuwazaki Seigo, Yamamoto Kimiko, Ohtani Toshio, Sugiyama Shigeru, (2006), Dissection and high-yield recovery of nanometre-scale chromosome fragments using an atomic-force microscope, *.9:>2059:7:4A, 17:1391-1396

186 Tsunoda Yuki, Sakai Nobuya, Kikuchi Koji, Kstoh Shizue, Akagi Kayo, Miura-Ohnuma Jun, Tashiro Yumiko, (2005), Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli, +<:>269 %@;<2==6:9 .91 +?<6360.>6:9, 42:268-277

187 Turuspekov Yerlan, Kawada Noriyuki, Honda Ichiro, Watanabe Yoshiro, Komatsuda Takao, (2005), Identification and mapping of QTL for rachis internode length associated with cleistogamy in barley., +7.9> #<221694, 124:542- 545

188 Uchiumi Takao, Komatsu Setsuko, Koshiba Tomokazu, Okamoto Takashi, (2006), Isolation of gamrtes and central cells from Oryza sativa L., -2@+7.9> ,2;<:1 , 19:37-45

189 Ueda Minoru, Tsutsumi Nobuhiro, Kadowaki Koh-ichi, (2005), Translocation of a 190-kb mitochondrial fragment into rice chromosome 12 followed by the integration of four retrotransposons, '9>2<9.>6:9.7 (:?<9.7 :3 #6:7:460.7 -062902=, 1(3):110-113

190 Ueda Tadamasa, Sato Tadashi, Hidema Jun, Hirouchi Tokuhisa, Yamamoto Kazuo, Kumagai Tadashi, Yano Masahiro, (2005), qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase., &292>60=, 171(4):1941-1950

191 Ueguchi-Tanaka Miyako, Ashikari Motoyuki, Nakajima Masatoshi, Itoh Hironori, Katoh Etsuko, Kobayashi Masatomo, Chow Teh-Yuan, Hsing Yue-ieb C.(Institute of Bo, (2005), GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin, 9.>?<2, 437:693-698

%# &++0(* ')-,./ "!!$ 192 Ueno Osamu, Agarie Sakae, (2005), Silica deposition in cell walls of the stomatal apparatus of rice leaves., +8/:? +=;2@1?7;: -173:13, 8(1):71-73

193 Ueno Osamu, Sentoku Naoki, (2006), Comparison of leaf structure and photosynthetic characteristics of C3 and C 4 Alloteropsis semialata subspecies, Plant, %388 /:2 &:A7=;:93:?, 29(2):257-268

194 Ueno Osamu, Yoshimura Yasuyuki, Sentoku Naoki, (2005), Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses., #::/8> ;4 $;?/:B, 96(5):863-869

195 Vassileva, V., Kouchi, H., Ridge, R.W., (2005), Microtubule dynamics in living root hairs: Transient slowing by Lipochitin Oligosaccharide Nodulation, +8/:? %388, 17:1777-1787

196 Vaughan Duncan Alexander, Kadowaki Koh-ichi, Kaga Akito, Tomooka Norihiko, (2005), On the phylogeny and biogeography onf the genus Oryza, $=3327:5 -173:13, 55(2):113-122

197 Vaughan Duncan Alexander, Kuroda Yosuke, Tomooka Norihiko, Kaga Akito, (2005), Genetic erosion in rice and its relatives: past, present and future, Expert Consultation on Genetic Erosion Methodologies and Indicators ICRISAT, Patancheru, Hyderabad, India, 19-21 december (2005),

198 Wang Pi Chao, Takezawa Toshiaki, (2005), Reconstruction of renal glomerular tissue using collagen vitrigel scaffold, );@=:/8 ;4 $7;>173:13 /:2 $7;3:57:33=7:5, 99(6):529-540

199 Wen Ying, Hatabayashi Hidemi, Arai Hatsue, Kitamoto K. Hiroko, Yabe Kimiko, (2005), Function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus, #<<8732 /:2 &:A7=;:93:?/8 *71=;07;8;5B, 71(6): 3192-3198

200 Yamada Manabu, Nakamura Kikuyasu, Saido-Sakanaka Hisako, Asaoka Ai, Yamakawa Minoru, Yamamoto Yu, Koyama Yukari, Hikosaka Kenji, Shimizu Akira, Hirota Yoshikazu, (2005), Therapeutic effect of modified oligopeptides from the beetle Allomirina dichotoma on Methicillin-resistant Staphylococcus aureus (MRSA) infection in mice, );@=:/8 ;4 .3?3=7:/=B *3271/8 -173:13>, 67(10):1005-1011

201 Yamaguchi Takahiro, Lee Dong Yeon, Miyao Akio, Hirochika Hirohiko, An Gynheung, Hirano Hiroyuki, (2006), Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa, +8/:? %388, 18:15-28

202 Yamaguchi Takeshi, Minami Eiichi, Ueki Jun, Shibuya Naoto, (2005), Elicitor-induced activation of phopholipases plays an important role for the induction of defense responses in suspension-cultured rice cells, +8/:? /:2 %388 +6B>7;8;5B, 46(4):579

203 Yamamoto Ryuji, Isobe Tamaki, Eguchi Tomoko, Tang Wei Ran, Kiyokawa Nobutaka, Amemiya Hiroshi, Fujimoto Junichiro, Sato Eimei, Takagaki Yohtaroh, Yasue Hiroshi, (2005), Porcine TCR CD3ζ-chain and η-chain, *;831@8/= (99@:;8;5B, 42:1485-1493

204 Yamamoto Ryuji, Uenishi Hiroshi, Hatsuse Hiromi, Sato Eimei, Awata Takashi, Yasue Hiroshi, Takagaki Yohtaroh, (2005), Jalpha-gene segment usage and the CDR3 diversity of porcine TCRalpha-chain cDNA clones from the PBL of a five-month-old pig and the thymus of a one-month-old pig., *;831@8/= (99@:;8;5B, 42:1375-1383

205 Yamamoto Ryuji, Uenishi Hiroshi, Hatsuse Hiromi, Sato Eimei, Awata Takashi, Yasue Hiroshi, Takagaki Yohtaroh, (2005), TRAV gene usage in pig T-cell receptor alpha cDNA, (99@:53:3?71>, 57:219-225

206 Yamanaka Naoki, Hua Yue Jin, Mizoguchi Akira, Watanaka Ken, Niwa Ryusuke, Tanaka Yoshiaki, Kataoka Hiroshi, (2005), Identification of a novel prothoracicostatic hormone and its receptor in the silkworm, Bombyx mori., );@=:/8 ;4 $7;8;571/8 %6397>?=B, 280(15):14684-14690

207 Yamasaki Chisato, Koyanagi O. Kanako, Fujii Yasuyuki, Itoh Takeshi, Roberto Barrero, Tamura Takuro, Yamaguchi- Kabata Yumi, Tanino Motohiko, Takeda Jun-ichi, Fukuchi Satoshi, Miyazaki Satoru, Nomura Nobuo, Sugano Sumio, Imanishi Tadashi, Gojobori Takashi, (2005), Investigation of protein functions through data-minig on integrated human transcriptome database, H-Invitational database (H-InvDB), '3:3, 364:99-107

208 Yamazaki Toshimasa, Furuya Hidemine, Watanabe Takeshi, Miyachi Sayaka, Nishiuti Yuji, Nishio Hideki, Abe Akihiro, (2005), Quantitative analysis of helix-coil transition of block copolypeptide, Glu12-Ala12, by combined use of CD and NMR spectroscopy, $7;<;8B93=>!+3

209 Yang Guangxiao, Inoue Akihiko, Takasaki Hironori, Kaku Hisatoshi, Akao Shoichiro, Komatsu Setsuko, (2005), A proteomic approach to analyze auxin- and zinc-responsive protein in rice, );@=:/8 ;4 +=;?3;93 ,3>3/=16, 4(2): 456-463

&++0(* ')-,./ "!!$ %# 210 Yasue Hiroshi, Kiuchi Sachiko, Hiraiwa Hideki, Ozawa Akihito, Hayashi Takeshi, (2006), Assignment of 101 genes localized in HSA10 to a swine RH (IMpRH) map to generate a dense human-swine comparative map., %GC?86>6C:4 2>5 (6>?=6 .6B62A49, 112:121-125

211 Yasui Hiroe, Wakamura Sadao, (2006), Behavioral response in feeding to green color as visual stimulus with two lepidopteran larvae, Spodoptera litura (Fabricius)(Noctuidae) and basalis pryeri Druce (Geometridae), #@@<:65 '>C?=?5 1??

212 Yasukochi Yuji, Banno Yutaka, Yamamoto Kohji, Goldsmith M. R., Fujii Hiroshi, (2005), Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28)., (6>?=6, 48(4):626-629

213 Yasuo Shinobu, Ohkura Satoshi, Okamura Hiroaki, Yoshimura Takashi, (2006), Long-day suppressed expression of type 2 deiodinase gene in the mediobasal hypothalamus of the Saanen goat, a short-day breeder: Implication for seasonal window of thyroid hormone action on reproductive neuroendocrine axis., '>5?4A:>?

214 Yoshido Atsuo, Bando Hisanori, Yasukochi Yuji,,Sahara Ken, (2005), The Bombyx mori karyotype and the assignment of linkage groups., (6>6C:4B, 170(6):675-685

215 Yoshii Tsuyoshi, Kuji Naoaki, Komatsu Setsuko, Iwahashi Kazuhiro, Tanaka Yudai, Yoshida Hiroyuki, Wada Akira, Yoshimura Yasunori, (2005), Fine resokution of human sperm nucleoproteins by two-dimensional electrophoresis, ,?<64D<2A )D=2> .6@A?5D4C:?>, 11:677-681

216 Yoshikawa Manabu, Angela Peragine, Mee-Yeon Park, R. Scott Poethig, (2005), A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis, (6>6B " &6E6C, 19(16):2164-2175

217 Yoza Koh-ichi, Imamura Taro, Karl J. Kramer, Thomas D. Morgan, Nakamura Sumiko, Akiyama Kohki, Kawasaki Shinji, Takaiwa Fumio, Ohtsubo Kenichi, (2005), Avidin Expressed in transgenic rice confers resistance to the stored-product insect pests, Tribolium confusum and Sitotroga cereallella, $:?B4:6>46 $:?C649>?5 $:?496=:BCAG, 69(5):966-971

218 Zhang Hong, Morikawa Kazuya, Ohta Toshiko, Kato Yusuke, (2005), In vitro resistance to the CS alpha beta-type antimicrobial peptide ASABF-alpha is conferred by overexpression of sigma factor sigB in Staphylococcus aureus., *?DA>2< ?7 #>C:=:4A?3:2< %96=?C96A2@G, 55(5):686-691

219 Zink Sabrina, Mehlgarten Constance, Kitamoto K. Hiroko, Nagase Junko, Jablonowski Daniel, Dickson C. Robert, Stark J. R. Michael, Schaffrath Raffael, (2005), Mannosyl-diinositolphospho-ceramide, the major yeast plasma membrane sphingolipid, governs toxicity of Kluyveromyces lactis zymocin, 'D;2AG?C:4 %6<<, 4(5):879-889

2)Japanese language with English Summary

1 Fujii Kiyoshi, Hayano-SaitoYuriko, Sugiura Naoki, Hayashi Nagao, Izawa Toshihiko, Iwasaki Mabito, (2005), Quantitative evalution bof protective effect of Pb1 gene, conferring field resistance to rice panicle blast, using near -isogenic lines, $A665:>8 A6B62A49, 7(2):75-85

2 Gotoh Yohko, Niimi Shingo, (2005), Effects of supplementation with insulin and dexamethasone on morphologies and maintenance of rat hepatocytes onto lactose-silk fibroin conjugates during primary culture, +?3D>B9: .?>3D>B9D, 62(7):326-330

3 Hinomoto Norihide, Maeda Taro, (2005), Isolation of Microsatellite Markers in Neoseiulus womersleyi Schicha (Acari: Phytoseiidae), *! #42A?., 14(1):25-30

4 Ishii Kazuo, Satoh Masahiro, Sasaki Osamu, Takeda Hisato, Furukawa Tsutomu, Nagamine Yoshitaka, (2005), Relationships of dams body weight at parturition and reproductive traits in Syrian hamsters selected by weaning weight, 'F@6A:=6>C2< )6AE:E?A2, 29:73-77

5 Iwao-Mushika Junko, You Takahisa, Taketa Shin, Miyao Akio, Hirochika Hirohiko, Ichii Masahiko, (2005), Molecular genetic analysis of a Tos17-tagged mutant line related to root morphology in rice, $A665:>8 .6B62A49,7 (4):171-178

6 Kinoshita Haruo, Iizuka Masayo, Watase Hisaya, Nagayasu Ken-ichi, Kosegawa Eiichi, Hirokawa Masahiko, Tatematsu Ken-ichiro, (2005), Building of the data retrieval system for the genetic resources of silkworm, *! /:<; /4:! 0649! *@>., 14:29-36

7 Murakami Ritsuko, Shirata Akira, (2005), Myrotoxin B detection from mulberry leaves infected with Myrothecium roridum, cause Myroyhecium leaf spot of mulberry, and possible roles in pathogenicity. *@>! *! -9GC?@2C9?<., 71(2): 91-100

$# %**/') &(,+-. "!!# 8 Murakami Ritsuko, Koyama Akio, Shirata Akira, (2005), Grouping of pathogens of Myrothecium leaf spot of mulberry disease (Myrothecium roridum and M. verrucaria) isolated from the soil of mulberry fields in Japan, based on the antimicrobial substances produced by the fungus, '! ;/:3.! +.3! '97., 74(2·3):65-72

9 Murakami Ritsuko, Hiradate Syuntaro, Shirata Akira, (2005), Detection of toxic substances from diseased mulberry leaves inoculated with Myrothecium verrucaria, cause of Myrothecium leaf spot of mulberry, '97! '! )2><89-<285. 71(3):166-178

10 Nakamura Masato, Morioka Rika, Yayou Ken-ichi, Ito Shuichi, (2005), Changes in claw shape and load after hoof trimming in dairy cattle housed in a free stall system, (3287 $234=;-7 &-44-328, 76(2):183-190

11 Ohtake Yuhko, Takamiya Tomoko, Okuizumi Hisato, (2005), Detection of clonal polymorphism of plant using RLGS method, %(" )8>68:923;6, 13:62-64

12 Okuizumi Hisato, Takamiya Tomoko, Ohtake Yuhko, Yamashita Hideji, (2005), Development of the DNA marker in mat rush, Juncus effusus, %(" )8>68:923;6, 13:77-79

13 Shimizu Kunimitu, Hirokawa Masahiko, Tatematsu Ken-ichiro, Kosegawa Eiichi, (2005), Effective induction of egg diapause in multivoltine silkworm races by low temperature and shortphotoperiod rearing condition, '! +/:3.! +.3! '97., 74(1):1-7

14 Shimizu Kunimitu, Hirokawa Masahiko, Tatematsu Ken-ichiro, Kosegawa Eiichi, (2005), Genetic analysis of the new mutant, maternal brown egg of Shimizu, in the silkworm, Bombix mori, '! +/:3.! +.3! '97., 74(2·3):53-57

15 Shimizu Kunimitu, Hirokawa Masahiko, Tatematsu Ken-ichiro, Kosegawa Eiichi, (2005), Influence of environmental condition in embrionic stage on diapause and hatchability of the multivol silkworm, Bombix mori, '! +/:3.! +.3! '97., 74(2·3):73-79

16 Shirai Kayo, Souma Jun, Sumino Akio, Aoki Takayuki, (2005), Identification of deoxynivalenol- and nivalenol- chemotypes and lineages of Fusarium graminearum-complex isolates from Central Hokkaido, "77! */9

17 Tomioka Keisuke, (2005), Demonstration and diagnosis of new fungal diseases on flowers and vegetables, #=55/<37 ("*$ 08: ,/;

18 Tsukamoto Kazumi, Kuwazaki Seigo, Yamamoto Kimiko, Shichiri Motoharu, Yoshino Tomoyuki, Ohtani Toshio, Sugiyama Shigeru, (2005), Dissection and recovery of chromosome fragments by atomic force microscopy, '! +=:0-./ +.3! '97., 26(7):404-409

19 Yamakawa Naomi, Okuizumi Hisato, Takamiya Tomoko, Sang-gun Roh, Kagami Hiroshi, (2005), Chicken polymorphism detection using RLGS and novel method, MONIC, %(" )8>68:923;6, 13:116-119

&++0(* ')-,./ "!!# %$ &)3,)42 '.( %/./*1'0+ !$. #.*-,2+"

1 Berville Andre, Breton Catherine, Cunliffe Ken, Darmency Henri, Good Allen, Gressel Jonathan, Hall Linda, McPherson Marc, Medail Frederic, Pinatel Christian, Vaughan Duncan, Warwick Suzanne, (2005), Issues of ferality or potential for ferality in Oats, Olives, the Vigna group, ryegrass species, safflower, and sugarcane, (IGH +>I:DBKN :F= 8GDLFK>>IBFJE, 231-255

2 Ishibashi Jun, Tomie Tetsuya, Sawahata Ryoko, Saido-Sakanaka Hisako, Yamakawa Minoru, (2005), Chitin binding activity of Oryctes rhinoceros antimicrobial peptides, 4>HKB=> 6F<> $##%, 255-258

3 Yazaki Junshi, Kikuchi Shoshi, (2005), The genomic view of genes responsive to the antagonistic phytopormones, abscisic acid, and gibberellin, 40&27 -35132*6,72

4 Kaewsuralikhit, S., Yokoyama, T., Kouchi, H., Arima, Y., (2005), Comprehensive analysis of plant gene expression in soybean root nodules at different growth stages., 6GBD 6F<> :F= 4D:FK 2LKIBKBGF, 51:535-547

5 Kobayashi Satoe, Asaoka Ai, Miyazawa Mitsuhiro, Ishibashi Jun, Yamakawa Minoru, (2005), Interaction of an antimicrobial peptide moricin with lipid layers, 4>HKB=> 6F<> 2004, 251-254

6 Komatsu Setsuko, (2005), Plant Proteomics Databases: Their status in (2005), (LII>FK 'BGBF?GIE:KB

7 Komatsu Setsuko, (2005), Rice proteomics: A step toward functional analysis of stress responses, (LII>FK 4IGK>GEB

8 Komatsu Setsuko, Konishi Hirosato, (2005), Proteomics analysis of rice root proteins regulated by gibberellin, ,>FGEBGEB

9 Mizumachi Koko, Kurisaki Jun-ichi, (2005), Milk proteins, 2LKI:<>LKB<:D 4IGK>BFJ :F= 4>HKB=>J BF ->:DKA :F= )BJ>:J>, 431-444

10 Okuno Kazutoshi, (2005), Germplasm enhancement and breeding strategies for crop quality in Japan, 4D:FK 4IG=LF<>, 8(3(Special)):320-325

11 Onishi Akira, (2006), Cloning pigs from somatic cells and its applications, *HB@>F>KB< IBJCJ G?

12 Paterson Andrew, Freeling Michael, Sasaki Takuji, (2005), Grains of knowledge: Genomics of model cereals, ,>FGE> 5>J>:I

13 Peter Christian, Eric Carstens, Leslie Domier, John Johnson, Karlyn Johnson, Nobuhiko Nakashima, Paul Scotti, Frank van der Wilk, (2005), Family Dicistroviridae, 8BILJ 7:MGFGEN 8..., 783-788

14 Peter Christian, Eric Carstens, Leslie Domier, John Johnson, Karlyn Johnson, Nobuhiko Nakashima, Paul Scotti, Frank van der Wilk, (2005), Genus Iflavirus, 8BILJ 7:MGFGEN 8..., 779-782

15 Saito Toshiyuki, (2005), Consideration of adult neurogenesis from physiological and patho-physiological aspects, 1>=B<:D 6F<> 1GFBKGIBF@, 11(10):LE15

16 Sasaki Takuji, (2005), Steaming ahead, 7A> /:H:F /GLIF:D, 2(1):26-26

17 Sasaki Takuji, (2005), The complete rice genome sequences and its application to breeding and genetics, Rice is life:scientific perspectives for the 21st century. 4IG<>>=BF@J G? KA> 9GID= 5B<> 5>J>:II>F<>! 7JLCL;:! /:H:F, 66-69

18 Satoh Masahiro, (2005), Use of the Syrian hamster, Mesocricetus auratus, in selection for fertility in animal breeding 1. Physiology of reproduction for selection experiment, /GLIF:D G? &FBE:D ,>F>KB

19 Satoh Masahiro, Ishii Kazuo, Furukawa Tsutomu, (2005), Use of the Syrian hamster, Mesocricetus auratus, in selection for fertility in animal breeding 2. Selection for reproductive performance, /GLIF:D G? &FBE:D ,>F>KB

%$ &++0(* ')-,./ "!!# 20 Todoroki Junichi, Kaneko Hiroyuki, (2006), Formation of follicular cysts in cattle and therapeutic effects of controlled internal drug release, /FKHE;C F? 5>GHF=KL>CFGD>EJ, 52(1):1-11

21 Tomooka Norihiko, Vaughan Duncan Alexander, Kaga Akito, (2005), Mungbean [Vigna radiata (L.) Wilczek], ,*2*7.( 5*6385(*6! (-5313631* *2,.2**5.2,! &2) (534.14539*1*279FCKD> $ ,H;BE 0>@KD>I, 1:325-345

22 Vaughan Duncan, Kadowaki Koh-ichi, Kaga Akito, Tomooka Norihiko, (2005), Eco-genetic diversification in the genus Oryza: implications for sustainable rice production, 5B<> BI CB?>" IEJB?B< G>HIG>I ?FH JA> %$IJ <>EJKHM, 44-46

23 Vaughan Duncan, Sanchez Paulino, Ushiki Jun, Kaga Akito, Tomooka Norihiko, (2005), Asian rice and weedy rice - evolutionary perspectives, (HFG +>H;CBJM ;E= 9FCKEJ>>HBID, 257-277

24 Voughan Duncan Alexander, Tomooka Norihiko, Kaga Akito, (2005), Azuki Bean [Vigna angularis (Willd.) Ohwi & Ohashi], ,*2*7.( 5*6385(*6! (-5313631* *2,.2**5.2,! &2) (534.14539*1*279FCKD> $ ,H;BE 0>@KD>I, 1:347-359

25 Watanabe Masahiko, (2006), Anhydrobiosis in invertebrates, &GGCB>= *EJFDFCF@M ;E= :FFCF@M, 41(1):15-31

26 Yamazaki Toshimasa, (2005), NMR study of HIV-1 protease/Inhibitor complex, -M=HF@>E# ;E= -M=H;JBFE# 6>EIBJBL> 6JHK

%**/') &(,+-. "!!# $$ Main meetings related NIAS

The 13th International Genetic Resources Workshop on Rice Genome and Plant Genetic Resources NIAS sponsored the 13th NIAS International Workshop on Genetic Resources as parts of symposium sessions during the 10th International Congress of SABRAO (The Society for the Advancement of Breeding Research in Asia and Oceania) which was held on August 22-23, 2005 in Tsukuba, In this WS, two research highlights on Rice Genome: Challenges and Opportunies and Perspectives of Utilization and Conservation of Plant Genetic resources was discussed by 16 contributors (7 from outside Japan).

The NIAS-COE International Symposium: Present Status of Studies for Utilization of Insect Properties The NIAS-COE International Symposium was held on Octover 4-5, 2005, at Tsukuba International Congress Center (Epocal Tsukuba). The purpose of this symposium was to discuss recent progress in the fields of utilization of insect properties involved in Baculoviruses, gene expression, cell lines, tissue engineering, silk hydrogel, insect immunity, trypanosoma, development, and gene expression, 13 oral presentations, 6 from overseas and 7 from Japan, were given. This symposium stimulated the activities of many participants on utilization of insect properties and gave several new ideas as to the next step.

International Workshop on Animal Genome Analysis 2005: Analysis of MHC, MHC-related genes and disease resistance for animal breeding and selection International workshop on animal genome analysis 2005 was held under the title Analysis of MHC, MHC-related genes and disease resistance for animal breeding and selection on November 9, 2005 at Jitensha Kaikan in Tokyo with 81 participants. Eight invited speakers (1 from overseas and 7 Japanese) gave presentations on genome structure and function in infectiondefence of MHC, MHC -related genes.

44th Gamma-Field Sympo (Mito, July 12-13, 2005)

2005 Silk Summer Seminar in Okaya (July 28-29, 2005)

Rice Genome Sympo (Tsukuba, March 22, 2006)

"!! %**/') &(,+-. #!!$ Executive Members and Research Staff Members

"*0 ., )*/+- '%! &$$(#

Executive Members President Ishige Teruo Vice President Sasaki Takuji Vice President Natori Shinji Auditor Horio Yosinori Auditor Motoi Yoshiko Research Staff Department of Research Planning and Director Shinbo Hiroshi Coordination Domonn Eiji Research Planner Hirai Kazuo Research Planner Mitsuhashi Hatsuhito 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 Ohura Masanobu Imai Tsuneo Office for GMO Research and Development Head Tabei Yutaka Okuizumi Hisato 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 Center Director Obata Taro Genome Research Department Director Research Leader Yasue Hiroshi Plant Genome Laboratory Head Matsumoto Takashi Katayose Yuuichi Wu Jianzhong Mizuno Hiroshi Animal Genome Laboratory Head Awata Takashi Hamajima Noriyuki Hayashi Takeshi Mikawa Satoshi Uenishi Hirohide Sato Shuji Insect Genome Laboratory Head Mita Kazuei Kadono Keiko

%**/') &(,+-. #!!$ "!" Yamamoto Kimiko Yasukochi Yuji Bioinfomatics Laboratory Head Ito Takeshi Maeda Miki Numa Hisataka Tanaka Tsuyoshi DNA Bank Head Nagamura Yoshiaki Baltazar Alcaraz Antonio Rice Genome Resource Center Nagamura Yoshiaki Miyao Akio Baltazar Alcaraz Antonio 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 Head Sato Masahiro Takeya Masaru Genebank Director Okuno Kazutoshi Research Leader Shirata Kazuto Research Leader Niino Takao Plant Genetic Resources Laboratory Head Kawase Makoto Ebana Kaoru Fukuoka Shuuichi Uga Yuusaku Microorganism Genetic Resources Laboratory Head Sato Toyozou 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 Developmental Mechanisms Laboratory Head Myohara Maroko Nakao Hajime Kato Yusuke Hatakeyama Masatsugu Development and Differentiation Laboratory Head Tokunaga Tomoyuki 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 Laboratory Takahashi Toru Hosoe Misa Molecular Biology and Immunology Department Director Dohi Hiroshi Research Leader Sakurai Michiharu 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 Department Director Kawasaki Kenjiro Research Leader Inouchi Jun Insect Life-Cycles and Physiology Laboratory Head Tanaka Seiji Okuda Takashi Kotaki Toyomi Watanabe Masahiko Insect Nutrition and Metabolism Laboratory Head Nakamura Masatoshi Hirayama Chikara Kikawada Takahiro Insect Neurobiology Laboratory Head Asaoka Kiyoshi Inoue Hisashi Ichikawa Akio Kihara Mami Insect Behavior Laboratory Head Wakamura Sadao Akino Toshiharu

&++0(* ')-,./ #!!% "!$ Yasui Hiroe Animal Gene Function Laboratory 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 Insect Genetics and Evolution Department Director 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 Department Director Takeda Satoshi 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 Insect Biotechnology and Sericology Department Director Machii Hiroaki

"!$ &++0(* ')-,./ #!!% 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 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 Sugimoto Kazuhiko Yamanouchi Utako Yamamoto Shin-ichi Ueda Tadamasa Yamamoto Shin-ichi Epigenetics Laboratory Head Izawa Takeshi Okuizumi Hisato Gene Expression Laboratory Head Kikuchi Shoushi Mori Masaki Kishimoto Naoki Gene Regulation Laboratory Head Komatsu Setsuko Yoshikawa Manabu Asano Takayuki Biochemistry Department Director Crystallography Laboratory Takase Kenji Momma Mitsuru Fujimoto Zui Wako Toshiyuki Biophysics Laboratory Yamazaki Toshimasa Katoh Etsuko Suzuki Rintaro Glycobiology Laboratory Head Minami Eiichi

&++0(* ')-,./ #!!% "!$ 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 Imaizumi Haruko 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 Takagi Hidenori Plant Cell Engineering Laboratory Head Tabei Yutaka Hagio Takashi Habu Yoshiki Mochizuki Atsuko Molecular Breeding Laboratory Koga-Ban Yasunori Otake Yuko Biosystems Laboratory Head Handa Hirokazu Ogawa Taiichi Kawahigashi Hiroyuki Institute of Radiation Breeding Director Nakagawa Hitoshi

"!$ %**/') &(,+-. #!!$ Mutation Genetics Laboratory Head Nishimura Minoru Morita Ryohei Radiation Technology Laboratory Head Morishita Toshikazu Shimizu Akemi Mutation Breeding Laboratory Head Yoshioka Terutaka Takyu Toshio Yamanouchi Hiroaki

&++0(* ')-,./ #!!$ "!% Members of NIAS Evaluation Comittee

"*0 ., )*/+- '%!&$$(#

Ueda Ryu National Institute of Genetics

Kiguchi Kenji Shinshu University

Kouno Tomohiro Tokyo University of Agriculture

Takeda Kazuyoshi 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

"!% &++0(* ')-,./ #!!$ +,/(/),(- 02*12,*3

+9=65: 475< %$$& "(;<9: %$$& ! .5<68 %$$'#

thousands of yen

TOTAL BUDGET #$!#&(!%"&

OPERATING COSTS %!(%$!&*'

Personnel(407)* &!#'#!"+*

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

Administrators ,(87)** Administrators -(42)*** Researchers(273) * Number of persons shown in ( ) ** General administration *** Field management and transportations Administrative costs &*)!"$# Facilities Improvement Expense #"&!&))

RESEARCH PROMOTION COSTS (!%#%!)"*

Research Grant from MAFF $!++"!*$# Entrusted Research Expenses from MAFF %!%'&!"'+ Entrusted Research Expenses from MEXT %+(!($' Entrusted Research Expenses from others ()%!%"&

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

Entrusted Research Entrusted Research Expenses from others 673,304 (5.5%) Expenses from MEXT Personnel 396,625 (3.3%) 4,151,098 (34.1%)

Entrusted Research Expenses from MAFF 3,354,059 (27.6%)

Administrative costs 487,021(4.0%)

Research Grant from MAFF Facilities Improvement Expense 2,990,821(24.6%) 104,477(0.9%)

&++0(* ')-,./ #!!$ "!% ""! %**/') &(,+-. #!!$ Annual Report 2006

(Apr. 2005Mar. 2006) No.5

Published by National Institute of Agrobiological Sciences Kannondai, Tsukuba, Ibaraki, 305-8602, Japan TEL : 029(838)7406 FAX : 029(838)7408 URL : http://www.nias.affrc.go.jp/index_e.html