USOO657007OB1 (12) United States Patent (10) Patent No.: US 6,570,070 B1 Nakajima et al. (45) Date of Patent: May 27, 2003

(54) PRODUCTION OF PLANTS EITHER Kruse, E. et al; Coproporphyrinogen III oxidase from barley TRANSFORMED WITH THE and tobacco-Sequence analysis and initial expression Stud PROTOPORPHYRINOGEN IX BINDING ies, PLANTA (Heidelberg) 1995, vol. 196, No. 4, 1995, pp. SUBUNIT OF A MAGNESIUM CHELATASE 796-8O3. OR A FERROCHELATASE HAVING Kruse, E. et al; Isolation and characterization of tobacco INCREASED HERBICIDE RESISTANCE (Nicotiana tabacum) cDNA clones encoding proteins involved in magnesium chelation into protoporphyrin IX, (75) Inventors: Hiroki Nakajima, Nishinomiya; Akitsu Plant Molecular Biology Dec., 1997, vol. 35, No. 6, pp. Nagasawa, Takarazuka, both of (JP) 1053-1056. Madsen, O. et al., A Soybean coproporphyrinogen oxidase (73) Assignee: Sumitomo Chemical Company, gene is highly expressed in root nodules, Plant Molecular Limited, Osaka-fu (JP) Biology 1993, vol. 23, No. 1, 1993, pp. 35–43. (*) Notice: Subject to any disclaimer, the term of this G. della-Cioppa et al., Targeting a Herbicide-Resistant patent is extended or adjusted under 35 Enzyme from Escherichia Coli to Chloroplasts of Higher U.S.C. 154(b) by 0 days. Plants, Bio/Technology, vol. 5, pp. 579–584 (Jun. 1987). M. A. W. Hinchee et al., Production of Transgenic Soybean Plants. Using Agrobacterium-Mediated DNA Transfer, Bio/ (21) Appl. No.: 09/302,357 Technology, vol. 6, pp. 915-922 (Aug. 1998). (22) Filed: Apr. 30, 1999 M. De Block et al., Engineering herbicide resistance in plants by expression of a detoxifying enzyme, The EMBO (30) Foreign Application Priority Data Journal, vol. 6, No. 9, pp. 2513–2518 (1987). Apr. 30, 1998 (JP) ...... 10-120553 L. C. D. Gibson et al., Magnesium-protoporphyrin chelatase Oct. 2, 1998 (JP) ...... 10-281127 of Rhodobacter Sphaeroides: Reconstitution of activity by Nov. 20, 1998 (JP) ...... 10-330.981 combining the products of the bchH, -I, and -D genes Mar. 2, 1999 (JP) ...... 11-054730 expressed in Escherichia coli, Proc. Natl. Acad. Sci. USA, (51) Int. Cl." ...... C12N 15/82; C12N 15/84; vol. 92, pp. 1941-1944 (Mar. 1995). C12N 15/31 * cited by examiner (52) U.S. Cl...... 800/300; 800/278; 800/288; 800/294 Primary Examiner David T. Fox (58) Field of Search ...... 435/69.1, 468, ASSistant Examiner David H Kruse 435/418, 419, 320.1; 800/300, 278, 294, (74) Attorney, Agent, or Firm-Birch, Stewart, Kolasch & 288; 530/300, 370; 536/23.2, 23.7, 23.6 Birch, LLP (56) References Cited (57) ABSTRACT FOREIGN PATENT DOCUMENTS The present invention relates to a method of producing a herbicide resistant transgenic plant by transforming a plant EP O77O682 A2 5/1997 WO 95.34659 12/1995 with a nucleic acid molecule encoding a protoporphyrin IX WO WO97.04.088 2/1997 binding Subunit of a plant or photosynthetic microorganism WO WO9732011 9/1997 magnesium chelatase or a deletion variant having the WO WO9833927 8/1998 organelle transit Signal deleted. The invention also relates to WO WO984933O 11/1998 a method of producing a herbicide resistant transgenic plant by transforming a plant with a nucleic acid molecule encod OTHER PUBLICATIONS ing a plant ferrochelatase or a ferrochelatase deletion variant Yuan et al. Modification of plant components. Current having the organelle transit Signal deleted. In addition, the Opinions in Biotechnology 1997, 8:227–233. see p. 231.* invention relates to a method of producing a herbicide Papenbrock et al. Decreased and increased expression of the resistant transgenic plant by transforming a plant with a subunit CHL 1 diminishes Mg chelatase activity and reduces nucleic acid molecule encoding a peptide polymer. chlorophyll Synthesis in transgenic tobacco plants. The Plant Journal 2000, 22(2): 155–164.* 18 Claims, 6 Drawing Sheets U.S. Patent May 27, 2003 Sheet 1 of 6 US 6,570,070 B1

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PUH US 6,570,070 B1 1 2 PRODUCTION OF PLANTS EITHER gene of the photosynthetic bacterium Rhodobacter Sphaeroi TRANSFORMED WITH THE deS. lac pro represents the promoter Sequence of a lactose PROTOPORPHYRINOGEN IX BINDING operon. Amp' is an amplicillin resistant gene and ori is the SUBUNIT OF A MAGNESIUM CHELATASE replication origin. OR A FERROCHELATASE HAVING FIG. 4 is the restriction map of plasmid pBIBCH. bchH is INCREASED HERBICIDE RESISTANCE magnesium chelatase protoporphyrin IX binding Subunit gene of the photosynthetic bacterium Rhodobacter Sphaeroi deS. NP is the promoter Sequence of a nopaline Synthase BACKGROUND OF THE INVENTION gene, NT is the terminator Sequence of the nopaline Synthase gene, and 35S is the 35S promoter of cauliflower mosaic 1. Field of the Invention virus. NPTII represents a kanamycin resistant gene, and RB The present invention relates to a method for giving and LB represent right and left border sequences of T-DNA, resistance to weed control compounds to plants. respectively. FIG. 5 is the restriction map of plasmid pNO. NP is the 2. Disclosure of the Related Art promoter Sequence of a nopaline Synthase gene, NT is the Weed control is very important work for improving yields 15 terminator Sequence of the nopaline Synthase gene, and 35S and quality of cultivated plants. For this purpose, weed is the 35S promoter of cauliflower mosaic virus. NPTII control compounds Such as herbicides are mainly used. represents a kanamycin resistant gene, and RB and LB However, for using weed control compounds, it is not represent right and left border Sequences of T-DNA, respec always easy to distinguish cultivated plants from Weeds of tively. allied species to Selectively control only weeds. Then, pro FIG. 6 is the restriction map of plasmid pTCHLH. duction of plants having resistance to weed control com TCHLH is protoporphyrin IX binding subunit gene of pounds (hereinafter referred to as weed control compound tobacco magnesium chelatase whose chloroplast transit Sig resistance) has been attempted and Some resistant plants nal has been deleted. lac pro represents the promoter have been put to practical use. Sequence of a lactose operon. Am is an amplicillin resistant Recently, gene engineering techniques have been utilized 25 gene, Km is a kanamycin resistant gene and ori is the for producing plants having weed control compound replication origin. resistance. AS Such a technique, for example, Hinchee, M. A. FIG. 7 is the restriction map of plasmid pBITCHLH. W. et al. disclose a method for producing a plant having TCHLH is protoporphyrin IX binding subunit gene of resistance to a herbicide, glyphosate, where in tobacco magnesium chelatase whose chloroplast transit Sig 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) nal has been deleted. NP is the promoter sequence of a gene which is a target enzyme of glyphosate is mutagenized nopaline Synthase, NT is the terminator Sequence of the So that an affinity for glyphosate is reduced, and the gene is nopaline synthase and 35S is the 35S promoter of cauli introduced into a plant Hinchee, M. A. W. et al., BIO/ flower mosaic virus. NPTII represents a kanamycin resistant TECHNOLOGY, 6: p. 915 (1988)). gene, and RB and LB represent right and left border 35 Sequences of T-DNA, respectively. OBJECTS OF THE INVENTION FIG. 8 is the restriction map of plasmid pTVGMP, GMP Varieties of known methods for giving weed control is Soybean protoporphyrinogen IX oxidase gene whose compound-resistance to plants are not necessarily Sufficient chloroplast transit Signal and FAD binding Sequence have and it has been desired to develop further various kinds of 40 been deleted. lac pro represents the promoter Sequence of a methods for giving weed control compound-resistance to lactose operon. Amp' represents an amplicillin resistant gene plants. and ori is the replication origin. FIG. 9 is the restriction map of plasmid pBIGMP, GMP is The main object of the present invention is to provide a Soybean protoporphyrinogen oxidase gene whose chloro new kind of a method for giving weed control compound plast transit Signal and FAD binding Sequence have been resistance to plants. 45 deleted. NP is the promoter Sequence of a nopaline Synthase This object as well as other objects and advantages of the and NT is the terminator Sequence of a nopaline Synthase, present invention will become apparent to those skilled in the art from the following description with reference to the and 35S is the 35S promoter of cauliflower mosaic virus. accompanying drawings. NPTII is a kanamycin resistant gene, and RB and LB are the 50 right and left border sequences of T-DNA, respectively. FIG. 10 is the restriction map of plasmid pTVCRP, CRP BRIEF DESCRIPTION OF THE DRAWINGS is protoporphyrinogen oxidase gene of Chlamydomonas FIG. 1 is the restriction map of plasmid pTBCH. bchH reinhardtii whose chloroplast transit signal and FAD binding is magnesium chelatase protoporphyrin IX binding Subunit Sequence have been deleted. lac pro represents the promoter gene of a photosynthetic bacterium Rhodobacter Sphaeroi 55 Sequence of a lactose operon. Amp' is an amplicillin resistant deS. T7 pro represents the promoter Sequence of T7 phage, gene and ori is the replication origin. and T7ter represents the terminator Sequence of T7 phage. FIG. 11 is the restriction map of plasmid, pBICRP CRP Amp' is an amplicillin resistant gene, lacIt is a repressor is protoporphyrinogen oxidase gene of Chlamydomonas protein gene of a lactose operon, and ori is the replication reinhardtii whose chloroplast transit signal and FAD binding origin. 60 Sequence have been deleted. NP is the promoter Sequence of FIG. 2 is the restriction map of plasmid pACYCSP PPO a nopaline Synthase and NT is the terminator Sequence of a is protoporphyrinogen IX oxidase gene of Soybean and lac nopaline synthase, and 35S is the 35S promoter of cauli pro represents the promoter Sequence of a lactose operon. flower mosaic virus. NPTII is a kanamycin resistant gene, Cm is a chloramphenicol resistant gene and ori is the and RB and LB are the right and left border sequences of replication origin. 65 T-DNA, respectively. FIG. 3 is the restriction map of plasmid pTVBCH. bchH FIG. 12 is the restriction map of plasmid pTVHVF1. HVF is magnesium chelatase protoporphyrin IX binding Subunit is barley ferrochelatase gene whose signal Sequence has US 6,570,070 B1 3 4 been deleted. lac pro represents the promoter Sequence of a (a) having a specific affinity for a Substance which is lactose operon. Amp' represents an amplicillin resistant gene concerned with the weed control activity of a weed and ori is the replication origin. control compound, (b) having Substantially no capability of modifying a FIG. 13 is the restriction map of plasmid pBIHVF. HVF Substance for which said protein has a Specific is barley ferrochelatase gene whose Signal Sequence has affinity, and been deleted. NP is the promoter Sequence of a nopaline (c) being Substantially free from framework regions of Synthase and NT is the terminator Sequence of a nopaline variable regions in an immunoglobulin, into a plant synthase, and 35S is the 35S promoter of cauliflower mosaic cell; and virus. NPTII is a kanamycin resistant gene, and RB and LB expressing the gene (hereinafter referred to as the first are the right and left border sequences of T-DNA, respec 1O aspect of the method of the present invention). tively. 2. The method according to the above 1, wherein the gene FIG. 14 is the restriction map of plasmid pTVCSF, CSF is introduced into the plant cell in the form that it is operably is cucumber ferrochelatase gene whose Signal Sequence has ligated to a promoter and a terminator both of which are been deleted. lac pro represents the promoter Sequence of a functional in the plant cell. lactose operon. Amp' is an amplicillin resistant gene, and ori 15 3. The method according to the above 1 or 2, wherein the is the replication origin. Substance which is concerned with the weed control activity of the weed control compound is the weed control com FIG. 15 is the restriction map of plasmid pBICSF, CSF is pound itself. cucumber ferrochelatase gene whose Signal Sequence has 4. The method according to the above 1 or 2, wherein the been deleted. NP is the promoter Sequence of a nopaline Substance which is concerned with the weed control activity Synthase and NT is the terminator Sequence of a nopaline of a weed control compound is an endogenous Substance in synthase, and 35S is the 35S promoter of cauliflower mosaic a plant. virus. NPTII is a kanamycin resistant gene, and RB and LB are the right and left border sequences of T-DNA, respec 5. The method according to the above 1 or 2, wherein the tively. weed control compound is that inhibiting porphyrin biosyn 25 thesis of a plant. FIG. 16 is the restriction map of plasmid pHEMF. HEMF 6. The method according to the above 1 or 2, wherein the is coproporphyrinogen III oxidase gene (hemF) of Escheri weed control compound is a protoporphyrinogen IX oxidase chia coli. lac pro is the promoter Sequence of a lactose inhibitory-type herbicidal compound. operon. Amp' is an amplicillin resistant gene, and ori is the 7. The method according to the above 5 or 6, wherein the replication origin. Substance which is concerned with the weed control activity FIG. 17 is the restriction map of plasmid pBIHEMF. of the weed control compound is protoporphyrin IX. HEMF is coproporphyrinogen III oxidase gene (hemF) of 8. The method according to the above 5 or 6, wherein the Escherichia coli. NP is the promoter Sequence of a nopaline protein is protoporphyrin IX binding Subunit protein of Synthase and NT is the terminator Sequence of a nopaline magnesium chelatase, or a variant of Said protein having a synthase, and 35S is the 35S promoter of cauliflower mosaic 35 specific affinity for protoporphyrin IX. virus. NPTII is a kanamycin resistant gene, and RB and LB 9. The method according to the above 8, wherein the are the right and left border sequences of T-DNA, respec protein is magnesium chelatase derived from a photosyn tively. thetic microorganism. FIG. 18 is the restriction map of plasmid pBIHASYS8. 10. The method according to the above 8, wherein the HASYS8 is a gene encoding MG(HASYS) protein. NP is 40 protein is magnesium chelatase derived from a plant. the promoter Sequence of a nopaline Synthase and NT is the 11. The method according to the above 8, wherein the terminator Sequence of a nopaline Synthase, and 35S is the protein is magnesium chelatase derived from tobacco. 35S promoter of cauliflower mosaic virus. NPTII is a 12. The method according to the above 5 or 6, wherein the kanamycin resistant gene, and RB and LB are the right and protein comprises the amino acid sequence of SEQ ID NO: left border sequences of T-DNA, respectively. 45 53. FIG. 19 is the restriction map of plasmid p3IRASSL8. 13. The method according to the above 5 or 6, wherein the RASSL8 is MG(RASSL) protein. NP is the promoter protein has the amino acid sequence of SEQ ID NO: 54. Sequence of a nopaline Synthase and NT is the terminator 14. The method according to the above 5 or 6, wherein the Sequence of a nopaline Synthase, and 35S is the 35S pro protein comprises the amino acid sequence of SEQ ID NO: moter of cauliflower mosaic virus. NPTII is a kanamycin 50 55. resistant gene, and RB and LB are the right and left border 15. The method according to the above 5 or 6, wherein the Sequences of T-DNA, respectively. protein has the amino acid sequence of SEQ ID NO: 56. 16. The method according to the above 5 or 6, wherein the SUMMARY OF THE INVENTION protein comprises the amino acid sequence of SEQ ID NO: 55 57. Under these circumstances, the present inventors have 17. The method according to the above 5 or 6, wherein the Studied intensively So as to develop a new kind of a method protein has the amino acid sequence of SEQ ID NO: 58. for giving weed control compound-resistance to plants. AS a 18. The method according to the above 5 or 6, wherein the result, it has been found that weed control compound protein comprises the amino acid sequence of SEQ ID NO: resistance can be given to plants by allowing the plants to 60 59. produce a certain protein in the plant cells. Thus, the present 19. The method according to the above 5 or 6, wherein the invention has been completed. protein has the amino acid sequence of SEQ ID NO: 60. That is, the present invention provides: 20. The method according to the above 5 or 6, wherein the 1. A method for giving weed control compound-resistance protein is composed of 4 to 100 amino acids. to a plant which comprises the Steps of: 65 21. The method according to the above 5 or 6, wherein the introducing a gene encoding a protein having the follow Substance which is concerned with the weed control activity ing characteristics (a) to (c): of the weed control compound is protoporphyrinogen IX. US 6,570,070 B1 S 6 22. The method according to the above 5 or 6, wherein the expressing the gene (hereinafter referred to as the third protein is a variant of protoporphyrinogen IX oxidase having aspect of the method of the present invention). no capability of oxidizing protoporphyrinogen IX and hav 39. The method according to the above 38, wherein the ing a Specific affinity for a protoporphyrinogen IX. gene is introduced into the plant cell in the form that it is 23. The method according to the above 5 or 6, wherein the protein is a variant of protoporphyrinogen IX oxidase having operably ligated to a promoter and a terminator both of no capability of oxidizing protoporphyrinogen IX and hav which are functional in the plant cell. ing a specific affinity for a protoporphyrin IX oxidase 40. The method according to the above 38 or 39, wherein inhibitory-type herbicidal compound. the protein is coproporphyrinogen III oxidase or a variant of 24. The method according to the above 22 or 23, wherein Said protein having a specific affinity for protoporphyrino the protein is a variant of protoporphyrinogen IX oxidase gen IX. derived from a plant. 41. The method according to the above 38 or 39, wherein 25. The method according to the above 22 or 23, wherein the protein is coproporphyrinogen III oxidase derived from the protein is a variant of protoporphyrinogen IX oxidase a microorganism. derived from soybean. 42 The method according to the above 38 or 39, wherein 26. The method according to the above 22 or 23, wherein 15 the protein is a variant of protoporphyrinogen IX oxidase the protein is coproporphyrinogen III oxidase derived from derived from an algae. Escherichia coli. 27. The method according to the above 22 or 23, wherein 43. A weed control compound-resistant plant whose resis the protein is a variant of protoporphyrinogen IX oxidase tance is given by the method of the above 1, 2, 28 or 29. derived from Chlamydomonas. 44. A weed control compound-resistant plant whose resis 28. A method for giving weed control compound tance is given by the method of the above 38 or 39. resistance to a plant which comprises the Steps of: 45. A method for protecting a plant which comprises introducing a gene encoding a protein having the follow applying the weed control compound to a growth area of the ing characteristics (a) to (c): plant of the above 43. (a) having a specific affinity for protoporphyrin IX, 25 46. A method for protecting a plant which comprises (b) having Substantially no capability of modifying applying the weed control compound to a growth area of the protoporphyrinogen IX, and plant of the above 44. (c) being Substantially free from framework regions of 47. A method for Selecting a plant which comprises variable regions in an immunoglobulin, into a plant applying a weed control compound to which the plant of the cell; and above 43 is resistant to a growth area of the plant of the expressing the gene (hereinafter referred to as the Second above 43 and other plants, and Selecting either plant on the aspect of the method of the present invention). basis of difference in growth between the plants. 29. The method according to the above 28, wherein the 48. A method for Selecting a plant which comprises gene is introduced in the plant cell in the form that it is applying a weed control compound to which the plant of the operably ligated to a promoter and a terminator both of 35 above 44 is resistant to a growth area of the plant of the which are functional in the plant cell. above 44 and other plants, and Selecting either plant on the 30. The method according to the above 28 or 29, wherein basis of difference in growth between the plants. the weed control compound is that inhibiting porphyrin 49. The method according to the above 47, wherein the biosynthesis of a plant. plants are plant cells. 31. The method according to the above 28 or 29, wherein 40 50. The method according to the above 48, wherein the the weed control compound is a protoporphyrinogen IX plants are plant cells. oxidase inhibitory-type herbicidal compound. 51. The method according to the above 1 or 2, wherein the 32. The method according to the above 30 or 31, wherein weed control compound is a protoporphyrinogen IX oxidase the protein is magnesium chelatase or a variant of Said inhibitory-type herbicidal compound Selected from the com protein having a specific affinity for protoporphyrin IX. 45 pounds of (1) to (3) below, and the Substance which is 33. The method according to the above 30 or 31, wherein concerned with the weed control activity of the weed control the protein is ferrochelatase or a variant of Said protein compound is protoporphyrin IX, protoporphyrinogen IX or having an Specific affinity for protoporphyrin IX. a protoporphyrinogen IX oxidase inhibitory-type herbicidal 34. The method according to the above 30 or 31, wherein compound: the protein is ferrochelatase derived from a plant. 50 (1) chlormethoxynil, bifenox, chlornitrofen (CNP), acif 35. The method according to the above 30 or 31, wherein luorfen (5-2-chloro-4-(trifluoromethyl)phenoxy-2- the protein is ferrochelatase derived from barley. nitorobenzoic acid) and its ethyl ester, acifluorfen 36. The method according to the above 30 or 31, wherein sodium, oxyfluorfen (2-chloro-1-(3-ethoxy-4- the protein is ferrochelatase derived from cucumber. nitrophenoxy)-4-trifluoromethylbenzene), oxadiazon 37. The method according to the above 30 or 31, wherein 55 (3-2,4-dichloro-5-(1-methylethoxy)phenyl-5-(1,1- the protein is a peptide composed of 4 to 100 amino acids. dimethylethyl)-1,3,4-oxadiazol-2(3H)-one), 2-4- 38. A method for giving weed control compound chloro-2-fluoro-5-(prop-2-ynyloxy)phenyl-2,3,4,5,6, resistance to a plant which comprises the Steps of: 7-hexahydro-1H-isoindol-1,3-dione, chlorphthalim (N- introducing a gene encoding a protein having the follow (4-chlorophenyl)-3,4,5,6-tetrahydrophtalimide), ing characteristics (a) to (c): 60 TNPP-ethyl (ethyl 2-1-(2,3,4-trichlorophenyl)-4- (a) having a specific affinity for protoporphyrinogen nitropy razolyl-5-oxypropionate), or N3-(1- IX, phenylethyl)-2,6-dimethyl-5-propyonylnicotinamide; (b) having the capability for modifying coproporphy (2) a compound represented by the general formula: J-G rinogen III, and (I), wherein G is a group represented by any one of the (c) being Substantially free from framework regions of 65 following general formulas G-1 to G-9 and J is a group variable regions in an immunoglobulin, into a plant represented by any one of the following general for cell; and mulas of J-1 to J-30:

US 6,570,070 B1 12 -continued group, CHROH group, CHRC(O)R group or J-26 OCHROC(O)NR'R' group, or, when G is G-2 or R13 O V G-6, R" and R may form C=O group together with N the carbon atom to which they are attached; R is C-C alkyl group, C-C haloalkyl group, R"-( / C-C alkoxyalkyl group, C-C alkenyl group or N C-C alkynyl group; R11 X is single bond, oxygen atom, Sulfur atom, NH J-27 group, N(C-C alkyl) group, N(C-C haloalkyl) O group or N(allyl) group; R’ is hydrogen atom, C-C alkyl group, C-C, y-( haloalkyl group, halogen atom, S(O)2(C-C alkyl) R-( N group or C(=O)R" group; R is hydrogen atom, C-Cs alkyl group, C-Cs S—(R11 15 cycloalkyl group, C-Cs alkenyl group, C-Cs alky J-28 nyl group, C-C haloalkyl group, C-C alkoxy R13 O alkyl group, C-C alkoxyalkoxyalkyl group, C-C, V haloalkynyl group, C-Cs haloalkenyl group, C-C N alkylsulfonyl group, C-C haloalkylsulfonyl group, R14 \ / C-C alkoxycarbonylalkyl group, S(O)NH(C-Cs N alkyl) group, C(O)R' group or benzyl group whose R11 phenyl ring may be substituted with R', J-29 in and m are independently 0, 1, 2 or 3 and m+n is 2 or R11 3; 25 Z is CR'R' group, oxygen atom, sulfur atom, S(O) group, S(O) group or N(C-C alkyl) group; / \ each R is independently hydrogen atom, C-C alkyl FN group, halogen atom, hydroxyl group, C-C alkoxy J-30 group, C-C haloalkyl group, C-C haloalkoxy R11 O group, C-C alkylcarbonyloxy group or C-C, haloalkylcarbonyloxy group; each R" is independently hydrogen atom, C-C alkyl R14 / N group, hydroxyl group or halogen atom; R" and R'' are independently hydrogen atom, halogen N { 35 atom, C-C alkyl group, C-C alkenyl group or \ Yv C-C haloalkyl group; T R" is hydrogen atom, C-C alkyl group, C-C, haloalkyl group, C-C alkenyl group, C-C, wherein the dotted lines in the formulas J-5, J-6, J-12 haloalkenyl group, C-C alkynyl group, C-C, and J-24 represent that the left hand ring contains only 40 haloalkynyl group, HC(=O) group, (C-C alkyl)C Single bonds, or one bond in the ring is a double bond between carbon atoms, (=O) group or NH group; X is oxygen atom or Sulfur atom; R" is C-C alkyl group, C-C alkylthio groups Y is oxygen atom or Sulfur atom; C-C haloalkyl group or N(CH) group; R" is hydrogen atom or halogen atom; W is nitrogen atom or CR'; R is hydrogen atom, C-Cs alkyl group, C-Cs 45 R" is hydrogen atom, C-C alkyl group, halogen haloalkyl group, halogen atom, OH glyp, OR27 atom, or phenyl group optionally Substituted with group, SH group, S(O).R. group, COR group, C-C alkyl group, one or two halogen atoms, C-C, COR7 group, C(O)SR-7 group, C(O)NR'R'' alkoxy group or CF group; group, CHO group, CR 7=NOR group, each Q is independently oxygen atom or Sulfur atom; CH=CR7COR7 group, CHCHR7COR7 50 Q' is oxygen atom or Sulfur atom; group, CON=CRR group, nitro group, cyano Z' is CR'R'' group, oxygen atom, sulfur atom, S(O) group, NHSOR group, NHSONHR group, group, S(O) group or N(C-C alkyl) group; NR7R group, NH group or phenyl group option each R' is independently hydrogen atom, halogen ally Substituted with one or more and the same or atom, hydroxyl group, C-C alkoxy group, C-C, different C-C alkyl groups; 55 haloalkyl group, C-C haloalkoxy group, C-C, p is 0, 1 or 2, alkylcarbonyloxy group or C-C haloalkylcarbony R is C-C alkyl group, C-Chaloalkyl group, OCH loxy group; group, SCH group, OCHF group, halogen atom, each R'' is independently hydrogen atom, hydroxyl cyano group or nitro group; group or halogen atom; R" is hydrogen atom, C-C alkyl group, C-C, 60 R" is C-C alkyl group, halogen atom or C-C, haloalkyl group or halogen atom; haloalkyl group; R is hydrogen atom, C-C alkyl group, halogen atom, R'' and R” are independently hydrogen atom, C-C, C-C haloalkyl group, cyclopropyl group, Vinyl alkyl group, or C-C haloalkyl group; group, C, alkynyl group, cyano group, C(O)R Z is oxygen atom, sulfur atom, NR' group or CR'R'' group, COR group, C(O) NR'R' group, 65 grOup, CRRCN group, CRRC(O)R group, R° and R’ are independently C-C alkyl group, CRR35 COR3s group, CR R5 C(O)NR'R'' C-Chaloalkyl group, C-C alkenyl group, C-C, US 6,570,070 B1 13 14 haloalkenyl group, C-C alkynyl group or C-C, R is C-C alkyl group, C-C alkenyl group, C-C, haloalkynyl group; alkynyl group or tetrahydrofuranyl group; R is hydrogen atom, halogen atom or cyano group; R” and R are independently hydrogen atom or R" is C-C alkylsulfonyl group, C-C alkyl group, C-C alkyl group; C-Chaloalkyl group, C-C alkenyl group, C-C, R" and R are independently C-C alkyl group or alkynyl group, C-C alkoxy group, C-C, phenyl group whose ring may be Substituted with at haloalkoxy group or halogen atom; least one Substituent Selected from the group con R’ is C-C alkyl group, C-Cs haloalkyl group, sisting of halogen atom, C-C alkyl group and C-C alkenyl group or C-C alkynyl group; C-C haloalkyl group; or, R’ is C-C alkyl group, C-C haloalkyl group or R’ and R together may form -(CH)-, phenyl group optionally Substituted with C-C, 1O -(CH-)- or -CHCHOCH2CH-, or the ring alkyl, one or two halogen atoms, one or two nitro thus formed may be substituted with at least one groups, C-C alkoxy group or CF group; Substituent Selected from the group consisting of W' is nitrogen atom or CH group; C-C alkyl group, phenyl group and benzyl group; T is a group represented by any one of the following Or, general formulas T-1, T-2 and T-3; 15 RandR may from C-Cs cycloalkyl group together T-1 with the carbon atom to which they are attached; El E2 E3 E4 R is C-C alkyl group, C-C haloalkyl group or V / V / C-C alkenyl group; -C C R" and R are independently hydrogen atom or T-2 E5 E6 E7 E8 E9 E10 C-C alkyl group; V / V / V / R is hydrogen atom, C-C alkyl group, C-C, -C C C alkenyl group or C-C alkynyl group; T-3 R" is hydrogen atom, C-C alkyl group or halogen atom, 25 R is hydrogen atom, C-C alkyl group, C-C, E11 E12 cycloalkyl group, C-C alkenyl group, C-C alky nyl group, C-C alkoxyalkyl group, C-C, (wherein E, E, E, E, E, E°, E7, E, E, E, El haloalkyl group, phenyl group whose ring may be and E' are independently hydrogen atom or C-C, Substituted with at least one Substituent selected from alkyl group); the group consisting of halogen atom, C-C alkyl R’ is CI-Cs alkyl group, Ca-Cs cycloalkyl group, group and C-C alkoxy group, —CH2CO(C-C, C-C alkenyl group, Ca-Calkynyl group, C-Cs alkyl) group or -CH(CH2)CO(C-C alkyl) group; haloalkyl group, C-C alkoxyalkyl group, C-Cs R’ is hydrogen atom, C-C alkyl group or C(O)O alkylthioalkyl group, C-C alkylsulfinylalkyl (C-C alkyl) group; group, C-C alkylsulfonylalkyl group, C-C alkyl 35 R" is hydrogen atom, C-C alkyl group, C-C, Sulfonyl group, phenylsulfonyl group whose phenyl alkoxy group or NH(C-C alkyl) group; ring may be Substituted with at least one Substituent R" is C-C alkyl group, C-C haloalkyl group, Selected from the group consisting of halogen atom C-C alkoxy group, NH(C-C alkyl) group, phe and C-C alkyl group, C-C alkoxyalkoxyalkyl nyl group whose ring may be Substituted with one group, Ca-Cs cycloalkylalkyl group, Co-Cs 40 substituent selected from the group consisting of R' cycloalkoxyalkyl group, C-C alkenyloxyalkyl group, benzyl group and C-C dialkylamino group; group, Ca-Cs alkynyloxyalkyl group, Ca-Cs haloalkoxyalkyl group, C-C haloalkenyloxyalkyl and group, C-C haloalkynyloxyalkyl group, Co-Cs R" is C-C alkyl group, one or two halogen atoms, cycloalkylthioalkyl group, C-C alkenylthioalkyl 45 C-C alkoxy group or CF group; group, C-C alkynylthioalkyl group, C-C alkyl (3) a compound of the formula (II): group Substituted with phenoxy group whose ring is Substituted with at least one Substituent selected from R45 the group consisting of halogen atom, C-C alkyl R44 N R46 e M s 50 group and C-C haloalkyl group, benzyloxy group N whose ring is Substituted with at least one Substituent NN / le Selected from the group consisting of halogen atom, R43 N R47 C-C alkyl group and C-C haloalkyl group, R48 C-C trialkylsilylalkyl group, C-C cyanoalkyl group, C-C halocycloalkyl group, C-Cs haloalk 55 or nipilacrofen, enyl group, Cs-Cs alkoxyalkenyl group, Cs-Cs haloalkoxyalkenyl group, C-C alkylthioalkenyl wherein R" is C-C alkyl group; group, C-Chaloalkynyl group, C-C alkoxyalky R" is C-C alkyl group, C-C alkylthio group, C-C, nyl group, Cs-C haloalkoxyalkynyl group, C-C, alkoxy group, C-C haloalkyl group, C-C haloalky alkylthioalkynyl group, C-C alkylcarbonyl group, 60 lthio group or C-C haloalkoxy group; benzyl group whose ring is Substituted with at least R" and R' together may form -(CH)- or one Substituent Selected from the group consisting of -(CH-)-; halogen atom, C-C alkyl group and C-C, R" is hydrogen atom or halogen atom; halo alkyl group, CHR COR° group, R" is hydrogen atom or C-C alkyl group; CHRCOOR group, CHRP(O)(OR) group, 65 R" is hydrogen atom, nitro group, cyano group, CHRP(S)(R) group, CHRC(O)NR2R30 -COOR' group, —C(=X)NR'R' group or group or CHRC(O)NH group; —C(=X)R group; US 6,570,070 B1 15 16 R" is hydrogen atom, halogen atom, cyano group, C-C, group, cyano group, C-C alkyl group, C-C, alkyl group optionally Substituted with at least one alkoxy group and halo-C-C alkyl group, Substituent Selected from the group consisting of halo -C(=X')R group, -(CH2)-(O))-R' gen atom and hydroxyl group, C-C alkoxy group, group, -(CH2)-O-(CH2)-R' group, phenyl group optionally Substituted with at least one -(CH) X21:76 grOup, Substituent Selected from the group consisting of halo R53 and R3, together with the nitrogen atom to which gen atom, nitro group, cyano group, C-C alkyl group, they are attached may form Saturated alicyclic 3, 5 or C-C alkoxy group and halo-C-C alkyl group, pyr 6 membered ring or aromatic 5 or 6 membered ring rolyl group, C2-Cs alkyl group, C-Cs alkenyl group, in which a carbon atom may be optionally replaced C-C alkynyl group, C-C alkoxy group, a group with oxygen atom; Selected from the group consisting of C-C alkyl R is hydrogen atom, C-C alkyl group, C-C, group, C-Cs alkenyl group, C-Cs alkynyl group and alkenyl group or C-C alkynyl group, or R55 and C-C alkoxy group into which at least one oxygen R together may form-(CH2); atom is inserted, or any one of groups represented by R and R7 are independently C-C alkyl group the following formulas: optionally Substituted with at least one halogen atom, 15 C-C alkenyl group optionally Substituted with at least one halogen atom, C-C alkynyl optionally Substituted with at least one halogen atom or phenyl l, l, group optionally Substituted with at least one halo X X gen atom, hydrogen atom, C-C cycloalkyl group, O O -XR" group or "NRGR6 grOup, R’ is hydrogen atom, C-C alkyl group, C-C, alkenyl group, C-C alkynyl group, C-C alkyl -OR58 -S(O)R59 -) - carbonyl group, cyano-C-C alkyl group, C-C, alkoxycarbonyl-C-C alkyl group, di-C-C, O O 25 alkoxycarbonyl-C-C alkyl group, benzyl group, O O O C-C alkoxy-C-C, alkynyl group,-(CH2)-R' group, -(CH), X-R' group, -(CH2)-X -N (CH) R' group or -(CH), X-(CH), X-(CH2)-R' group; R’ is hydrogen atom, C-C alkyl group, C-C, O O O alkenyl group, C-C alkynyl group, cyano-C-C- O alkyl group, C-C alkylcarbonyl-C-C alkyl group -OX D -NR(CH),CR57 -(CH)-A or phenyl group, O R" is C-C alkyl group optionally substituted with at 35 least one halogen atom; o (CH2) R'' and Rare, the same or different, hydrogen atom -COR66 or C-C alkyl group; R" is C-C alkyl group optionally substituted with at least one halogen atom, C-C alkoxy-C-C alkyl wherein R', R and R are, the same or different, 40 group, C-C alkylthio-C-C alkyl group, C-C, hydrogen atom or C-C alkyl group; cycloalkyl group, phenyl group whose ring may be R'' and R may form saturated alicyclic 5 or 6 Substituted with one Substituent selected from the membered ring together with the nitrogen atom to group consisting of halogen atom, nitro group, cyano which they are attached; group, C-C alkyl group, C-C alkoxy group and R is hydrogen atom, C-C alkyl group or C-C, 45 halo-C-C alkyl group, -NR'R'' group or alkyl group Substituted with at least one halogen -(CH2)-(O).-R" group; atom, R" is C-C alkoxycarbonyl group or carboxyl group; R is hydrogen atom, C-C alkyl group optionally R is chloromethyl group, cyanomethyl group, Substituted with at least one halogen atom, C-C, C-C cycloalkyl group into which at least one alkenyl group optionally Substituted with at least one 50 oxygen atom may be inserted, or C-C, halogen atom, C-C alkynyl group optionally Sub alkoxycarbonyl-C-C alkyl group; Stituted with at least one halogen atom, phenyl group R is hydroxyl group or -NR7R group; optionally Substituted with at least one halogen atom, A is -NR'R' group or —S(O)-R" grOup, C-C cycloalkyl group, cyanomethyl group, or R7 and Rare, the same or different, hydrogen atom RCO-group; 55 or C-C alkyl group; R" is hydrogen atom, C-C alkyl group optionally R” is C-C alkyl group or C-C haloalkyl group; Substituted with at least one halogen atom, C-C, R" is hydrogen atom, hydroxyl group, halogen atom, alkenyl group optionally Substituted with at least one C-C alkyl group optionally Substituted with at halogen atom, C-C alkynyl group optionally Sub least one C-C alkoxy group, C-C cycloalkyl Stituted with at least one halogen atom, phenyl group 60 group into which at least one oxygen atom may be optionally Substituted with halogen atom, C-Cs inserted, C-C cycloalkyl group optionally Substi cycloalkyl group, cyanomethyl group, C-C, tuted with one or two methyl groups, furyl group, alkoxy-C-C alkyl group, di-C-C alkylamino thienyl group or -C(=O)R’ group; C-C alkyl group, tetrahydrofurfurylmethyl group, R'' and R' are, the same or different, C-C alkyl C-C alkynyloxy-C-C alkyl group, benzyl whose 65 group or C-C alkoxy group; ring may be Substituted with Substituent Selected R7 and R7 are, the same or different, C-C alkyl from the group consisting of halogen atom, nitro group or phenyl group; US 6,570,070 B1 17 18 R’ is C-C cycloalkyl into which at least one oxygen the compounds inhibiting amino acid biosynthesis, for atom may be inserted, C-C cycloalkyl group example, there are compounds inhibiting EPSPS, acetolac optionally substituted with one or two methyl tate synthase (EC 4.1.3.18; hereinafter referred to as ALS), groups, furyl group, thienyl group or -C(=O)R’ glutamine Synthetase (EC 6.3.1.2, hereinafter referred to as grOup, GS), dihydropteroate synthase (EC 2.5.1.15; hereinafter R" is C-C alkyl group; referred to as DHP), and the like. As the compounds a, b and c is independently 1, 2 or 3; inhibiting lipid biosynthesis, for example, there are com d is 0 or 1; pounds inhibiting acetyl CoA carboxylase (EC 6.4.1.2, e is 2 or 3; hereinafter referred to as ACC), and the like. As the com f is 1 or 2; and pounds inhibiting cell wall biosynthesis, for example, there X is oxygen atom or Sulfur atom. are compounds inhibiting cellulose biosynthesis, and the DETAILED DESCRIPTION OF THE like. AS the compounds influencing protein biosynthesis, INVENTION nucleic acid biosynthesis or cell division, for example, there In the method of the present invention, Substances which 15 are compounds inhibiting formation of microtubules, and are concerned with weed control activities of weed control the like. compounds (hereinafter referred to as weed control Examples of the compounds having plant growth regula Substances) are those constituting a part of metabolic reac tion Systems in organisms which are responsible for weed tor activities include compounds having antagonistic activi control activities upon applying the compounds to plants. ties against plant hormones which enhance cell elongation Examples thereof include weed control compounds and differentiation, and the like. Specifically, for example, themselves, endogenous Substances in plants, and the like. there are 2,4-D, phenoxyalkane carboxylic acid, derivatives Specifically, as Such endogenous Substances in plants, for of benzoic acid, derivatives of picolinic acid, and the like. example, there are Substrates of target enzymes on which As the above-described PPO inhibitory-type herbicidal weed control compounds act, or precursors or metabolites of 25 compounds, for example, there are the compounds disclosed the Substrates which cause cellular dysfunction upon accu in Duke, S. O., Rebeiz, C. A., ACS Symposium Series 559, mulating in plant cells; Substances produced by the above Porphyric Pesticides, Chemistry, Toxicology, and Pharma Substances in plant cells which cause cellular dysfunction; ceutical Applications, American Chemical Society, Wash and the like. More Specifically, it has been known that, when ington D.C. (1994), and the like. Specifically, examples a compound having herbicidal activity (hereinafter referred thereof include the following compounds: to as herbicidal compound) which inhibits the activity of (1) chlormethoxynil, bifenox, chlornitrofen (CNP), acifluo protoporphyrinogen IX oxidase (EC 1.3.3.4, hereinafter rfen (5-2-chloro-4-(trifluoromethyl)phenoxy-2- referred to as PPO) is applied to a plant, protoporphyrinogen nitorobenzoic acid) and its ethyl ester, acifluorfen IX which is the substrate of PPO is accumulated in the plant sodium, oxyfluorfen (2-chloro-1-(3-ethoxy-4- cells and it is metabolized to form protoporphyrin X, fol 35 nitrophenoxy)-4-trifluorobenzene), oxadiazon (3-2,4- lowed by formation of active oxygen in the presence of both dichloro-5-(1-methylethoxy) phenyl-5-(1,1- protoporphyrin X and light in the cells, which damageS cell dimethylethyl)-1,3,4-oxydiazol-2-(3H)-one), 2-4-chloro functions Junshi MIYAMOTO ed., Atarashii Noyaku no 2-fluoro-5-(prop-2-y nyloxy)phenyl)-2,3,4,5,6,7- Kagaku (Chemistry of New Agrochemicals), Chapter 3, hexahydro-1H-isoindol-1,3-dione, chlorphthalim, (N-(4- Section 3.3, p. 106 (1993), Hirokawa Shoten, Tokyo). Thus, 40 chlorophenyl)-3,4,5,6-tetrahydrophtalimide), TNPP-ethyl protoporphyrinogen IX, protoporphyrin IX and active oxy (ethyl 2-1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5- gen in these Systems, and the like can be exemplified as these oxypropionate), or N3-(1-phenylethyl)-2,6-dimethyl-5- Substances. propyonylnicotinamide; In the method of the present invention, weed control (2) a compound represented by the general formula: J-G (I), compounds include compounds having herbicidal activities, 45 wherein G is a group represented by any one of the plant growth regulator activities, and the like. following general formulas G-1 to G-9 and J is a group Examples of the herbicidal compounds include com represented by any one of the following general formulas pounds inhibiting porphyrin biosynthesis, compounds inhib J-1 to J-30: iting electron transfer in photosynthesis, compounds inhib iting carotenoid biosynthesis, compounds inhibiting amino 50 G-1 acid biosynthesis, compounds inhibiting lipid biosynthesis, compounds inhibiting cell wall biosynthesis, compounds influencing protein biosynthesis, nucleic acid biosynthesis and cell division, compounds having auxin antagonistic activity, and the like. More specifically, as the compounds 55 inhibiting porphyrin biosynthesis, for example, there are compounds inhibiting PPO activity (PPO inhibitory-type herbicidal compound), and the like. As the compounds inhibiting electron transfer in photosynthesis, for example, there are compounds inhibiting electron transfer of photo 60 chemical System I or II, compounds inhibiting 4-hydroxyphenyl pyruvate dioxygenase (EC 1.13.11.27; hereinafter referred to as 4-HPPD) which influences biosyn thesis of plastoquinone which transferS electrons, and the like. AS the compounds inhibiting carotenoid biosynthesis, 65 for example, there are compounds inhibiting phytoene desaturase (hereinafter referred to as PDS), and the like. As

US 6,570,070 B1 24 -continued alkyl) group, C(O)R' group or benzyl group whose J-29 phenyl ring may be substituted with R', R11 in and m are independently 0, 1, 2 or 3 and m+n is 2 or 3; Z is CRR' group, oxygen atom, sulfur atom, S(O) / \ group, S(O)-group or N(C-C alkyl) group; FN each R is independently hydrogen atom, C-C alkyl J-30 group, halogen atom, hydroxyl group, C-C alkoxy R11 O group, C-C haloalkyl group, C-C haloalkoxy group, C-C alkylcarbonyloxy group or C-C, R14 / N haloalkylcarbonyloxy group; each R" is independently hydrogen atom, C-C alkyl N { group, hydroxyl group or halogen atom; \ r1W1 R'' and R'' are independently hydrogen atom, halogen 15 atom, C-C alkyl group, C-C alkenyl group or wherein the dotted lines in the formulas J-5, J-6, J-12 and C-C haloalkyl group; J-24 represent that the left hand ring contains only Single R" is hydrogen atom, C-C alkyl group, C-C, bonds, or one bond in the ring is a double bond between haloalkyl group, C-C alkenyl group, C-C haloalk carbon atoms, enyl group, C-C alkynyl group, C-C haloalkynyl X is oxygen atom or Sulfur atom; group, HC(=O) group, (C-C alkyl)C(=O) group or Y is oxygen atom or Sulfur atom; NH group; R" is hydrogen atom or halogen atom; R" is C-C alkyl group, C-C alkylthio group, C-C, R is hydrogen atom, C-Cs alkyl group, C-Cs haloalkyl haloalkyl group or N(CH) group; group, halogen atom, OH group, OR group, SH 25 W is nitrogen atom or CR"; group, S(O)R’ group, COR group, COR-7 group, R" is hydrogen atom, C-C alkyl group, halogen atom, C(O)SR 7 group, C(O)NR'R'' group, CHO group, or phenyl group optionally Substituted with C-C, CR-7=NOR group, CH=CR7COR7 group, alkyl group, one or two halogen atoms, C-C alkoxy CHCHR7COR7 group, CON=CRR group, group or CF group; nitro group, cyano group, NHSO2R group, NHSONHR group, NR7R group, NH group or each Q is independently oxygen atom or Sulfur atom; phenyl group optionally Substituted with one or more Q is oxygen atom or Sulfur atom; and the same or different C-C alkyl groups; Z' is CRR 'group, oxygen atom, sulfur atom, S(O) p is 0, 1 or 2, 35 group, S(O) group or N(C-C alkyl) group; R is C-C alkyl group, C-C haloalkyl group, OCH each R' is independently hydrogen atom, halogen atom, group, SCH group, OCHF group, halogen atom, hydroxyl group, C-C alkoxy group, C-C haloalkyl cyano group or nitro group; group, C-C haloalkoxy group, C-C alkylcarbony R" is hydrogen atom, C-C alkyl group, C-C haloalkyl loxy group or C-C haloalkylcarbonyloxy group; group or halogen atom; 40 each R'' is independently hydrogen atom, hydroxyl group R is hydrogen atom, C-C alkyl group, halogen atom, or halogen atom; C-C haloalkyl group, cyclopropyl group, Vinyl R" is C-C alkyl group, halogen atom or C-C, group, C alkynyl group, cyano group, C(O)R group, haloalkyl group; COR group, C(O)NR'R' group, CRRCN R'' and R are independently hydrogen atom, C-C, group, CRRC(O)R group, CRR COR group, 45 alkyl group, or C-C haloalkyl group; CR'R'C(O)NR'R' group, CHROH group, Z is oxygen atom, sulfur atom, NR' group or CR'R'' CHROC(O)R group or OCHROC(O)NR'R'' grOup, group, or, when G is G-2 or G-6, R' and R may form R° and Rf are independently C-C alkyl group, C-C, C=O group together with the carbon atom to which 50 haloalkyl group, C-C alkenyl group, C-C haloalk they are attached; enyl group, C-C alkynyl group or C-C haloalkynyl R is C-C alkyl group, C-C haloalkyl group, C-C, grOup, alkoxyalkyl group, C-C alkenyl group or C-C, R is hydrogen atom, halogen atom or cyano group; alkynyl group; R" is C-C alkylsulfonyl group, C-C alkyl group, X" is single bond, oxygen atom, Sulfur atom, NH group, 55 C-C haloalkyl group, C-C alkenyl group, C-C, N(C-C alkyl) group, N(C-C haloalkyl) group or alkynyl group, C-C alkoxy group, C-C haloalkoxy N(allyl) group; group or halogen atom; R" is hydrogen atom, C-C alkyl group, C-Chaloalkyl R’ is C-C alkyl group, C-C haloalkyl group, C-C, group, halogen atom, S(O)2(C-C alkyl) group or alkenyl group or C-C alkynyl group; C(=O)R" group; 60 R is hydrogen atom, C-Cs alkyl group, Ca-Cs R is C-C alkyl group, C-C haloalkyl group or cycloalkyl group, C-C alkenyl group, C-C alkynyl phenyl group optionally Substituted with C-C alkyl, group, C-C aloalkyl group, C-C alkoxyalkyl one or two halogen atoms, one or two nitro groups, group, Ca-Cs alkoxyalkoxyalkyl group, Ca-Cs C-C alkoxy group or CF group; haloalkynyl group, C-C haloalkenyl group, C-C, 65 W' is nitrogen atom or CH group; alkylsulfonyl group, C-Cs haloalkylsulfonyl group, T is a group represented by any one of the following C-C alkoxycarbonylalkyl group, S(O)NH(C-Cs general formulas T-1, T-2 and T-3; US 6,570,070 B1 25 26 RandR may from C-Cs cycloalkyl group together T-1 with the carbon atom to which they are attached; El E2 E3 E4 R is C-C alkyl group, C-C haloalkyl group or V / V / -C C C-C alkenyl group; T-2 R" and R are independently hydrogen atom or E5 E6 E7 E8 E9 E10 C-C alkyl group; V / V / V R is hydrogen atom, C-C alkyl group, C-C, -C C C alkenyl group or C-C alkynyl group; T-3 R’ is hydrogen atom, C-C alkyl group or halogen atom, R is hydrogen atom, C-C alkyl group, C-C, E11 E12 cycloalkyl group, C-C alkenyl group, C-C alky nyl group, C-C alkoxyalkyl group, C-C, (wherein E, E, E, E, E, E, E7, E, E, E, E' and haloalkyl group, phenyl group whose ring may be E" are independently hydrogen atom or C-C alkyl Substituted with at least one Substituent selected from group); 15 the group consisting of halogen atom, C-C alkyl R’ is CI-Cs alkyl group, Ca-Cs cycloalkyl group, group and C-C alkoxy group, —CH2CO(C-C, C-C alkenyl group, C-C alkynyl group, C-Cs alkyl) group or -CH(CH2)CO(C-C alkyl) group; haloalkyl group, C-C alkoxyalkyl group, C-Cs R’ is hydrogen atom, C-C alkyl group or C(O)O alkylthioalkyl group, C-C alkylsulfinylalkyl (C-C alkyl) group; group, C-C alkylsulfonylalkyl group, C-C alkyl R" is hydrogen atom, C-C alkyl group, C-C, Sulfonyl group, phenylsulfonyl group whose phenyl alkoxy group or NH(C-C alkyl) group; ring may be Substituted with at least one Substituent R" is C-C alkyl group, C-C haloalkyl group, Selected from the group consisting of halogen atom C-C alkoxy group, NH(C-C alkyl) group, phe and C-C alkyl group, C-C alkoxyalkoxyalkyl nyl group whose ring may be Substituted with one group, C-C cycloalkylalkyl group, Co-Cs 25 substituent selected from the group consisting of R' cycloalkoxyalkyl group, C-C alkenyloxyalkyl group, benzyl group and C-C dialkylamino group; group, C-C alkynyloxyalkyl group, C-Cs haloalkoxyalkyl group, C-C haloalkenyloxyalkyl and group, C-C haloalkynyloxyalkyl group, C-C, R" is C-C alkyl group, one or two halogen atoms, cycloalkylthioalkyl group, C-C alkenylthioalkyl C-C alkoxy group or CF group; group, C-C alkynylthioalkyl group, C-C alkyl (3) a compound of the formula (II): group Substituted with phenoxy group whose ring is Substituted with at least one Substituent Selected from R45 R44 N R46 the group consisting of halogen atom, C-C alkyl e M NS group and C-C haloalkyl group, benzyloxy group 35 N whose ring is Substituted with at least one Substituent N-4 le Selected from the group consisting of halogen atom, R43 N R47 C-C alkyl group and C-C haloalkyl group, R48 C-C trialkylsilylalkyl group, C-C cyanoalkyl group, C-C halocycloalkyl group, C-C haloalk 40 or nipilacrofen, enyl group, Cs-Cs alkoxyalkenyl group, Cs-Cs wherein R" is C-C alkyl group; haloalkoxyalkenyl group, C-C alkylthioalkenyl R" is C-C alkyl group, C-C alkylthio group, C-C, group, C-Chaloalkynyl group, C-C alkoxyalky alkoxy group, C-C haloalkyl group, C-Chaloalky nyl group, Cs-Cs haloalkoxyalkynyl group, Cs-Cs lthio group or C-C haloalkoxy group; alkylthioalkynyl group, C-C alkylcarbonyl group, 45 benzyl group whose ring is Substituted with at least R" and R" together may form -(CH2)4- or one Substituent Selected from the group consisting of -(CH-)-; halogen atom, C-C alkyl group and C-C, R" is hydrogen atom or halogen atom; halo alkyl group, CHRCOR group, R" is hydrogen atom or C-C alkyl group; CHRCOOR group, CHRP(O)(OR), group, 50 R" is hydrogen atom, nitro group, cyano group, CHRP(S)O(R) group, CHRC(O)NR'R'' -COOR' group, —C(=X)NR'R' group or group or CHRC(O)NH group; —C(=X)R group; R is C-C alkyl group, C-C alkenyl group, C-C, R" is hydrogen atom, halogen atom, cyano group, C-C, alkynyl group or tetrahydrofuranyl group; alkyl group optionally Substituted with at least one R” and R are independently hydrogen atom or 55 Substituent Selected from the group consisting of halo C-C alkyl group; gen atom and hydroxyl group, C-C alkoxy group, R and R° are independently C-C alkyl group or phenyl group optionally Substituted with at least one phenyl group whose ring may be Substituted with at Substituent Selected from the group consisting of halo least one Substituent Selected from the group con gen atom, nitro group, cyano group, C-C alkyl group, Sisting of halogen atom, C-C alkyl group and 60 C-C alkoxy group and halo-C-C alkyl group, pyr C-C haloalkyl group; or, rolyl group, C2-Cs alkyl group, C-Cs alkenyl group, R’ and R together may form -(CH2)5-, C-C alkynyl group, C-C alkoxy group, a group -(CH-)- or -CHCHOCH2CH-, or the ring Selected from the group consisting of C-C alkyl thus formed may be substituted with at least one group, Cs-Cs alkenyl group, C-Cs alkynyl group and Substituent Selected from the group consisting of 65 C-C alkoxy group into which at least one oxygen C-C alkyl group, phenyl group and benzyl group; atom is inserted, or any one of groups represented by Or, the following formulas: US 6,570,070 B1 27 28 C-C alkenyl group optionally Substituted with at -NR5RS -NRSSCR56 -N(CR57) -N(CH2).R. least one halogen atom, C-C alkynyl optionally | Substituted with at least one halogen atom or phenyl X2 X2 X2 group optionally Substituted with at least one halo O O 5 gen atom, hydrogen atom, C-C cycloalkyl group, -XR" group or -NR'R' group; -OR58 -S(O)R59 -N -N R’ is hydrogen atom, C-C alkyl group, C-C, alkenyl group, C-C alkynyl group, C-C alkyl carbonyl group, cyano-C-C alkyl group, C-C, O O 1O alkoxycarbonyl-C-C alkyl group, di-C-C, O O O alkoxycarbonyl-C-C alkyl group, benzyl group, C-C alkoxy-C-C, alkynyl group,-(CH2)-R'

-N -N -N X-(CH2)-Rgroup, (CH.), X-R' group,group -(CH.), O 15 (CH), X-(CH), X-(CH2)-R' group; O O O R’ is hydrogen atom, C-C alkyl group, C-C, O alkenyl group, C-C alkynyl group, cyano-C-C- -N D -NR(CH),CR57 -(CH2). A alkyl group, C-C alkylcarbonyl-C-C alkyl group or phenyl group; O R55 x2 2O R" is C-C alkyl group optionally substituted with at -(CH)-O-(CH2)-R" -(CH)-O-R6 least one halogen atom; -COR66 R'' and Rare, the same or different, hydrogen atom or C-C alkyl group; R" is C-C alkyl group optionally substituted with at wherein R', R and R are, the same or different, least one halogen atom, C-C alkoxy-C-C alkyl hydrogen atom or C-C alkyl group; group, C-C alkylthio-C-C alkyl group, C-C, R and R may form saturated alicyclic 5 or 6 cycloalkyl group, phenyl group whose ring may be membered ring together with the nitrogen atom to Substituted with one Substituent selected from the which they are attached; group consisting of halogen atom, nitro group, cyano R is hydrogen atom, C-C alkyl group or C-C 30 group, C-C alkyl group, C-C alkoxy group and alkyl group Substituted with at least one halogen halo-C-C alkyl group, -NR'R'' group or atom, -(CH2)-(O).-R" group; R is hydrogen atom, C-C alkyl group optionally R" is C-C alkoxycarbonyl group or carboxyl group; Substituted with at least one halogen atom, C-C, R is chloromethyl group, cyanomethyl group, C-C, alkenyl group optionally Substituted with at least one 35 cycloalkyl group into which at least one oxygen halogen atom, C-C alkynyl group optionally Sub- atom may be inserted, or C-C alkoxycarbonyl Stituted with at least one halogen atom, phenyl group C-C alkyl group; optionally Substituted with at least one halogen atom, R is hydroxyl group or -NR'R' group; C-C cycloalkyl group, cyanomethyl group, or A is -NR'R' group or —S(O)-R" grOup, RCO-group; 40 R7 and Rare, the same or different, hydrogen atom R" is hydrogen atom, C-C alkyl group optionally or C-C alkyl group; Substituted with at least one halogen atom, C-C, R” is C-C alkyl group or C-C haloalkyl group; alkenyl group optionally Substituted with at least one R" is hydrogen atom, hydroxyl group, halogen atom, halogen atom, C-C alkynyl group optionally Sub- C-C alkyl group optionally Substituted with at Stituted with at least one halogen atom, phenyl group 45 least one C-C alkoxy group, C-C cycloalkyl optionally Substituted with halogen atom, C-Cs group into which at least one oxygen atom may be cycloalkyl group, cyanomethyl group, C-C, inserted, C-C cycloalkyl group optionally Substi alkoxy-C-C alkyl group, di-C-C alkylamino- tuted with one or two methyl groups, furyl group, C-C alkyl group, tetrahydrofurfurylmethyl group, thienyl group or -C(=O)R’ group; C-C alkynyloxy-C-C alkyl group, benzyl whose 50 R'' and R' are, the same or different, C-C alkyl ring may be Substituted with Substituent Selected group or C-C alkoxy group; from the group consisting of halogen atom, nitro R7 and R7 are, the same or different, C-C alkyl group, cyano group, C-C alkyl group, C-C, group or phenyl group; alkoxy group and halo-C-C alkyl group, R’ is C-C cycloalkyl into which at least one oxygen -C(=X)R group, -(CH)-(O) R' group, 55 atom may be inserted, C-C cycloalkyl group -(CH2)-O-(CH2)-R" group, -(CH2)- optionally substituted with one or two methyl X-R7 group; groups, furyl group, thienyl group or -C(=O)R’ R and R together with the nitrogen atom to which grOup, they are attached may form Saturated alicyclic 3, 5 or R7 is C-C alkyl group; 6 membered ring or aromatic 5 or 6 membered ring 60 a, b and c is independently 1, 2 or 3; in which a carbon atom may be optionally replaced d is 0 or 1; with oxygen atom; e is 2 or 3; R is hydrogen atom, C-C alkyl group, C-C, f is 1 or 2; and alkenyl group or C-C alkynyl group, or R and X is oxygen atom or Sulfur atom. R together may form -(CH2)-; 65 In addition, as other N-Substituted pyrazoles, there are the R and R7 are independently C-C alkyl group 3-substituted-2-aryl-4,5,6,7-tetrahydroindazoles described optionally Substituted with at least one halogen atom, in Lyga et al., Pesticide Sci., 42. p 29 (1994), and the like. US 6,570,070 B1 29 30 AS Specific examples of the compounds inhibiting elec -continued tron transfer in photochemical System I, for example, there Structure 6 are paraduat, diguat, and the like. AS Specific examples of COOCH the compounds inhibiting electron transfer in photochemical System II, for example, there are triazine compounds (e.g., atrazine, etc.), urea compounds (e.g., diuron, etc.), nitrile compounds (e.g., bromoxynil and ioxynil) and the like. AS specific examples of the compounds inhibiting 4-HPPD, for Cl example, there are isoxazoles (e.g., isoxaflutole), pyrazoles, 1O triketones, and the like. AS Specific examples of the com Structure 7 pounds inhibiting PDS, for example, there are norflurazon, Cl flurochloridone, fluridone, flurtamone, diflufenican, and the like. AS Specific examples of the compounds inhibiting EPSPS, for example, there are glyphosate, and the like. As 15 -()– Specific examples of the compounds inhibiting ALS, for -- O example, there are Sulfonylureas, imidazolinones, Cl pyrimidinylthiobenzoates, triazolopyrimidines, and the like. AS Specific examples of the compounds inhibiting GS, for Structure 8 example, there are bialaphos, glufosinate, and the like. AS F specific examples of the compounds inhibiting DHP, for example, there are asulam, and the like. AS Specific examples of the compounds inhibiting ACC, for example, there are cyclohexanediones, aryloxyphenoxypropionates, 25 and the like. AS Specific examples of the compounds inhib 2O iting cellulose, for example, there are dichlobenil, and the \ = like. Structure 9 Various examples of the weed control compounds useful in the present invention are shown by the following chemi cal Structures:

Structure 1 Cl COONa 35 O O ) = Structure 10 - - - F O Structure 2 40 C CONHSOCH

CF O NO2 O O O 45 Structure 3 \- COOC5H11 Structure 11 Cl COOCH(CH3)COOCHs F O

50 CF O NO2

Structure 4 O C COOCH2COOCHs 55 Structure 12 CF O NO2

Structure 5 NOCH2COOCH 60 O C CCHOCH

S (3- NO \- COOCH US 6,570,070 B1 31 32 -continued -continued Structure 13 Structure 19 Cl O F Br CHF CF X 1 s Cl N Cl M e N CH N-NNoH, HN O \-soch, 1O O

Structure 14 F O Structure 20 15 1 CHF X- N F S Cl N M e N CH O - N COOCHs C I O NO-E

25 Structure 15 O CH3 Structure 21 / F O )- N CF X ON C N O O / 35

Structure 22 F O Structure 16 F O CH3 40 X Cl N

- - CF 45 \ O O COOCHs \ = C Structure 17 Structure 23 F O CH Cl O - - X-x CF 50 N O 55 O o Structure 18 Structure 24 F O CH N X- M 60 Cl N / CF C

O O -( 65 US 6,570,070 B1 33 34 -continued -continued Structure 32 Structure 25 F O X- N X Cl N C

N O

1O Fo Structure 33 Structure 26

15 Cl

Structure 34 Cl

Structure 27 Cl N

25

Structure 35 F

Structure 28

-N -O CH- Cl 35 ( \ -= )- Structure 36 CH

40 Structure 29 CH

Structure 37 45 NH2 Cl NO e N CF Structure 30 N N 50 Cl

In the first aspect of the method of the present invention, the genes to be used are those encoding proteins having the 55 following characteristics (a) to (c) (hereinafter Sometimes referred to as the objective proteins): (a) having a specific affinity for weed control Substances; (b) having Substantially no capability of modifying Sub Structure 31 stances for which Said protein has a specific affinity; 60 and (c) being Substantially free from framework regions of Variable regions of an immunoglobulin. The term “a specific affinity” for weed control substances of the above characteristic (a) means that an enzyme (the 65 objective protein) and a Substrate (the weed (control Substance), or an enzyme (the objective protein) and an inhibitor or a regulator of an activity of the enzyme (the US 6,570,070 B1 35 36 weed control Substance) bind to each other, enzymatically; binant antibody genes, for example, Heavy primer mix or or that the objective protein and the weed control Substance Light primer mix of Recombinant Phage Antibody System bind to each other on the basis of affinity and Specificity, (Pharmacia Biotech) to analyze presence or absence of Such as those shown in a receptor-chemical bond, for amplification of DNA having a given length. Examples of example, a bond between a receptor and a ligand, and the the binding proteins having a specific affinity for weed like. The objective proteins may be naturally occurring control Substances also include peptides having an affinity proteins, variants thereof obtained by introduction of amino for the weed control Substances. acid Substitution, addition, deletion, modification and the Specific examples of the objective proteins having the like into naturally occurring proteins, and artificially Syn above characteristics of (a) to (c) include inactive-type thesized proteins having random amino acid Sequences binding. proteins having an affinity for protoporphyrin IX selected with the guidance of their affinity for weed control e.g., inactive-type magnesium chelatase whose Substrate is Substances, in So far as they have structures Specifically protoporphyrin IX (the weed control Substance), inactive binding to weed control Substances. type ferrochelatase (protoheme ferrolyase; EC 4.9.9.1), The term “having substantially no capability of modify inactive-type cobalt chelatase which catalyzes a chelating ing in the characteristic (b) means that enzymatic reactivity 15 reaction of a cobalt ion with a compound having tetrapyrrole with Substances for which Said protein has a Specific affinity ring as a Substrate, peptides having an affinity for protopor is Substantially inactive or not existed (except the specific phyrin IX, i.e., proteins composed of 4 to 100 amino acids affinity for weed control Substances in the characteristic (a)). (for example, peptide HASYS having an affinity for proto Examples of this include a case that the objective protein porphyrin IX, e.g., a protein comprising the amino acid does not have any capability of converting a Substance for sequence of SEQID NO: 53 and a protein having the amino which said protein has a specific affinity Such as a certain acid sequence of SEQID NO: 54, peptide RASSL having an weed control Substance or a Substance having an essential affinity for protoporphyrin IX, e.g., a protein comprising the part of the structure of the substrates on the basis of a amino acid sequence of SEQID NO:55 and a protein having Specific affinity for said protein, and the like to a Substance the amino acid sequence of SEQ ID NO: 56; peptide YAGY having a chemical structure different from that of the Sub 25 having an affinity for porphyrin compounds, e.g., a protein stance for which Said protein has a specific affinity. The comprising the amino acid sequence of SEQ ID NO: 57 and protein “having Substantially no capability of modifying a protein having the amino acid sequence of SEQ ID NO: can be, for example, identified by checking non-recovery of 58; peptide YAGF having affinity for porphyrin compounds, the growth of a microorganism whose gene encoding the e.g., a protein comprising the amino acid Sequence of SEQ Said protein is deleted and thus cannot grow under a usual ID NO. 59 and a protein having the amino acid sequence of condition in a case where the gene encoding the Said protein SEQ ID NO: 60; and the like), inactive-type binding is introduced into the microorganism in Such a State that the proteins having an affinity for protoporphyrinogen IX (e.g., introduced gene is expressed in the microorganism. inactive-type PPO, inactive-type coproporphyrinogen III The term “substantially free from the framework regions oxidase), and the like. of variable regions of an immunoglobulin' in the charac 35 The above inactive-type binding proteins include variants teristic (c) mean that the objective protein does not form a thereof whose activities have been lost by amino acid Stereostructure Specific for the variable regions of an immu Substitution, addition, deletion, modification and the like of noglobulin. The term “framework regions of variable naturally occurring active proteins under natural or artificial regions of an immunoglobulin' mean regions remaining conditions. after removing the hyperVariable regions from the variable 40 Cellular dysfunction caused by Weed control Substances regions of H chain and L chain which are the constituents of can be prevented by binding of these binding proteins to the the immunoglobulin molecule. In these regions, conserva weed control Substances in plant cells to exhibit the desired tion of the amino acid Sequences is relatively high and these weed control compound-resistance. regions function for maintaining the highly conserved Ste The inactive-type magnesium chelatase is protoporphyrin reostructure of the variable regions. Due to formation of the 45 IX binding Subunit protein of magnesium chelatase, or its above Stereostructure, the hyperVariable regions Separately variant having a specific affinity for protoporphyrin IX, and located at three sites on respective H chain and L chain are Specific examples thereof include the Subunit protein from collected to one site on the Stereostructure to form an antigen which its organelle transit Signal Sequence has been deleted, binding site Alberts, B., et al. ed. (1983), Molecular Biol and the like. ogy of the Cell, p. 979, Garland Publishing, Inc., New York. 50 The inactive-type ferrochelatase is its variant having no The objective protein having the above characteristic (c) capability of modifying protoporphyrin IX and having a can be Selected on the basis of, for example, the amino acid Specific affinity for protoporphyrin IX, and Specific Sequences of the proteins. AS Specific examples of the examples thereof include a ferrochelatase variant in which a protein, there are a protein which does not contain any region presumed to be a Fe ion binding Site of ferrochelatse amino acid Sequence composed of about 30 amino acids or 55 has been modified, and the like. more and having about 60% or more homology with the The inactive-type cobalt chelatase is a Substrate binding known amino acid Sequences of the framework regions of Subunit protein of cobalt chelatase, or its variant having no the variable regions of an immunoglobulin, and the like. For capability of modifying protoporphyrin IX and having a example, the presence or absence of the above framework specific affinity for protoporphyrin IX. regions can be confirmed by PCR using a gene encoding the 60 The inactive-type PPO is its variant having no capability protein as a template and DNAS having nucleotide of oxidizing protoporphyrinogen IX and having a specific Sequences encoding the variable regions derived from H affinity for protoporphyrinogen IX, and Specific examples chain or L chain of the immunoglobulin as amplification thereof include a PPO variant in which a region presumed to primers, for example, the primers VH1 BACK and be FAD binding site of PPO (a region having the amino acid VH1 FOR-2, or VK2BACK and VK4FOR described by 65 Sequence GXGXXG wherein X is any amino acid, e.g., a Clackson, T. et al., Nature 352; p 624 (1991), or primers region comprising the 63rd to 68th amino acids from the contained in a commercially available kit for cloning recom N-terminus of chloroplast localized PPO of mouse-ear cress US 6,570,070 B1 37 38 (Arabidopsis thaliana) and having the amino acid sequence like have been known. For isolating Such a known gene (its of GGGISG) has been deleted, and the like. The inactive nucleotide sequence has been known), PCR can be carried type coproporphyrinogen III oxidase is its variant having no out by using genomic DNA or cDNA of an organism having capability of oxidizing protoporphyrinogen IX and having a the desired gene as a template and primerS produced on the Specific affinity for protoporphyrinogen IX. basis of nucleotide Sequences corresponding to those about The genes encoding the above proteins can be obtained the N- and C-termini of the protein encoded by the gene to by, for example, as follows. amplify the desired gene. Further, for obtaining other genes AS the genes encoding protoporphyrin IX binding Subunit encoding ferrochelatase, for example, first, a cDNA library protein of magnesium chelatase, for example, those derived is constructed by obtaining mRNA from the desired from the photosynthetic bacterium, Rhodobacter capsulatus organism, Synthesizing cDNA by using the mRNA as a (Genebank accession M74001), mouse-ear cress (Genebank template with a reverse transcriptase, and integrating the accession Z68495), barley (Genebank accession U96216), cDNA into a phage vector such as ZAPII, etc. or a plasmid Snapdragon (Antirrhinum majus) (Genebank accession vector such as puC, etc. The cDNA library can be intro U26916), Synechocystis P.C.C. 6803 (Genebank accession duced into ferrochelatase deficient mutant strain of Escheri U29131) and the like have been known. For isolating such 15 chia coli VS200 described by Miyamoto, K, et al., Plant a known gene (its nucleotide sequence has been known), Physiol., 105; p 769 (1994), followed by subjecting a PCR can be carried out by using genomic DNA or cDNA of complementation test to Select clones containing ferroche an organism having the desired gene as a template and latase gene derived from the desired organism. Further, for primerS produced on the basis of nucleotide Sequences amplifying a DNA fragment, PCR can be carried out by corresponding to those about the N- and C-termini of the using the above-constructed cDNA library as a template and protein encoded by the gene to amplify the desired gene. primerS prepared on the basis of nucleotide Sequences well Further, genes encoding protoporphyrin IX binding Subunit conserved among known genes Such as the above-described protein of magnesium chelatase can be obtained from pho genes. Screening of the cDNA library can be carried out by tosynthetic organisms other than the above. For example, using the DNA fragment thus obtained as a probe to Select first, a cDNA library is constructed by obtaining mRNA 25 positive clones. The desired ferrochelatase gene can be from the desired photosynthetic organism, Synthesizing confirmed by Sequence determination of the nucleotide cDNA by using the mRNA as a template with a reverse Sequence of the Selected clone. transcriptase, and integrating the cDNA into a phage vector For obtaining the gene encoding a variant of ferroche such as ZAPII, etc. or a plasmid vector such as puC, etc. For latase having no capability of modifying protoporphyrin IX amplifying a DNA fragment containing at least a part of the and having a specific affinity for protoporphyrin IX (for gene encoding protoporphyrin IX binding Subunit protein of example, the gene encoding a ferrochelatase variant in magnesium chelatase, PCR can be carried out by using the which the region presumed to be a Fe ion binding Site of above-constructed cDNA library as a template and primers ferrochelatase is modified), PCR can be carried out by designed and Synthesized on the basis of nucleotide preparing a mutagenesis primer for introduction of mutation Sequences well conserved among known genes Such as the 35 into the region on the basis of nucleotide Sequence encoding above-described genes. Screening of the cDNA library can the amino acid Sequence about the region, and using a be carried out by using the DNA fragment thus obtained as commercially available Site-directed mutagenesis kit a probe to Select positive clones. The desired gene of (Mutan-Super Express, Takara Shuzo) to obtain the gene protoporphyrin IX binding Subunit protein of magnesium encoding the above variant. Specifically, a wild type ferro chelatase can be confirmed by Sequence determination of the 40 chelatase gene is inserted into the cloning Site of plasmid nucleotide Sequence of the Selected clone. vector pKF19K and PCR is carried out by using the resultant For obtaining the gene encoding a variant of protoporhy plasmid DNA as a template, the above-described mutagen rin IX binding Subunit protein of magnesium chelatase esis primer and a Selection primer for restoration of amber having an Specific affinity for protoporphyrin IX, for mutation located on kanamycin resistant gene of pKF19K. example, the gene encoding the Subunit protein is 45 The gene amplified by PCR is introduced into Escherichia mutagenized by introduction of nucleotide Substitution, coli MV1184 (suppressor free strain) and the transformants addition, deletion, modification and the like, followed by are Screened according to kanamycin resistance to isolate introducing the resultant gene into Escherichia coli BL21 Escherichia coli having ferrochelatase gene in which the (DE3) strain according to the method described by Gibson, nucleotide Sequence corresponding to the amino acid L. C. D. et al., Proc. Natl. Acad. Sci. USA, 92; p 1941 (1995) 50 Sequence which constitutes the desired region has been and the like to obtain transformants, and culturing the modified. The isolated gene can be confirmed as the gene transformants under conditions that high expression of the encoding the desired protein by analyzing the nucleotide gene thus introduced occurs. The desired gene encoding a sequence of the plasmid DNA of the Escherichia coli. variant of the Subunit protein having a specific affinity for The genes encoding the peptides having an affinity for protoporphyrin IX can be obtained by Selecting a Strain 55 protoporphyrin IX, i.e., the proteins composed of 4 to 100 whose cultured cells have turned red and have the fluores amino acids can be obtained by Synthesizing a peptide cence absorption showing accumulation of protoporphyrin library according to, for example, the combinatorial chem IX (excitation wavelength 405 nm, emission wavelength istry method as described by Sugimoto, N., Nakano, S., 630 nm). Chem., Lett., p. 939 (1997) and the like, selecting a peptide AS the genes encoding ferrochelatase, for example, those 60 having an affinity for the weed control Substance, analyzing derived from Escherichia coli (Genebank accession the amino acid Sequence of the peptide thus Selected with a D90259), Bacillus Subtilis (Genebank accession M97208), peptide Sequencer, designing a gene containing a nucleotide Bradyrhizobium japonicum (Genebank accession M92427), Sequence encoding the amino acid Sequence, and Synthesiz yeast Saccharomyces cerevisiae (Genebank accession ing the nucleotide Sequence with a DNA Synthesizer or the J05395), mouse (Genebank accessionJ05697), human being 65 like. (Genebank accession D00726), barley (Genebank accession Further, a phase clone displaying a peptide having an D26105), cucumber (Genebank accession D26106), and the affinity for the weed control substance can be selected from US 6,570,070 B1 39 40 a phage library according to phage display method. For obtaining the gene encoding a variant of PPO having Specifically, for example, a phage library displaying a no capability of oxidizing protoporphyrinogen IX and hav protein having a random amino acid Sequence on the Surface ing a specific affinity for protoporphyrinogen IX, for of M13 phage particles is constructed by inserting a nucle example, PPO gene is mutagenized by introducing nucle otide Sequence encoding the protein having the random otide Substitution, addition, deletion, modification, etc. and amino acid Sequence into the upstream from the region the resultant modified gene is introduced into the above encoding the coat protein pl of M13 phage gene. On the Escherichia coli whose growth is inhibited light other hand, the weed control Substance labeled with biotin is dependently by treatment with a PPO inhibitory-type her bound to a plate coated with avidin or Streptoavidin to bicidal compound. A gene encoding a protein having pro prepare a Support coated with the weed control Substance. A toporphyrinogen IX binding capability can be selected by phage displaying the desired protein having an affinity for culturing the Escherichia coli thus obtained in the presence the weed control Substance can be isolated by Screening the of hemin, aminolevulinic acid and a PPO inhibitory-type above phage library on the plate coated with the weed herbicidal compound to Select a clone which can grow even control Substance and the gene of the desired protein can be in the light. A gene encoding a protein having no capability obtained from the isolated phage. 15 of oxidizing protoporphyrinogen IX can be Selected by The gene encoding a protein containing the repetition of expressing the modified gene thus Selected in a host Such as the amino acid sequence represented by SEQID NO: 53,55, Escherichia coli etc. to prepare a protein encoded by the 57 or 59 four times or eight times can be produced by, for gene, and measuring its capability of oxidizing protopor example, Selecting a nucleotide Sequence in which the phyrinogen IX according to the method described by Jacobs, nucleotide Sequence encoding the above amino acid N.J. and Jacobs, J. M. (1982) Enzyme, 28, 206–219 and the Sequence is repeated the given times after the initiation like. More Specifically, the above modified gene is inserted codon ATG, Synthesizing an oligonucleotide comprising the into an expression vector for Escherichia coli and intro Selected nucleotide Sequence and an oligonucleotide com duced into PPO gene (hemC locus) deficient mutant of prising a nucleotide Sequence complementary to the Selected Escherichia coli Such as Escherichia coli BT3 strain nucleotide Sequence by a DNA synthesizer, and then Sub 25 described by Yamamoto, F., et al., Japanese J. Genet., 63; p jecting them to annealing. Further, the genes encoding the 237 (1988) and the like. The Escherichia coli is cultured in amino acid sequence represented by SEQ ID NO: 54, 56, 58 a culture medium containing hemin and aminolevulinic acid or 60 can be produced by Selecting a nucleotide Sequence in addition to the cell growth inhibitor corresponding to the encoding the amino acid Sequence, Synthesizing an oligo Selection marker of the vector introduced into the Escheri nucleotide comprising the Selected nucleotide Sequence and chia coli to obtain transformants. The protein encoded by the another oligonucleotide comprising a nucleotide Sequence modified gene can be produced from the transformant. complementary to the Selected nucleotide Sequence by a Further, a gene which does not complement PPO gene DNA synthesizer, and then Subjecting them to annealing. In deficiency of its host cell can be obtained by culturing the this respect, for Selecting the nucleotide Sequence encoding transformant in a culture medium Substantially free from the given amino acid Sequence, for example, it is preferred 35 hemin and aminolevulinic acid to identify a Strain which to Select codons frequently used in genes derived from does not grow. This latter method can also be used for plants. Selection of the gene encoding a protein having no capability As PPO genes, for example, those derived from Escheri of oxidizing protoporphyrinogen IX. chia coli (Genebank accession X68660), Bacillus Subtilis Further, for obtaining the gene encoding a variant of PPO (Genebank accession M97208), Haemophilus influenzae 40 in which the region presumed to be a FAD binding site of (Genebank accession L42023), mouse (Genebank accession PPO (the region having the amino acid sequence GXGXXG, D45185), human being (Genebank accession D38537), wherein X is any amino acid) is deleted, first, a mutagenesis mouse-ear cress (Genebank accession D83139), tobacco primer for introduction of deletion mutation of the region is (Genebank accession Y13465, Y13466) and the like have prepared on the basis of the nucleotide Sequence encoding been known. For isolating Such a known gene (its nucleotide 45 the amino acid Sequence about the region. Then, PCR is Sequence has been known), PCR is carried out by using carried out by using the mutagenesis primer and a commer genomic DNA or cDNA of an organism having the desired cially available site-directed mutagenesis kit (Mutan-Super gene as a template and primerS produced on the basis of Express, Takara Shuzo) as described above to obtain the nucleotide Sequences corresponding to those about the N gene encoding the above variant protein in which the region and C-termini of the protein encoded by the gene to amplify 50 has been deleted. the desired gene. Further, for obtaining other PPO genes, for The genes encoding peptide proteins Such as the peptides example, first, a cDNA library is constructed from an HASYS (SEQ ID NO: 53) and RASSL (SEQ ID NO: 55) organism having the desired gene according to the above having an affinity for protoporphyrin IX, and the peptides described method. The cDNA library can be introduced into YAGA (SEQ ID NO: 57) and YAGF (SEQ ID NO: 59) Escherichia coli PPO deficient mutant Strain VSR800 55 having an affinity for prophyrin compounds, and the like can described by Narita, S., et al., Gene, 182; p 169 (1996), be obtained by Subjecting oligonucleotides Synthesized by a followed by Subjecting a complementation test to Select DNA Synthesizer to annealing. clones containing PPO gene derived from the desired organ Furthermore, genes encoding unknown peptide proteins ism. Further, for amplifying a DNA fragment, PCR can be having affinities for other weed control Substances can be carried out by using the above-constructed cDNA library as 60 produced by the following methods and the like. For a template and primers prepared on the basis of nucleotide example, various peptide libraries can be constructed Sequences well conserved among known genes Such as the according to, for example, the combinatorial chemistry above-described genes. Screening of the cDNA library can method as described by Sugimoto, N., Nakano, S., Chem., be carried out by using the DNA fragment thus obtained as Lett., p. 939 (1997), and the like. Peptides are selected from a probe to select positive clones. The desired PPO gene can 65 the peptide libraries thus constructed with the guidance of be confirmed by Sequence determination of the nucleotide affinities for weed control Substances, followed by analyzing Sequence of the Selected clone. the amino acid Sequences of the peptides with a peptide US 6,570,070 B1 41 42 Sequencer. Thus, genes encoding the peptides can be Syn and a PPO inhibitory-type herbicide to select a clone which thesized by a DNA synthesizer. Alternatively, phase clones can grow even in the light. A gene encoding a protein having displaying peptides having affinities for weed control Sub no capability of oxidizing protoporphyrinogen IX can be stances can be obtained by Selecting phage libraries accord Selected by expressing the modified gene thus Selected in a ing to phage display method. Specifically, for example, a host Such as Escherichia coli, etc. to prepare a protein phage library displaying a protein having a random amino encoded by the gene, and measuring its capability of oxi acid Sequence on the Surface of M13 phage particles is dizing protoporphyrinogen IX according to the method constructed by inserting a nucleotide Sequence encoding the described by Jacobs, N.J. and Jacobs, J. M. (1982) Enzyme, protein having the random amino acid Sequence into the 28, 206-219 and the like. upstream from the region encoding the coat protein pII of The genes which is used in the Second aspect of the M13 phage gene. On the other hand, a weed control Sub method of the present invention are those encoding proteins stance labeled with biotin is bound to a plate coated with having the following characteristics (a) to (c): avidin or Streptoavidin to prepare a Support coated with the (a) having a specific affinity for protoporphyrin IX, weed control Substance. A phage displaying the desired (b) having Substantially no capability of modifying pro protein having an affinity for the weed control Substance can 15 toporphyrinogen IX, and be isolated by Screening the above phage library on the plate (c) being Substantially free from framework regions of coated with the weed control Substance and the gene of the Variable regions of immunoglobulins. desired protein can be obtained from the isolated phage. The term “a specific affinity” for protoporphyrin IX in the AS the genes encoding coproporphyrinogen III oxidase, characteristic (a) is Substantially the same as that in the for example, those derived from Escherichia coli (Genebank above first aspect of the method of the present invention and accession X75413), Salmonella typhimurium (Genebank means that the protein and protoporphyrin IX bind to each accession L19503), yeast Saccharomyces cerevisiae other, enzymatically or the protein and protoporphyrin IX (Genebank accession J03873), mouse (Genebank accession bind to each other on the basis of affinity and Specificity as D1633), human being (Genebank accession D16333), soy those shown in receptor chemical bond Such as a bond bean (Genebank accession X71083), barley (Genebank 25 between a receptor and a ligand and the like. The proteins accession X82830), tobacco (Genebank accession X82831) may be naturally occurring proteins, variants thereof in and the like have been known. For isolating Such a known which amino acid Substitution, addition, deletion, modifica gene (its nucleotide Sequence has been known), PCR is tion and the like are introduced into naturally occurring carried out by using genomic DNA or cDNA of an organism proteins, and artificially Synthesized proteins having random having the desired gene as a template and primers produced amino acid Sequences which are Selected with the guidance on the basis of nucleotide Sequences corresponding to those of an affinity for protoporphyrin IX in so far as they have about the N- and C-termini of the protein encoded by the Structures Specifically binding to protoporphyrin IX. gene to amplify the desired gene. Further, for obtaining other The term “having Substantially no capability of modify coproporphyrinogen III oxidase genes, for example, first, a ing protoporphyrinogen IX in the characteristic (b) means cDNA library is constructed from an organism having the 35 that enzymatic reactivity with protoporphyrinogen IX of the desired gene by preparing mRNA from the desired protein is Substantially inactive or not existed. For example, organism, Synthesizing cDNA using the mRNA as template this means that the protein does not have capability of with a reverse transcriptase and integrating this into a converting protoporphyrinogen IX into a Substance having a plasmid vector such as pRS313 described by Sikorski, R. S., chemical Structure different from that of protoporphyrinogen et al., Genetics, 122; p 19 (1989), and the like. The cDNA 40 IX. library can be introduced into yeast coproporphyrinogen III The term “substantially free from framework regions of oxidase deficient mutant strain HEM13 described by Troup, variable regions of immunoglobulins' means the Same as B., et al., Bacteriol., 176; p. 673 (1994), followed by sub that in the above first aspect of the method of the present jecting a complementation test to Select clones containing invention and the protein does not form the Stereostructure coproporphyrinogen III oxidase derived from the desired 45 Specific for the variable regions in the immunoglobulin as is organism. Further, for amplifying a DNA fragment, PCR can described hereinabove. be carried out by using the above-constructed cDNA library AS Specific examples of the proteins having the above as a template and primerS prepared on the basis of nucleotide characteristics (a) to (c), there are active or inactive-type Sequences well conserved among known genes Such as the binding proteins having an affinity for protoporphyrin IX above-described genes. Screening of the CDNA library can 50 e.g., active or inactive-type magnesium chelatase whose be carried out by using the DNA fragment thus obtained as Substrate is protoporphyrin IX, active or inactive-type a probe to Select positive clones. The desired coproporphy ferrochelatase, active or inactive-type cobalt chelatase rinogen III oxidase gene can be confirmed by Sequence which catalyzes a chelating reaction of a cobalt ion with a determination of the nucleotide Sequence of the Selected compound having tetrapyrrole ring as a Substrate, peptides, clone. 55 i.e., proteins composed of 4 to 100 amino acids, having an For obtaining the gene encoding a variant of coporphy affinity for protoporphyrin IX (for example, proteins con rinogen III oxidase having no capability of oxidizing pro taining at least one peptide selected from peptide HASYS toporphyrinogen IX and having a specific affinity for pro having an affinity for protoporphyrin IX, e.g., a protein toporphyrinogen IX, for example, coproporphyrinogen III comprising the amino acid sequence of SEQ ID NO: 53 and oxidase gene is mutagenized by introducing nucleotide 60 a protein having the amino acid sequence of SEQ ID NO: Substitution, addition, deletion, modification, etc. and the 54; peptide RASSL having an affinity for protoporphyrin IX, resultant gene is introduced into the above Escherichia coli i.e., a protein comprising the amino acid Sequence of SEQ whose growth is inhibited light-dependently by treatment ID NO: 55 and a protein having the amino acid sequence of with a PPO inhibitory-type herbicidal compound. A gene SEQ ID NO: 56; peptide YAGY having an affinity for encoding a protein having protoporphyrinogen IX binding 65 porphyrin compounds, e.g., a protein comprising the amino capability can be selected by culturing the Escherichia coli acid sequence of SEQ ID NO: 57 and a protein having the thus obtained in the presence of hemin, aminolevulinic acid amino acid sequence of SEQ ID NO: 58; peptide YAGF US 6,570,070 B1 43 44 having affinity for porphyrin compounds, i.e., a protein above first or Second aspect of the method of the present comprising the amino acid sequence of SEQ ID NO. 59 and invention and means that the protein and protoporphyrino a protein having the amino acid sequence of SEQ ID NO: gen IX bind to each other, enzymatically or the protein and 60; and the like), and the like. protoporphyrinogen IX are bound to each other on the basis The genes encoding the above proteins can be obtained 5 of affinity and Specificity as those shown in receptor by, for example, as follows. chemical bond Such as a bond between a receptor and a Active-type magnesium chelatase are composed of three ligand and the like. The proteins may be naturally occurring heterogenous Subunit proteins, i.e., protoporhyrin IX bind ing Subunit protein (HSubunit protein), I Subunit protein and proteins, variants thereof in which amino acid Substitution, D Subunit protein, all of them are essential for catalytic addition, deletion, modification and the like are introduced acitivity. Three independent Subunit proteins are encoded by into naturally occurring proteins, and artificially Synthesized different genes. The genes of protoporphyrin IX binding proteins having random amino acid Sequences which are subunit protein can be obtained by PCR or screening of Selected with the guidance of an affinity for protoporphy cDNA library as described hereinabove. rinogen IX in So far as they have structures Specifically AS the gene encoding I Subunit protein of a magnesium binding to protoporphyrinogen IX. The term “having the chelatase, for example, those derived from photosynthetic 15 capability of modifying coproporphyrinogen III in the bacterium, Rhodobacter Sphaeroides (Genebank accession characteristic (b) means that enzymatical reactivity with AF017642), Rhodobacter capsulatus (Genebank accession coproporphyrinogen III of the proteins is active. For Z11165), Arabidopsis (Genebank accession D49426), barley example, this means that the protein has the capability of (Genebank accession U26545), Soybean (Genebank acces converting coproporphyrinogen III into a Substance having sion D45857), tobacco (Genebank accession AF14053), a chemical Structure different from that of coproporphyrino Synechocystis P.C.C.6803 (Genebank accession U35144) gen III. and the like have been known. For isoltaing Such a known The term “substantially free from framework regions of gene (its nucleotide sequence has been known), PCR can be variable regions of immunoglobulins' means the Same as carried out by using genomic DNA or cDNA of an organism that in the above first or second aspect of the method of the having the desired gene as a template and primers produced 25 present invention and the protein does not form the Stereo on the basis of nucleotide Sequences corresponding to those Structure Specific for the variable regions in the immuno about the N- and C-termini of the protein encoded by the globulin as is described hereinabove. desired gene. Further, genes encoding I Subunit protein of a AS Specific examples of the proteins having the above magnesium chelatase can be obtained from photosynthetic characteristics (a) to (c), there are active or inactive-type organisms other than the above. For example, first, a cDNA binding proteins having an affinity for proporphyrinogen IX, library is constructed by obtaining mRNA from the desired for example, active-type coproporphyrinogen III oxidase photosynthetic organisms, Synthesizing cDNA by using the whose Substrate is proporphyrinogen IX, and the like. mRNA as a template with a reverse transcriptase, and AS a reference, the activity of a magnesium chelatase, a integrating the cDNA into a phage vector Such as ZAPII, etc. ferrochelatase or a coproporphyrinogen III oxidase is, for or plasmid vector such as puC, etc. For amplifying a DNA 35 example, measured by using the following method. fragment containing at least a part of the gene encoding I (1) A Magnesium Chelatase: Subunit protein of a magnesium chelatase, PCR can be The genes encoding independent three Subunit proteins carried out by using the above-constructed cDNA library as are used to detect a magnesium chelatase activity according a template and primerS designed and Synthesized on the to the method by Gibson, L. C. D., et al. (Proc. Natl. Acad. basis of nucleotide Sequences well conserved among known 40 Sci. USA, 92; p 1941 (1995)) and the like. genes Such as the above described genes. Screening of the (2) A Ferrochelatse: cDNA library can be carried out by using the DNA fragment A ferrochelatase activity can, for example, be detected thus obtained as a probe to Select positive clones. The according to the method by Porra, R.J. (Anal. Biochem., 68; desired gene of I Subunit protein of a magnesium chelatase p 289 (1975)) and the like. can be confirmed by determination of the nucleotide 45 (3) A Coproporphyrinogen III Oxidase: Sequence of the Selected clone. A coproporphyrinogen III oxidase activity can, for AS the gene encoding D Subunit protein of a magnesium example, be detected according to the method by Yoshinaga, chelatase, for example, those derived from photosynthetic T., Sano, S., et al. (J. Biol. Chem., 255; p 4722 (1980)) and bacterium, Rhodobacter Sphaeroides (Genebank accession the like. AJ001690), Rhodobacter capsulatus (Geneband accession 50 In the method (including the above first to third aspects) Z11165), pea (Genebank accession AF014399), tobacco of the present invention, for introducing the gene encoding (Genebank accession Y10022), Synechocystis P.C.C.6803 the protein having the characteristics of (a) to (c) into a plant (Genebank accession X96599) and the like have been cell, a gene encoding one protein can be introduced. Further, known. The isolation of Such a known gene (its nucleotide plural genes encoding different proteins can be introduced Sequence has been known) or genes other than the above can 55 into a plant cell. Such gene introduction into plant cells can be carried out in the same manner as described in that of the be carried out by conventional gene engineering techniques, gene encoding I Subunit protein of magnesium chelatase. for example, Agrobacterium infection (JP-B 2-58917 and The genes used in the third aspect of the method of the JP-A 60-70070), electroporation into protoplasts (JP-A present invention are those encoding proteins having the 60-251887 and JP-A 5-68575), particle gun methods (JP-A following characteristics (a) to (c): 60 5-508316 and JP-A 63-258525), and the like. Preferably, the (a) having a specific affinity for protoporphyrinogen IX, gene to be introduced into a plant cell is integrated into a (b) having the capability of modifying coproporphyrino vector having a Selection marker gene Such as a gene which gen III; and can give cell growth inhibitor resistance to the plant cell. (c) being Substantially free from framework regions of For expression of the gene in the plant cell, the gene can variable regions of immunoglobulins. 65 be introduced into the chromosome of a plant cell by The term “a specific affinity” for protoporphyrinogen IX homologous recombination Fraley, R. T. et al., Proc. Natl. in the characteristic (a) is Substantially the same as that in the Acad. Sci. USA, 80; p 4803 (1983) to select the plant cell US 6,570,070 B1 45 46 expressing the gene. Alternatively, the gene can be intro expressing the gene encoding the above protein can be duced into a plant cell in the form that it is operably ligated obtained by introducing the gene into Sterile-cultured wheat to a promoter and a terminator both of which can function immature Scutellum with a particle gun according to a in the plant cell. conventional method described by TAKUMI et al., Journal The term “operably ligated” used herein means that the of Breeding Society (1995), 44: Extra Vol. 1, p 57. Likewise, above promoter and terminator are joined in Such a State that according to a conventional method described by HAGIO, et the introduced gene is expressed in the plant cell under al., Journal of Breeding Society (1995), 44; Extra Vol. 1, p control of the promoter and the terminator. 67, barley expressing the gene encoding the above protein AS the promoter which can function in a plant cell, for can be obtained by introducing the gene into Sterile-cultured example, there are constitutive promoters derived from barley immature Scutellum with a particle gun. T-DNA Such as nopaline Synthase gene promoter, octopine For confirmation of weed control compound-resistance of Synthase gene promoter, etc., promoters derived from plant the plant expressing the gene encoding the above protein, viruses such as 19S and 35S promoters derived from cau preferably, the plant is reproduced with applying the weed liflower mosaic virus, etc., inductive promoterS Such as control compound to which resistance is given to evaluate phenylalanine ammonia-lyase gene promoter, chalcone Syn 15 the degree of reproduction of the plant. For more quantita thase gene promoter, pathogenesis-related protein gene tive confirmation, for example, in case of resistance to a promoter, etc., and the like. The promoter is not limited these compound having PPO inhibitory-type herbicidal activity, promoters and other plant promoters can be used. preferably, pieces of leaves of the plant are dipped in AS the terminator which can function in a plant cell, for aqueous Solutions containing the compound having PPO example, there are terminators derived from T-DNA such as inhibitory-type herbicidal activity at various concentrations, nopaline Synthase terminator, etc., terminators derived from or the aqueous Solutions containing the compound having plant Viruses Such as terminators derived from garlic viruses herbicidal activity are sprayed on pieces of leaves of the GV1, GV2, etc., and the like. The terminator is not limited plant, followed by allowing to Stand on an agar medium in to these terminators and other plant terminators can be used. the light at room temperature. After Several days, chloro AS the plant cells into which the genes are introduced, for 25 phyll is extracted from the plant leaves according to the example, there are plant tissues, whole plants, cultured cells, method described by Mackenney, G., J. Biol. Chem., 140; p Seeds and the like. Examples of the plant species into which 315 (1941) to determine the content of chlorophyll. the genes are introduced include dicotyledones Such as Since the weed control compound-resistant plants (e.g., tobacco, cotton, rapeseed, Sugar beet, mouse-ear creSS, plant tissues, whole plants, cultured cells, seeds, etc.) canola, flax, Sunflower, potato, alfalfa, lettuce, banana, obtained by the method of the present invention (including Soybean, pea, legume, pine, poplar, apple, grape, citrus the first to third aspects) show resistance to weed control fruits, nuts, etc.; and monocotyledones Such as corn, rice, compounds, even in case that a weed control compound is Wheat, barley, rye, oat, Sorghum, Sugar cane, lawn, etc. applied to a growth area (e.g., cultivation area, proliferation The transformant plant cells expressing the gene encoding area, etc.), the plant can grow. Therefore, when a weed the binding protein having the characteristics of (a) to (c) can 35 control compound is applied to a growth area of the desired be obtained by culturing cells into which the gene is trans weed control compound resistant-plant, the desired plant ferred in a Selection culture medium corresponding to a can be protected from plants without resistance to the weed Selection marker joined to the locus on the gene, for control plant. For example, weeds can be controlled effi example, a culture medium containing a cell growth ciently by applying a weed control compound on a growth inhibitor, or the like, and isolating a clone capable of 40 area of the plant having resistance to the weed control growing in the culture medium. Alternatively, the above compound. transformant plant cells can be Selected by culturing plant Further, by applying a weed control compound to a cells into which the gene is introduced in a culture medium growth area of the weed control compound-resistant plant containing the weed control compound to which the resis obtained by the method of the present invention (including tance is given, and isolating clones capable of growing in the 45 the first to third aspects) and other plants (e.g., those having culture medium. The desired weed control compound no or weak resistance to the weed control compound), one resistant plant can be obtained from the transformant cells of the plants can be selected on the basis of the difference in thus obtained by regenerating the whole plant according to growth between the plants. For example, by applying a conventional plant cell culture method, for example, that (adding) a weed control compound to a cultivation area described in Plant Gene Manipulation Manual, Method for 50 (culture medium) of the weed control compound-resistant Producing Transgenic Plants, UCHIMIYA, Kodansha Sci plant cells obtained by the method of the present invention entific (1996). Thus, the transformed plants such as plant and other plant cells (e.g., those having no or weak resis tissues, whole plants, cultured cells, Seeds and the like can tance to the weed control compound), one of the plant cells be obtained. can be selected efficiently on the basis of the difference in For example, rice and mouse-ear creSS expressing the 55 growth between the plants. gene encoding the above protein can be obtained according The following Examples further illustrate the present to the method described Experimental Protocol of Model invention in detail but are not to be construed to limit the Plants, Rice and Mouse-Ear Cress Edition, (Supervisors: Scope thereof. Koh SHIMAMOTO and Kiyotaka OKADA, Shujun-sha, 1996), Chapter 4. Further, according to the method 60 EXAMPLE 1. described in JP-A 3-291501, soybean expressing the gene encoding the binding protein by introducing the gene into Isolation of Protoporphyrin IX Binding Subunit Soybean adventitious embryo with a particle gun. Likewise, Protein Gene of Magnesium Chelatase according to the method described by Fromm, M. E., et al., Bio/Technology, 8; p. 838 (1990), corn expressing the gene 65 Genomic DNA of photosynthetic bacterium Rhodobacter encoding the above protein can be obtained by introducing Sphaeroides ATCC17023 was prepared using ISOPLANT the gene into adventitious embryo with a particle gun. Wheat kit for genomic DNA preparation (manufactured by Nippon US 6,570,070 B1 47 48 Gene). Then, according to the description of Gibson, L. C. collected. The collected green leaves were frozen with liquid D. et al., Proc. Natl. Acad. Sci. USA, 92; p 1941 (1995), nitrogen and the frozen leaves were ground with pestle and PCR was carried out by using about 1 lug of Said genomic mortar. From the ground leaves, RNA were extracted by DNA as a template, and 10 pmol of oligonucleotide com using RNA extracting reagent ISOGEN (manufactured by posed of nucleotide sequence represented by SEQ ID NO: 1 Nippon Gene) according to the manual attached thereto. The and 10 pmol of oligonucleotide composed of nucleotide resultant RNA liquid extract was Subjected to ethanol pre sequence represented by SEQ ID NO: 2 as primers to cipitation to collect total RNA, then the total RNA was amplify the DNA fragment containing protoporphyrin IX fractionated by using poly (A) RNA fractionating kit BIOMAG mRNA Purification Kit (manufactured by Per binding Subunit protein gene bchh of magnesium chelatase. ceptive Bio System) according to the manual attached The oligonucleotides were prepared with a DNA synthesizer thereto to collect poly (A) RNA fraction. Using 1 lug of this (PE Applied Biosystems; Model 394 DNA/RNA poly(A) RNA fraction as a template, cDNA was synthesized Synthesizer) and purified with an oligonucleotide purifica with the cDNA synthetic reagent contained in Marathon tion cartridge (PE Applied Biosystems; OPC cartridge). The cDNA amplification kit (manufactured by Clontech) accord PCR was carried out by maintaining at 94 C. for 2 minutes, ing to the manual attached thereto. PCR was carried out by at 96° C. for 40 seconds and then at 68 C. for 7 minutes, 15 using the resultant CDNA as a template, and oligonucleotide repeating a cycle for maintaining at 96° C. for 40 Seconds composed of nucleotide sequence of SEQ ID NO: 3 and and then at 68 C. for 7 minutes 28 times, and finally oligonucleotide composed of nucleotide Sequence of SEQ maintaining at 96° C. for 40 seconds, at 68 C. for 7 minutes ID NO. 4 as primers to amplify the DNA fragment contain and then at 72 C. for 10 minutes. ing chloroplast-type protoporphyrinogen IX oxidase gene. The above oligonucleotides were prepared with a DNA EXAMPLE 2 synthesizer (PE Applied Biosystems; Model 394 DNA/RNA Synthesizer) and purified with a oligonucleotide purification Expression of Protoporphyrin IX Binding Subunit cartridge (PE Applied Biosystems; OPC cartridge). The Protein Gene of Magnesium Chelatase in PCR was carried out by maintaining at 94 C. for 1 minutes Escherichia coli (Hereinafter Abbreviated to E. 25 and then at 65 C. for 5 minutes, repeating a cycle for coli) maintaining at 94 C. for 15 seconds and then at 65 C. for 5 minutes 29 times. After the PCR, the amplified DNA According to the description of Gibson, L. C. D. et al., fragment was purified by filtering the reaction mixture with Proc. Natl. Acad. Sci. USA, 92; p 1941 (1995), the DNA MicroSpin S-400HR (manufactured by Pharmacia Biotech), fragment containing bchH gene prepared in Example 1 was and the DNA fragment was ligated to plasmid pCR2.1 digested with restriction enzymes Nde and BglII. The (manufactured by Invitrogen) cleaved by restriction enzyme resultant DNA fragment was inserted between Nde restric SalI to obtain plasmid pSPPO-P. Then, the plasmid was tion site and BamHI restriction site of expression vector introduced into competent cells of E. coli INVCF strain pET11a (manufactured by Stratagene) to obtain plasmid (manufactured by Invitrogen) and amplicillin resistant strains pETBCH (FIG. 1). This plasmid pETBCH was introduced were Selected. Then, the plasmid contained in Selected into E. coli BL21 (DE3) strain competent cells 35 ampicillin resistant Strains was Sequenced by using Dye (manufactured by Stratagene) according to the manual terminator cycle sequencing kit (manufactured by PE attached to the competent cells to obtain E. coli BL21(DE3)/ applied Biosystems) and DNA sequencer 373S pETBCH strain. The strain was inoculated into 1.5 ml LB (manufactured by PE applied Biosystems). As a result, the liquid culture medium (1% tryptone, 0.5% yeast extract, nucleotide sequence of SEQID NO: 5 was revealed, thereby 0.5% NaCl) containing 100 tug/ml amplicillin in a tube 40 confirming that plasmid pSPPO-P contained chloroplast (14x10 mm), and the tube was covered with aluminum foil type protoporphyrinogen IX oxidase gene of Soybean. (hereinafter referred to as dark conditions), cultured with The plasmid pSPPO-P was digested with restriction shaking at 37° C. under light of fluorescent lamp (about enzyme PshBI, the resultant DNA fragment was blunted by 8000 lux). When the absorbance at 600 nm of the liquid using T4DNA polymerase and further digested with SphI to culture medium became about 0.6, isopropyl B-D- 45 isolate the DNA fragment containing chloroplast-type PPO thiogalactopyranoside (IPTG) was added to the liquid cul gene of Soybean and lac promoter. Then, the plasmid ture medium So that the final concentration was 0.4 mM, and PACYC184 (manufactured by Nippon Gene) was digested the culture was continued for about additional 20 hours. At with restriction enzymes NruI and SphI to remove a frag ment of 410 bp and the above DNA fragment was inserted that time, the Escherichia coli turned red and fluorescent instead to obtain plasmid paCYCSP (FIG. 2). Then, the absorbance (excitation wavelength 405 nm, emission wave 50 plasmid paCYCSP was introduced into PPO gene (hemC length 630 nm) which showed the accumulation of proto gene locus) deficient mutant E. coli BT3 strain (described in porphyrin IX in E. coli was observed. When E. coli BL21 Yamamoto, F. et al., Japanese J. Genet., 63; p. 237 (1988) (DE3)/pETBCH strain was cultured according to the same etc.) according to the method described in Hanahan, D. J., manner except that IPTG was not added, E. coli did not Mol. Biol., 166; p 557 (1983). The resultant E. coli were turned red and the above fluorescent absorbance did not 55 cultured in YPT medium (5 g/liter yeast extract, 5 g/liter detected. In contrast to this, when E. coli BL21(DE3)/ tryptone, 5 g/liter peptone, 10 g/liter NaCl, pH 7.0) contain pETBCH Strain was cultured according to the same manner ing 15 lug/ml chloramphenicol and 10 ug/ml kanamycin to (with IPTG) except that the tube was not covered with select E. coli BT3/pACYCSP strain resistant to chloram aluminum foil (hereinafter referred to as light conditions), E. phenicol and kanamycin whose hemC gene deficiency was coli grew and turned red as above. 60 complemented by PPO gene derived from soybean. EXAMPLE 3 EXAMPLE 4 Test of Protoporphyrin IX Binding Subunit Protein Expression of PPO Gene Derived from Soybeans in of Magnesium Chelatase for Capability of Giving hemC Gene Deficient E. coli 65 Weed Control Compound-Resistance Soybeans (Glycine max var. Williams82) were seeded and E. coli BT3/pACYCSP strain prepared in Example 3 was cultivated at 25 C. for 20 days and green leaves were inoculated into YPT medium containing 10 or 1 ppm of PPO US 6,570,070 B1 49 inhibitory type herbicidal compound represented by the above Structure 8, 10 ug/ml hemin, 50 tug/ml aminolevulinic TABLE 2 acid, 15 lug/ml chloramphenicol and 10 ug/ml kanamycin, Relative absorbance cultured under dark conditions or light conditions according Concentration of test to the same manner as in Example 2. As a control, E. coli Culture compound BT3/pACYCSP strain was cultured in the same medium as E. coli strain conditions 10 ppm 1 ppm 0 ppm above without the herbicidal compounds under the same BT3/pACYCSP + p.TVBCH in the light O.80 O.77 1.O conditions. Then, 18 hours after initiation of culture, the BT3/pACYCSP + p.TVBCH in the dark O.90 1.06 1.O absorbance of the liquid culture medium was measured at BT3/pACYCSP + p.TV118N in the light O.18 O31 1.O 600 nm. By taking the absorbance of the medium without BT3/pACYCSP + p.TV118N in the dark O.68 O.77 1.O the herbicidal compound as 1, the relative value of the absorbance of the medium containing the herbicidal com Further, these strains were inoculated into YPT medium pound was calculated. The results are shown in Table 1. containing PPO inhibitory-type herbicidal compounds 5 15 represented by the above Structures 1, 14, 15, 18-22, 29, 32, TABLE 1. 33, 34 and 36, respectively, 100 tug/ml ampicillin, 15 lug/ml Relative absorbance chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml hemin and E. coli Culture Concentration of test compound 50 ug/ml aminolevulinic acid, cultured under dark condi tions or light conditions similar to the Example 2. Then, 18 strain conditions 10 ppm 1 ppm 0 ppm hours after initiation of culture, the absorbance of liquid BT3/pACYCSP in the light O.10 O.25 1.O culture medium was measured at 600 nm. By taking the BT3/pACYCSP in the dark O.73 0.95 1.O absorbance of the medium without the herbicidal compound as 1, the relative value of the absorbance of the medium containing the herbicidal compound was calculated. The 25 results are shown in Table 3.

Plasmid pTVBCH (FIG. 3) was constructed by amplifi TABLE 3 cation of the DNA fragment containing bchH gene derived from photosynthetic bacterium Rhodobacter Sphaeroides Relative absorbance using the oligonucleotide composed of the nucleotide Test BT3/ BT3/pACYCSP + sequence of SEQ ID NO: 1 and the oligonucleotide com compound Test pACYCSP + p TVBCH pTV11SN posed of the nucleotide sequence of SEQ ID NO: 2 accord Structure Concent- in the in the in the in the ing to the same manner as in Example 1, digestion of the No. ration light dark light dark resultant DNA fragment with restriction enzymes NcoI and 35 Structure 5.0 O.88 O.88 O31 0.87 BglII and introducing the digested DNA fragment between 1. NcoI restriction site and BamHI restriction site of plasmid Structure 14 1O O.47 O.93 O.12 O.81 pTV118N (manufactured by Takara Shuzo Co., Ltd.). Structure 15 0.5 O.94 O.94 O.38 O.82 40 Structure Plasmids PTVBCH and pTV118N respectively were 18 2.O O.68 1.O O.33 O.91 Structure introduced into E. coli BT3/pACYCSP strain prepared in 19 5.0 O.78 O.89 O40 0.71 Example 3 according to the method described in Hanahan, Structure 2O 5.0 0.57 O.88 O.11 O.75 D. J., Mol. Biol., 166; p 557 (1983). The resultant E. coli 45 Structure were cultured in YPT medium containing 100 tug/ml 2 1O O.88 O.91 O.25 O.85 ampicillin, 15 lug/ml chloramphenicol and 10 ug/ml kana Structure 1O 0.55 O.93 O.29 O.94 22 mycin to obtain E. coli BT3/pACYCSP+pTVBCH strain Structure bearing plasmids pACYCSP and pTVBCH, and E. coli 29 0.5 O.64 O.90 O.22 O.77 BT3/pACYCSP+pTV 118N strain bearing plasmids 50 Structure 32 2.O O.70 O.94 O.37 0.87 pACYCSP and pTV118N. Structure 33 2.O O.81 O.92 O41 O.91 Structure 34 1.O O41 O.94 O.19 O.86 These strains were inoculated into YPT medium contain Structure ing 10 or 1 ppm of the PPO inhibitory-type herbicidal 55 36 0.5 0.55 0.95 O.28 O.96 compound represented by the above Structure 8, 100 tug/ml ampicillin, 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured EXAMPLE 5 under dark conditions or light conditions according to the 60 Same manner as in Example 2. Then, 18 hours after initiation Introduction of Gene Encoding Protoporphyrin IX of culture, the absorbance of the liquid culture medium was Binding Subunit Protein of Magnesium Chelatase measured at 600 nm. By taking the absorbance of the into Tobacco medium without the herbicidal compound as 1, the relative Aplasmid was constructed for introducing bchH gene into value of the absorbance of the medium containing the 65 a plant by Agrobacterium infection method. First, binary herbicidal compound was calculated. The results are shown vector pBI121 (manufactured by Clontech) was digested in Table 2. with restriction enzyme SacI, and Kpn I linker US 6,570,070 B1 S1 52 (manufactured by Takara Shuzo Co., Ltd.) was inserted to Example 5 were collected and each leaf was divided into the prepare plasmid PBIK wherein SacI recognition site of right and left equivalent pieces along the main vein, respec pBI121 was removed and Kpn I recognition site was added. tively. To one piece was applied an aqueous Solution con On the other hand, according to the same manner as described in Example 1, PCR was carried out by using the taining 0.3 ppm PPO inhibitory-type herbicidal compound genomic DNA of photosynthetic bacterium Rhodobacter of Structure 8, while, to the other piece was not applied the Sphaeroides as a template, and the oligonucleotide primer compound. These leaf pieces were placed on MS medium composed of the nucleotide sequence of SEQ ID NO: 7 and containing 0.8% agar and allowed to Stand at room tem the oligonucleotide primer composed of the nucleotide perature for 7 days in light place. Then, each leaf piece was sequence of SEQ ID NO: 8 to amplify the DNA fragment containing bchH gene. Then, the above plasmid pBIK was ground with pestle and mortar in 5 ml of 80% aqueous digested with restriction enzymes Xbal and Kpnl to remove acetone Solution to extract chlorophyll. The extract liquid B-glucuronidase gene, and instead thereof, a DNA fragment was diluted with 80% aqueous acetone solution and the which was obtained by digesting the above DNA fragment absorbance was measured at 750 nm, 663mm and 645 nm to containing bchH gene with restriction enzymes Xbal and calculate total chlorophyll content according to the method KpnI was inserted to produce plasmid pBIBCH (FIG. 4) in described by Mackinney G., J. Biol. Chem. (1941) 140, p which bchH gene was joined downstream from 35S pro 15 moter. Binary vector pBI121 (manufactured by Clontech) 315. The results obtained from 4 clones of tobacco into was also digested with restriction enzymes BamHI and SacI which bchH gene was introduced (BCH1 to 4) and control to remove B-glucuronidase gene, the resultant DNA frag recombinant tobacco is shown in Table 4. In the table, the ment was blunted by using T4 DNA polymerase, followed resistant level to the herbicidal compound was represented by self-cyclization with T4DNA ligase to construct plasmid by percentages of the total chlorophyll content of leaf pieces pNO (FIG. 5). The plasmid was used as a vector control of treated with herbicidal compound to that of untreated leaf bchH expression plasmid PBIBCH. pieces. The plasmid pBIBCH and pNO were introduced into Agrobacterium tumefaciens LBA4404, respectively. Abro TABLE 4 bacterium strain bearing pBIBCH and that bearing pNO 25 were isolated by culturing the resultant transformants in a Total chlorophyll content medium containing 300 lug/ml Streptomycin, 100 ug/ml mg S-fresh weight Resistant rifampicin and 25 ug/ml kanamycin and Selecting the desired transformants. Recombinant untreated- level to test Then, according to the method described in Manual for tobacco leaf treated-leaf compound (%) Gene Manipulation of Plant (by Hirofumi UCHIMIYA, control 2.49 O.19 7.63 Kodan-sha Scientific, 1992), the gene was introduced into BCH-1 1.35 1.70 126 tobacco. Agrobacterium strain bearing plasmid pBIBCH BCH-2 2.06 2.14 104 was cultured at 28°C. overnight in LB medium and then leaf BCH-3 1.93 1.57 813 pieces of tobacco cultured Sterilely were dipped in the liquid BCH-4 1.51 1.06 70.2 culture medium. The leaf pieces were cultured at room temperature for 2 days in Murashige-Skoog medium (MS 35 medium, described in Murasige T. and Skoog F., Physiol. The tobacco clone into which bchH gene was introduced Plant. (1962) 15, p. 473) containing 0.8% agar, 0.1 mg/liter and control recombinant tobacco were also treated in the naphthalene acetic acid and 1.0 mg/liter benzyl aminopu same manner with the solution containing PPO inhibitory rine. Then, the leaf pieces were washed with sterilized water type herbicidal compound represented by the above Struc and cultured for 7 days on MS medium containing 0.8% 40 ture 3, 7, 10, 11, 13, 17, 23, 24, 25, 27, 28, 30 or 35, and the agar, 0.1 mg/liter naphthalene acetic acid, 1.0 mg/liter resistant level to each herbicidal compound was measured. benzyl aminopurine and 500 lug/ml cefotaxime. The leaf The results are shown in Table 5. In the table, the resistant pieces were transplanted onto MS medium containing 0.8% levels to the herbicidal compound were represented by agar, 0.1 mg/liter naphthalene acetic acid, 1.0 mg/liter percentages of the total chlorophyll content of leaf pieces benzyl aminopurine, 500 ug/ml cefotaxime and 100 ug/ml 45 kanamycin (hereinafter referred to as selective MS medium) treated with the herbicidal compound to that of untreated and cultured on the medium continuously for 4 months with leaf pieces. transplanting the tobacco leaf pieces onto fresh Selective MS medium every 1 month. During culture, Stem-leaf differen TABLE 5 tiated Shoots were appeared from the tobacco leaf pieces, these shoots were transplanted to MS medium containing 50 Resistant level to test 0.8% agar, 300 tug/ml cefotaXime and 50 lug/ml kanamycin compound (% to induce roots to obtain regenerated plants. The resultant Test bchEH control regenerated plant was transplanted and cultured on MS Test compound concentration recombinant recombinant medium 0.8% agar and 50 ug/ml kanamycin to obtain Structure No. (ppm) tobacco tobacco 55 tobacco plant into which bchH gene was introduced. Structure 3 1O 114 9.94 Similarly, tobacco leaf pieces were infected with Agrobac Structure 7 3O 89.3 8.62 terium Strain bearing pNO to obtain regenerated plant from Structure 10 1O 84.O 14.9 Structure 11 O.30 78.1 5.51 the tobacco leaf pieces and tobacco plant (hereinafter Structure 13 3O 95.2 14.8 referred to as control recombinant tobacco). Structure 17 O.30 80.4 14.3 60 EXAMPLE 6 Structure 23 3.0 106 5.58 Structure 24 1O 129 5.18 Test of Tobacco Bearing Introduced Gene Encoding Structure 25 1O 104 16.O Protoporphyrin IX Binding Subunit Protein of Structure 27 1O 86.8 16.8 Structure 28 O.30 72.2 8.79 Magnesium Chelatase for Resistance to Herbicidal Structure 30 3.0 102 4.24 Compounds 65 Structure 35 O.30 83.3 17.4 The tobacco leaves into which bchH gene was introduced and control recombinant tobacco leaves obtained in US 6,570,070 B1 S3 S4 EXAMPLE 7 ampicillin, 15 lug/ml chloramphenicol, 50 lug/ml kanamycin, 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured Isolation of Gene Encoding Variant Protein of under dark conditions or light conditions according to the Protoporphyrin IX Binding Subunit Protein of Same manner as in Example 2. Then, 18 hours after initiation Tobacco Magnesium Chelatase of culture, the absorbance of the liquid culture medium was measured at 600 nm. By taking the absorbance of the Total RNAS were prepared from leaf tissues of tobacco medium without the herbicidal compound as 1, the relative (Nicotiana tabacum cv. SR1) by using RNeasy Plant Kit value of the absorbance of the medium containing the (manufactured by QIAGEN) according to the manual herbicidal compound was calculated. The results are shown attached thereto. The DNA fragment containing the gene in Table 6. encoding protoporphyrin IX binding Subunit protein of tobacco magnesium chelatase whose chloroplast transit Sig TABLE 6 nal had been deleted (hereinafter referred to as the variant Relative absorbance tobacco chelatase subunit) was obtained. by using RNALA concentration of test PCR Kit (AMV) Ver 1.1 (manufactured by Takara Shuzo Culture compound Co., Ltd.) according to the manual attached thereto. First, 1st 15 strand cDNA was synthesized by using tobacco total RNAS E. coli strain conditions 10 ppm 1 ppm 0 ppm as templates and Oligo dT-Adaptor Primer contained in the BT3/pACYCSP + p.TCHLH1 in the light O.69 O.89 1.O above kit as the primer with the reverse transcriptase con BT3/pACYCSP + p.TCHLH1 in the dark O.92 O.93 1.O tained in the above kit. Then, PCR was carried out by using BT3/pACYCSP + pCR2.1 in the light O.O3 O.08 1.O the 1st Strand cDNA as a template and LA Taq polymerase BT3/pACYCSP + pCR2.1 in the dark 1.O 1.O 1.O contained in the above kit to amplify the DNA fragment containing the gene encoding the variant tobacco chelatase subunit protein. In this PCR, oligonucleotide primer com EXAMPLE 9 posed of the nucleotide sequence of SEQ ID NO: 9 and the Introduction of Gene Encoding Variant Tobacco oligonucleotide primer composed of the nucleotide 25 Magnesium Chelatase Subunit Protein into Tobacco sequence of SEQ ID NO: 10 were used. These oligonucle A plasmid for introducing the gene encoding a variant otides were synthesized by using a DNA synthesizer (PE tobacco magnesium chelatase Subunit protein into tobacco Applied Biosystems; Model 394 DNA/RNA Synthesizer) by Agrobacterium infection method was constructed. First, and purified with an oligonucleotide purification cartridge the DNA fragment containing the gene encoding the variant (PE Applied Biosystems; OPC cartridge). The PCR was tobacco magnesium chelatase Subunit protein was prepared carried out by maintaining at 94 C. for 2 minutes and then by digesting plasmid pTCHLH1 prepared in Example 7 with repeating a cycle for maintaining at 94 C. for 30 Seconds, restriction enzymes Kipnl and SalI. On the other hand, binary at 50° C. for 30 seconds and then at 72° C. for 7 minutes 30 vector pBI121 (manufactured by Clonetech) was digested times. After the PCR, the DNA fragment amplified by the with restriction enzyme SmaI and Kipni linker PCR was cloned into plasmid pCR2.1 by using TA Cloning 35 (manufactured by Takara Shuzo Co., Ltd.) was inserted into Kit (manufactured by Invitrogen) according to the manual this portion to prepare plasmid p3I121K in which SmaI attached thereto. The resultant plasmid was digested with recognition Site of pBI121 was removed and KpnI recogni restriction enzyme Kpn and analyzed by agarose gel elec tion site was added. The plasmid pBI121K was digested trophoresis. The plasmid from which 8.0 kb DNA fragment with restriction enzyme SacI followed by blunting the DNA 40 by adding nucleotides to the double-stranded DNA gap with was detected was named pTCHLH. The plasmid had the DNA polymerase I. Then, the DNA was dephosphorylated Structure that the gene encoding the variant tobacco che with alkaline phosphatase derived from calf intestine and latase Subunit has been inserted in the direction expressible cyclized by inserting phosphorylated Sall linker (4680P, under the control of lac promoter. Plasmid pTCHLH was manufactured by Takara Shuzo Co., Ltd.) to construct plas digested with restriction enzyme KpnI followed by self 45 mid p3I121KS. The binary vector pBI121KS was digested ligaiton to obtain plasmid pTCHLH1 (FIG. 6) in which DNA with restriction enzymes KpnI and SalI to remove fragment composed of about 60 nucleotides had been B-glucuronidase gene and the gene encoding the variant deleted from plasmid pTCHLH. tobacco magnesium chelatase Subunit protein was inserted into this portion to prepare plasmid pBITCHLH (FIG. 7). EXAMPLE 8 The plasmid pBITCHLH was introduced into Agrobac 50 terium tumefaciens LBA4404. The resultant transformants Test of Variant Tobacco Magnesium Chelatase were cultured in a medium containing 300 lug/ml Subunit Protein for Capability of Giving Resistance Streptomycin, 100 lug/ml rifampicin and 25 ug/ml to Herbicidal Compounds kanamycin, followed by Selecting the desired transformants to isolate a Agrobacterium strain bearing pBITCHLH. The plasmid pTCHLH1 and pCR2.1 prepared in Example 55 Leaf pieces of tobacco cultured Sterilely are infected with 7 were introduced into E. coli BT3/pACYCSP strain pre the Agrobacterium Strain and, according to the same manner pared in Example 3, respectively according to the method as in Example 5, tobacco into which the gene encoding the described in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). variant tobacco magnesium chelatase Subunit protein is E. coli BT3/pACYCSP+pTCHLH1 strain bearing plasmids introduced is obtained. pACYCSP and pTCHLH1, and E. coli BT3/pACYCSP+ 60 pCR2.1 strain bearing plasmids paCYCSP and pCR2.1 EXAMPLE 10 were obtained by culturing the above strains in YPT medium Confirmation of Resistance to Herbicidal containing 100 lug/ml amplicillin, 15 lug/ml chloramphenicol Compounds of Tobacco Bearing Introduced Gene and 50 lug/ml kanamycin, respectively. Encoding Variant Tobacco Magnesium Chelatase These E. coli strains were inoculated into YPT medium 65 Subunit Protein containing 10 or 1 ppm of the PPO inhibitory-type herbi The levels of resistance to herbicidal compounds are cidal compound represented by Structure 8, 100 ug/ml confirmed quantitatively by testing tobacco introduced with US 6,570,070 B1 SS the gene encoding the variant tobacco magnesium chelatase Subunit protein prepared in Example 9 according to the same TABLE 7 manner as in Example 6. Relative absorbance Concentration of test EXAMPLE 11 Culture Compound E. coli strain conditions 10 ppm 1 ppm 0 ppm Isolation of Gene Encoding Variant Protein of Soybean PPO Having No Capability of Oxidizing BT3/pACYCSP + p.TVGMP in the light O.33 O.85 1.O BT3/pACYCSP + p.TVGMP in the dark O.91 O.94 1.O Protoporphyrinogen IX and Having Specific BT3/pACYCSP + p.TV118N in the light O.OS O.09 1.O Affinity for Protoporphyrinogen IX BT3/pACYCSP + p.TV118N in the dark O.89 O.91 1.O PCR was carried out by using plasmid pSPPO-P prepared in Example 3 as a template and an oligonucleotide com EXAMPLE 13 posed of the nucleotide sequence of SEQ ID NO: 11 and an 15 oligonucleotide composed of the nucleotide Sequence of SEQ ID NO: 12 as primers to amplify the DNA fragment Introduction of the Gene Encoding Variant Soybean encoding Soybean PPO whose chloroplast transit Signal and PPO into Tobacco FAD binding sequence had been deleted (hereinafter referred to as the variant soybean PPO). The oligonucle A plasmid for introducing the gene encoding the variant otides were prepared with a DNA synthesizer (PE Applied soybean PPO into a plant by Agrobacterium infection Biosystems; Model 394 DNA/RNA synthesizer) and puri method was constructed. PCR was carried out by using the fied with an oligonucleotide purification cartridge (PE plasmid pSPPO-P prepared in Example 3 as a template, an Applied Biosystems; OPC cartridge). The PCR was carried oligonucleotide primer composed of the nucleotide out by repeating a cycle for maintaining at 94 C. for 1 25 sequence of SEQ ID NO: 13 and an oligonucleotide primer minute, at 55 C. for 2 minutes and the 72 C. for 3 minutes composed of the nucleotide sequence of SEQ ID NO: 14 to 30 times. The amplified DNA fragments were digested with amplify the DNA fragment containing the gene encoding the restriction enzymes NcoI and SalI, and introduced between variant soybean PPO. Then, plasmid pBI121K prepared in NcoI restriction site and SalI restriction site of plasmid Example 9 was digested with restriction enzymes Kipnl and pTV118N (manufactured by Takara Shuzo Co., Ltd.) to SacI to remove B-glucuronidase gene, and the DNA frag construct plasmid pTVGMP (FIG. 8). ment which was obtained by digesting the DNA fragment containing the above gene encoding the variant Soybean The plasmid pTVGMP was introduced into E. coli PPO PPO with restriction enzymes Kipn and Sac I was inserted gene deficient mutant BT3 Strain according to the method into this portion to prepare plasmid pBIGMP (FIG. 9) in described in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). 35 which the gene was joined downstream from 35S promoter. When the resultant E. coli were cultured in YPT medium containing 100 lig/ml amplicillin and 10 ug/ml kanamycin, The plasmid pBIGMP was introduced into Agrobacterium no growth complemented clone was obtained. tumefaciens LBA4404. The resultant transformants were cultured in a medium containing 300 lig/ml Streptomycin, EXAMPLE 12 40 100 lig/ml rifampicin and 25 ug/ml kanamycin, followed by Selecting the desired transformants to isolate Agrobacterium Test for Effect of Giving Resistance to Herbicidal strain bearing pBIGMP Compounds of Variant Soybean PPO Leaf pieces of tobacco cultured Sterilely were infected 45 with the Agrobacterium Strain and, according to the same Plasmids pTVGMP and pTV118N prepared in Example manner as in Example 5, tobacco into which the gene 11 were introduced into E. coli BT3/pACYCSP strain pre encoding the variant soybean PPO was introduced was pared in Example 3 respectively according to the method obtained. described in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+pTVGMP strain bearing plasmids pACYCSP and PTVGMP, and E. coli BT3/pACYCSP+ 50 EXAMPLE 1.4 pTV118N strain bearing plasmids pACYCSP and pTV118N were obtained by culturing the above strains in YPT medium Confirmation of Resistance to Herbicidal containing 100 lug/ml amplicillin, 15 lug/ml chloramphenicol Compounds of Tobacco Bearing Introduced Gene and 10 ug/ml kanamycin. Encoding Variant Soybean PPO 55 These E. coli strains were inoculated into YPT medium The level of resistance to PPO inhibitory type herbicidal containing 10 or 1 ppm of PPO inhibitory-type herbicidal compound represented by Structure 8 was confirmed quan compound represented by Structure 8, 100 lug/ml amplicillin, titatively by testing tobacco into which the gene encoding 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml the variant soybean PPO prepared in Example 13 was hemin and 50 lug/ml aminolevulinic acid, cultured under 60 introduced according to the same manner as in Example 6. dark conditions or light conditions according to the same The results obtained from 4 clones (GMP 1-4) of tobacco manner as in Example 2. Then, 18 hours after initiation of introduced with the gene encoding the variant soybean PPO culture, the absorbance of liquid culture medium was mea and control recombinant tobacco are shown in Table 8. In the sured at 600 nm. By taking the absorbance of the medium table, the resistant level to herbicidal compound is repre without the herbicidal compound as 1, the relative value of 65 Sented by percentage of the total chlorophyll content of leaf the absorbance of the medium containing the herbicidal pieces treated with the herbicidal compound to that of compound was calculated. The results are shown in Table 7. untreated leaf pieces. US 6,570,070 B1 57 58 EXAMPLE 16 TABLE 8 Total chlorophyll content Isolation of Gene Encoding Variant Protein of mg g-fresh weight Resistant Chlamydomonas reinhardtii PPO Having No Capability of Oxidizing Protoporphyrinogen IX and Recombinant untreated- level to test tobacco leaf treated-leaf compound (%) Specific Affinity for Protoporphyrinogen IX control 3.49 O.35 1.O.O PCR was carried out by using plasmid pCPPO prepared in GMP-1 1.89 2.55 135 GMP-2 O.89 O.96 108 Example 15 as a template, and an oligonucleotide composed GMP-3 1.50 1.49 99.3 of the nucleotide sequence of SEQ ID NO: 19 and an GMP-4 2.91 2.34 80.4 oligonucleotide composed of the nucleotide SEQ ID NO: 20 as primers to amplify the DNA fragment encoding Chlamy EXAMPLE 1.5 domonas reinhardtii PPO whose chloroplast transit signal 15 and FAD binding sequence had been deleted (hereinafter Isolation of PPO Gene of Chlamydomonas referred to as the variant Chlamydomonas reinhardtili PPO). Chlamydomonas reinhardtii CC407 strain was obtained The oligonucleotides were prepared with a DNA synthesizer from Chlamydomonas Genetics Center (address: DCMB (PE Applied Biosystems; Model 394 DNA/RNA Group, Department of Botany, Box 91000, Duke University, Synthesizer) and purified with an oligonucleotide purifica Durham, NC 27708-1000, USA), cultured under 200 tion cartridge (PE Applied Biosystems; OPC cartridge). The uE/m°/s photosynthesis active light for 5 days in TAP liquid PCR was carried out by repeating a cycle for maintaining at culture medium (E. H. Harris, The Chlamydomonas 94 C. for 1 minute, at 55° C. for 2 minutes and then at 72 Sourcebook, Academic Press, San Diego, 1989, p. 576-577) containing 7 mM NHCl, 0.4 mM MgSO.7H2O, 0.34 mM C. for 3 minutes 30 times. The amplified DNA fragment was CaCl2.H2O, 25 mM potassium phosphate, 0.5 mM Tris (pH 25 digested with restriction enzymes BamHI and SacI, and 7.5), 1 ml/liter Hatner miner element and 1 ml/liter glacial inserted between BamHI restriction site and SacI restriction acetic acid to obtain 200 ml (10x10 cells/ml) liquid culture site of plasmid pTV119N (manufactured by Takara Shuzo medium containing early Stationary growth phase cells. Co., Ltd.) to construct plasmid pTVCRP (FIG. 10). Total RNAS were prepared from these cells by using The plasmid pTVCRP was introduced into E. coli PPO ISOGEN (manufactured by Nippon Gene) according to the gene deficient mutant BT3 Strain according to the method manual attached thereto. Also, poly(A)RNA was fraction described in Hanahan, D.J., Mol. Biol., 166; p 557 (1983). ated using BioMag mRNA Purification Kit (manufactured When the resultant E. coli were cultured in YPT medium by Perceptive Bio System) according to the manual attached containing 100 lig/ml amplicillin and 10 ug/ml kanamycin, thereto. cDNA was synthesized from the resultant poly(A) 35 RNA by using Marathon cDNA Amplification Kit no growth complemented clone was obtained. (manufactured by Clontech) according to the manual attached thereto and the cDNA was used as a template for EXAMPLE 1.7 PCR. AS PCR primers, an oligonucleotide composed of the 40 Test of Variant Modified Chlamydomonas nucleotide sequence of SEQ ID NO: 15 and an oligonucle otide composed of the nucleotide sequence of SEQ ID NO: reinhardtii PPO for Capability of Giving Resistance 16 were prepared. The oligonucleotides were prepared with to Herbicidal Compounds a DNA synthesizer (PE Applied Biosystems; Model 394 Plasmids pTVCRP and pTV118N prepared in Example 16 DNA/RNA synthesizer) and purified with an oligonucle 45 otide purification cartridge (PE Applied Biosystems; OPC were introduced into E. coli BT3/pACYCSP strain prepared cartridge). in Example 3 respectively according to the method PCR was carried out by preparing a reaction liquid using described in Hanahan, D.J., Mol. Biol., 166; p 557 (1983). Advantage cDNA PCR kit (manufactured by Clontech) E. coli BT3/pACYCSP+pTVCRP strain bearing plasmids according to the manual attached thereto, and then, after 50 pACYCSP and pTVCRP, and E. coli BT3/pACYCSP+ maintaining at 94 C. for 1 minute and then at 65° C. for 5 pTV118N strain bearing plasmids pACYCSP and pTV118N minutes, repeating a cycle for maintaining at 94 C. for 15 were obtained by culturing the above strains in YPT medium seconds and the 65 C. for 5 minutes 29 times. After the containing 100 lug/ml amplicillin, 15 lug/ml chloramphenicol PCR, the amplified DNA fragments were purified by filter and 10 ug/ml kanamycin. ing the reaction liquid with MicroSpin S-400 HR 55 (manufactured by Pharmacia Biotech), and the DNA frag These E. coli strains were inoculated into YPT medium ment was cloned into plasmid pCR2.1 by using TA Cloning containing 10 or 1 ppm of the PPO inhibitory-type herbi Kit (manufactured by Invitrogen) according to the manual cidal compound represented by Structure 8, 100 ug/ml attached thereto to construct plasmid PCPPO. ampicillin, 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, The nucleotide Sequence of DNA fragment contained in 60 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured the resultant plasmid pCPPO was determined by using Dye under dark conditions or light conditions in the same manner terminator cycle sequencing kit (manufactured by PE as in Example 2. Then, 18 hours after initiation of culture, applied Biosystems) and DNA sequencer 373S the absorbance of liquid culture medium was measured at (manufactured by PE applied Biosystems). As a result, the 600 nm. By taking the absorbance of the medium containing nucleotide sequence of SEQ ID NO: 17 was revealed, 65 no herbicidal compound as 1, the relative value of the thereby confirming that plasmid pCPPO contained the full absorbance of the medium containing the herbicidal pound length PPO cDNA of Chlamydomonas reinhardtii. was calculated. The results are shown in Table 9. US 6,570,070 B1 60

TABLE 9 TABLE 10 Relative absorbance Total chlorophyll content Concentration of test mg g-fresh weight Resistant Culture compound Recombinant untreated- level to test E. coli strain conditions 10 ppm 1 ppm 0 ppm tobacco leaf treated-leaf compound (%) BT3/pACYCSP + pTVCRP in the light O.23 O42 1.O control 2.28 O42 18.4 BT3/pACYCSP + pTVCRP in the dark O.81 O.82 1.O CRP-1 1.27 1.54 121 BT3/pACYCSP + pTV118N in the light O.12 O.24 1.O CRP-2 1.50 1.67 111 BT3/pACYCSP + pTV118N in the dark O.8O O.91 1.O CRP-3 1.10 1.11 101 CRP-4 1.58 1.57 99.4

EXAMPLE 1.8 15 EXAMPLE 2.0 Introduction of Gene Encoding Variant Test of Variant Protein of Barley Ferrochelatase Chlamydomonas reinhardtii PPO into Tobacco Having Affinity for Protoporphyrin IX Specifically A plasmid for introducing the gene encoding the variant for Capability of Giving Resistance to Herbicidal Chlamydomonas reinhardtii PPO into a plant by Agrobac Compounds terium infection method was constructed. The DNA frag A plasmid bearing barley ferrochelatase gene was pre ment containing the gene encoding the variant Chlamy pared by the method described in Miyamoto, K. et al., Plant domonas reinhardtii PPO was prepared by digesting plasmid 25 Physiol. 105; p 769 (1994). The resultant plasmid was pTVCRP prepared in Example 16 with restriction enzymes digested with restriction enzymes NspI and EcoRI to obtain BamHI and SacI. Binary vector pBI121 (manufactured by the DNA fragment containing the gene encoding barley Clontech) was digested with restriction enzymes BamHI and ferrochelatase whose Signal Sequence had been deleted SacI to remove B-glucuronidase gene and the above gene (hereinafter referred to as the variant barley ferrochelatase). encoding the variant Chlamydomonas reinhardtii PPO was This DNA fragment was inserted between SphI restriction inserted into this portion to prepare plasmid pBICRP (FIG. site and EcoRI restriction site of plasmid pTV119N 11). (manufactured by Takara ShuZO Co., Ltd.) to construct plasmid pTVHVFI (FIG. 12). The plasmid pBICRP was introduced into Agrobacterium The plasmids pTVHVF1 and pTV118N were introduced tumefaciens LBA4404. The resultant transformants were 35 into E. coli BT3/pACYCSP strains prepared in Example 3 cultured in a medium containing 300 lig/ml Streptomycin, respectively according to the method described in Hanahan, 100 lig/ml rifampicin and 25 ug/ml kanamycin, followed by D. J., Mol. Biol., 166; p 557 (1983). E. coli BT3/ Selecting the desired transformants to isolate Agrobacterium pACYCSP+pTVHVF strain bearing plasmid pACYCSP strain bearing pBICRP. and pTVHVF1, and E. coli BT3/pACYCSP+pTV118N 40 strain bearing plasmid paCYCSP and pTV118N were Leaf pieces of tobacco cultured Sterilely were infected obtained by culturing the above strains in YPT medium with the Agrobacterium Strain and, according to the same containing 100 lug/ml amplicillin, 15 lug/ml chloramphenicol manner as in Example 5, tobacco into which the gene and 10 ug/ml kanamycin. encoding the variant Chlamydomonas reinhardtii PPO was introduced was obtained. 45 These E. coli strains were inoculated into YPT medium containing 10 or 1 ppm of the PPO inhibitory-type herbi cidal compound represented by Structure 8, 100 ug/ml EXAMPLE 1.9 ampicillin, 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured Confirmation of Resistance to Herbicidal 50 under dark conditions or light conditions according to the Compounds of Tobacco Bearing Introduced Gene Same manner as in Example 2. Then, 18 hours after initiation Encoding Variant Chlamydomonas reinhardtii PPO of culture, the absorbance of liquid culture medium was measured at 600 nm. By taking the absorbance of the The level of resistance to the PPO-inhibitory type herbi medium without the herbicidal compound as 1, the relative cidal compound represented by Structure 8 was confirmed 55 value of the absorbance of the medium containing the quantitatively by testing tobacco into which the gene encod herbicidal compound was calculated. The results are shown ing the variant Chlamydomonas reinhardtii PPO prepared in in Table 11. Example 18 was introduced according to the Same manner TABLE 11 as in Example 6. The results obtained from 4 clones (CRP 60 1-4) of tobacco into which the gene encoding the variant Relative absorbance Concentration of test Chlamydomonas reinhardtii PPO was introduced and con Culture compound trol recombinant tobacco is shown in Table 10. In the table, the resistant levels to the herbicidal compound are repre E. coli strain conditions 10 ppm 1 ppm 0 ppm Sented by percentages of the total chlorophyll content of leaf 65 BT3/pACYCSP + p.TVHVF1 in the light O.39 O.94 1.O pieces treated with the herbicidal compound to that of BT3/pACYCSP + p.TVHVF1 in the dark O.94 O.96 1.O untreated leaf pieces. US 6,570,070 B1 61 62 table 12. In the table, the resistant levels to the herbicidal TABLE 11-continued compound are represented by percentages of the total chlo rophyll content of leaf pieces treated with herbicidal com Relative absorbance Concentration of test pound to that of untreated leaf pieces. Culture compound TABLE 12 E. coli strain conditions 10 ppm 1 ppm 0 ppm Total chlorophyll content BT3/pACYCSP + pTV118N in the light O.12 O.24 1.O mg g-fresh weight Resistant BT3/pACYCSP + pTV118N in the dark O.8O O.91 1.O Recombinant untreated- level to test tobacco leaf treated-leaf compound (%) EXAMPLE 21 control 1.93 O-160 8.29 HVF-1 O.876 O.930 106 Introduction of the Gene Encoding Variant Barley HVF-2 1.14 1.16 102 15 HVF-3 1.06 1.04 98.1 Ferrochelatase into Tobacco HVF-4 148 1.42 95.9 A plasmid for introducing the gene encoding barley ferrochelatase into tobacco by Agrobacterium infection method was constructed. The plasmid pTVHVFl described EXAMPLE 23 in Example 20 was digested with restriction enzyme Nco I followed by blunting the DNA with DNA polymerase I by Test of Variant Protein of Cucumber Ferrochelatase adding nucleotides to the double-Stranded DNA gap. Then, Having Specific Affinity for Protoporphyrin IX for the DNA was dephosphorylated with alkaline phosphatase Capability of Giving Resistance to Herbicidal derived from calf intestine and cyclized by inserting phos Compounds phorylated BamHI linker (4610P, manufactured by Takara 25 PCR was carried out by using cucumber ferrochelatase Shuzo Co., Ltd.) to construct plasmid pTVHVF2. Then, cDNA clone isolated by the method described in Miyamoto, pTVHVF2 was digested with restriction enzyme EcoRI K. et al., Plant Physiol., 105; p 769 (1994) as a template, an followed by blunting of the DNA with DNA polymerase I by oligonucleotide composed of the nucleotide Sequence of adding nucleotides to the double-Stranded DNA gap. SEQ ID NO: 21 and an oligonucleotide composed of the Further, the DNA was dephosphorylated with alkaline phos nucleotide sequence of SEQ ID NO: 22 as primers to phatase derived from calf intestine and cyclized by inserting amplify the DNA fragment encoding cucumber ferroche phosphorylated SalI linker (4680P, manufactured by Takara latase whose signal sequence had been deleted (hereinafter Shuzo Co., Ltd.) to construct plasmid pTVHVF3. Plasmid referred to as the variant cucumber ferrochelatase). The pBI121KS prepared in Example 9 was digested with restric oligonucleotides were prepared with a DNA synthesizer (PE tion enzymes BamHI and SalI to remove f3-glucuronidase 35 Applied Biosystems; Model 394 DNA/RNA synthesizer) gene. The DNA fragment containing the gene encoding the and purified with an oligonucleotides purification cartridge variant barley ferrochelatase was prepared by digesting the (PE Applied Biosystems; OPC cartridge). The PCR was above pTVHVF3 with restriction enzymes BamHI and SalI. carried out by repeating a cycle for maintaining at 94 C. for The resultant DNA fragment was inserted into plasmid 1 minute, at 55 C. for 2 minutes and then at 72° C. for 3 pBI121KS with replacing B-glucuronidase gene to prepare 40 minutes 30 times. The amplified DNA fragments were plasmid pPIHVF (FIG. 13) in which variant barley gene digested with restriction enzymes BamHI and SacI, and joined downstream from 35S promoter. inserted between BamHI restriction site and SacI restriction The plasmid pBIHVF was introduced into Agrobacterium site of plasmid pTV119N (manufactured by Takara Shuzo tumefaciens LBA4404. The resultant transformants were Co., Ltd.) to construct plasmid pTVCSF (FIG. 14). cultured in a medium containing 300 lig/ml Streptomycin, 45 The plasmids pTVCSF and pTV118N were introduced 100 lig/ml rifampicin and 25 ug/ml kanamycin, followed by into E. coli BT3/pACYCSP strain prepared in Example 3 Selecting the desired transformants to isolate Agrobacterium respectively according to the method described in Hanahan, strain bearing pBIHVF. D. J., Mol. Biol., 166; p 557 (1983). E. coli BT3/ Leaf pieces of tobacco cultured Sterilely were infected pACYCSP+pTVCSF strain bearing plasmid pACYCSP and with Said Agrobacterium Strain and, according to the same 50 pTVCSF, and E. coli BT3/pACYCSP+pTV118N strain manner as in Example 5, tobacco into which the gene bearing plasmid paCYCSP and pTV118N were obtained by encoding the variant barley ferrochelatase was introduced culturing the above strains in YPT medium containing 100 was obtained. tug/ml amplicillin, 15 lug/ml chloramphenicol and 10 ug/ml kanamycin. EXAMPLE 22 55 These E. coli strains were inoculated into YPT medium containing 10 or 1 ppm of the PPO inhibitory-type herbi Confirmation of Resistance to Herbicidal cidal compound represented by Structure 8, 100 ug/ml Compounds of Tobacco Bearing Introduced Gene ampicillin, 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, Encoding Variant Barley Ferrochelatase 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured The level of resistance to the PPO inhibitory-type herbi 60 under dark conditions or light conditions according to the cidal compound represented by Structure 8 was confirmed Same manner as in Example 2. Then, 18 hours after initiation quantitatively by testing tobacco into which the gene encod of culture, the absorbance of liquid culture medium was ing the variant barley ferrochelatase prepared in Example 21 measured at 600 nm. By taking the absorbance of the was introdued according to the same manner as in Example medium without the herbicidal compound as 1, the relative 6. The results obtained from 4 clones (HVF 1-4) of tobacco 65 value of the absorbance of the medium containing the introduced with the gene encoding the variant barley ferro herbicidal compound was calculated. The results are shown chelatase and control recombinant tobacco are shown in in Table 13. US 6,570,070 B1 63 64 and purified with an oligonucleotides purification cartridge TABLE 13 (PE Applied Biosystems; OPC cartridge). PCR was carried out by using about 1 lug of E. coli LE392 Strain genomic Relative absorbance Concentration of test DNA as a template and the above oligonucleotides (each 10 Culture compound pmol) as primers to amplify the DNA fragment containing E. coli hemF gene. The PCR was carried out by repeating a E. coli strain conditions 10 ppm 1 ppm 0 ppm cycle for maintaining at 96° C. for 1 minute, at 55 C. for 2 BT3/pACYCSP + pTVCSF in the light O.73 O.78 1.O minutes and then at 72 C. for 3 minutes 30 times. BT3/pACYCSP + pTVCSF in the dark O.89 O.92 1.O BT3/pACYCSP + pTV118N in the light O.O6 O.08 1.O EXAMPLE 27 BT3/pACYCSP + pTV118N in the dark O.81 O.91 1.O Test of E. coli hemF Protein for Capability of Giving Resistance to Herbicidal Compounds EXAMPLE 24 The DNA fragment containing hemF gene amplified by 15 the method described in Example 26 was digested with Introduction of the Gene Encoding Variant restriction enzymes Fbal and Pst, and inserted between Cucumber Ferrochelatase into Tobacco BamHI restriction site and Pst restriction site of commer Aplasmid for introducing the gene encoding the modified cially available plasmid pUC118N (manufactured by Takara cucumber ferrochelatase into tobacco by Agrobacterium Shuzo Co., Ltd.) to construct plasmid pHEMF (FIG. 16). infection method was constructed. Plasmid pBI 121 The plasmid pHEMF and pTV118N were introduced into (manufactured by Colntech) was digested with restriction E. coli BT3/pACYCSP strain prepared in Example 3 respec enzymes BamHI and SacI to remove B-glucuronidase gene. tively according to the method described in Hanahan, D. J., A DNA fragment containing the gene encoding the variant Mol. Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+ cucumber ferrochelatase was prepared by digesting plasmid pHEMF strain bearing plasmid pACYCSP and pHEMF, and pTVCSF described in Example 23 with restriction enzymes 25 E. coli BT3/pACYCSP+pTV118N strain bearing plasmid BamHI and SacI. The resultant DNA fragment was intro pACYCSP and PTV118N were obtained by culturing the duced into plasmid pBI121 with replacing B-glucuronidase above strains in YPT medium containing 100 tug/ml gene to prepare plasmid pBICSF (FIG. 15) in which variant ampicillin, 15 lug/ml chloramphenicol and 10 ug/ml kana cucumber ferrochelatase gene was joined downstream from mycin. 35S promoter. These E. coli strains were inoculated into YPT medium The plasmid PBICSF was introduced into Agrobacterium containing 10 or 1 ppm of the PPO inhibitory-type herbi tumefaciens LBA4404. The resultant transformants were cidal compound represented by Structure 8, 100 ug/ml cultured in a medium containing 300 lig/ml Streptomycin, ampicillin, 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 100 lig/ml rifampicin and 25 ug/ml kanamycin, followed by 35 10 ug/ml hemin and 50 lug/ml aminolevulinic acid, cultured Selecting the desired transformants to isolate Agrobacterium under dark conditions or light conditions according to the strain bearing pBICSF. Same manner as in Example 2. Then, 18 hours after initiation Leaf pieces of tobacco cultured Sterilely were infected of culture, the absorbance of liquid culture medium was with Said Agrobacterium Strain to obtain tobacco introduced measured at 600 nm. By taking the absorbance of the with the gene encoding the modified cucumber ferroche 40 medium without the herbicidal compound as 1, the relative latase according to the same manner as in Example 5. value of the absorbance of the medium containing the herbicidal compound was calculated. The results are shown EXAMPLE 25 in Table 14.

Confirmation of Resistance to Herbicidal TABLE 1.4 Compounds of Tobacco Bearing Introduced Gene 45 Encoding Variant Cucumber Ferrochelatase Relative absorbance Concentration of test The level of resistance to PPO inhibitory-type herbicidal Culture Compound compounds is confirmed quantitatively by testing tobacco E. coli strain conditions 10 ppm 1 ppm 0 ppm introduced with the gene encoding the modified cucumber 50 ferrochelatase prepared in Example 24 according to the BT3/pACYCSP + pHEMF in the light O.48 1.O 1.O Same manner as in Example 6. BT3/pACYCSP + pHEMF in the dark O.94 0.95 1.O BT3/pACYCSP + p.TV118N in the light O.O6 O16 1.O EXAMPLE 26 BT3/pACYCSP + p.TV118N in the dark O.96 O.98 1.O Isolation of E. coli Coproporphyrinogen III 55 Oxidase (hemF) Gene EXAMPLE 28 Genomic DNA was prepared from E. coli LE392 strain using Kit ISOPLANT for genome DNA preparation Introduction of E. coli hemF gene into Tobacco (manufactured by Nippon Gene). An oligonucleotide primer 60 A plasmid for introducing E. coli hemF gene into a plant composed of the nucleotide sequence of SEQID NO: 23 and by Agrobacterium infection method was constructed. First, an oligonucleotide primer composed of the nucleotide for obtaining E. coli hemF gene, an oligonucleotide primer sequence of SEQ ID NO: 24 were synthesized according to composed of the nucleotide sequence of SEQID NO: 25 and nucleotide sequences of its 5' and 3' regions of E. coli hemF an oligonucleotide primer composed of the nucleotide gene registered in GenBank (Accession X75413). The oli 65 sequence of SEQ ID NO: 26 were synthesized with a DNA gonucleotides were prepared with a DNA synthesizer (PE synthesizer (PE Applied Biosystems; Model 394 DNA/RNA Applied Biosystems; Model 394 DNA/RNA synthesizer) Synthesizer) and purified with an oligonucleotide purifica US 6,570,070 B1 65 66 tion cartridge (PE Applied Biosystems; OPC cartridge). by cooling slowly to room temperature at rate of 0.5 PCR was carried out by using the oligonucleotide primers C./minute. Plasmid pCANTAB5E (manufactured by Phar according to the same manner as in Example 26 to amplify macia Biotech) was digested with restriction enzymes Sfil the DNA fragment containing E. coli hemf gene. and Not to remove the recombinant antibody gene ScFv. The above phosphorylated and annealed oligonucleotide Plasmid pBI121 (manufactured by Clontech) was pair was inserted into the portion of the above recombinant digested with restriction enzymes BamHI and SacI to antibody gene ScFV to prepare a plasmid containing a remove B-glucuronidase gene. The DNA fragment contain nucleotide Sequence encoding a protein composed of a 5 ing the gene encoding the E. coli hemF gene was prepared random amino acid Sequence upstream from a protein com by digesting the above PCR-amplified DNA fragment with prising an amino acid Sequence of M13 phage coat protein. restriction enzymes BamHI and SacI. The resultant DNA The plasmid was introduced into E. coli TG-1 strain accord fragment was introduced into plasmid pBI121 with replac ing to the method described in Hanahan, D. J., Mol. Biol. ing 3-glucuronidase gene to prepare plasmid pBIHEMF 166; p 557 (1983) and cultured in 2xYT medium (10 g/liter (FIG. 17) in which E. coli hemF gene was joined down yeast extract, 15 g/liter tryptone and 5 g/liter NaCl, pH 7.2) stream from 35S promoter. containing 100 ug/ml amplicillin to obtain recombinant E. The plasmid pBIHEMF was introduced into Agrobacte 15 coli TG-1 strain. The recombinant E. coli TG-1 strain was rium tumefaciens LBA4404. The resultant transformants inoculated into 2xYT medium containing 100 lug/ml ampi were cultured in a medium containing 300 lug/ml cillin and cultured with shaking at 37 C. Then, 1 hour after Streptomycin, 100 lug/ml rifampicin and 25 ug/ml initiation of culture, 6x10" pfu helper-phage M13K07 kanamycin, followed by Selecting the desired transformants (manufactured by Pharmacia Biotech) was inoculated to the to isolate Agrobacterium strain bearing pBIHEMF. medium, and culture was continued for additional 18 hours with Shaking. Then, the liquid culture medium was centri Leaf pieces of tobacco cultured Sterilely were infected fuged at 1,000xg for 20 minutes to collect the phage library with the Agrobacterium Strain to obtain tobacco introduced displaying a protein containing the amino acid Sequence with E. coli hemF gene according to the same manner as in composed of 5 random amino acids. Example 5. 25 For preparing the phage clone displaying a protein con taining the amino acid sequence HASYS (SEQ ID NO. 53), EXAMPLE 29 an oligonucleotide composed of the nucleotide Sequence of Confirmation of Resistance to Herbicidal SEQ ID NO: 29 and an oligonucleotide composed of the nucleotide sequence of SEQ ID NO: 30 were synthesized. Compounds of Tobacco Introduced with the E. coli And, for preparing the phage clone displaying a protein hemF Gene containing the amino acid sequence RASSL (SEQ ID NO: The level of resistance to the PPO inhibitory-type herbi 55), an oligonucleotide composed of the nucleotide cidal compounds is confirmed quantitatively by testing sequence of SEQ ID NO: 31 and an oligonucleotide com tobacco introduced with the E. coli hemF gene (prepared in posed of the nucleotide sequence of SEQ ID NO: 32 were Example 28) according to the same manner as in Example 35 Synthesized. These oligonucleotides were Synthesized with a 6. DNA synthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) and purified with an oligonucle EXAMPLE 30 otide purification cartridge (PE Applied Biosystems; OPC Binding Test of Porphyrin Compound-Binding cartridge). The phage clone displaying the protein contain 40 ing the amino acid sequence HASYS (SEQ ID NO: 53) or Protein to Protoporphyrin IX RASSL (SEQ ID NO: 55) was obtained by the same A phage library presenting a protein containing an amino operation as the above that for obtaining the phage library acid Sequence composed of 5 random amino acids and a displaying a protein containing the amino acid Sequence phage clone displaying a protein containing an amino acid composed of 5 random amino acids. sequence HASYS (SEQ ID NO: 53) or RASSL (SEQ ID 45 A phage Suspension containing the phage clone display NO: 55) (wherein H is histidine, A is alanine, S is serine, Y ing the protein containing the amino acid Sequence HASYS is tyrosine, R is arginine and L is leucine) which can (SEQ ID NO: 53), the phage clone displaying the protein Specifically bind to porphyrin compound 5, 10, 15, containing the amino acid sequence RASSL (SEQ ID NO: 20-tetrakis (N-methylpyridinium-4-yl)-21H,23H-porphine 55) or the phage library displaying the protein containing the (HTMpyP) were prepared according to the method 50 amino acid Sequence consisting of 5 random amino acids described in KITANO et al., Nihon Kagakukai (Chemical (titer 10 pfu) was respectively spotted to nitro cellulose Society of Japan) 74th Spring Annual Meeting Pre filter (manufactured by Schleicher & Schuell), and then the Published Abstracts of Presentation II, p 1353, 4G511 nitro cellulose filter was blocked by shaking it in PBT buffer (1998). (137 mM NaCl, 8.10 mM NaHPO, 2.68 mM KCl, 1.47 First, the phage library displaying a protein containing an 55 mM KHPO, 0.05% Tween 20, pH 7.2) containing 1% amino acid Sequence composed of 5 random amino acids bovine serum albumin. The nitro cellulose filter was washed was constructed. Mixed oligonucleotides composed of the with PBT buffer and shaken for 18 hours in 2XSSC buffer nucleotide sequence of SEQ ID NO: 27 and mixed oligo (0.3 M NaCl, 0.03M sodium citric acid) containing 10 uM nucleotides composed of the nucleotide sequence of SEQID protoporphyrin IX. Further, said nitro cellulose filter was NO: 28 were synthesized. The mixed oligonucleotides were 60 washed with 2xSSC buffer, dried, and fluorescence derived synthesized with a DNA synthesizer (PE Applied Biosys from protoporphyrin IX was detected under ultraviolet light tems; Model 394 DNA/RNA synthesizer) and purified with (365 nm). an oligonucleotide purification cartridge (PE Applied Bio The spots of the phage library did not show fluorescence, systems; OPC cartridge). The above mixed oligonucleotides while the Spots of both phage clones displaying the protein (each 50 pmol) were phosphorylated at 51 end by treating 65 containing the amino acid sequence HASYS (SEQ ID NO: with T4 DNA kinase respectively. They were mixed and, 53) and that containing the amino acid sequence RASSL after heating at 70° C. for 10 minutes, subjected to annealing (SEQ ID NO: 55) showed clear fluorescence. US 6,570,070 B1 67 68 EXAMPLE 31 gene encoding protein MGYAGF (SEQ ID No. 60) were prepared by the same procedure as that for plasmid pHA Test of Protoporphyrin IX Binding Protein for SYS. Capability of Giving Resistance to Herbicidal The above plasmids pHASYS, pRASSL, pYAGY, Compounds pYAGF and pTV118N were introduced into E. coli BT3/ First, a plasmid which could express the gene encoding pACYCSP strain prepared in Example 3 respectively the protein containing the amino acid Sequence HASYS according to the method described in Hanahan, D.J., Mol. (SEQ ID NO:53), or the amino acid sequence RASSL (SEQ Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+pHASYS ID NO: 55) was prepared. For preparing the plasmid capable strain bearing plasmid paCYCSP and pHASYS, E. coli of expressing the gene encoding the protein composed of the BT3/pACYCSP+p RASSL strain bearing plasmid amino acid sequence of SEQID NO:54 (hereinafter referred pACYCSP and pRASSL, E. coli BT/pACYCSP+pYAGY to as the protein MGHASYS), an oligonucleotide composed strain bearing plasmid paCYCSP and pYAGY, E. coli of the nucleotide sequence of SEQ ID NO: 33 and an BT3/pACYCSP+pYAGF strain bearing plasmid pACYCSP oligonucleotide composed of the nucleotide Sequence of and pYAGF and E. coli BT3/pACYCSP+pTV118N strain SEQ ID NO: 34 were synthesized. The oligonucleotides 15 bearing plasmid paCYCSP and pTV118N were obtained by were synthesized with a DNA synthesizer (PE Applied culturing the above strains in YPT medium containing 100 Biosystems; Model 394 DNA/RNA synthesizer) and puri tug/ml amplicillin, 15 lug/ml chloramphenicol and 10 ug/ml fied with an oligonucleotide purification cartridge (PE kanamycin. Applied Biosystems; OPC cartridge). The above oligonucle These E. coli strains were inoculated into YPT medium otides (each 50 pmol) were phosphorylated at 5' end by containing 1 ppm of the PPO inhibitory-type herbicidal treating with T4DNAkinase, respectively. They were mixed compound represented by Structure 8, 100 lug/ml amplicillin, and then, after heating for 10 minutes at 70° C., Subjected to 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml annealing by cooling slowly to room temperature at rate of hemin and 50 lug/ml aminolevulinic acid, cultured under 0.5° C./minute. Plasmid pTV118N was digested with dark conditions or light conditions according to the same restriction enzymes NcoI and EcoRI to remove the gene 25 manner as in Example 2. Then, 18 hours after initiation of fragment consisting of 16 base pairs. Plasmid pHASYS culture, the absorbance of liquid culture medium was mea capable of expressing the gene encoding protein MGHA sured at 600 nm. By taking the absorbance of the medium SYS (SEQ ID No. 54) was prepared by inserted the above without the herbicidal compound as 1, the relative value of phosphorylated and annealed oligonucleotide pairs into the the absorbance of the medium containing the herbicidal position of the above 16 base pairs. compound was calculated. The results are shown in Table Then, for preparing the plasmid capable of expressing the 15. gene encoding the protein consisting of amino acid Sequence of SEQ ID NO: 56 (hereinafter referred to as protein TABLE 1.5 MGRASSL), an oligonucleotide composed of the nucleotide Relative absorbance sequence of SEQ ID NO: 35 and an oligonucleotide com 35 posed of the nucleotide sequence of SEQ ID NO: 36 were Concentration of Synthesized. The oligonucleotides were Synthesized with a Culture test compound DNA synthesizer (PE Applied Biosystems; Model 394 E. coli strain conditions 1 ppm 0 ppm DNA/RNA synthesizer) and purified with an oligonucle BT3/paCYCSP + pHASYS in the light O.65 1.O otide purification cartridge (PE Applied Biosystems; OPC 40 BT3/paCYCSP + pHASYS in the dark O.96 1.O cartridge). Plasmid pRASSL capable of expressing the gene BT3/pACYCSP + pRASSL in the light 0.59 1.O BT3/pACYCSP + pRASSL in the dark 1.O 1.O encoding protein MGRASSL(SEQID No. 56) was prepared BT3/paCYCSP + pYAGY in the light O.48 1.O by the same procedure as that for plasmid pHASYS. BT3/paCYCSP + pYAGY in the dark O.99 1.O A plasmid capable of expressing the gene encoding the BT3/paCYCSP + pYAGF in the light O.62 1.O protein containing the amino acid sequence YAGY or YAGF 45 BT3/paCYCSP + pYAGF in the dark O.96 1.O (wherein Y is tyrosine, A is alanine, G is glycine, F is BT3/paCYCSP + pTV118N in the light O.O7 1.O phenylalanine) (Sugimoto, N., Nakano. S., Chem. Lett. p BT3/paCYCSP + pTV118N in the dark O.93 1.O 939, 1997) capable of binding to porphyrin compound HTMPyP was prepared. For preparing the plasmid capable Further, a plasmid capable of expressing a gene encoding of expressing the gene encoding the protein consisting of the 50 a protein containing an amino acid Sequence in which one amino acid sequence of SEQID NO:58 (hereinafter referred unit of the amino acid sequences HASYS (SEQ ID No. 53) to as protein MGYAGY), an oligonucleotide composed of or RASSL (SEQ ID No. 55) were repeatedly joined. For the nucleotide sequence of SEQ ID NO:37 and an oligo preparing the plasmid capable of expressing the gene encod nucleotide composed of the nucleotide sequence of SEQ ID ing the protein composed of the amino acid Sequence of SEQ NO:38 were synthesized. For preparing the plasmid capable 55 ID NO: 61 (hereinafter referred to as protein MG(HASYS), of expressing the gene encoding the protein composed of the (HASYS), referred to as a sequence in which peptide amino acid sequence of SEQID NO: 60 (hereinafter referred HASYS (SEQ ID No. 53) was repeatedly joined to each to as protein MGYAGF), an oligonucleotide composed of other n times), an oligonucleotide composed of the nucle the nucleotide sequence of SEQ ID NO: 39 and an oligo otide sequence of SEQID NO:41, SEQID NO:42, SEQ ID nucleotide composed of the nucleotide sequence of SEQ ID 60 NO: 43 or SEQ ID NO: 44 was synthesized. These oligo NO: 40 were also synthesized. These oligonucleotides were nucleotides were synthesized with a DNA synthesizer (PE synthesized with a DNA synthesizer (PE Applied Biosys Applied Biosystems; Model 394 DNA/RNA synthesizer) tems; Model 394 DNA/RNA synthesizer) and purified with and purified with an oligonucleotide purification cartridge an oligonucleotide purification cartridge (PE Applied Bio (PE Applied Biosystems; OPC cartridge). First, the oligo systems; OPC cartridge). Plasmid pYAGY capable of 65 nucleotide composed of the nucleotide sequence of SEQ ID expressing the gene encoding the protein MGYAGY (SEQ NO. 42 and the oligonucleotide composed of the nucleotide ID No. 58) and plasmid pYAGF capable of expressing the sequence of SEQ ID NO: 43 were phosphorylated respec US 6,570,070 B1 69 70 tively at 5' end by treating with T4DNA kinase. Thereafter, nucleotide purification cartridge (PE Applied BioSystems; the oligonucleotide composed of the nucleotide Sequence of OPC cartridge). SEQ ID NO: 41 and the oligonucleotide composed of the Plasmid pRASSL4 capable of expressing protein phosphorylated nucleotide sequence of SEQ ID NO: 42 or MG(RASSL) (SEQ ID No: 63) were prepared according to the oligonucleotide composed of the phosphorylated nucle the same manner as that for the above plasmid pHASYS4. otide sequence of SEQ ID NO: 43 and the oligonucleotide Plasmid pRASSL8 capable of expressing protein composed of the nucleotide sequence of SEQ ID NO: 44 MG(RASSL) (SEQID No. 64) were also prepared accord were mixed (each 300 pmol), and after heating for 5 minutes ing to the same manner as that for the above plasmid at 70° C., annealed by cooling slowly to room temperature pHASYS8. at rate of 0.5 C./minute. The above two annealed oligo The above plasmids pHASYS4, pHASYS8, pRASSL4, nucleotide pairs were mixed and ligated with T4 DNA pRASSL8 and pTV118N were introduced into E. coli BT3/ ligase, then the resultant DNA fragment was phosphorylated pACYCSP strain prepared in Example 3 respectively with T4 DNA kinase at 5' end. On the other hand, vector according to the method described in Hanahan, D.J., Mol. pTV118N was digested with restriction enzymes NcoI and Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+pHASYS4 EcoRI to remove a DNA fragment of 16 base pairs and the 15 strain bearing plasmid paCYCSP and pHASYS4, E. coli above phosphorylated DNA fragment was inserted into this BT3/pACYCSP+pHASYS8 strain bearing plasmid portion to obtain plasmid pHASYS4 expressing the gene pACYCSP and pHASYS8, E. coli BT3/pACYCSP+ encoding protein MG(HASYS) (SEQ ID No. 61). pRASSL4 strain bearing plasmid paCYCSP and pRASSL4, Further, for preparing the plasmid capable of expressing E. coli BT3/pACYCSP+pRASSL8 strain bearing plasmid the gene encoding the protein composed of the amino acid pACYCSP and pRASSL8 and E. coli BT3/pACYCSP+ sequence of SEQ ID NO: 62 (hereinafter referred to as pTV118N strain bearing plasmid pACYCSP and pTV118N protein MG(HASYS)), an oligonucleotide composed of the were obtained by culturing the above strains in YPT medium nucleotide sequence of SEQ ID NO: 45 and an oligonucle containing 100 lug/ml amplicillin, 15 lug/ml chloramphenicol otide composed of the nucleotide sequence of SEQ ID NO: and 10 ug/ml kanamycin. 46 were Synthesized. These oligonucleotides were Synthe 25 sized with a DNA synthesizer (PE Applied Biosystems; These E. coli strains were inoculated into YPT medium Model 394 DNA/RNA synthesizer) and purified with an containing 1 ppm of the PPO inhibitory-type herbicidal oligonucleotide purification cartridge (PE Applied BioSys compound represented by Structure 8, 100 lug/ml amplicillin, tems; OPC cartridge). First, the above oligonucleotides were 15 lug/ml chloramphenicol, 10 ug/ml kanamycin, 10 ug/ml phosphorylated at 5' end by treating with T4 DNA kinase. hemin and 50 lug/ml aminolevulinic acid, cultured under Thereafter, an oligonucleotide composed of the nucleotide dark conditions or light conditions according to the same sequence of SEQ ID NO: 41 and an oligonucleotide com manner as in Example 2. Then, 18 hours after initiation of posed of the phosphorylated nucleotide sequence of SEQ ID culture, the absorbance of the liquid culture medium was NO: 42 were mixed (each 300 pmol), an oligonucleotide measured at 600 nm. By taking the absorbance of the culture composed of the phosphorylated nucleotide Sequence of 35 medium without the herbicidal compound as 1, the relative SEQ ID NO: 43 and an oligonucleotide composed of the value of the absorbance of the culture medium containing nucleotide sequence of SEQ ID NO: 44 were mixed (each the herbicidal compound was calculated. The results are 300 pmol), and further, an oligonucleotide composed of the shown in Table 16. phosphorylated nucleotide sequence of SEQ ID NO: 45 and an oligonucleotide composed of the phosphorylated nucle 40 TABLE 16 otide sequence of SEQ ID NO: 46 were mixed (each 600 Relative absorbance pmol). These three mixtures were heated for 5 minutes at Concentration of 70° C., and annealed by cooling slowly to room temperature Culture test compound at rate of 0.5 C./minute, respectively. The above three E. coli strain condition 1 ppm 0 ppm annealed oligonucleotide pairs were mixed, and ligated with 45 T4 DNA ligase, and then the resultant DNA fragment was BT3/pACYCSP + pHASYS4 in the light O.91 1.O phosphorylated with T4 DNA kinase at 5' end. Plasmid BT3/pACYCSP + pHASYS4 in the dark 1.O 1.O BT3/pACYCSP + pHASYS8 in the light 0.57 1.O pHASYS8 capable of expressing protein MG(HASYS) BT3/pACYCSP + pHASYS8 in the dark 1.O 1.O (SEQ ID No. 62) were prepared in the same manner as that BT3/pACYCSP + pRASSL4 in the light 1.1 1.O for the above plasmid pHASYS4. 50 BT3/pACYCSP + pRASSL4 in the dark O.98 1.O Then, for preparing a plasmid capable of expressing the BT3/pACYCSP + pRASSL8 in the light 0.79 1.O BT3/pACYCSP + pRASSL8 in the dark 1.O 1.O gene encoding the protein composed of the amino acid BT3/pACYCSP + pTV118N in the light O.15 1.O sequence of SEQ ID NO: 63 (hereinafter referred to as BT3/pACYCSP + pTV118N in the dark O.81 1.O protein MG(RASSL), (RASSL), referred to as a sequence in which peptide RASSL (SEQ ID No. 55) was repeatedly 55 joined to each other n times), an oligonucleotide composed of the nucleotide sequence of SEQ ID NO:47, SEQ ID NO: EXAMPLE 32 48, SEQ ID NO: 49 or SEQ ID NO: 50 were synthesized. Introduction of the Gene Encoding Protoporphyrin Also, for preparing a plasmid capable of expressing the gene IX Binding Peptide into Tobacco encoding the protein composed of the amino acid Sequence 60 of SEQ ID NO: 64 (hereinafter referred to as protein A plasmid for introducing the gene encoding the proto MG(RASSL)), an oligonucleotide composed of the nucle porphyrin IX binding peptide into tobacco by Agrobacte otide sequence of SEQ ID NO: 51 and an oligonucleotide rium method was constructed. The plasmid pHASYS8 pre composed of the nucleotide sequence of SEQ ID No. 52 pared in Example 31 was digested with restriction enzyme were Synthesized. These oligonucleotides were Synthesized 65 NcoI followed by blunting the DNA with DNA polymerase with a DNA synthesizer (PE Applied Biosystems; Model I with addition of nucleotides to the double-stranded DNA 394 DNA/RNA synthesizer) and purified with an oligo gap. Then, the DNA was dephosphorylated with alkaline US 6,570,070 B1 71 72 phosphatase derived from calf intestine and cyclized by SEO ID NO: 9 inserting phosphorylated BamH I linker (4610P, manufac Designed oligonucleotide primer to amplify DNA frag tured by Takara Syuzo Co., Ltd.) to construct plasmid ment having partial Sequence of tobacco chlh gene pHASYS8B. Plasmid pBI121 (manufactured by Clonetech) SEO ID NO: 10 was digested with restriction enzymes BamHI and SacI to Designed oligonucleotide primer to amplify DNA frag remove B-glucuronidase gene. On the other hand, plasmid ment having partial Sequence of tobacco chill gene pHASYS8B was digested with restriction enzymes BamHI SEO ID NO: 11 and SacI to prepare the DNA fragment containing the gene Designed oligonucleotide primer to amplify DNA frag encoding protein MG(HASYS), (SEQ ID NO: 62) the ment having partial Sequence of Soybean PPO gene resultant DNA fragment was inserted into plasmid pBI121 with replacing B-glucuronidase gene to prepare plasmid SEO ID NO: 12 pBIHASYS8 (FIG. 18) in which the gene encoding proto Designed oligonucleotide primer to amplify DNA frag porphyrin IX binding protein MG(HASYS). (SEQ ID NO: ment having partial Sequence of Soybean PPO gene 62) was joined downstream from 35S promoter. SEO ID NO: 13 A plasmid for introducing the gene encoding the proto 15 Designed oligonucleotide primer to amplify DNA frag porphyrin IX binding peptide MG(RASSL). (SEQ ID NO: ment having partial Sequence of Soybean PPO gene 62) into a plant by Agrobacterium infection method was SEO ID NO: 14 constructed. Plasmid pBIRASSL8 (FIG. 19) in which the Designed oligonucleotide primer to amplify DNA frag gene encoding protoporphyrin IX binding protein ment having partial Sequence of Soybean PPO gene MG(RASSL). (SEQ ID NO: 62) was joined downstream SEO ID NO: 15 from 35S promoter was prepared from pRASSL8 according Designed oligonucleotide primer to amplify Chlamy to the same procedure as that for pBIHASYS8. domonas PPO gene The above plasmid pBIHASYS8 and pBIRASSL8 were SEO ID NO: 16 introduced into Agrobacterium tumefaciens LBA4404 Designed oligonucleotide primer to amplify Chlamy respectively. The resultant transformants were cultured in a 25 domonas PPO gene medium containing 300 lug/ml Streptomycin, 100 ug/ml SEO ID NO: 19 rifampicin and 25 ug/ml kanamycin, followed by Selecting Designed oligonucleotide primer to amplify DNA frag the desired transformants to isolate Agrobacterium Strains ment having partial Sequence of Chlamydomonas bearing pBIHASYS8 and pBIRASSL8, respectively. PPO gene Leaf pieces of tobacco cultured Sterilely are infected with SEO ID NO: 20 Said Agrobacterium Strains to obtain tobacco introduced Designed oligonucleotide primer to amplify DNA frag with the gene encoding protoporphyrin IX binding protein ment having partial Sequence of Chlamydomonas MG(HASYS), (SEQ ID NO: 64) and the tobacco intro PPO gene duced with the gene encoding protoporphyrin IX binding SEO ID NO: 21 protein MG(RASSL), in the same manner as in Example 5. 35 Designed oligonucleotide primer to amplify DNA frag ment having partial Sequence of cucumber ferroche EXAMPLE 33 latase gene Confirmation of Resistance to Herbicidal SEO ID NO: 22 Compounds of Tobacco Bearing Introduced Gene 40 Designed oligonucleotide primer to amplify DNA frag Encoding the Protoporphyrin IX Binding Peptide ment having partial Sequence of cucumber ferroche latase gene The level of resistance to herbicidal compounds is con SEO ID NO: 23 firmed quantitatively by testing tobacco introduced with the Designed oligonucleotide primer to amplify Escheri gene encoding the protoporphyrin IX binding peptide pre 45 chia coli hemF gene pared in Example 32 according to the same manner as in SEO ID NO: 24 Example 14. Designed oligonucleotide primer to amplify Escheri AS described hereinabove, according to the present chia coli hemF gene invention, weed control compound-resistant plant can be SEO ID NO: 25 produced. 50 Designed oligonucleotide primer to amplify Escheri SEQUENCE LISTING FREETEXT chia coli hemF gene SEO ID NO: 26 SEO ID NO: 1 Designed oligonucleotide primer to amplify Escheri Designed oligonucleotide primer to amplify bchH gene chia coli hemF gene 55 SEO ID NO: 2 SEO ID NO: 27 Designed oligonucleotide primer to amplify bchH gene Designed oligonucleotides to Synthesize genes encod SEO ID NO: 3 ing random peptides comprising 5 amino acids Designed oligonucleotide primer to amplify Soybean SEO ID NO: 28 PPO gene 60 Designed oligonucleotides to Synthesize genes encod SEO ID NO: 4 ing random peptides comprising 5 amino acids Designed oligonucleotide primer to amplify Soybean SEO ID NO: 29 PPO gene Designed oligonucleotide to Synthesize the gene encod SEO ID NO: 7 ing the peptide HASYS Designed oligonucleotide primer to amplify bchH gene 65 SEO ID NO:30 SEO ID NO: 8 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide primer to amplify bchH gene ing the peptide HASYS US 6,570,070 B1 73 74 SEO ID NO:31 SEO ID NO: 46 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide RASSL ing the peptide MG(HASYS)8 SEO ID NO:32 SEO ID NO: 47 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide RASSL ing the peptide MG(RASSL)4 SEO ID NO: 33 SEO ID NO: 48 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide MGHASYS ing the peptide MG(RASSL)4 SEO ID NO:34 SEO ID NO: 49 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide MGHASYS ing the peptide MG(RASSL)4 SEO ID NO:35 SEO ID NO: 50 Designed oligonucleotide to Synthesize the gene encod 15 Designed oligonucleotide to Synthesize the gene encod ing the peptide MGRASSL ing the peptide MG(RASSL)4 SEO ID NO:36 SEO ID NO: 51 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide MGRASSL ing the peptide MG(RASSL)8 SEO ID NO:37 SEO ID NO: 52 Designed oligonucleotide to Synthesize the gene encod Designed oligonucleotide to Synthesize the gene encod ing the peptide MGYAGY ing the peptide MG(RASSL)8 SEO ID NO:38 SEO ID NO: 53 Designed oligonucleotide to Synthesize the gene encod Protoporphyrin IX binding protein HASYS ing the peptide MGYAGY 25 SEO ID NO: 54 SEO ID NO:39 Protoporphyrin IX binding protein MGHASYS Designed oligonucleotide to Synthesize the gene encod SEO ID NO: 55 ing the peptide MGYAGF Protoporphyrin IX binding protein RASSL SEO ID NO: 40 SEO ID NO: 56 Designed oligonucleotide to Synthesize the gene encod Protoporphyrin IX binding protein MGRASSL ing the peptide MGYAGF SEO ID NO: 57 SEO ID NO: 41 HTMpyP binding protein YAGY Designed oligonucleotide to Synthesize the gene encod SEO ID NO: 58 ing the peptide MG(HASYS)4 35 HTMpyP binding protein MGYAGY SEO ID NO: 42 SEO ID NO: 59 Designed oligonucleotide to Synthesize the gene encod HTMpyP binding protein YAGF ing the peptide MG(HASYS)4 SEO ID NO: 60 SEO ID NO: 43 HTMpyP binding protein MGYAGF Designed oligonucleotide to Synthesize the gene encod 40 SEO ID NO: 61 ing the peptide MG(HASYS)4 Protoporphyrin IX binding protein MG(HASYS) SEO ID NO: 44 SEO ID NO: 62 Designed oligonucleotide to Synthesize the gene encod Protoporphyrin IX binding protein MG(HASYS) ing the peptide MG(HASYS)4 45 SEO ID NO: 63 SEO ID NO: 45 Protoporphyrin IX binding protein MG(RASSL) Designed oligonucleotide to Synthesize the gene encod SEO ID NO: 64 ing the peptide MG(HASYS)8 Protoporphyrin IX binding protein MG(RASSL)

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 65 <210> SEQ ID NO 1 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &22O > FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify bchH gene

<400 SEQUENCE: 1 gacatctaga ggagacg acc atatgcacgg togaagttcto 39 US 6,570,070 B1 75

-continued <210> SEQ ID NO 2 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify bchH gene <400 SEQUENCE: 2 acggaagctt agatcttcac toggcggcaa t 31

<210> SEQ ID NO 3 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify soybean PPO gene <400 SEQUENCE: 3 to gag citcca tagtttcc.gt cittcaacgag atcctatto 39

<210> SEQ ID NO 4 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify soybean PPO gene

<400 SEQUENCE: 4 ttgtcg acaa citgctact at ttgtacactc tatttg 36

<210 SEQ ID NO 5 &2 11s LENGTH 1632 &212> TYPE DNA <213> ORGANISM: Glycine max var. Williams82 &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (1) . . (1632) <400 SEQUENCE: 5 atg gtt toc gtc titc aac gag atc cta titc cog ccg aac caa acc citt 48 Met Wal Ser Wall Phe Asn. Glu Ile Leu Phe Pro Pro Asn Glin Thr Leu 1 5 10 15 citt cqc coc toc citc cat toc coa acc tot titc titc acc tot coc act 96 Leu Arg Pro Ser Leu. His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr 2O 25 30 cga aaa titc cct cqc tot cqc cot aac cot att cita cqc toc toc att 144 Arg Llys Phe Pro Arg Ser Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile 35 40 45 gcq gag gala toc acc gcg tot cog ccc aaa acc aga gac toc goc coc 192 Ala Glu Glu Ser Thr Ala Ser Pro Pro Llys Thr Arg Asp Ser Ala Pro 5 O 55 60 gtg gac toc gtc gtc gtc. g.gc gga ggc gtc agc ggc citc toc atc goc 240 Val Asp Cys Val Val Val Gly Gly Gly Val Ser Gly Lieu. Cys Ile Ala 65 70 75 8O cag gCC citc goc acc aaa cac goc aat gcc aac gtc gtc gtc. acg gag 288 Glin Ala Lieu Ala Thr Lys His Ala Asn Ala Asn Val Val Val Thr Glu 85 90 95 gcc cqa gaC cqC gtc. g.gc ggC aac atc acc acg atg gag agg gaC gga 336 Ala Arg Asp Arg Val Gly Gly Asn. Ile Thir Thr Met Glu Arg Asp Gly 100 105 110 tac citc tog gala gaa gqc coc aac agc titc cag cct tct gat coa atg 384 Tyr Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp Pro Met

US 6,570,070 B1 79 80

-continued Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys Thr Asp Ser Glu 435 4 40 4 45 citt gtg gala aca gtt gat cqa gat ttg agg aaa atc citt ata aac coa 392 Leu Val Glu Thr Val Asp Arg Asp Leu Arg Lys Ile Lieu. Ile Asn Pro 450 455 460 aat goc cag gat coa titt gta gt g g g g g to aga citg togg cct caa gCt 4 40 Asn Ala Glin Asp Pro Phe Val Val Gly Val Arg Leu Trp Pro Glin Ala 465 470 475 480 att coa cag titc tta gtt ggc cat citt gat citt cita gat gtt got aaa 488 Ile Pro Glin Phe Leu Val Gly. His Lieu. Asp Leu Lleu. Asp Wall Ala Lys 485 490 495 gct tct atc aga aat act g g g titt gala ggg citc titc citt ggg ggit aat 536 Ala Ser Ile Arg Asn Thr Gly Phe Glu Gly Lieu Phe Leu Gly Gly Asn 5 OO 505 510 tat gtg tot ggt gtt gcc titg gga cqa toc gtt gag gga gCC tat gag 584 Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu 515 52O 525 gta gCa gct gala gta aac gat titt citc aca aat aga gtg tac aaa tag 632 Val Ala Ala Glu Val Asn Asp Phe Lieu. Thir Asn Arg Val Tyr Lys 530 535 540

<210> SEQ ID NO 6 &2 11s LENGTH 543 &212> TYPE PRT <213> ORGANISM: Glycine max var. Williams82 <400 SEQUENCE: 6

Met Wal Ser Wall Phe Asn. Glu Ile Leu Phe Pro Pro Asn Glin Thr Leu 1 5 10 15 Leu Arg Pro Ser Leu. His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr 2O 25 30 Arg Llys Phe Pro Arg Ser Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile 35 40 45 Ala Glu Glu Ser Thr Ala Ser Pro Pro Llys Thr Arg Asp Ser Ala Pro 5 O 55 60 Val Asp Cys Val Val Val Gly Gly Gly Val Ser Gly Lieu. Cys Ile Ala 65 70 75 8O Glin Ala Lieu Ala Thr Lys His Ala Asn Ala Asn Val Val Val Thr Glu 85 90 95 Ala Arg Asp Arg Val Gly Gly Asn. Ile Thir Thr Met Glu Arg Asp Gly 100 105 110 Tyr Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp Pro Met 115 120 125 Lieu. Thir Met Val Val Asp Ser Gly Lieu Lys Asp Glu Lieu Val Lieu Gly 130 135 1 4 0 Asp Pro Asp Ala Pro Arg Phe Wall Leu Trp Asn Arg Lys Lieu Arg Pro 145 15 O 155 160 Val Pro Gly Lys Leu Thr Asp Leu Pro Phe Phe Asp Leu Met Ser Ile 1.65 170 175 Gly Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu Gly Ile Arg Pro Pro 18O 185 190 Pro Pro Gly His Glu Glu Ser Val Glu Glu Phe Val Arg Arg Asn Leu 195 200 2O5 Gly Asp Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser Gly Val 210 215 220 Tyr Ala Gly Asp Pro Ser Lys Lieu Ser Met Lys Ala Ala Phe Gly Lys 225 230 235 240 US 6,570,070 B1 81

-continued

Val Trp Llys Lieu Glu Lys Asn Gly Gly Ser Ile Ile Gly Gly. Thir Phe 245 250 255 Lys Ala Ile Glin Glu Arg Asn Gly Ala Ser Lys Pro Pro Arg Asp Pro 260 265 27 O Arg Lieu Pro Llys Pro Lys Gly Glin Thr Val Gly Ser Phe Arg Lys Gly 275 280 285 Lieu. Thir Met Leu Pro Asp Ala Ile Ser Ala Arg Lieu Gly Asn Lys Wal 29 O 295 3OO Lys Lieu Ser Trp Lys Lieu Ser Ser Ile Ser Lys Lieu. Asp Ser Gly Glu 305 310 315 320 Tyr Ser Leu Thr Tyr Glu Thr Pro Glu Gly Val Val Ser Leu Glin Cys 325 330 335 Lys Thr Val Val Leu Thir Ile Pro Ser Tyr Val Ala Ser Thr Leu Leu 340 345 350 Arg Pro Leu Ser Ala Ala Ala Ala Asp Ala Leu Ser Lys Phe Tyr Tyr 355 360 365 Pro Pro Val Ala Ala Val Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg 370 375 38O Ser Glu Cys Lieu. Ile Asp Gly Glu Lieu Lys Gly Phe Gly Glin Lieu. His 385 390 395 400 Pro Arg Ser Glin Gly Val Glu Thr Leu Gly. Thir Ile Tyr Ser Ser Ser 405 410 415 Leu Phe Pro Asn Arg Ala Pro Pro Gly Arg Val Lieu Lleu Lieu. Asn Tyr 420 425 430 Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys Thr Asp Ser Glu 435 4 40 4 45 Leu Val Glu Thr Val Asp Arg Asp Leu Arg Lys Ile Lieu. Ile Asn Pro 450 455 460 Asn Ala Glin Asp Pro Phe Val Val Gly Val Arg Leu Trp Pro Glin Ala 465 470 475 480 Ile Pro Glin Phe Leu Val Gly. His Lieu. Asp Leu Lleu. Asp Wall Ala Lys 485 490 495 Ala Ser Ile Arg Asn Thr Gly Phe Glu Gly Lieu Phe Leu Gly Gly Asn 5 OO 505 510 Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu 515 52O 525 Val Ala Ala Glu Val Asn Asp Phe Lieu. Thir Asn Arg Val Tyr Lys 530 535 540

<210 SEQ ID NO 7 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify bchH gene <400 SEQUENCE: 7 gacatctagt citagacg acc atatgcacgg talagtotc

<210 SEQ ID NO 8 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify bchH gene US 6,570,070 B1 83 84

-continued

SEQUENCE: 8 acggaagctt got accitcac toggcggcaa t 31

SEQ ID NO 9 LENGTH 35 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of tobacco ch1H gene

<400 SEQUENCE: 9 ccaatgtaat gctatggtac citatgttatt cactc 35

SEQ ID NO 10 LENGTH 34 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of tobacco ch1H gene

<400 SEQUENCE: 10 gag atcattc tttittgctgt cq acttatcg atcg 34

SEQ ID NO 11 LENGTH 36 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of soybean PPO gene

<400 SEQUENCE: 11 ggcggaggcg to accatggit citgcatc.gcc caggcc 36

SEQ ID NO 12 LENGTH 36 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of soybean PPO gene

<400 SEQUENCE: 12 gcct gcaggit cqacaactgc tactatttgt acactic 36

SEQ ID NO 13 LENGTH 33 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of soybean PPO gene

<400 SEQUENCE: 13 cacaggaaag gtaccatggit citgcatc.gcc cag 33

SEQ ID NO 14 LENGTH 33 TYPE DNA US 6,570,070 B1 85

-continued <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of soybean PPO gene <400 SEQUENCE: 14 cctgcagotc gagagctcct act atttgta cac 33

<210 SEQ ID NO 15 &2 11s LENGTH 2.8 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Chlamydomonas PPO gene <400 SEQUENCE: 15 aatgatgttg accoag acto citggg acc 28

<210> SEQ ID NO 16 &2 11s LENGTH 27 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Chlamydomonas PPO gene <400 SEQUENCE: 16 tact acacat cocagoaa.gc gccaatg 27

<210 SEQ ID NO 17 &2 11s LENGTH 1838 &212> TYPE DNA <213> ORGANISM: Chlamydomonas reinhardtii CC407 &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (2) ... (1693) <400 SEQUENCE: 17 a at g at g ttg acc cag act cot ggg acc gcc acg gct tct agc cqg cqg 49 Met Met Leu Thr Gln Thr Pro Gly Thr Ala Thr Ala Ser Ser Arg Arg 1 5 10 15 tog cag atc cqc to g g cit gcg cac gtc. tcc goc aag gito gog cct cqg 97 Ser Glin Ile Arg Ser Ala Ala His Val Ser Ala Lys Val Ala Pro Arg 2O 25 30 ccc acg cca titc. tcg gtc gcg agc ccc gog acc gct gcg agc ccc gog 145 Pro Thr Pro Phe Ser Wall Ala Ser Pro Ala Thr Ala Ala Ser Pro Ala 35 40 45 acc gog gC g gCC cqc cqc aca citc. cac cqc act gct gcg gC g gcc act 193 Thr Ala Ala Ala Arg Arg Thr Lieu. His Arg Thr Ala Ala Ala Ala Thr 5 O 55 60 ggit gct coc acg gcg toc gga gCC ggc gtc goc aag acg ctic gac aat 241 Gly Ala Pro Thr Ala Ser Gly Ala Gly Val Ala Lys Thr Lieu. Asp Asn 65 70 75 8O gtg tat gac gtg atc gtg gito ggt gga ggit citc. tcg ggc ct g g to acc 289 Val Tyr Asp Val Ile Val Val Gly Gly Gly Leu Ser Gly Leu Val Thr 85 90 95 ggc cag gCC ct g g cq got cag cac aaa att cag aac titc citt gtt acg 337 Gly Glin Ala Lieu Ala Ala Gln His Lys Ile Glin Asn. Phe Lieu Val Thr 100 105 110 gag gCt c gc gag cqc gtc. g.gc ggc aac att acg toc atg to g g g c gat 385 Glu Ala Arg Glu Arg Val Gly Gly Asn. Ile Thr Ser Met Ser Gly Asp

US 6,570,070 B1 89 90

-continued Ser Ser Ser Leu Phe Pro Gly Arg Ala Pro Glu Gly His Met Leu Leu 435 4 40 4 45 citc aac tac atc ggc ggc acc acc aac cqc ggc atc gtc. aac cag acc 393 Leu Asn Tyr Ile Gly Gly. Thir Thr Asn Arg Gly Ile Val Asin Glin Thr 450 455 460 acc gag cag ct g g to gag cag gtg gac aag gac citg cqc aac at g g to 441 Thr Glu Gln Leu Val Glu Glin Val Asp Lys Asp Leu Arg Asn Met Val 465 470 475 480 atc aag coc gac gog ccc aag coc cqt gtg gtg ggc gtg cqc gtg togg 489 Ile Llys Pro Asp Ala Pro Llys Pro Arg Val Val Gly Val Arg Val Trp 485 490 495 cc.g. cqc gcc atc cc.g. cag titc aac ct g g g c cac citg gag cag citg gac 537 Pro Arg Ala Ile Pro Glin Phe Asn Lieu Gly His Leu Glu Glin Lieu. Asp 5 OO 505 510 aag gC g cqC aag gcq Ctg gaC gCg gC g g g g Ctg Cag ggc gtg cac Ctg 585 Lys Ala Arg Lys Ala Lieu. Asp Ala Ala Gly Lieu Glin Gly Wal His Lieu 515 52O 525 ggg ggC aac tac gtc. agc ggt gtg gCC ct g g gC aag gtg gtg gag CaC 633 Gly Gly Asn Tyr Val Ser Gly Val Ala Leu Gly Lys Val Val Glu His 530 535 540 ggc tac gag toc goa gcc aac citg gcc aag agc gtg toc aag gCC goa 681 Gly Tyr Glu Ser Ala Ala Asn Lieu Ala Lys Ser Val Ser Lys Ala Ala 545 550 555 560 gto: aag gCC taa goggctgcag cagtag cagc agcago atcg ggct gtagct 733 Wall Lys Ala ggtaaatgcc gcagtggcac cqg cagoagc aattggcaag cacttggggc aag.cggagtg 793 gaggcgaggg gggggctacc attgg.cgctt gctgggatgt gtagt 838

<210> SEQ ID NO 18 &2 11s LENGTH 563 &212> TYPE PRT <213> ORGANISM: Chlamydomonas reinhardtii CC407 <400 SEQUENCE: 18 Met Met Leu Thr Gln Thr Pro Gly Thr Ala Thr Ala Ser Ser Arg Arg 1 5 10 15 Ser Glin Ile Arg Ser Ala Ala His Val Ser Ala Lys Val Ala Pro Arg 2O 25 30

Pro Thr Pro Phe Ser Wall Ala Ser Pro Ala Thr Ala Ala Ser Pro Ala 35 40 45 Thr Ala Ala Ala Arg Arg Thr Lieu. His Arg Thr Ala Ala Ala Ala Thr 5 O 55 60 Gly Ala Pro Thr Ala Ser Gly Ala Gly Val Ala Lys Thr Lieu. Asp Asn 65 70 75 8O Val Tyr Asp Val Ile Val Val Gly Gly Gly Leu Ser Gly Leu Val Thr 85 90 95 Gly Glin Ala Lieu Ala Ala Gln His Lys Ile Glin Asn. Phe Lieu Val Thr 100 105 110 Glu Ala Arg Glu Arg Val Gly Gly Asn. Ile Thr Ser Met Ser Gly Asp 115 120 125 Gly Tyr Val Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Asn Asp Ser 130 135 1 4 0 Met Leu Glin Ile Ala Wall Asp Ser Gly Cys Glu Lys Asp Leu Val Phe 145 15 O 155 160 Gly Asp Pro Thr Ala Pro Arg Phe Val Trp Trp Glu Gly Lys Lieu Arg 1.65 170 175 US 6,570,070 B1 91 92

-continued Pro Wall Pro Ser Gly Teu Asp Ala Phe Thr Phe Asp Leu Met Ser Ile 18O 185 190

Pro Gly Lys Ile Arg Ala Gly Telu Gly Ala Ile Gly Teu Ile Asn Gly 195 200 2O5

Ala Met Pro Ser Phe Glu Glu Ser Wall Glu Glin Phe Ile Arg Asn 210 215 220

Teu Gly Asp Glu Wall Phe Phe Arg Telu Ile Glu Pro Phe Ser Gly 225 230 235 240

Wall Ala Gly Asp Pro Ser Telu Ser Met Ala Ala Phe Asn 245 250 255

Arg Trp Ile Teu Glu Asn Gly Gly Ser Teu Wall Gly Gly Ala 260 265 27 O

Ile Telu Phe Glin Glu Arg Glin Ser Asn Pro Ala Pro Pro Asp 275 280 285

Pro Arg Telu Pro Pro Pro Gly Glin Thr Wall Gly Ser Phe 29 O 295

Lys Gly Telu Met Teu Pro Asp Ala Ile Glu Arg Asn Ile Pro Asp 305 310 315 320

Ile Arg Wall Asn Trp Telu Wall Ser Teu Gly Glu Ala Asp 325 330 335

Gly Gly Teu Wall Asp Thr Pro Glu Gly Wall Lys Wall 340 345 350

Phe Ala Arg Ala Wall Ala Teu Thr Ala Pro Ser Wall Wall Ala Asp 355 360 365

Teu Wall Glu Glin Ala Pro Ala Ala Ala Glu Ala Teu Ser Phe 370 375 380

Asp Pro Pro Wall Gly Ala Wall Thr Telu Ser Pro Teu Ser Ala 385 390 395 400

Wall Arg Glu Glu Arg Ala Ser Asp Gly Ser Wall Pro Phe Gly 405 410 415

Glin Telu His Pro Arg Thr Glin Gly Ile Thr Thr Teu Gly Thr Ile Tyr 420 425 430

Ser Ser Ser Telu Phe Pro Gly Arg Ala Pro Glu Gly His Met Telu Telu 435 4 40 4 45

Teu Asn Ile Gly Gly Thr Thr Asn Arg Gly Ile Wall Asn Glin Thr 450 455 460

Thr Glu Glin Telu Wall Glu Glin Wall Asp Asp Teu Arg Asn Met Wall 465 470 475 480

Ile Pro Asp Ala Pro Pro Arg Wall Wall Gly Wall Arg Wall Trp 485 490 495

Pro Arg Ala Ile Pro Glin Phe Asn Telu Gly His Teu Glu Glin Telu Asp 5 OO 505 510

Ala Arg Ala Teu Asp Ala Ala Gly Teu Glin Gly Wall His Telu 515 525

Gly Gly Asn Wall Ser Gly Wall Ala Telu Gly Lys Wall Wall Glu His 530 535 540

Gly Tyr Glu Ser Ala Ala Asn Telu Ala Ser Wall Ser Ala Ala 545 550 555 560

Wall Lys Ala

SEQ ID NO 19 LENGTH 32 TYPE DNA ORGANISM: Artificial Sequence US 6,570,070 B1 93 94

-continued

&220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of Chlamydomonas PPO gene <400 SEQUENCE: 19 ggtoggtgga ggggat.ccga tectggtgac cq 32

<210> SEQ ID NO 20 &2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of Chlamydomonas PPO gene

<400 SEQUENCE: 20 gctact gctg. c.gagct citta ggccttgact gc 32

<210> SEQ ID NO 21 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of cucumber ferrochelatase gene

<400 SEQUENCE: 21 gctittagaat cqgatccitat ggcagtggat gac 33

<210> SEQ ID NO 22 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify DNA fragment having partial sequence of cucumber ferrochelatase gene

<400 SEQUENCE: 22 ggtgaactitc tatttgagct citcaggtaaa tataag 36

<210> SEQ ID NO 23 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Escherichia coli hemF gene <400 SEQUENCE: 23 gctgaaggcg tdatcagtta titt.co. 25

<210> SEQ ID NO 24 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Escherichia coli hemF gene <400 SEQUENCE: 24 US 6,570,070 B1 95 96

-continued catcagcctg cagtgc galaa agtg 24

SEQ ID NO 25 LENGTH 26 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Escherichia coli hemF gene

<400 SEQUENCE: 25 cgaaaaaggg atc.cgittatgaaacco 26

SEQ ID NO 26 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide Primer to amplify Escherichia coli hemF gene

<400 SEQUENCE: 26 gctgtttitcc gagctoccgt cac 23

SEQ ID NO 27 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotides to synthesize genes encoding random peptides comprising 5 amino acids FEATURE: OTHER INFORMATION: any n = a, g, c, or t (unknnown or other) <400 SEQUENCE: 27 tggc.cnnkinn knnknniknink go 22

EQ ID NO 28 ENGTH 29 YPE DNA GANISM: Artificial Sequence ATURE THER INFORMATION: Description of Artificial Sequence: Synthetic ligonucleotides to synthesize genes encoding andom peptides comprising 5 amino acids EATURE i. THER INFORMATION: any n = a, c, g, or t (unknown or other) <400 SEQUENCE: 28 ggcc.gcmnnm nnmnnmnnmn nggccagot 29

SEQ ID NO 29 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide HASYS <400 SEQUENCE: 29 tggcc catgc tagttagt cq go 22

SEQ ID NO 30 LENGTH 29 US 6,570,070 B1 97 98

-continued

TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide HASYS <400 SEQUENCE: 30 tggcgc.cgac talactago at gggccagot 29

SEQ ID NO 31 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide RASSL <400 SEQUENCE: 31 tggCCCgggC gtcgtcgttg gC 22

SEQ ID NO 32 LENGTH 29 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide RASSL <400 SEQUENCE: 32 ggcc.gc.caac gacgacgc.cc gg.gc.cagot 29

SEQ ID NO 33 LENGTH 26 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGHASYS <400 SEQUENCE: 33 catgggtoac gottcttact cotaag 26

SEQ ID NO 34 LENGTH 26 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGHASYS <400 SEQUENCE: 34 aattcttagg agtaagaagc gtgacc 26

SEQ ID NO 35 LENGTH 26 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGRASSL <400 SEQUENCE: 35 US 6,570,070 B1 99 100

-continued catgggtogt gcttctitc.cc totaag 26

<210 SEQ ID NO 36 &2 11s LENGTH 26 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGRASSL <400 SEQUENCE: 36 aattcttaca gggaagaagc acg acc 26

<210 SEQ ID NO 37 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGYAGY <400 SEQUENCE: 37 catgggttac gotggctact aag 23

<210 SEQ ID NO 38 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGYAGY <400 SEQUENCE: 38 aattcttagt agc.ca.gcgta acc 23

<210 SEQ ID NO 39 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGYAGF <400 SEQUENCE: 39 catgggttac gotggcttct aag 23

<210> SEQ ID NO 40 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MGYAGF <400 SEQUENCE: 40 aattcttaga agc.ca.gcgta acc 23

<210> SEQ ID NO 41 <211& LENGTH: 34 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 6,570,070 B1 101 102

-continued Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 4 SEQUENCE: 41 catgggtoac gottcttact cocatgcatc titac 34

SEQ ID NO 42 LENGTH 36 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 4 <400 SEQUENCE: 42 gtgggagtaa gatgcatggg agtaagaa.gc gtgacC 36

SEQ ID NO 43 LENGTH 37 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 4 <400 SEQUENCE: 43 toccacgctt cittacticcca to catcttac toctaag 37

SEQ ID NO 44 LENGTH 35 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 4 <400 SEQUENCE: 44 aattcttagg agtaagatgc atgggagtaa gaagc 35

SEQ ID NO 45 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 8 <400 SEQUENCE: 45 toccacgctt cittacticcca to catcttac 30

SEQ ID NO 46 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (HASYS) 8 <400 SEQUENCE: 46 gtgggagtaa gatgcatggg agtaagaa.gc 30

SEQ ID NO 47 US 6,570,070 B1 103 104

-continued

LENGTH 34 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 4 <400 SEQUENCE: 47 catggg togt gcttctitccc tdcgc.gcatc titcc 34

SEQ ID NO 48 LENGTH 36 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 4 <400 SEQUENCE: 48 acgcagggaa gatgcgc.gca gggaagaa.gc acgaCC 36

SEQ ID NO 49 LENGTH 37 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 4 <400 SEQUENCE: 49 ctg.cgtgctt cittccctg.cg cqcatctitcc citgitaag 37

SEQ ID NO 50 LENGTH 35 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 4 <400 SEQUENCE: 50 aattcttaca gggaagatgc gcgcagggaa gaagc 35

SEQ ID NO 51 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 8 <400 SEQUENCE: 51 ctg.cgtgctt cittccctg.cg cqcatctitcc 30

SEQ ID NO 52 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic Oligonucleotide to synthesize the gene encoding the peptide MG (RASSL) 8 <400 SEQUENCE: 52 US 6,570,070 B1 105 106

-continued acgcagggaa gatgcgc.gca gggaagaa.gc 30

SEQ ID NO 53 LENGTH 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein HASYS

<400 SEQUENCE: 53 His Ala Ser Tyr Ser 1 5

SEQ ID NO 54 LENGTH 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MGHASYS

<400 SEQUENCE: 54 Met Gly His Ala Ser Tyr Ser 1 5

SEQ ID NO 55 LENGTH 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein RASSL

<400 SEQUENCE: 55 Arg Ala Ser Ser Lieu 1 5

SEQ ID NO 56 LENGTH 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MGRASSL

<400 SEQUENCE: 56 Met Gly Arg Ala Ser Ser Lieu 1 5

SEQ ID NO 57 LENGTH 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: H(2) TMpyP binding protein YGAY

<400 SEQUENCE: 57 Tyr Ala Gly Tyr 1

SEQ ID NO 58 LENGTH 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: US 6,570,070 B1 107 108

-continued OTHER INFORMATION: Description of Artificial Sequence: H(2) TMpyP binding protein MGYAGY

<400 SEQUENCE: 58 Met Gly Tyr Ala Gly Tyr 1 5

SEQ ID NO 59 LENGTH 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: H(2) TMpyP binding protein YAGF

<400 SEQUENCE: 59 Tyr Ala Gly Phe 1

SEQ ID NO 60 LENGTH 6 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: H(2) TMpyP binding protein MGYAGF

<400 SEQUENCE: 60 Met Gly Tyr Ala Gly Phe 1 5

SEQ ID NO 61 LENGTH 22 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MG (HASYS) 4

<400 SEQUENCE: 61 Met Gly His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr 1 5 10 15 Ser His Ala Ser Tyr Ser 2O

SEQ ID NO 62 LENGTH 42 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MG (HASYS) 8

<400 SEQUENCE: 62 Met Gly His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr 1 5 10 15 Ser His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr Ser 2O 25 30 His Ala Ser Tyr Ser His Ala Ser Tyr Ser 35 40

SEQ ID NO 63 LENGTH 22 TYPE PRT ORGANISM: Artificial Sequence FEATURE: US 6,570,070 B1 109 110

-continued <223> OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MG (RASSL) 4 <400 SEQUENCE: 63 Met Gly Arg Ala Ser Ser Leu Arg Ala Ser Ser Lieu Arg Ala Ser Ser 1 5 10 15 Leu Arg Ala Ser Ser Lieu 2O

<210> SEQ ID NO 64 <211& LENGTH 42 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Protoporphyrin IX binding protein MG (RASSL) 8 <400 SEQUENCE: 64 Met Gly Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu Arg Ala Ser Ser 1 5 10 15 Leu Arg Ala Ser Ser Lieu Arg Ala Ser Ser Leu Arg Ala Ser Ser Lieu 2O 25 30 Arg Ala Ser Ser Leu Arg Ala Ser Ser Lieu 35 40

<210 SEQ ID NO 65 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400s. SEQUENCE: 65 Gly Gly Gly Ile Ser Gly 1 5

What is claimed is: followed by repeating a cycle of maintaining (a) at 96 1. A method for increasing resistance to weed control 40 C. for 40 seconds, then (b) at 68 C. for 7 minutes, 28 compounds in plants, Said method comprising: times, followed by maintaining at 96° C. for 40 introducing into a plant, a nucleic acid encoding a pro seconds, at 68 C. for 7 minutes and then at 72 C. for toporphyrin IX binding Subunit protein of a plant or 10 minutes, and photosynthetic microorganism magnesium chelatase or a nucleic acid encoding a deletion variant protoporphyrin a deletion variant protoporphyrin IX binding Subunit 45 IX binding Subunit protein of a magnesium chelatase protein of a plant or photosynthetic microorganism comprising a deletion of the organelle transit Signal, magnesium chelatase comprising a deletion of the wherein Said nucleic acid encoding the deletion variant organelle transit Signal. protoporphyrin IX binding Subunit protein of a mag nesium chelatase is amplifiable via PCR with a primer 2. The method according to claim 1 wherein the nucleic consisting essentially of the nucleotide Sequence of acid is operably ligated to a promoter and a terminator both 50 SEQ ID NO: 9 and a primer consisting essentially of of which are functional in the plant. the nucleotide sequence of SEQ ID NO: 10, wherein 3. The method according to claim 1, wherein the weed the PCR entails maintaining at 94 C. for 2 minutes, control compound inhibits porphyrin biosynthesis of a plant. followed by repeating a cycle of maintaining (a) at 94 4. The method according to claim 1, wherein the weed C. for 30 seconds, then (b) at 50° C. for 30 seconds, and control compound is a protoporphyrinogen IX oxidase 55 then (c) at 72° C. for 7 minutes, 30 times. inhibitory-type herbicidal compound. 6. A method for increasing resistance to weed control 5. The method according to claim 1, wherein the nucleic compounds in plants, Said method comprising: acid is: introducing into a plant, a nucleic acid encoding a peptide a nucleic acid encoding a protoporphyrin IX binding wherein Said peptide comprises an amino acid Sequence Subunit protein of a magnesium chelatase, wherein Said 60 selected from the group consisting of SEQ ID NO: 53 nucleic acid encoding the protoporphyrin IX binding and SEQID NO:55, in which the sequence is repeated Subunit protein of a magnesium chelatase is amplifiable at least four times. via PCR with a primer consisting essentially of the 7. A method for increasing resistance to weed control nucleotide sequence of SEQ ID NO: 1 and a primer compounds in plants, Said method comprising: consisting essentially of the nucleotide Sequence of 65 introducing into a plant, a nucleic acid encoding a plant SEQ ID NO: 2, wherein the PCR entails maintaining at ferrochelatase or a deletion variant plant ferrochelatase 94 C. for 40 seconds and then at 68 C. to 7 minutes, comprising a deletion of the organelle transit Signal. US 6,570,070 B1 111 8. The method according to claim 7, wherein the nucleic -continued acid is operably ligated to a promoter and a terminator both G-5 of which are functional in the plant. R4 9. The method according to claim 7, wherein the weed NZ R5 control compound inhibits porphyrin biosynthesis in a plant. 10. The method according to claim 7, wherein the weed O control compound is a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound. R1 11. The method according to claim 7, wherein the nucleic acid encodes the barley ferrochelatase or the cucumber R6 Y ferrochelatase. V 12. The method 7, wherein the nucleic acid encodes the N R4 cucumber ferrochelatase. R5 13. The method according to claim 1, 6 or 7, wherein the 15 X weed control compound is a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound Selected from the com pounds of (1), (2) or (3) below: R1 (1) chlormethoxynil, bifenox, chlornitrofen, acifluorfen and its ethyl ester, acifluorfen-Sodium, oxyfluorfen, R4 oxadiazon, 2-4-chloro-2-fluoro-5-(prop-2-ynyloxy) O y< phenyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1,3-dione, O chlorphthalim, TNPP-ethyl, or N3-(1-phenylethyl)-2,6- dimethyl-5-propyonylnicotinamide, (2) a compound represented by the general formula: J-G (I), 25 R3 wherein G is a group represented by any one of the following general formulas G-1 to G-9 and J is a group R1 represented by any one of the following general formulas G-8 O. O

R \/ R N N1 R4

35 R3

R1

R8 O O 40 \N-S \/

X 7 X1 R 45 R1 J-1 O R 50 YS 7- N 10 R i.

O 55 O

60

65 US 6,570,070 B1 113 114 -continued -continued J-13 R13se Y NS J-14 J-5 10

15 J-15

J-16

25

J-17

35 J-18

40

J-19 J-10

45

J-20 50

J-11

55

J-21

J-12 60

65 US 6,570,070 B1 116 -continued C(O)SR 7 group, C(O)NR'R'' group, CHO group,

J-23 CR-7=NOR group, CH=CR7COR7 group, CHCHRCOR7 group, CON=CRR group, nitro group, cyano group, NHSO2R group, NHSONHR group, NR7R group, NH group or phenyl group optionally Substituted with one or more and the same or different C-C alkyl groups; p is 0, 1 or 2, J-24 R is C-C alkyl group, C-C haloalkyl group, OCH 1O group, SCH group, OCHF group, halogen atom, cyano group or nitro group; R" is hydrogen atom, C-C alkyl group, C-C haloalkyl J-25 group or halogen atom; 15 R is hydrogen atom, C-C alkyl group, halogen atom, C-C haloalkyl group, cyclopropyl group, Vinyl group, C, alkynyl group, cyano group, C(O)R group, COR group, C(O)NR'R' group, CR'R'CN group, CRRC(O)R group, CRR COR group, CRR(O)NR'R' group, CHROH group, J-26 CHROC(O)R group or OCHROC(O)NR'R'' group, or, when G is G-2 or G-6, R" and R may form C=O group together with the carbon atom to which they are attached; 25 R is C-C alkyl group, C-C haloalkyl group, C-C, alkoxyalkyl group, C-C alkenyl group or C-C, alkynyl group; J-27 X is single bond, oxygen atom, Sulfur atom, NH group, N(C-C alkyl) group, N(C-C haloalkyl) group or N(allyl) group; R’ is hydrogen atom, C-C alkyl group, C-Chaloalkyl group, halogen atom, S(O)(C-C alkyl) group or C(=O)R" group; J-28 35 R is hydrogen atom, C-Cs alkyl group, Ca-Cs R13 O cycloalkyl group, C-C alkenyl group, C-C alkynyl V group, C-C haloalkyl group, C-C alkoxyalkyl N group, C-C alkoxyalkoxyalkyl group, C-Cs haloalkynyl group, C-C haloalkenyl group, C-Cs R14 \ / alkylsulfonyl group, C-Cs haloalkylsulfonyl group, N 40 C-C alkoxycarbonylalkyl group, S(O)NH(C-Cs R11 J-29 alkyl) group, C(O)R' group or benzyl group whose R11 phenyl ring may be substituted with R', in and m are independently 0, 1, 2 or 3 and m+n is 2 or 3; 45 Z is CR'R' group, oxygen atom, sulfur atom, S(O) / \ group, S(O)-group or N(C-C alkyl) group; FN each R is independently hydrogen atom, C-C alkyl J-30 group, halogen atom, hydroxyl group, C-C alkoxy R11 O group, C-C haloalkyl group, C-C haloalkoxy 50 group, C-C alkylcarbonyloxy group or C-C, R14 / N haloalkylcarbonyloxy group; each R's independently hydrogen atom, C-C alkyl N { group, and hydroxyl group or halogen atom; \ W1 R'' and R'' are independently hydrogen atom, halogen 1 55 atom, C-C alkyl group, C-C alkenyl group or C-C haloalkyl group; wherein the dotted lines in the formulas J-5, J-6, J-12 and R" is hydrogen atom, C-C alkyl group, C-C, J-24 represent that the left hand ring contains only Single haloalkyl group, C-C alkenyl group, C-C haloalk bonds, or one bond in the ring is a double bond between enyl group, C-C alkynyl group, C-C haloalkynyl carbon atoms, 60 group, HC(=O) group, (C-C alkyl)C(=O) group or X is oxygen atom or Sulfur atom; NH group; Y is oxygen atom or Sulfur atom; R" is C-C alkyl group, C-C alkylthio group, C-C, R" is hydrogen atom or halogen atom; haloalkyl group or N(CH) group; R is hydrogen atom, C-s alkyl group, C-Cs haloalkyl 65 W is nitrogen atom or CR'; group, halogen atom, OH group, OR group, SH R" is hydrogen atom, C-C alkyl group, halogen atom, grouproup, S(O).R7K group,group COR7 groupgroup, COR72R grOupgroup, or phenyl group optionally Substituted with C-C, US 6,570,070 B1 117 118 alkyl group, one or two halogen atoms, C-C alkoxy group, C-C haloalkynyloxyalkyl group, Co-C group or CF-group; cycloalkylthioalkyl group, C-C alkenylthioalkyl each Q is independently oxygen atom or Sulfur atom; group, C-C alkynylthioalkyl group, C-C alkyl group Substituted with phenoxy group whose ring is Q' is oxygen atom or Sulfur atom; Substituted with at least one Substituent selected from Z' is CR'R'' group, oxygen atom, sulfur atom, S(O) the group consisting of halogen atom, C-C alkyl group, S(O) group or N(C-C alkyl) group; group and C-C haloalkyl group, benzyloxy group each R' is independently hydrogen atom, halogen atom, whose ring is Substituted with at least one Substituent hydroxyl group, C-C alkoxy group, C-C haloalkyl Selected from the group consisting of halogen atom, group, C-C haloalkoxy group, C-C alkylcarbony C-C alkyl group and C-C haloalkyl group, loxy group or C-C haloalkylcarbonyloxy group; 1O C-C trialkylsilylalkyl group, C-C cyanoalkyl each R'' is independently hydrogen atom, hydroxyl group group, C-C halocycloalkyl group, C-C haloalk enyl group, Cs-Cs alkoxyalkenyl group, Cs-Cs or halogen atom; haloalkoxyalkenyl group, C-C alkylthioalkenyl R" is C-C alkyl group, halogen atom or C-C, group, C-C haloalkynyl group, C-C alkoxyalky haloalkyl group; 15 nyl group, Cs-C haloalkoxyalkynyl group, C-C, R'' and R'' are independently hydrogen atom, C-C, alkylthioalkynyl group, C-C alkylcarbonyl group, alkyl group, or C-C haloalkyl group; benzyl group whose ring is Substituted with at least Z is oxygen atom, sulfur atom, NR' group or CR'R'' one Substituent Selected from the group consisting of halogen atom, C-C alkyl group and C-C, grOup, halo alkyl group, CHRCOR group, R° and R’ are independently C-C alkyl group, C-C, CHRCOOR group, CHRP(O)(OR), group, haloalkyl group, C-C alkenyl group, C-C haloalk CHRP(S)(OR) group, CHRC(O)NR'R'' enyl group, C-C alkynyl group or C-C haloalkynyl group or CHRC(O)NHgroup; grOup, R is C-C alkyl group, C-C alkenyl group, C-C, R is hydrogen atom, halogen atom or cyano group; 25 alkynyl group or tetrahydrofuranyl group; R" is C-C alkylsulfonyl group, C-C alkyl group, R” and R are independently hydrogen atom or C-C haloalkyl group, C-C alkenyl group, C-C, C-C alkyl group; alkynyl group, C-C alkoxy group, C-C haloalkoxy R and R' are independently C-C alkyl group or group or halogen atom; phenyl group whose ring may be Substituted with at R’ is C-C alkyl group, C-C haloalkyl group, C-C, least one Substituent Selected from the group con alkenyl group or C-C alkynyl group; Sisting of halogen atom, C-C alkyl group and R" is C-C alkyl group, C-C haloalkyl group or C-C haloalkyl group; or, phenyl group optionally Substituted with C-C alkyl, R’ and R together may form -(CH) ,-(CH)- one or two halogen atoms, one or two nitro groups, or -CHCHOCHCH-, or the ring thus formed C-C alkoxy group or CF group; 35 may be substituted with at least one substituent selected W' is nitrogen atom or CH group; from the group consisting of C-C alkyl group, phenyl T is a group represented by any one of the following group and benzyl group; or, general formulas T-1, T-2 and T-3; RandR may from C-Cs cycloalkyl group together with the carbon atom to which they are attached; 40 R is C-C alkyl group, C-C haloalkyl group or C-C alkenyl group; R" and R are independently hydrogen atom or C-C alkyl group; R is hydrogen atom, C-C alkyl group, C-C, 45 alkenyl group or C-C alkynyl group; R’ is hydrogen atom, C-C alkyl group or halogen atom, R is hydrogen atom, C-C alkyl group, C-C, cycloalkyl group, C-C alkenyl group, C-C alky 50 nyl group, C-C alkoxyalkyl group, C-C, haloalkyl group, phenyl group whose ring may be (wherein E, E, E, E, E, E, E7, E, E, E, E' and Substituted with at least one Substituent selected from E" are independently hydrogen atom or C-C alkyl the group consisting of halogen atom, C-C alkyl group); group and C-C alkoxy group, —CH2CO(C-C, R’ is CI-Cs alkyl group, Ca-Cs cycloalkyl group, 55 alkyl) group or -CH(CH2)CO(C-C alkyl) group; C-Cs alkenyl group, C-Cs alkynyl group, C-Cs R’ is hydrogen atom, C-C alkyl group or C(O)O haloalkyl group, C-C alkoxyalkyl group, C-Cs (C-C alkyl) group; alkylthioalkyl group, C-C alkylsulfinylalkyl R" is hydrogen atom, C-C alkyl group, C-C, group, C-C alkylsulfonylalkyl group, C-C alkyl alkoxy group or NH(C-C alkyl) group; Sulfonyl group, phenylsulfonyl group whose phenyl 60 R" is C-C alkyl group, C-C haloalkyl group, ring may be Substituted with at least one Substituent C-C alkoxy group, NH(C-C alkyl) group, phe Selected from the group consisting of halogen atom nyl group whose ring may be Substituted with one and C-C alkyl group, C-C alkoxyalkoxyalkyl substituent selected from the group consisting of R' group, C-C cycloalkylalkyl group, Co-Cs group, benzyl group and C-C dialkylamino group; cycloalkoxyalkyl group, C-C alkenyloxyalkyl 65 and group, C-C alkynyloxyalkyl group, C-Cs R" is C-C alkyl group, one or two halogen atoms, haloalkoxyalkyl group, C-C haloalkenyloxyalkyl C-C alkoxy group or CF group; US 6,570,070 B1 119 120 (3) a compound of the formula (II): R is hydrogen atom, C-C alkyl group or C-C, alkyl group Substituted with at least one halogen R45 atom, R is hydrogen atom, C-C alkyl group optionally Substituted with at least one halogen atom, C-C, alkenyl group optionally Substituted with at least one halogen atom, C-C alkynyl group optionally Sub Stituted with at least one halogen atom, phenyl group optionally Substituted with at least one halogen atom, 1O C-C cycloalkyl group, cyanomethyl group, or or nipilacrofen, RCO-group; wherein R" is C-C alkyl group; R" is hydrogen atom, C-C alkyl group optionally R" is C-C alkyl group, C-C alkylthio group, C-C, alkoxy group, C-C haloalkyl group, C-C haloalky Substituted with at least one halogen atom, C-C, lthio group or C-C haloalkoxy group; alkenyl group optionally Substituted with at least one R" and R' together may form -(CH2)- or 15 halogen atom, C-C alkynyl group optionally Sub -(CH-)-; Stituted with at least one halogen atom, phenyl group R" is hydrogen atom or halogen atom; optionally Substituted with halogen atom, C-Cs R" is hydrogen atom or C-C alkyl group; cycloalkyl group, cyanomethyl group, C-C, R" is hydrogen atom, nitro group, cyano group, alkoxy-C-C alkyl group, di-C-C alkylamino -COOR'group, —C(=X)NR'R' group or C-C alkyl group, tetrahydrofurfurylmethyl group, —C(=X)R group; C-C alkynyloxy-C-C alkyl group, benzyl whose R" is hydrogen atom, halogen atom, cyano group, C-C, ring may be Substituted with Substituent Selected alkyl group optionally Substituted with at least one from the group consisting of halogen atom, nitro Substituent Selected from the group consisting of halo group, cyano group, C-C alkyl group, C-C, gen atom and hydroxyl group, C-C alkoxy group, 25 alkoxy group and halo-C-C alkyl group, phenyl group optionally Substituted with at least one -C(=X)R group, -(CH-)-(O)-R' group, Substituent Selected from the group consisting of halo -(CH-)-O-(CH2)-R' group, -(CH2)- gen atom, nitro group, cyano group, C-C alkyl group, X-R7 group; C-C alkoxy group and halo-C-C alkyl group, pyr R and R together with the nitrogen atom to which rolyl group, C2-Cs alkyl group, C-Cs alkenyl group, they are attached may form Saturated alicyclic 3, 5 or C-C alkynyl group, C-C alkoxy group, a group 6 membered ring or aromatic 5 or 6 membered ring Selected from the group consisting of C-C alkyl in which a carbon atom may be optionally replaced group, C-C alkenyl group, C-C alkynyl group and With oxygen atom; C-C alkoxy group into which at least one oxygen R is hydrogen atom, C-C alkyl group, C-C, atom is inserted, or any one of groups represented by 35 alkenyl group or C-C alkynyl group, or R and the following formulas: R together may form -(CH2)-; R and R7 are independently C-C alkyl group optionally Substituted with at least one halogen atom, C-C alkenyl group optionally Substituted with at 40 least one halogen atom, C-C alkynyl optionally Substituted with at least one halogen atom or phenyl group optionally Substituted with at least one halo O O O gen atom, hydrogen atom, C-C cycloalkyl group, -XR" group or -NR'R' group; 45 R’ is hydrogen atom, C-C alkyl group, C-C, alkenyl group, C-C alkynyl group, C-C alkyl – HN - C carbonyl group, cyano-C-C alkyl group, C-C, O O O alkoxycarbonyl-C-C alkyl group, di-C-C, O O alkoxycarbonyl-C-C alkyl group, benzyl group, 50 C-C alkoxy-C-C, alkynyl group, -(CH2)-R' group, -(CH), X-R' group, -(CH),X- -N -N (CH) R' group or -(CH-)-X-(CH-)- X-(CH2)-R' group; O O R’ is hydrogen atom, C-C alkyl group, C-C, O 55 alkenyl group, C-C alkynyl group, cyano-C-C- alkyl group, C-C alkylcarbonyl-C-C alkyl group -OX D -NR55(CH),CR57 or phenyl group; O R" is C-C alkyl group optionally substituted with at - (CH), - (CH2) least one halogen atom; 60 o (CH2) O-R65 -COR66 R'' and Rare, the same or different, hydrogen atom or C-C alkyl group; R" is C-C alkyl group optionally substituted with at wherein R', R and R are, the same or different, least one halogen atom, C-C alkoxy-C-C alkyl hydrogen atom or C-C alkyl group; group, C-C alkylthio-C-C alkyl group, C-C, R and R may form saturated alicyclic 5 or 6 65 cycloalkyl group, phenyl group whose ring may be membered ring together with the nitrogen atom to Substituted with one Substituent selected from the which they are attached; group consisting of halogen atom, nitro group, cyano US 6,570,070 B1 121 122 group, C-C alkyl group, C-C alkoxy group and 14. The method according to claim 7, wherein the nucleic halo-C-C alkyl group, -NR'R'' group or acid is: -(CH2)-(O).-R" group; a nucleic acid encoding a deletion variant of ferroche R" is C-C alkoxycarbonyl group or carboxyl group; latase comprising a deletion of the organelle transit R is chloromethyl group, cyanomethyl group, C-C, Signal, wherein Said nucleic acid encoding the deletion cycloalkyl group into which at least one oxygen variant ferrochelatase is amplifiable via PCR with a atom may be inserted, or C-C alkoxycarbonyl primer consisting essentially of the nucleotide C-C alkyl group; Sequence of SEQ ID: 21 and a primer consisting R is hydroxyl group or -NR7R group; essentially of the nucleotide sequence of SEQ ID: 22 wherein the PCR entails repeating a cycle of maintain A is -NR'R' group or —S(O)-R" grOup, 1O ing (a) at 94° C. for 1 minute, (b) at 55° C. for 2 R7 and Rare, the same or different, hydrogen atom minutes, and then (c) at 72° C. for 3 minutes, 30 times. or C-C alkyl group; 15. The method according to claim 1, wherein the nucleic R" is C-C alkyl group or C-C haloalkyl group; acid encodes a protoporphyrin IX binding Subunit protein of R" is hydrogen atom, hydroxyl group, halogen atom, a Rhodobacter capsulatuS magnesium chelatase, a protopor C-C alkyl group optionally Substituted with at 15 phyrin IX binding subunit protein of a Rhodobacter Sphaeri least one C-C alkoxy group, C-C cycloalkyl Odes magnesium chelatase, a protoporphyrin IX binding group into which at least one oxygen atom may be Subunit protein of a Snapdragon magnesium chelatase, a inserted, C-C cycloalkyl group optionally Substi protoporphyrin IX binding Subunit protein of a Synechocytis tuted with one or two methyl groups, furyl group, P.C.C. 6803 magnesium chelatase, a protoporphyrin IX thienyl group or -C(=O)R’ group; binding Subunit protein of a mouse-ear creSS magnesium R7 and R'' are, the same or different, C-C alkyl chelatase or a protoporphyrin IX binding Subunit protein of group or C-C alkoxy group; a barley magnesium chelatase. R7 and R7 are, the same or different, C-C alkyl 16. The method according to claim 7, wherein the nucleic group or phenyl group; acid encodes a barley ferrochelatase or a cucumber ferro R’ is C-C cycloalkyl into which at least one oxygen 25 chelatase. atom may be inserted, C-C cycloalkyl group 17. The method according to claim 1, 6 or 7, wherein the optionally substituted with one or two methyl introducing Step is conducted by Agrobacterium infection. groups, furyl group, thienyl group or -C(=O)R’ 18. A method of increasing resistance to weed control grOup, compounds in plants, Said method comprising: R7 is C-C alkyl group; introducing into a plant, a nucleic acid encoding a protein a, b and c is independently 1, 2 or 3; consisting essentially of the amino acid Sequence of d is 0 or 1; SEQ ID NO: 53, wherein said sequence is repeated at e is 2 or 3; least four times. f is 1 or 2; and X is oxygen atom or Sulfur atom.