JP2001190168A - Herbicide resistant plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a plant having resistance against plural compounds having respectively different herbicidal action mechanisms. SOLUTION: This plant has at least one enzymatic activity selected from (1) and (2) shown below by which its resistance against a herbicide is exhibited at amounts inhibiting plant natural enzymatic activity for synthesizing 5-enol pyruvylshikimic acid-3-phosphate. (1) Enzymatic activity for synthesizing 5-enol pyruvylshikimic acid-3-phosphate practically different from plant natural enzymatic activity for synthesizing 5-enol pyruvylshikimic acid-3-phosphate. (2) Glyphosate oxide reductase activity different from plant natural glyphosate oxide reductase activity. Wherein a gene encoding a protein having characters of (3), (4) and (5) shown below is transduced into the plant and the plant expresses the gene, and so on. (3) Specifically binding to materials involved in herbicidal activity of protoporphyrinogen IX oxidase inhibitory-type herbicide. (4) Practically having no modifying action on materials specifically bound with the above protein. (5) Practically including no framework region of variable region of immunoglobulin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a herbicide-tolerant plant.

[0002]

2. Description of the Related Art Weed control is an important work for improving crop yield and quality, and herbicides are mainly used for improving the efficiency of weed control work. Since there are many types of weeds to be controlled and their occurrence is long-term, a plurality of compounds acting by different herbicidal action mechanisms may be used for weed control as necessary.

[0003]

In such a situation where a herbicide is used, it is sometimes difficult to clearly distinguish crops from closely related weeds and selectively control only weeds. There is a strong need for the development of crops that are resistant to multiple compounds acting by different herbicidal mechanisms.

[0004]

Under these circumstances, the present inventors have conducted intensive studies. As a result, by producing certain proteins in plant cells, resistance to glyphosate and its salts and prototyping have been improved. Porphyrinogen IX
The present inventors have found that it is possible to produce plants having resistance to oxidase-inhibiting herbicides, and have reached the present invention. That is, the present invention provides: 1) one or more enzyme activities selected from the following (1) and (2), and the enzyme activity allows natural 5-enolpyruvylshikimate-3-phosphate of a plant A plant that is resistant to an acid synthase-inhibiting amount of a herbicide, and (1) is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant 5-Enolpyruvylshikimate-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A plant into which a gene encoding a protein having the following properties (3), (4) and (5) has been introduced and which expresses the gene (hereinafter referred to as plant 1 of the present invention); It specifically binds to substances related to the herbicidal activity of porphyrinogen IX oxidase-inhibiting herbicides. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 2) The above item 1), wherein the protein has a property of specifically binding to a substance contained as an active ingredient in a protoporphyrinogen IX oxidase-inhibiting herbicide.
3) The protein according to the above 1), wherein the protein has a property of specifically binding to a plant cell endogenous substance involved in the expression of a weed controlling activity of a protoporphyrinogen IX oxidase-inhibiting herbicide in a plant cell. 4) The plant according to the above 1) or 3), wherein the protein is a protein having a property of specifically binding to protoporphyrin IX. 5) The protein is a protoporphyrin IX binding subunit protein of magnesium keratase. Or the modified protein of the above-mentioned protein, which is a protein having the property of specifically binding to protoporphyrin IX, the plant according to the above 1), 3) or 4), 6) the protein is protoporphyrinogen 1) or 3) above, which is a protein having the property of specifically binding to IX. 7) The protein is a modified protein of protoporphyrinogen IX oxidase and has no property of oxidizing protoporphyrinogen IX and has a property of specifically binding to protoporphyrinogen IX. The plant according to the above 1), 3) or 6), 8) the protein is a modified protein of protoporphyrinogen IX oxidase, and does not have the ability to oxidize protoporphyrinogen IX and has a protoporphyrinogen IX oxidase inhibitory type The plant according to the above 1), 2) or 6), which is a protein having a property of specifically binding to a substance contained as an active ingredient in a herbicide; 9) 1 selected from the following (1) and (2): Having the above enzyme activity, the enzyme activity allows the natural synthesis of 5-enolpyruvylshikimate-3-phosphate in plants A plant that exhibits tolerance to herbicides in an amount that inhibits elementary activity, wherein (1) a plant that is substantially different from the plant's natural 5-enolpyruvylshikimate-3-phosphate synthase activity. Enolpyruvylshikimate-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A plant into which a gene encoding a protein having the following properties (3), (4) and (5) has been introduced and which expresses the gene (hereinafter referred to as plant 2 of the present invention); Binds specifically to porphyrin IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 10) The plant according to the above 9), wherein the protein is ferrochelatase or a modified protein of the protein, which is a protein having a property of specifically binding to protoporphyrin IX. 11) The following (1) and (2): Having at least one enzymatic activity selected from the group consisting of a herbicide resistant to an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzymatic activity. The plant shown, wherein: (1) a 5-enolpyruvylshikimate-3-phosphate synthase activity substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A plant into which a gene encoding a protein having the following properties (3), (4) and (5) has been introduced and which expresses the gene (hereinafter referred to as plant 3 of the present invention); It specifically binds to porphyrinogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 12) The plant according to the above item 11), wherein the protein is coproporphyrinogen III oxidase or a modified protein of the protein, which is a protein having a property of specifically binding to protoporphyrinogen IX. 1) A method for producing a herbicide-tolerant plant, which comprises growing the plant according to any one of 1) to 12). 14) Applying a herbicide to the cultivation area of the plant according to any of 1) to 12) above. A method for controlling weeds to be sprayed; 15) a method for selecting a herbicide-tolerant plant that sprays a herbicide on a cultivation area of a plant according to any one of the above items 1) to 12); A method for selecting a herbicide-tolerant plant cell in which a herbicide is added to a culture area of a plant cell described in 17), and 17) a gene encoding one or more proteins selected from the following (1) and (2): (1) a protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (2) A protein having substantially glyphosate oxidoreductase activity. A step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. It specifically binds to substances related to the herbicidal activity of porphyrinogen IX oxidase-inhibiting herbicides. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 18) It has one or more enzyme activities selected from the following (1) and (2), and inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of plants by the enzyme activities. (1) 5-enolpyruvylshikimate which is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant. Acid-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. (3) a method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding a protein having the properties of (3), (4) and (5) in a plant cell; It specifically binds to substances related to the herbicidal activity of linogen IX oxidase-inhibiting herbicides. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 19) To a plant cell into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene: (1) Protoporphyrinogen IX oxidase-inhibiting herbicide Specifically binds to substances related to herbicidal activity. (2) the protein has substantially no ability to alter the substance to which it specifically binds; (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) 5-enolpyruvir A protein having substantially shikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity. 20) a step of introducing and expressing a gene encoding one or more proteins selected from the following (1) and (2) into plant cells, and (1) synthesizing 5-enolpyruvylshikimate-3-phosphate A protein having substantially enzymatic activity. (2) A protein having substantially glyphosate oxidoreductase activity. A step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. Binds specifically to porphyrin IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 21) It has one or more enzyme activities selected from the following (1) and (2), and inhibits natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzyme activities. (1) 5-enolpyruvylshikimate which is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant. Acid-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. The following (3), (4) and (5) a method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding a protein having the properties of a plant cell into a plant cell, (3) protoporphyrin Binds specifically to IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 22) (1) Specific binding to protoporphyrin IX to plant cells into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene. (2) It does not have the ability to transform protoporphyrinogen IX. (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) 5-enolpyruvir A protein having substantially shikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity. 23) a step of introducing a gene encoding one or more proteins selected from the following (1) and (2) into a plant cell to express the gene, and (1) synthesizing 5-enolpyruvylshikimate-3-phosphate A protein having substantially enzymatic activity. (2) A protein having substantially glyphosate oxidoreductase activity. A step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. It specifically binds to porphyrinogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 24) has one or more enzyme activities selected from the following (1) and (2), and inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzyme activities: (1) 5-enolpyruvylshikimate which is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant. Acid-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. (3) a method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding a protein having the properties of (3), (4) and (5) in a plant cell; Binds specifically to Linogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 25) Specific binding to protoporphyrinogen IX to a plant cell into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene: I do. (2) It has the ability to transform coproporphyrinogen III. (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) 5-enolpyruvir A protein having substantially shikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity. Is provided.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. Protoporphyrinogen IX oxidase-inhibiting herbicide is protoporphyrinogen IX oxidase
(protoporphyrinogen IX oxidase; EC 1.3.3.4.
Notated as PPO. )) (Hereinafter referred to as PP
O-inhibited herbicidal compounds. ) As an active ingredient.

[0006] In the present invention, the substance relating to the herbicidal activity of a PPO-inhibiting herbicide (hereinafter referred to as the present substance) refers to PPO.
The term refers to an inhibitory herbicidal compound and a plant cell endogenous substance involved in the expression of the herbicidal activity of the herbicide in a plant cell when the PPO inhibitory herbicide is applied to a plant. Compounds included as active ingredients in PPO-inhibiting herbicides include Duke,
SO, Rebeiz, CA ACS Symposium Series 559, Porph
yric Pesticides, Chemistry, Toxicology, and Pharma
ceutical Applications. American Chemical Society,
Examples include compounds described in Washington DC (1994) and the like. PPO-inhibiting herbicidal compounds encompass many different structural molecular species [Duke et al., Weed Sci. 39: p465 (199).
1); Nandihalli et al., Pesticide Biochem. Physiol.
43: p193 (1992); Matringe et al., FEBS Lett. 245:
p35 (1989); Yanase, Andoh, Pesticide Biochem. Physi
ol. 35: p70 (1989)], specifically, diphenyl ethers such as chloromethoxynil, bifenox, chloronitrophen (CNP), acifluorfen {5- [2-chloro-
4- (trifluoromethyl) phenoxy] -2-nitrobenzoic acid}, ethyl ester of acifluorfen, acifluorfen-sodium, oxyfluorfen [2-chloro-1- (3-ethoxy-4-nitrophenoxy)- 4-trifluoromethylbenzene], oxadiazole [eg oxadiazone {3- [2,4-dichloro-5- (1-methylethoxy) phenyl] -5- (1,1-dimethylethyl) -1,3, 4-oxadiazole-2 (3H) -one)}], cyclic imide [eg, S-23142 [N-
(4-chloro-2-fluoro-5-propargyloxyphenyl)-
3,4,5,6-tetrahydrophthalimide], chlorophthalim [N- (4-chlorophenyl) -3,4,5,6-tetrahydrophthalimide], phenylpyrazole [eg TNPP-ethyl
{Ethyl 2- [1- (2,3,4-trichlorophenyl) -4-nitropyrazolyl-5-oxy] propionate}], pyridine derivative [for example, LS82-556 [N3- (1-phenylethyl) -2, 6-dimethyl-5-propionylnicotinamide]], phenopyrates, O-phenylpyrrolidinocarbamate analogs of phenopyrates, piperidinocarbamate analogs of phenopyrates and the like.

Particularly important diphenyl ethers include:
Examples include compounds represented by the following structural formulas 1 to 7 [Structural formula 4: Maigrot et al., Brighton Crop
See Protection Conference-Weeds: 47-51 (1989); Structural formula 5: Hayashi et al., Brighton Crop Protection C
onference-Weeds: 53-58 (1989); Structural formula 6: Bifenox, Dest et al., Proc. Northeast Weed Sci.
Conf. 27:31 (1973)].

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Further, other particularly important PPO-inhibiting herbicidal compounds include compounds represented by the following general formula (2), and more specifically, compounds represented by the following chemical formulas (7) to (10): Compounds represented by structural formulas 8 to 37 are exemplified.

Wherein G is a group represented by G-1 to G-9 shown in the following Chemical Formula 3, and J is a J shown in the following Chemical Formulas 4 to 6.
And groups represented by -1 to 30.

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Where the formulas J-5, J-6, J-12 and J-24
Indicates that the ring on the left contains only a single bond.
Or one bond in a ring is a double bond between carbon atoms
X represents an oxygen atom or a sulfur atom, and Y represents
Represents an oxygen or sulfur atom, R¹ Is a hydrogen atom or
Represents a halogen atom, R^Two Is a hydrogen atom, C₁-C₈ Alkyl
Group, C₁-C₈ Haloalkyl group, halogen atom, hydroxyl group, -O
R²⁷ Group, -SH group, -S (O) pR²⁷ Group, -COR²⁷ Group, -CO_TwoR²⁷ 
Group, -C (O) SR²⁷Group, -C (O) NR²⁹R³⁰ Group, -CHO group, -CR²⁷= N
OR³⁶ Group, -CH = CR³⁷CO_TwoR²⁷ Group, -CH_TwoCHR ³⁷CO_TwoR²⁷ Group, -C
O_TwoN = CR³¹R³² Group, nitro group, cyano group, -NHSO_TwoR³³ Group,
-NHSO_TwoNHR³³ Group, -NR²⁷R³⁸ Group, -NH_Two Group or one
Substituted with the same or different C1-C4 alkyl groups
Represents an optionally substituted phenyl group, p is 0, 1 or 2
And R^Three Is C₁-C_Two Alkyl group, C₁-C_Two Haloalkyl
Group, -OCH_Three Group, -SCH_Three Group, -OCHF_TwoGroup, halogen atom,
Represents an ano group or a nitro group, R^Four Is a hydrogen atom, C₁-C_Three
 Alkyl group, C₁-C_Three Haloalkyl group or halogen
Represents an atom, R^Five Is a hydrogen atom, C₁-C_Three Alkyl group, halo
Gen atom, C₁-C_Three Haloalkyl group, cyclopropyl group,
Vinyl group, C_Two Alkynyl group, cyano group, -C (O) R³⁸ Group,
-CO_TwoR^{Three 8} Group, -C (O) NR³⁸R³⁹ Group, -CR³⁴R³⁵CN group, -CR³⁴R
³⁵C (O) R³⁸ Group, -CR³⁴R³⁵CO_TwoR³⁸Group, -CR³⁴R³⁵C (O) NR³⁸R
³⁹ Group, -CHR³⁴OH group, -CHR³⁴OC (O) R³⁸ Group, or -OCH
R³⁴OC (O) NR³⁸R³⁹ Represents a group, or G is G-2
Or R for G-6^FourAnd R^FiveAnd these are combined
And a carbon atom may represent a C = O group;⁶ Is C₁-C₆
 Alkyl group, C₁-C₆ Haloalkyl group, C_Two-C₆ Alkoki
Silalkyl group, C_Three-C₆ Alkenyl group, or C_Three-C₆ A
X represents a rukinyl group;¹ Is a direct bond, an oxygen atom, a sulfur atom
, -NH group, -N (C₁-C_Three Alkyl) group, -N (C₁-C_ThreeHaloal
Kill) group, or -N (allyl) group, R⁷ Is hydrogen field
Child, C₁-C₆ Alkyl group, C₁-C₆ Haloalkyl group, halogen
Atom, -S (O)_Two(C₁-C₆Alkyl) group, or -C (= O) R⁴⁰ 
Represents a group, R⁸ Is a hydrogen atom, C₁-C₈ Alkyl group, C_Three-C₈ 
Cycloalkyl group, C_Three-C₈ Alkenyl group, C_Three-C₈ Archi
Nyl group, C₁-C₈ Haloalkyl group, C_Two-C₈ Alkoxyal
Kill group, C_Three-C₈ Alkoxyalkoxyalkyl group, C_Three-C
₈ Haloalkynyl group, C_Three-C₈Haloalkenyl group, C₁-C₈ 
Alkylsulfonyl group, C₁-C₈ Haloalkylsulfonyl
Group, C_Three-C₈ Alkoxycarbonylalkyl group, -S (O)_TwoNH
(C₁-C₈ Alkyl) group, -C (O) R⁴¹ Group or phenyl ring
On R⁴² Represents a benzyl group optionally substituted with
 And m are each independently 0, 1, 2 or 3.
And m + n represents 2 or 3, and Z is -CR⁹R^Ten 
Group, oxygen atom, sulfur atom, -S (O) group, -S (O)_Two Group, also
Is -N (C₁-C _Four Alkyl) group, each R⁹ Is German
Standing hydrogen atom, C₁-C_Three Alkyl group, halogen atom, ar
Droxy group, C₁-C₆ Alkoxy group, C₁-C₆ Haloalkyl
Group, C₁-C₆ Haloalkoxy group, C_Two-C₆ Alkyl carboni
Roxy group, or C_Two-C₆ Haloalkylcarbonylo
Represents a xy group, each R^Ten Is independently a hydrogen atom, C
₁-C_Three Alkyl group, hydroxy group, or halogen atom
And R¹¹ And R¹² Are independently hydrogen sources
Child, halogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Alkene
Or C₁-C₆ Represents a haloalkyl group, R¹³ Is water
Elementary atom, C₁-C₆ Alkyl group, C₁-C₆ Haloalkyl group, C_Three
-C₆ Alkenyl group, C_Three-C₆ Haloalkenyl group, C_Three-C₆ A
Rukinyl group, C_Three-C₆ Haloalkynyl group, HC (= 0) group, (C₁-
C_Four Alkyl) C (= O) group, or -NH_Two Represents a group, R¹⁴ Is
 C₁-C₆ Alkyl group, C₁-C₆ Alkylthio group, C₁-C₆ C
Loalkyl group, or -N (CH_Three)_Two W represents a nitrogen atom
Child or -CR^Fifteen Represents a group, R^Fifteen Is a hydrogen atom, C₁-C₆ A
Alkyl group, halogen atom, or C₁-C₆ Alkyl group,
1 or 2 halogen atoms, C₁-C₆ Alkoxy groups
Or -CF_Three Phenyl group which may be substituted
And each Q is independently an oxygen or sulfur atom
And Q¹ Represents an oxygen atom or a sulfur atom; Z¹ Is-
CR¹⁶R¹⁷ Group, oxygen atom, sulfur atom, -S (O) group, -S (O)_Two 
Group, or -N (C₁-C_Four Alkyl) group,
R¹⁶ Is independently a hydrogen atom, a halogen atom, hydroxy
Group, C₁-C₆ Alkoxy group, C₁-C₆ Haloalkyl group, C₁-C
₆ Haloalkoxy group, C_Two-C₆ Alkylcarbonyloxy
Group or C_Two-C₆ A haloalkylcarbonyloxy group
Represent each R¹⁷ Is independently hydrogen atom, hydroxy
Or a halogen atom, R¹⁸ Is C₁-C₆ Al
Kill group, halogen atom, or C₁-C₆ Haloalkyl group
And R¹⁹ And R²⁰ Are independently hydrogen sources
Child, C₁-C₆ Alkyl group, or C₁-C ₆ Haloalkyl group
And Z^Two Is an oxygen atom, a sulfur atom, -NR⁹ Group, or -C
R⁹R^Ten Represents a group, R^{twenty one} And R^{twenty two} Are independent of each other
C₁-C₆ Alkyl group, C₁-C₆ Haloalkyl group, C_Three-C₆ Al
Kenyl group, C_Three-C₆ Haloalkenyl group, C_Three-C₆ Alkynyl
Group, or C_Three-C₆ Represents a haloalkynyl group, R^{twenty three} Is hydrogen
Represents an atom, a halogen atom, or a cyano group; R^{twenty four} Is C
₁-C₆ Alkylsulfonyl group, C₁-C₆ Alkyl group, C₁-
C₆ Haloalkyl group, C_Three-C₆ Alkenyl group, C_Three-C₆ Al
Quinyl group, C₁-C₆ Alkoxy group, C₁-C₆Haloalkoxy
Represents a group or a halogen atom, R^{twenty five} Is C₁-C₆ Archi
Group, C₁-C₆ Haloalkyl group, C_Three-C₆ Alkenyl group,
Or C_Three-C₆ Represents an alkynyl group, R²⁶ Is C₁-C₆ Archi
Group, C₁-C₆ Haloalkyl group or C on the ring₁-C₆ 
An alkyl group, one or two halogen atoms, one to
Two nitro groups, C₁-C₆ Alkoxy group, and CF_Three Base
Substituted with a substituent selected from the group consisting of
Represents a phenyl group which may be¹ Is a nitrogen atom or
Represents a CH group, where T is the following general formula T-1, T-2, or T-3
Represents any group of(Where E¹, E^Two, E^Three, E^Four, E^Five, E⁶, E⁷, E⁸, E⁹, E^Ten, E
¹¹, And E¹² Is a hydrogen atom or C₁-C_Three Al
Represents a kill group. ) R²⁷ Is C₁-C₈ Alkyl group, C_Three-C₈ Cycloalkyl group, C
_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl group, C₁-C₈ Halo
Alkyl group, C_Two-C₈ Alkoxyalkyl group, C_Two-C₈Al
Quilthioalkyl group, C_Two-C₈ Alkylsulfinyl al
Kill group, C_Two-C₈ Alkylsulfonylalkyl group, C₁-C
₈ Alkyl sulfonyl group, halogen source on phenyl ring
Child and C₁-C_Four From the group consisting of alkyl groups
Substituted with at least one selected substituent
Phenylsulfonyl group, C_Four-C₈ Alkoxyal
Coxyalkyl group, C_Four-C₈ Cycloalkylalkyl group,
C₆-C₈ Cycloalkoxyalkyl group, C_Four-C₈ Alkenyl
Oxyalkyl group, C_Four-C₈Alkynyloxyalkyl
Group, C_Three-C₈ Haloalkoxyalkyl group, C_Four-C₈ Haloal
Kenyloxyalkyl group, C_Four-C₈ Haloalkynyloxy
Alkyl group, C₆-C₈ Cycloalkylthioalkyl group, C_Four
-C₈ Alkenylthioalkyl group, C_Four-C₈ Alkynylthio
Alkyl group, halogen atom on the ring, C₁-C_Three Alkyl group
And C₁-C_Three From the group consisting of haloalkyl groups
Substituted with at least one selected substituent
Substituted with a good phenoxy group₁-C_Four Alkyl group,
Halogen atom on the ring, C₁-C_Three Alkyl group and C₁-C_Three 
Selected from the group consisting of haloalkyl groups
Benzy optionally substituted with at least one substituent
C substituted with a ruoxy group₁-C_Four Alkyl group, C_Four-C₈bird
Alkylsilylalkyl group, C_Three-C₈ Cyanoalkyl group,
C_Three-C₈ Halocycloalkyl group, C_Three-C₈ Haloalkenyl
Group, C_Five-C₈ Alkoxyalkynyl group, C_Five-C₈ Halo alco
Xylalkynyl group, C_Five-C₈ Alkylthioalkenyl group,
C_Three-C₈ Haloalkynyl group, C_Five-C₈ Alkoxyalkynyl
Group, C_Five-C₈ Haloalkoxyalkynyl group, C_Five-C₈ Archi
Luthioalkynyl group, C_Two-C₈ Alkylcarbonyl group, ring
A halogen atom, C₁-C_Three Alkyl group and C₁-C_Three C
Selected from the group consisting of
Benzyl optionally substituted with at least one substituent
Group, -CHR³⁴COR²⁸ Group, -CHR³⁴COOR²⁸ Group, -CHR³⁴P (O) (OR
²⁸)_Two Group, -CHR³⁴P (S) (OR²⁸)_TwoGroup, -CHR³⁴C (O) NR²⁹R³⁰ 
Group, or -CHR³⁴C (O) NH_Two Represents a group, R²⁸ Is C₁-C₆ A
Lucyl group, C_Two-C₆ Alkenyl group, C_Three-C₆ Alkynyl group,
Or a tetrahydrofuranyl group, R²⁹ And R³¹
 Is independently a hydrogen atom, or C₁-C_Four Table showing alkyl groups
Then R³⁰ And R³² Is independently C₁-C_Four Alkyl group
Or a halogen atom on the ring, C₁-C_Three Alkyl group and C₁-
C_Three Selected from the group consisting of haloalkyl groups
A group optionally substituted with at least one substituent
Represents a phenyl group, or R²⁹And R³⁰And in-(CH_Two)_Five-,
-(CH_Two)_Four-Or -CH_TwoCH_TwoOCH_TwoCH_Two-May be represented
In each ring thus formed, C₁-
C_Three Consists of alkyl, phenyl and benzyl groups
Even if substituted with a substituent selected from the group
Good or R³¹And R³²And these are combined
C with carbon atoms_Three-C₈ Even if it represents a cycloalkyl group
Well, R³³ Is C₁-C_Four Alkyl group, C₁-C_Four Haloalkyl
Group or C_Three-C₆ Represents an alkenyl group, R³⁴ And R
³⁵ Is independently a hydrogen atom or C₁-C_Four Table showing alkyl groups
Then R³⁶ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Arche
Nyl group, or C_Three-C₆ Represents an alkynyl group, R³⁷ Is water
Elementary atom, C₁-C_Four Displays an alkyl group or halogen atom
Then R³⁸ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Cyclo
Alkyl group, C_Three-C₆ Alkenyl group, C_Three-C₆ Alkynyl
Group, C_Two-C₆ Alkoxyalkyl group, C₁-C₆ Haloalkyl
Group, halogen atom on the ring, C₁-C_Four Alkyl group and C₁-
C_Four Selected from the group consisting of alkoxy groups
Fe which may be substituted with at least one substituent
Nyl group, -CH_TwoCO_Two(C₁-C_Four Alkyl) group, or -CH (CH_Three)
CO_Two(C₁-C_Four Alkyl) group, R³⁹ Is a hydrogen atom, C₁-C_Two
 Alkyl group or C (O) O (C₁-C_Four Alkyl) group
Then R⁴⁰ Is a hydrogen atom, C₁-C₆ Alkyl group, C₁-C₆ Arco
Xyl group, or NH (C₁-C₆ Alkyl) group, R⁴¹ Is
C₁-C₆ Alkyl group, C₁-C₆ Haloalkyl group, C₁-C₆ Al
Coxy group, NH (C₁-C₆ Alkyl) group, R⁴² Substituted with a group
Optionally phenyl, benzyl, or C_Two-C₈ The
Represents an alkylamino group, R⁴² Is C₁-C₆ Alkyl group, 1
 Or two halogen atoms, C₁-C₆ An alkoxy group
Or CF_Three Represents a group.

[0010]

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Further, as PPO-inhibiting herbicidal compounds, N-substituted pyrazoles represented by the following general formula (see International Patent Publications WO 94/08999, WO 93/10100 and US Pat. No. 5,405,829 to Schering) and the like: Is raised.

Embedded imageWhere R⁴³ Is C₁-C_Four Represents an alkyl group, R⁴⁴ Is C₁-C
_Four Alkyl group, C₁-C_Four Alkylthio group, C₁-C_Four Arco
Xyl group, C₁-C_FourHaloalkyl group, C₁-C_Four Haloalkyl
Thio group, or C₁-C_Four Represents a haloalkoxy group;
Well, R⁴³ And R⁴⁴And in-(CH_Two)_Three-Or- (CH_Two)_Four-Represents
May be R⁴⁵ Represents a hydrogen atom or a halogen atom
Then R⁴⁶ Is a hydrogen atom or C₁-C_Four Represents an alkyl group, R
⁴⁷ Is hydrogen atom, nitro group, cyano group, -COOR⁴⁹ Group, -C
(= X) NR⁵⁰R⁵¹ Group, or -C (= X^Two) R⁵² Represents a group, R⁴⁸ Is
Hydrogen atom, halogen atom, cyano group, halogen atom and
At least selected from the group consisting of
May also be substituted with one substituent₁-C_Four Archi
Group, C₁-C_Four An alkoxy group, a halogen atom on the ring,
Toro, cyano, C₁-C_Four Alkyl group, C₁-C_Four Arco
Xy group and halo-C₁-C_Four Group consisting of alkyl groups
Substituted with at least one substituent selected from
Optionally substituted phenyl, pyrrolyl, C_Two-C₈ A
Ruquil group, C_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl
Group, C_Three-C₈ An alkoxy group, (the C_Two-C₈ An alkyl group,
C _Three-C₈ An alkenyl group, the C_Three-C₈ An alkynyl group and
C_Three-C₈ An alkoxy group contains at least one oxygen source
Or a group shown below.
AndR⁴⁹ R⁵⁰ And R⁵¹ May be the same or different,
Hydrogen atom or C₁-C_FourRepresents an alkyl group, or R
⁵⁰ And R⁵¹Is a 5-member or a nitrogen atom to which these bind
May form a 6-membered saturated ring, R⁵² Is hydrogen
Atom, C₁-C_Four An alkyl group, or at least one or more
C substituted with a halogen atom₁-C_Four Represents an alkyl group, R
⁵³ Is a hydrogen atom, C₁-C_Four Alkyl group, C_Two-C₆ Alkene
Group, C_Three-C₆ Alkynyl group, phenyl group (C₁-C_Four A
Alkyl group, the C_Two-C₆ Alkenyl group, the C_Three-C₆ Alkini
And the phenyl group is substituted with a halogen atom.
May be present), C _Three-C₈ Cycloalkyl group, cyanomethy
Or R⁶³Represents a CO- group, R⁵⁴ Is a hydrogen atom, halo
C optionally substituted with a gen atom₁-C₆ Alkyl group,
C optionally substituted with a halogen atom_Two-C₆ Alkene
C, which may be substituted with_Three-C₆ A
Alkynyl group,
Nyl group, C_Three-C₈ Cycloalkyl group, cyanomethyl group,
C₁-C_Four Alkoxy C ₁-C₆ Alkyl group, di C₁-C_Four Al
Killamino C₁-C_Four Alkyl group, tetrahydrofurfury
Methyl group, C_Three-C₆ Alkynyloxy C₁-C_Four Alkyl
Group, benzyl group, halogen atom on the ring, nitro group, cyano
No group, C₁-C_Four Alkyl group, C₁-C_Four An alkoxy group and
Halo C₁-C_Four Select from groups consisting of alkyl groups
Benzyl group which may be substituted with a substituent represented by -C (=
X^Two) R⁶³Group,-(CH_Two)_a-(O)_d-R⁷⁰ Group,-(CH_Two)_a-O- (CH_Two)_b-R
⁷⁰ Group, or-(CH_Two)_a-X^Two-R⁷⁶Represents a group, or R⁵³
 And R⁵⁴Is the nitrogen atom to which they are attached, 3 members, 5 members
6 or 6 membered saturated ring or 5 or 6 membered ring
Aromatic ring (in the saturated ring and the aromatic ring, one carbon
Element atom may be replaced by one oxygen atom)
May be formed, R⁵⁵ Is a hydrogen atom, C₁-C_Four Alkyl
Group, C_Two-C₆ Alkenyl group, or C_Three-C₆ Alkynyl group
Or R⁵⁵ And R⁵⁶ And in-(CH_Two)_e-Form a group
May be, R⁵⁶ And R⁵⁷ Is C₁-C_Four Alkyl
Group, C_Two-C₆ Alkenyl group, C_Three-C₆ Alkynyl group or
Is a phenyl group (the C₁-C_Four An alkyl group, the C_Two-C₆ Arche
Nyl group, the C_Three-C₆ An alkynyl group and the phenyl group are
Hydrogen atom, C_Three
-C₆ Cycloalkyl group, -X^TwoR⁶⁰ Group, or -NR⁶¹R⁶²Base
And R⁵⁸ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Two-C₆ 
Alkenyl group, C_Three-C₆ Alkynyl group, C₁-C_Four Alkyl
Carbonyl group, cyano C₁-C_Three Alkyl group, C₁-C_Four Al
Coxycarbonyl C₁-C_Four Alkyl group, di C₁-C_Four Arco
Xycarbonyl C₁-C_FourAlkyl group, benzyl group, C₁-C_Four
Alkoxy-C₁-C_FourAlkynyl group,-(CH_Two)_a-R⁷⁵Group,-(CH
_Two)_a-X^Two-R⁷²Group,-(CH_Two)_a-X^Two-(CH_Two)_b-R⁷²Group, or-(C
H_Two)_a-X^Two-(CH_Two)_b-X ^Two-(CH_Two)_c-R⁷²Represents a group, R⁵⁹ Is hydrogen field
Child, C₁-C_Four Alkyl group, C_Two-C₆ Alkenyl group, C_Three-C₆
 Alkynyl group, cyano C₁-C_Three Alkyl group, C₁-C_Four A
Lucylcarbonyl C₁-C_Three Alkyl group or phenyl
Represents a group, R⁶⁰ Is replaced by at least one halogen atom
May be C₁-C_Four Represents an alkyl group, R⁶¹ And R⁶²
 May be the same or different and represent a hydrogen atom, or
C₁-C_Four Represents an alkyl group, R⁶³ Is at least one
C optionally substituted with a halogen atom₁-C_Four Alkyl
Group, C₁-C_Four Alkoxy C₁-C_Four Alkyl group, C₁-C_Four A
Lucircio C₁-C_Four Alkyl group, C_Three-C₆ Cycloalkyl
Group, halogen atom on the ring, nitro group, cyano group, C₁-C_Four
 Alkyl group, C₁-C_Four Alkoxy group and halo C₁-C_Four 
One selected from the group consisting of alkyl groups
A phenyl group optionally substituted with a substituent of -NR⁷³R⁷⁴
Group, or-(CH_Two)_a-(O)_d-R⁷⁵Represents a group, R⁶⁴ Is C₁-C_Four
Displays an alkoxycarbonyl group or carboxyl group
Then R⁶⁵ Is a chloromethyl group, a cyanomethyl group, at least
May also have one or more oxygen atoms inserted C_Three-C₆ Shi
Chloroalkyl group, or C₁-C_Four Alkoxycarbonyl
C₁-C_Four Represents an alkyl group, R⁶⁶ Is a hydroxyl group, or -NR⁶⁷R
⁶⁸And A is -NR⁶⁷R⁶⁸, Or -S (O)_f-R⁶⁹Table
Then R⁶⁷ And R⁶⁸ May be the same or different, and hydrogen
Atom, or C₁-C_Four Represents an alkyl group, R⁶⁹ Is C₁-C_Four 
Alkyl group, or C₁-C_Four Represents a haloalkyl group, R⁷⁰
 Is a hydrogen atom, hydroxyl group, halogen atom, at least one
C on₁-C_Four C optionally substituted with an alkoxy group₁-C
_Four Alkyl group, at least one oxygen atom is inserted
May be C_Three-C₆1 or 2 cycloalkyl groups
C optionally substituted with a methyl group_Three-C₆Cycloalkyl
Group, furyl group, thienyl group, or -C (= O) R⁷¹Table
Then R⁷¹ And R⁷² May be the same or different and C₁-
C_Four Alkyl group, or C₁-C _Four Represents an alkoxy group, R
⁷³ And R⁷⁴ May be the same or different and C₁-C_Four A
Represents a alkyl group or a phenyl group;⁷⁵ Is at least
One or more oxygen atoms may be inserted C_Three-C₆Shiku
Substituted with 1 or 2 methyl groups,
Good C_Three-C₆Cycloalkyl group, furyl group, thienyl
Group, or -C (= O) R⁷¹Represents a group, R⁷⁶ Is C₁-C_Four Archi
A, b, and c are each independently 1, 2,
Or 3; d represents 0 or 1; e represents 2 or 3
Represents, f represents 1 or 2, X^Two Is an oxygen atom or sulfur
Represents an atom.

Other N-substituted pyrazoles include
Nipiraclofen represented by Structural Formula 38 described in Chemical Formula 12 {"The Pesticide Manual", 9th ed., CR Worthing
Ed., British Crop Protection Council, Surrey (199
1), p. 621}, and 3-substituted-2-aryl-4,5,6,
7-tetrahydroindazole {Lyga et al., PesticideS
ci. 42: p29 (1994)}.

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When the PPO-inhibiting herbicide is applied to a plant, the plant cell-endogenous substance involved in the expression of the herbicidal activity of the herbicide in the plant cells may be, for example, the active ingredient of the PPO-inhibiting herbicide. A substrate of the target enzyme or a precursor or metabolite of the substrate, which causes cell dysfunction when accumulated in a plant cell, or a substance produced in a plant cell by such a substance. Substances that cause cell dysfunction are listed. More specifically, P
When a plant is treated with a PO-inhibiting herbicidal compound, protoporphyrinogen, a substrate of PPO, is contained in the cells of the plant.
It is known that IX accumulates and is metabolized in the cell to produce protoporphyrin IX, and furthermore, active oxygen is generated in the cell in the presence of protoporphyrin IX and light, which results in impaired cell function. Tori (Junno Miyamoto, 1993)
Year, New Science of Pesticides Chapter 3, Section 3.3, p106, Hirokawa Shoten
Tokyo), protoporphyrinogen IX in this system,
Protoporphyrin IX and active oxygen can be mentioned.

On the other hand, glyphosate is a general name for N- (phosphonomethyl) glycine. Glyphosate is generally used as an active ingredient of a herbicide in the form of various salts such as ammonium salt, sodium salt, isopropylamine salt, trimesium salt and potassium salt. Such glyphosate and a salt thereof (hereinafter collectively referred to as a glyphosate compound) have an activity of inhibiting amino acid biosynthesis, and specifically, 5-enolpyruvylshikimic acid.
3-phosphate synthase (5-enolpyruvylshikimate-3-phospha
te synthase. Hereinafter, it is referred to as EPSPS. ). In the present invention, glyphosate resistance means resistance to a glyphosate compound as described above.

The plant 1 of the present invention has one or more enzyme activities selected from the following (1) and (2), and is used in an amount of a herbicide that inhibits the natural EPSPS activity of the plant by the enzyme activity. Shows resistance to. (1) EPSPS activity substantially different from the natural EPSPS activity of the plant. (2) Plant natural glyphosate oxidoreductase
(glyphosate oxidoreductase; hereinafter referred to as GOX) GOX activity substantially different from the activity. In the present invention, the “natural EPSPS activity of a plant” refers to the EPSPS activity naturally possessed by a plant that has not been subjected to artificial manipulation, directly or indirectly. The “GOX activity” refers to an activity of converting glyphosate to aminomethyl phosphonate and glyoxylate. And
"Natural GOX activity of a plant" refers to the GOX activity of a natural plant that has not been subjected to direct or indirect artificial manipulation. The plant 1 of the present invention has one or more enzyme activities selected from the above (1) and (2), ie, the activity of (1) or the activity of (2), or the activity of (1) and (2). By having, it is resistant to herbicides in amounts that inhibit the plant's natural EPSPS activity. Here, "plant natural EPSP
Shows tolerance to herbicides in amounts that inhibit S activity. '' Means that the inhibition of natural EPSPS activity affects the growth of normal plants, and the inhibition of growth received by an amount of herbicide that can function as a herbicide is smaller than that of a naturally occurring plant of the same species. This means, for example, that the growth is not substantially affected by the above-mentioned amount of herbicide. The term “substantially different” in the above (1) and (2) means that they differ to such an extent that they can make some contribution to the resistance of the plant to the herbicide in an amount that inhibits the natural EPSPS activity. Examples of a method for imparting one or more enzyme activities selected from the above (1) and (2) to a plant include, for example, a method of highly expressing a gene encoding a natural wild-type EPSPS in plant cells, Method for expressing a gene encoding a mutant EPSPS having higher glyphosate resistance than type EPSPS in plant cells, plant EPSPS
Bacterial EPSP with higher glyphosate resistance than
A method for expressing a gene encoding S in plant cells,
A method for expressing a gene encoding EPSPS linked to a chloroplast translocation signal sequence in a plant cell, a method for expressing a gene encoding GOX linked to a chloroplast translocation signal sequence in a plant cell, Alternatively, there may be mentioned a method of expressing a gene encoding EPSPS and a gene encoding GOX in plant cells. More specifically, for example, the cauliflower mosaic virus 35S promoter (hereinafter referred to as CaMV35S), which has a stronger promoter activity in plants than the EPSPS native promoter.
(Mitchell diploid petunia) EPSP downstream
A method of introducing a chimeric gene linked to a gene encoding S into a cultivated plant [EP218571], or a method of introducing a gene encoding EPSPS with increased glyphosate resistance by amino acid substitution into a cultivated plant [Hinchee, MAW
et al., BIO / TECHNOLOGY, 6: p915 (1988), EP389066,
EP409815, WO9206201, US5312910 etc.] and EPSPS of plants
Agrobacterium (A
grobacterium tumefaciens sp.strain CP4) EPSPS (cla
ss II) is a petunia (Petunia hyb
rida) a chimeric gene linked downstream of the base sequence encoding the chloroplast translocation signal sequence of EPSPS, CaMV35
Method for introducing into cultivated plants by connecting downstream of S [WO920444
9, US5633435, etc.] and a chloroplast translocation signal sequence of the small subunit of sunflower ribulose-1,5-bisphosphate carboxidase (hereinafter referred to as rubisco) downstream of the double CaMV35S sequence. Nucleotide sequence, a nucleotide sequence encoding the N-terminal 22 amino acids of the corn rubisco small subunit, a nucleotide sequence encoding the chloroplast translocation signal sequence of the corn rubisco small subunit, and Salmonella typhimurium
A method of introducing a chimeric gene comprising a gene linked to EPSPS into a cultivated plant [EP508909], and encoding a glyphosate-degrading enzyme GOX in a nucleotide sequence encoding a chloroplast translocation signal sequence of Rubisco in Arabidopsis thaliana A fungus (Aspergillus nid
ulans) derived from the promoter of alcohol dehydrogenase A and introduced into cultivated plants [WO97062
69] and Agrobacterium tumefacien
The gene encoding EPSPS (class II) of s sp.strain CP4) is linked to the downstream of the base sequence encoding the chloroplast translocation signal sequence of Petunia (Petunia hybrida) EPSPS, a tobacco mosaic CaMV35S with enhanced promoter activity by viral omega sequences
To the cultivated plant together with the GOX [WO9706269].

The gene which can be used in the above-mentioned method for imparting one or more enzyme activities selected from the above (1) and (2) to a plant is a "protein having substantially EPSPS activity". Alternatively, it is a gene encoding “a protein having substantially GOX activity”. Examples of the gene encoding the “protein having substantially EPSPS activity” include, for example, Petunia Petunia x hybrida (Genebank
accession M37029), Tomato (strain VF36) p
istil (Genebank accession M21071), Arabidopsis
Arabidopsis thaliana (Genebank accession X06613),
Soybean, corn Zea mays. (Genebank accession
X63374), Escherichia coli (Genebank accession X00557), and EPSPS genes derived from Agrobacterium and the like. Such a gene having a known base sequence is prepared by using a genomic DNA or cDNA of an organism having the target gene as a template, and a base sequence corresponding to the vicinity of the amino terminal and a base corresponding to the vicinity of the carboxy terminal of the protein encoded by the gene. It can be amplified and isolated by performing PCR using primers prepared based on the sequence. Also,
A gene encoding EPSPS can be obtained from an organism other than the above. First, mRNA is prepared from a target organism, cDNA is synthesized using the mRNA as a template by using a reverse transcriptase, and the cDNA is incorporated into a phage vector such as ZAPII or a plasmid vector such as pUC to prepare a cDNA library. . Using this cDNA library as a template, a primer was designed and synthesized based on a base sequence well-conserved among genes whose base sequence was known as described above.
By performing R, a DNA fragment containing at least a part of the EPSPS gene can be amplified. Using this DNA fragment as a probe, a cDNA library is screened to select a positive clone. By determining the nucleotide sequence of the DNA of the selected clone, it can be confirmed that the gene is the target EPSPS gene. In order to obtain a gene encoding EPSPS with increased glyphosate resistance due to mutation, for example, a base substitution mutation is introduced into the gene encoding EPSPS, and the resulting modified gene is reduced to a MOPS minimum containing no aromatic amino acid. It is introduced into a mutant Escherichia coli deficient in the EPSPS gene (aroA locus) whose growth is inhibited in a nutrient medium. The Escherichia coli, into which the modified gene has been introduced, is cultured in the presence of glyphosate at a concentration sufficient to inhibit the growth of the Escherichia coli, into which the EPSPS gene having no mutation has been introduced, and a clone capable of growing is selected. It is possible to obtain a gene encoding EPSPS with increased resistance to acetate. Further, the modified gene thus obtained is expressed in a host cell such as Escherichia coli to prepare a protein encoded by the gene, and the resulting protein has an ability to synthesize 5-enolpyruvylshikimate-3-phosphate. For example, by measuring according to the method described in EP409815 etc., it is also possible to select a gene of a protein having a high glyphosate resistance and a high ability to synthesize 5-enolpyruvylshikimate-3-phosphate. .

"Protein having substantially GOX activity"
Examples of the gene encoding Pseudomonas s
Examples include genes derived from p.strains LBAA, Pseudomonas sp.strains LBr, Agrobacterium Agrobacterium sp.strain T10, and the like. Such a gene having a known base sequence is prepared by using a genomic DNA or cDNA of an organism having the target gene as a template, and a base sequence corresponding to the vicinity of the amino terminal and a base corresponding to the vicinity of the carboxy terminal of the protein encoded by the gene. It can be amplified and isolated by performing PCR using primers prepared based on the sequence. Further, a gene encoding GOX can be obtained from an organism other than the above. First, mRNA is prepared from a target organism, cDNA is synthesized using the mRNA as a template by using a reverse transcriptase, and the cDNA is incorporated into a phage vector such as ZAPII or a plasmid vector such as pUC to prepare a cDNA library. . By using this cDNA library as a template and performing PCR using primers designed and synthesized based on the base sequence well-conserved among genes whose base sequence is known as described above, the gene encoding GOX A DNA fragment containing at least a portion can be amplified. Using this DNA fragment as a probe, a cDNA library is screened to select a positive clone. By determining the nucleotide sequence of the DNA of the selected clone, it can be confirmed that the gene is the target GOX gene. To obtain a GOX-encoding gene or to confirm the glyphosate resolution of GOX encoded by the gene, for example, Pseudomonas sp.strai
ns LBAA cosmid vector cDNA library or GOX
Is introduced into a mutant strain Escherichia coli (E. coli SR2000 Mpu +) capable of growing on a MOPS minimal nutrient medium using a phosphate compound as a sole nitrogen source. The gene-introduced Escherichia coli is cultured under conditions in which glyphosate is used as the sole nitrogen source at a concentration that inhibits the growth of the Escherichia coli not transfected with the gene encoding GOX, and a viable clone is obtained. By selecting, a gene encoding GOX can be obtained. Further, in Escherichia coli capable of growing in this way, the protein encoded by the gene introduced into the Escherichia coli decomposes glyphosate into aminomethylphosphate, and the phosphate compound is used as the only nitrogen source by the Escherichia coli. It is thought that it is growing. Actually, the Escherichia coli was cultured in a medium to which glyphosate labeled with radioactive 3- ¹⁴ C was added.
It can be confirmed that glyphosate labeled with ¹⁴ C is taken up and degraded by Escherichia coli.

The plant 1 of the present invention also comprises the following (1-3), (1-
A gene encoding a protein having the properties of 4) and (1-5) is introduced, and the gene is expressed. (1-3) Specific binding to the substance. (1-4) The protein has substantially no ability to alter a substance to which it specifically binds. (1-5) It does not substantially contain a framework region of an immunoglobulin variable region. In the present invention, the term “specifically binds” a substance to a substance refers to, for example, an enzyme-substrate or an enzyme-chemical bond between an enzyme and an inhibitor or an activity regulator, a receptor-ligand. Means that a protein binds to a substance based on affinity and specificity as shown in the receptor chemical binding and the like. "Substantially does not have the property of altering the substance to which the protein specifically binds" means that the enzymatic reactivity with the substance is inactive. Excluding specific binding to a substance}, for example, the ability to catalyze an enzymatic chemical reaction that converts the substance into a substance represented by a chemical structural formula different from the chemical structural formula representing the substance. There are cases where they do not have them substantially. A protein having "substantially no metamorphic properties" can express, for example, a gene encoding the protein in a microorganism in which the gene encoding the protein having the metamorphic property has been deleted and cannot grow under normal conditions. May be selected as an indicator that the growth of the microorganism is not complemented when introduced in a proper state. In the present invention, "substantially not containing the framework region of the immunoglobulin variable region" means that a three-dimensional structure specific to the immunoglobulin variable region is not formed.
Here, "the framework region of the immunoglobulin variable region" refers to an H chain and an L chain constituting an immunoglobulin molecule.
Of the variable regions of the chain, the hypervariable region (hypervariable regi
The remaining region excluding on) is a region in which the conservation of the amino acid sequence is relatively high and serves to maintain the highly conserved three-dimensional structure of the variable region. By taking the three-dimensional structure of the variable region, hypervariable regions scattered at three positions of each of the H chain and the L chain are united at one position on the three-dimensional structure to form an antigen binding site [Alberts, B ., et al. ed. (19
83) Molecular Biology of the Cell p979, Garland Pub
lishing, Inc. New York]. A protein “substantially free of the framework regions of the immunoglobulin variable region” can be selected, for example, based on the amino acid sequence of the protein. Specifically, such proteins include, for example, immunoglobulin variable regions. Proteins which do not contain an amino acid sequence consisting of about 30 amino acids or more, which show about 60% or more homology with the known amino acid sequence of the framework region of the region. Whether or not the above-described framework region is contained is determined, for example, by using the gene encoding the protein as a template and
A primer capable of amplifying a DNA having a nucleotide sequence encoding a variable region derived from an H chain or an L chain, specifically, for example, a primer VH1BACK described in Clackson, T. et al., Nature 352; p624 (1991) And VH1FOR-2 or V
K2BACK and VK4FOR, or commercially available kits for cloning recombinant antibody genes, such as Recombinant Phage
Primer Heavy primers mix or Light primer mix contained in Antibody System (Pharmacia Biotech)
It can also be confirmed by performing PCR using, for example, and analyzing the presence or absence of amplification of DNA of a specific length.

The above (1-3), (1-4) and (1-5) produced by expressing the gene in the plant 1 of the present invention.
The protein having the above-mentioned properties may be a naturally-occurring protein, a protein in which amino acid substitutions, additions, deletions, etc. are introduced into a natural protein. It may be a protein that is selected from proteins that have been specifically produced by using the binding to the substance as an index. In the present invention, the term “protein” refers to a substance in which two or more amino acids are bound by peptide bonds, for example, 4 to 1
Also included are substances formed by binding about 100 amino acids by peptide bonds. The above properties (1-3), (1-4) and (1
Specific examples of the protein having -5) include the following proteins. Protoporphyrin
As a protein that specifically binds to IX, for example, magnesium keratase (magnesium protoporphyrin IX che
latase) protoporphyrin IX binding subunit protein, or a modified protein of the protein, which specifically binds to protoporphyrin IX, and more specifically, an organelle translocation signal from the subunit protein. Examples include proteins from which sequences have been removed. Also, ferrochelatase (p
rotoheme ferrolyase; EC 4.9.9.1) modified proteins that do not have the ability to alter protoporphyrin IX and specifically bind to protoporphyrin IX.More specifically, iron ions in ferrokeratase And the like, in which the region presumed to be the binding site to is modified and has no ability to alter protoporphyrin IX. Furthermore, a substrate binding subunit protein of cobalt chelatase (cobaltochelatase) or a modified protein of the protein, which specifically binds to protoporphyrin IX can also be mentioned. The protein that specifically binds to protoporphyrinogen IX is a modified protein of PPO, such as protoporphyrinogen IX.
A protein that does not have the ability to oxidize the protein and specifically binds to protoporphyrinogen IX, and more specifically, a region presumed to be a FAD binding site in PPO (a region having the amino acid sequence GXGXXG, Here, X represents an arbitrary amino acid, for example, a protein comprising the amino acids 63 to 68 from the amino terminus of Arabidopsis thaliana chloroplast-localized PPO and lacking the amino acid sequence GGGISG). be able to. In addition, proteins that bind to protoporphyrinogen IX include coproporphyrinogen III oxidase (coprop
orphyrinogen III oxidase (EC 1.3.3.3), which does not have the ability to oxidize protoporphyrinogen IX and coproporphyrinogen III and specifically binds to protoporphyrinogen IX. it can. By binding of the protein as described above to the substance in the cell, cell dysfunction caused by the substance is prevented, and herbicide resistance can be expressed. A gene encoding such a protein can be obtained, for example, as follows.

The gene encoding the protoporphyrin IX binding subunit protein of magnesium keratase includes a photosynthetic bacterium Rhodobacter capsulatus (Geneba
nk accession M74001), Arabidopsis (Genebank acce
ssion Z68495), barley (Genebank accession U9621)
6), snapdragon (Genebank accession X73144), Synec
The nucleotide sequences of genes derived from hocystis PCC6803 (Genebank accession U29131) and the like are known. Such a gene whose base sequence is known is the genome of the organism having the gene of interest.
Using DNA or cDNA as a template, amplification is performed by performing PCR using primers prepared based on the nucleotide sequence near the amino terminus and the nucleotide sequence near the carboxy terminus of the protein encoded by the gene. This can be isolated. In addition, from the photosynthetic organisms other than the above, protoporphyrin of magnesium keratase
Genes encoding IX binding subunit proteins can also be obtained. First, from the target photosynthetic organism
Prepare mRNA, using reverse transcriptase with this mRNA as template
A cDNA is synthesized and inserted into a phage vector such as ZAPII or a plasmid vector such as pUC to prepare a cDNA library. By using this cDNA library as a template and performing PCR using primers designed and synthesized based on a base sequence well-conserved among genes whose base sequence is known as described above, protoporphyrin of magnesium keratase A DNA fragment containing at least a part of the IX binding subunit protein gene can be amplified. Using this DNA fragment as a probe, a cDNA library is screened to select a positive clone. By determining the nucleotide sequence of the DNA of the selected clone, it can be confirmed that the gene is the protoporphyrin IX-binding subunit protein gene of the target magnesium keratase. In order to obtain a gene encoding a protein that specifically binds to protoporphyrin IX, which is a modified protein of the protoporphyrin IX binding subunit protein of magnesium keratase, for example, substituting a base for the gene of the subunit protein , Deletion, addition, etc., to introduce the gene into Gi
bson, LCD etal., Proc. Acad. Sci. USA, 92; p194
According to the method described in 1 (1995), a transformed strain is obtained by introducing the strain into Escherichia coli BL21 (DE3), and the transformed strain is cultured under conditions in which the introduced gene is highly expressed. Cultured cells turn red,
Fluorescence absorption indicating the accumulation of protoporphyrin IX (excitation wavelength
By selecting a strain in which 405 nm and a fluorescence wavelength of 630 nm are observed, a gene encoding a protein that specifically binds to protoporphyrin IX, which is a modified protein of the subunit protein, can be obtained.

As a gene encoding ferrochelatase, Escherichia coli (Genebank acc.
ession D90259), Bacillus subtilis (Genebank
accession M97208), Bradyrhizobium japonicum (Geneb
ank accession M92427), yeast Saccharomyces cerevisia
e (Genebank accession J05395), mouse (Genebankacce
ssion J05697), human (Genebank accession D00726), barley (Genebank accession D26105), cucumber (Geneba
nk accession D26106) and the like are known. Such a gene having a known base sequence is prepared by using a genomic DNA or cDNA of an organism having the gene of interest as a template, and a base sequence corresponding to the vicinity of the amino terminal and a base corresponding to the vicinity of the carboxy terminal of the protein encoded by the gene. PC using primers prepared based on the sequence
By performing R, it can be amplified and isolated. To obtain a ferrochelatase gene whose base sequence is not known, first, mRNA is prepared from the target organism, cDNA is synthesized using the mRNA as a template by using a reverse transcriptase, and this is used as a ZAPII or the like. A cDNA library is prepared by incorporating the plasmid into a phage vector or a plasmid vector such as pUC. This cDNA library was transferred to Miyamoto, K., et a
l. Plant Physiol., 105; p769 (1994), by introducing into a ferrochelatase deletion mutant VS200 of Escherichia coli and performing a complementation test, a clone having a ferrochelatase gene derived from the target organism was selected from the cDNA library. can do. Further, using the cDNA library as a template, a DNA fragment is amplified by performing PCR using primers prepared based on a nucleotide sequence well-conserved between genes having a known nucleotide sequence as described above, DNA
The cDNA library may be screened using the fragment as a probe to select a positive clone. By determining the nucleotide sequence of the DNA of the clone thus selected, it can be confirmed that the clone is the target ferrochelatase gene. A modified protein of ferrochelatase that does not have the ability to alter protoporphyrin IX and specifically binds to protoporphyrin IX, e.g., a region that is presumed to be a binding site for iron ions in ferrochelatase has been modified to produce protoporphyrin IX In order to obtain a gene encoding a protein that does not have the ability to alter the region, a primer for introducing a mutation into the region is prepared based on the base sequence encoding the amino acid sequence near the region, and this mutation-introducing primer is prepared. A gene encoding a mutant in the above region can be prepared by performing PCR using a commercially available site-specific mutagenesis kit (Mutan-Super Express Km, manufactured by Takara Shuzo). Specifically, the wild-type ferrochelatase gene is inserted into the cloning site of the plasmid vector pKF19k, and the amber mutation on the kanamycin resistance gene of the above-described mutagenesis primer and pKF19k is restored using the resulting plasmid DNA as a template. PCR using the selection primers for PCR. By introducing the gene amplified by the PCR into Escherichia coli MV1184 strain (suppressor-free) and selecting the transfected strain with kanamycin resistance, a ferrokeratase gene having a modified base sequence corresponding to the amino acid constituting the target region was obtained. Can be isolated. Plasmid DNA of the Escherichia coli
By analyzing the base sequence of the above, it can be confirmed that the isolated gene is a gene encoding the desired modified protein.

The PPO gene has been used for Escherichia coli Esheri.
chia coli (Genebank accession X68660), Bacillus subtilis Bacil
lus subtilis (Genebank accession M97208), Haemophi
lusinflunzae (Genebank accession L42023), mouse (G
enebank accession D45185), human (Genebank accession
D38537), Arabidopsis (Genebank accession D8313)
9), base sequences of genes derived from tobacco (Genebank accession Y13465, Y13466) and the like are known. Such a gene whose base sequence is known is the genome of the organism having the gene of interest.
Using DNA or cDNA as a template, amplification is performed by performing PCR using primers prepared based on the nucleotide sequence near the amino terminus and the nucleotide sequence near the carboxy terminus of the protein encoded by the gene. Can be isolated. To obtain a PPO gene whose base sequence is not known, first, a cDNA library is prepared from an organism having a target gene by the above-described method. This cDNA library was purchased from Narita, S., et.
al., Gene, 182; PP of Escherichia coli described in p169 (1996) and the like.
By introducing the gene into the O-deletion mutant VSR800 and performing a complementation test, a clone having the PPO gene derived from the target organism can be selected from the cDNA library. Also,
Using the cDNA library as a template, amplify a DNA fragment by performing PCR using primers prepared based on a base sequence well-conserved among genes whose base sequence is known as described above, Using the above cD
The NA library may be screened to select positive clones. By determining the nucleotide sequence of the selected clone, it can be confirmed that it is the target PPO gene. To obtain a gene for a PPO modified protein that does not have the ability to oxidize protoporphyrinogen IX and specifically binds to protoporphyrinogen IX, for example, PPO
Mutations such as substitution, deletion and addition of bases are introduced into the gene, and the modified gene is introduced into the above-mentioned Escherichia coli whose growth is inhibited in a light-dependent manner by treatment with a PPO-inhibiting herbicide. Selecting a gene encoding a protein having binding ability to protoporphyrinogen IX by culturing the Escherichia coli in the presence of hemin, aminolevulinic acid and a PPO-inhibiting herbicide and selecting a clone capable of growing in the light; Can be. The modified gene thus selected is expressed in a host cell such as Escherichia coli to prepare a protein encoded by the gene, and the oxidizing ability of the obtained protein to protoporphyrinogen IX is determined, for example, by Jacob, NJand Ja.
By measuring according to the method described in cobs, JM (1982) Enzyme, 28, 206-219, etc., a gene of a protein having no oxidizing ability to protoporphyrinogen IX can be selected. More specifically, the above modified gene was inserted into an expression vector for Escherichia coli, and Yamamoto, F.et
al., Japanese J. Genet., 63; p237 (1988) and the like, a vector introduced into a PPO gene (hemG locus) -deficient mutant strain Escherichia coli such as the Escherichia coli BT3 strain and introduced into the E. coli. A transformant is obtained by culturing the Escherichia coli in a medium containing hemin and aminolevulinic acid in addition to the cell growth inhibitor corresponding to the above selectable marker to obtain a transformant, and preparing a protein encoding the modified gene from the transformant Is also good. Further, by culturing the transformant in a medium substantially free of hemin and aminolevulinic acid, and confirming the strain that does not grow, obtaining a gene that does not complement the PPO gene deficiency of the host cell from the transformant Such a method may be used to select a gene encoding a protein having no ability to oxidize protoporphyrinogen IX. Also, to obtain a gene encoding a protein in which a region presumed to be a FAD binding site in PPO (a region having the amino acid sequence GXGXXG, where X represents any amino acid) is deleted. ,
First, a primer for introducing a deletion mutation in the region is prepared based on the nucleotide sequence encoding the amino acid sequence in the vicinity of the region. Using this mutagenesis primer, a commercially available site-directed mutagenesis kit (Mutan-Super
Express Km, manufactured by Takara Shuzo), and a gene encoding a deletion mutant in the region can be prepared.

The gene encoding coproporphyrinogen III oxidase has so far been Escherichia coli Eshe
richia coli (Genebank accession X75413), Salmonell
a typhimurium (Genebank accession L19503), yeast Sac
charomyces cerevisiae (Genebank accession J0387
3), mouse (Genebank accession D16333), human (Geneba
nk accession D16611), soybean (Genebank accession X7
1083), barley (Genebank accession X82830), tobacco
(Genebank accession X82831) and the like are known. Such a gene whose base sequence is known is a genomic DNA or cDNA of an organism having the gene of interest.
Amplified and isolated by performing PCR using primers prepared based on the nucleotide sequence near the amino terminal and the nucleotide sequence near the carboxy terminal of the protein encoded by the gene as a template can do. To obtain a coproporphyrinogen III oxidase gene whose base sequence is not known,
First, mRNA is prepared from the target organism, cDNA is synthesized using the mRNA as a template using reverse transcriptase, and
pRS described in i, RS et al. Genetics, 122; p19 (1989)
A cDNA library is prepared by incorporating it into a plasmid vector such as 313. This cDNA library was transferred from Troup, B.
et al. J. Bacteriol., 176; p673 (1994), introduced into the yeast coproporphyrinogen III oxidase deletion mutant HEM13, performed a complementation test, and selected a viable clone to obtain the cDNA. From the library, a clone having the coproporphyrinogen III oxidase gene derived from the target organism can be selected.
Further, using the cDNA library as a template, a DNA fragment is amplified by performing PCR using primers prepared based on a nucleotide sequence well-conserved between genes having a known nucleotide sequence as described above, The cDNA library may be screened using a DNA fragment as a probe to select a positive clone. By determining the nucleotide sequence of the DNA of the clone selected in this way, it can be confirmed that the gene is the target coproporphyrinogen III oxidase gene. To obtain a gene for a protein that is a modified protein of coproporphyrinogen III oxidase and does not have the ability to oxidize protoporphyrinogen IX and coproporphyrinogen III and specifically binds to protoporphyrinogen IX, For example, a mutation such as substitution, deletion, or addition of a base is introduced into the coproporphyrinogen III oxidase gene, and the modified gene is introduced into the above-described Escherichia coli whose growth is inhibited in a light-dependent manner by treatment with a PPO-inhibited herbicide. I do. The Escherichia coli is treated with hemin, aminolevulinic acid and PPO.
By selecting clones that can grow in the light by culturing in the presence of inhibitory herbicides, protoporphyrinogen
A gene encoding a protein having IX binding ability can be selected. The modified gene thus selected is expressed, for example, in a host cell such as Escherichia coli to prepare a protein encoded by the gene, and the oxidizing ability of the obtained protein to protoporphyrinogen IX is determined, for example, by Jacob. , NJand Jacobs, JM (1982) Enzyme, 28,
By measuring according to the method described in 206-219 etc.,
A gene of a protein having no oxidizing ability to protoporphyrinogen IX can be selected. further,
For proteins prepared from E. coli as described above, the ability to convert coproporphyrinogen III to protoporphyrinogen IX (coproporphyrinogen III oxidase activity), for example, Yoshinaga, T., Sa
no, S., J. Biol. Chem., 255; p4722 (1980), etc., to select genes for proteins that do not have the ability to oxidize coproporphyrinogen III. it can.

[0024] Genes of proteins that can specifically bind to this substance are described, for example, in Sugimoto, N., Nakano, S., Chem.
As described in tt., p939 (1997), etc., a peptide showing a binding property to the substance was selected from a peptide library synthesized using a combinatorial chemistry method,
It can also be obtained by analyzing the amino acid sequence of the selected peptide with a peptide sequencer, designing a gene containing a base sequence encoding the amino acid sequence, and synthesizing it with a DNA synthesizer or the like. Further, a phage clone displaying a protein having a binding property to the present substance can be selected from a phage library and obtained by using a phage display method. Specifically, for example, by inserting a nucleotide sequence encoding a protein having a random amino acid sequence upstream of the region encoding the coat protein pIII of the M13 phage gene, a random amino acid sequence on the surface of the M13 phage particle To construct a phage library displaying a protein having On the other hand, a biotin-labeled substance is bound to a plate coated with avidin or streptavidin to prepare a support coated with the substance. By screening the phage library described above on a plate coated with this substance, it is possible to isolate a phage displaying a target protein that binds to this substance, and to isolate the target protein from the isolated phage. Gene can be obtained.

The plant 1 of the present invention can be prepared, for example, using the above-mentioned “EP
A step of introducing into a plant cell and expressing a gene encoding one or more proteins selected from a protein having substantially SPS activity and a protein having substantially GOX activity, ), A step of introducing a gene encoding a protein having the properties of (1-4) and (1-5) into a plant cell and expressing the gene. Specifically, for example, a gene encoding "a protein having substantially EPSPS activity" and / or a gene encoding "a protein having substantially GOX activity", and (1-3), (1-4) And a gene encoding a protein having the properties of (1-5) may be simultaneously introduced into a plant cell, and the plant 1 of the present invention expressing these genes may be produced from the obtained cell. (1-3), (1-4) and (1-4) to a plant cell into which a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity” has been introduced. (1-
A gene encoding a protein having the property of 5) may be introduced, and the plant 1 of the present invention expressing these genes may be produced from the obtained cells. (1-3), into a plant cell into which a gene encoding a protein having the properties of (1-4) and (1-5) has been introduced, a gene encoding "a protein having substantially EPSPS activity" and / or A gene encoding a "protein having substantially GOX activity" may be introduced, and the plant 1 of the present invention expressing these genes may be produced from the obtained cells. Further, a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity”, and (1-3), (1-4) and (1 The present invention, which expresses these genes by introducing genes encoding proteins having the properties of -5) into plant cells to produce plants expressing the respective genes, and crossing the obtained plants. Plant 1 may be produced. further,
It has one or more enzyme activities selected from the above (1) and (2), and inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzyme activity. (1-3), to plant cells showing resistance to the amount of herbicide,
A gene encoding a protein having the properties of (1-4) and (1-5) may be introduced, and the plant 1 of the present invention expressing the gene may be produced from the obtained cells. 3), (1-4)
And a gene encoding a protein having the properties of (1-5) is introduced into a plant cell that expresses the gene, by adding `` EP
A plant encoding the protein 1 of the present invention, which expresses these genes from the obtained cells, is introduced with a gene encoding a protein substantially having SPS activity and / or a gene encoding a protein substantially having GOX activity. May be created. In each case, a gene encoding a “protein having substantial EPSPS activity”, a gene encoding a “protein having substantial GOX activity”, (1-3),
Regarding any gene among the genes encoding the proteins having the properties of (1-4) and (1-5), two or more genes may be introduced into a plant cell.

The plant 2 of the present invention has at least one enzyme activity selected from the above (1) and (2) similarly to the plant 1 of the present invention, and the enzyme activity reduces the natural EPSPS activity of the plant. A plant that is resistant to an inhibitory amount of a herbicide,
In addition, it is a plant into which a gene encoding a protein having the following properties (2-3), (2-4) and (2-5) has been introduced and expresses the gene. (2-3) Specific binding to protoporphyrin IX. (2-4) It does not have the ability to transform protoporphyrinogen IX. (2-5) It does not substantially contain a framework region of an immunoglobulin variable region. Here, "specifically binds to protoporphyrin IX"
As described above, for example, as indicated in an enzymatic chemical bond between an enzyme and a substrate or an enzyme and an inhibitor or an activity modulator, a receptor chemical bond between a receptor and a ligand, and the like. Means that the protein binds to protoporphyrin IX based on high affinity and specificity. Also, `` has no metamorphic property to protoporphyrinogen IX '' means that the enzymatic chemical reactivity with protoporphyrinogen IX is inactive, for example, protoporphyrinogen IX, There may be mentioned a case where the compound does not substantially have the ability to catalyze an enzymatic chemical reaction for converting into a substance represented by a chemical structural formula different from the chemical structural formula. "Substantially free of the framework regions of the immunoglobulin variable regions", as described above,
It does not form a conformation unique to the variable region of an immunoglobulin. In the plant 2 of the present invention, the protein having the above-mentioned properties (2-3), (2-4) and (2-5) produced by expressing the gene is a naturally occurring protein. It may be a protein in which amino acid substitutions, additions, deletions, etc. have been introduced into a natural protein, and may be used to determine the binding to the substance from artificially created proteins having random amino acid sequences. It may be a protein selected as an indicator. Specific examples of such a protein include, for example, magnesium keratase and a protein which is a modified protein of magnesium keratase and which does not specifically bind to protoporphyrin IX and does not have the ability to denature protoporphyrinogen IX. I can give it. In addition, ferrochelatase and modified proteins of ferrochelatase, which specifically bind to protoporphyrin IX and do not have the ability to denature protoporphyrinogen IX, can also be mentioned.

The gene encoding the above protein is
For example, it can be obtained as follows. Magnesium keratase has the ability to coordinate magnesium ions to protoporphyrin IX to generate magnesium porphyrin IX, and is composed of protoporphyrin IX binding subunit protein, I subunit protein and D subunit protein. To express magnesium keratase in plants, genes encoding these three subunit proteins are obtained. The gene encoding the protoporphyrin IX binding subunit protein of magnesium keratase can be obtained by the above-described method. As a gene encoding the I subunit protein of magnesium keratase, a photosynthetic bacterium Rhodobacter sphaerides (Genebank accession A
F017642), photosynthetic bacterium Rhodobacter capsulatus (Geneba
nk accession Z11165), Arabidopsis (Genebank acce
ssion D49426), barley (Genebank accession U2654)
5), soybean (Genebank accession D45857), tobacco (Gene
bank accession AF14053), Synechocystis PCC6803
(Genebank accession U35144) and the like are known. Such a known gene (having a known base sequence)
Using a genomic DNA or cDNA of the organism having the gene of interest as a template, a primer prepared based on the nucleotide sequence near the amino terminus and the nucleotide sequence near the carboxy terminus of the protein encoded by that gene is used. Amplification and isolation can be performed by performing PCR. In addition, a gene encoding a magnesium keratase I subunit protein can be obtained from a photosynthetic organism other than those described above. First, mRNA is prepared from the desired photosynthetic organism, cDNA is synthesized using the mRNA as a template using reverse transcriptase, and this is incorporated into a phage vector such as ZAPII or a plasmid vector such as pUC to obtain cDN.
Create A library. Using this cDNA library as a template, PCR is performed using primers designed and synthesized based on the base sequence well-conserved between the above-mentioned known genes, and the I-subunit of magnesium keratase is obtained. DNA containing at least a part of the gene of the unit protein can be amplified. Using this DNA as a probe, a cDNA library is screened to select positive clones. By determining the nucleotide sequence of the DNA of the selected clone, it can be confirmed that the gene is the gene for the target I subunit protein of magnesium keratase. In addition, as a gene encoding the D subunit protein of magnesium keratase, a photosynthetic bacterium Rhodobacter sphaerides (Genebank
accession AJ001690), photosynthetic bacterium Rhodobacter capsul
atus (Genebank accession Z11165), peas (Geneban
k accession AF014399), tobacco (Genebank accession Y
10022), Synechocystis PCC6803 (Genebank accessio
n X96599) and the like. The gene encoding the magnesium keratase D subunit protein can be obtained by the same method as the method for obtaining the gene encoding the magnesium keratase I subunit protein. Detection of the activity of magnesium keratase consisting of these three subunit proteins is described, for example, in Gibson, LCD et al., Proc.Acad.
Sci. USA, 92; p1941 (1995) and the like. The gene encoding ferrochelatase can be obtained by the method described above. Detection of the activity of ferrochelatase can be performed, for example, by using Porra, RJ, Anal.
iochem., 68; p289 (1975) and the like.

The plant 2 of the present invention can be prepared, for example, using the above-mentioned “EP
A step of introducing a gene encoding one or more proteins selected from among a protein having substantially SPS activity and a protein having substantially GOX activity into a plant cell and expressing the gene, ), A step of introducing a gene encoding a protein having the properties of (2-4) and (2-5) into plant cells and expressing the gene. Specifically, for example, a gene encoding "a protein having substantially EPSPS activity" and / or a gene encoding "a protein having substantially GOX activity", and (2-3), (2-4) And a gene encoding a protein having the property of (2-5) may be simultaneously introduced into a plant cell, and the plant 2 of the present invention expressing these genes may be produced from the obtained cell. (2-3), (2-4) and (2-4) to a plant cell into which a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity” has been introduced. (2-
A gene encoding a protein having the property of 5) may be introduced, and the plant 2 of the present invention expressing these genes may be produced from the obtained cells. (2-3), into a plant cell into which a gene encoding a protein having the properties of (2-4) and (2-5) has been introduced, a gene encoding "a protein having substantially EPSPS activity" and / or A gene encoding a "protein having substantially GOX activity" may be introduced, and the plant 2 of the present invention expressing these genes may be produced from the obtained cells. Further, a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity”, and (2-3), (2-4) and (2 The present invention, which expresses these genes by introducing genes encoding proteins having the properties of -5) into plant cells to produce plants expressing the respective genes, and crossing the obtained plants. Plant 2 may be produced. further,
It has one or more enzyme activities selected from the above (1) and (2), and inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzyme activity. (2-3), to plant cells showing resistance to the amount of herbicide,
A gene encoding a protein having the properties of (2-4) and (2-5) may be introduced, and the plant 2 of the present invention expressing the gene may be produced from the obtained cells. 3), (2-4)
And a gene encoding a protein having the properties of (2-5) is introduced into a plant cell that expresses the gene, by adding "" EP
A plant encoding the protein 2 of the present invention which expresses these genes from the cells obtained by introducing a gene encoding a protein having substantially SPS activity and / or a gene encoding a protein substantially having GOX activity is introduced. May be created. In each case, a gene encoding a “protein having substantial EPSPS activity”, a gene encoding a “protein having substantial GOX activity”, (2-3),
Two or more genes may be introduced into plant cells for any of the genes encoding the proteins having the properties of (2-4) and (2-5).

The plant 3 of the present invention has one or more enzyme activities selected from the above (1) and (2) similarly to the plant 1 of the present invention, and the enzyme activity reduces the natural EPSPS activity of the plant. A plant that is resistant to an inhibitory amount of a herbicide,
Further, it is a plant into which a gene encoding a protein having the following properties (3-3), (3-4) and (3-5) has been introduced and expresses the gene. (3-3) specifically binds to protoporphyrinogen IX. (3-4) It has a strange ability to coproporphyrinogen III. (3-5) It does not substantially contain a framework region of an immunoglobulin variable region. "Specifically binds to protoporphyrinogen IX" means, as described above, for example, an enzyme and a substrate, or an enzymatic chemical bond between an enzyme and an inhibitor or an activity regulator, a receptor and a ligand. Means that the protein binds to protoporphyrinogen IX on the basis of affinity and specificity as shown in receptor chemical binding and the like. "Having an ability to transform coproporphyrinogen III" means that there is enzymatic chemical reactivity with coproporphyrinogen III, for example,
Coproporphyrinogen III may be substantially capable of catalyzing an enzymatic chemical reaction that converts coproporphyrinogen III into a substance represented by a chemical structural formula different from its chemical structural formula. Specifically, for example, coproporphyrinogen
The ability to convert III to protoporphyrinogen IX (coproporphyrinogen III oxidase activity) can be given, for example, Yoshinaga, T., San
o, S., J. Biol. Chem., 255; p4722 (1980) and the like. The expression "substantially free of the framework region of the immunoglobulin variable region" means that a three-dimensional structure specific to the immunoglobulin variable region is not formed, as described above. In the plant 3 of the present invention, the protein having the above-mentioned properties (3-3), (3-4) and (3-5) produced by expressing the gene is a naturally occurring protein. Well,
It may be a protein in which amino acid substitutions, additions, deletions, etc. are introduced into a natural protein, and selected from artificially created proteins having random amino acid sequences based on the binding to this substance as an index It may be a protein that has been prepared. As such a protein, specifically, coproporphyrinogen III oxidase, and a modified protein of coproporphyrinogen III oxidase, which oxidizes coproporphyrinogen III to produce protoporphyrinogen IX And proteins having the ability. The gene encoding coproporphyrinogen III oxidase is, for example,
It can be obtained by the method described above.

The plant 3 of the present invention can be prepared, for example, using the above-mentioned “EP
Introducing a gene encoding one or more proteins selected from among a protein having substantially SPS activity and a protein having substantially GOX activity into a plant cell and expressing the gene, ), (3-4) and (3-5) a step of introducing a gene encoding a protein having the properties described in (3-5) into plant cells and expressing the gene. Specifically, for example, a gene encoding "a protein having substantially EPSPS activity" and / or a gene encoding "a protein having substantially GOX activity", and (3-3), (3-4) And a gene encoding a protein having the property of (3-5) may be simultaneously introduced into a plant cell, and the plant 3 of the present invention expressing these genes may be produced from the obtained cell. (3-3), (3-4) and (3-4) to a plant cell into which a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity” has been introduced. (3-
A gene encoding a protein having the property of 5) may be introduced, and the plant 3 of the present invention expressing these genes may be produced from the obtained cells. (3-3), into a plant cell into which a gene encoding a protein having the properties of (3-4) and (3-5) has been introduced, a gene encoding "a protein having substantially EPSPS activity" and / or A gene encoding "a protein having substantially GOX activity" may be introduced, and the plant 3 of the present invention expressing these genes may be produced from the obtained cells. Also, a gene encoding a “protein having substantial EPSPS activity” and / or a gene encoding a “protein having substantial GOX activity”, and (3-3), (3-4) and (3-3) The present invention, which expresses these genes by introducing genes encoding proteins having the properties of -5) into plant cells to produce plants expressing the respective genes, and crossing the obtained plants. Plant 3 may be produced. further,
It has one or more enzyme activities selected from the above (1) and (2), and inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant by the enzyme activity. (3-3), to plant cells showing resistance to the amount of herbicide,
A gene encoding a protein having the properties of (3-4) and (3-5) may be introduced, and the plant 3 of the present invention expressing the gene may be produced from the obtained cells. 3), (3-4)
And a gene encoding a protein having the properties of (3-5) is introduced into plant cells expressing
A plant encoding the protein 3 having the SPS activity and / or a gene encoding the protein having a substantial GOX activity is introduced, and the plant 3 of the present invention expressing these genes is obtained from the obtained cells. May be created. In each case, a gene encoding a “protein having substantial EPSPS activity”, a gene encoding a “protein having substantial GOX activity”, (3-3),
Regarding any gene among the genes encoding the proteins having the properties of (3-4) and (3-5), two or more kinds of genes may be introduced into a plant cell.

In preparing the plants 1 to 3 of the present invention, a method for introducing a gene into plant cells includes the Agrobacterium infection method (Japanese Patent Publication No. 2-58917 and JP-A-60-7008).
0), electroporation method to protoplasts
(JP-A-60-251887 and JP-A-5-68575), or a particle gun method (Japanese Patent Laid-Open Publication No. 5-508316 and JP-A-63-25852).
Known means such as 5) can be used. The gene to be introduced into the cell is preferably incorporated into a vector having a selectable marker gene such as a gene capable of imparting cell growth inhibitor resistance to the cell. To express the gene of interest in plant cells, homologous recombination [Fraley, RT et al., P.
USA, 80; p4803 (1983)] to select cells expressing the gene by introducing the gene into the chromosome of the plant, or to allow the gene to function in plant cells in advance. It may be operably linked to a possible promoter and a terminator operable in a plant cell and introduced into the cell. Here, "in a functional way"
The expression means that the gene encoding the present protein is linked to these promoters and terminators so as to be expressed under the control of the promoters and terminators in plant cells to be introduced. Promoters that can function in plant cells include:
For example, T-DNA derived constitutive promoters such as nopaline synthase gene promoter, octopine synthase gene promoter, and cauliflower mosaic virus-derived
Examples include plant virus-derived promoters such as the 19S promoter and 35S promoter, inducible promoters such as the phenylalanine ammonia-lyase gene promoter, the chalcone synthase gene promoter, and the Pathogenesis-related protein gene promoter. Further, other plant promoters not limited to these may be used. Examples of the terminator that can function in a plant cell include, for example, a T-DNA-derived terminator such as a nopaline synthase gene terminator,
Terminators derived from plant viruses, such as garlic virus GV1 and GV2 terminators, can be mentioned. Further, other plant terminators not limited to these may be used.

As the cells into which such a gene is introduced, plant tissues, plant individuals, cultured cells, seeds and the like can be used. Examples of plant species into which such genes are introduced include, for example, tobacco, cotton, rape, sugar beet, Arabidopsis, canola, flax, sunflower, potato, alfalfa, lettuce, banana, soybean, pea, other legumes, pine, poplar Dicotyledonous plants such as apples, grapes, oranges, lemons, other citrus fruits, almonds, walnuts, other nuts, corn, rice,
Monocotyledonous plants such as wheat, barley, rye, oat, sorghum, oats, sugarcane, turf and the like can be mentioned. A transformed plant cell that expresses the introduced gene may be a selection medium corresponding to a selection marker gene linked to the gene-transfected cell, for example, a medium containing a cell growth inhibitor. And a clone capable of growing in the medium is isolated. The transformed plant cells can also be selected by culturing the cells into which the gene has been introduced in a medium containing a herbicide to which tolerance is to be imparted or an active ingredient thereof, and isolating a clone capable of growing.
From the transformed plant cells thus obtained, for example, a plant gene manipulation manual: How to make a transgenic plant (by Uchimiya, Kodansha Scientific 1990)
Years), pp. 27-55, etc., a plant expressing the introduced gene can be obtained by regenerating the plant by the plant cell culture method. More specifically, for example, experimental protocols for model plants, edited by Rice and Arabidopsis (Isao Shimamoto, supervised by Kiyotaka Okada, Shujunsha 1996)
Years), the rice and Arabidopsis thaliana expressing the introduced gene can be obtained by the method described in Chapter 4.
Further, a gene can be introduced into a soybean somatic embryo using a particle gun by a method described in JP-A-3-291501, and a soybean expressing the introduced gene can be obtained.
Similarly, Fromm, ME, et al. Bio / Technology, 8; p838
According to the method described in (1990), a gene can be introduced into a maize somatic embryo using a particle gun, and maize expressing the introduced gene can be obtained.Takami et al., Journal of the Japanese Society of Breeding, 1995 Year, Volume 44, Separate Volume 1
No., p. 57, a gene can be introduced into a wheat immature scutellum aseptically cultured using a particle gun using a particle gun to obtain a wheat expressing the introduced gene. Similarly, Hagio et al., Journal of Japanese Society of Breeding, 1995
Year, Vol. 44, Separate Volume 1, p. 67, A barley expressing the introduced gene by introducing a gene into a barley immature scutellum aseptically cultured using a particle gun using a particle gun. Can be obtained.

In order to confirm the herbicide resistance of a plant expressing the introduced gene, for example, the herbicide to be evaluated may be sprayed on the cultivation area of the plant, and the growth of the plant may be measured. For more quantitative confirmation, for example, when confirming the resistance to a PPO-inhibiting herbicide, the leaf pieces of the plant are immersed in an aqueous solution containing various concentrations of the PPO-inhibiting herbicide, or The herbicide aqueous solution is sprayed on the leaf pieces of the plant and left at room temperature in the light on an agar medium.
It is preferable to extract chlorophyll and measure the chlorophyll content according to the method described in Macknney, G., J. Bol. Chem., 140; p315 (1941).

Since the plants 1 to 3 of the present invention show resistance to both the glyphosate compound and the PPO-inhibiting herbicidal compound, they can be favorably used even when a herbicide containing these compounds as an active ingredient is sprayed. Can grow. Therefore, the method of the present invention imparts a resistance to a glyphosate compound and a resistance to a PPO-inhibited herbicidal compound to a target cultivated plant, cultivates the plant, and cultivates the plant. By spraying a herbicide containing a herbicidal compound as an active ingredient, weeds can be efficiently removed, and labor for weed control, improvement in the yield of the cultivated plant, high quality, and the like can be achieved.

[0035]

The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited to these examples. Reference Example 1 Isolation of glyphosate-resistant gene After seeding glyphosate-resistant soybean (Glycine max),
After cultivation at 7 ° C. for 30 days, the first leaves of the germinated individuals were collected.
The collected green leaves were frozen with liquid nitrogen, ground with a mortar and pestle, and a genomic DNA extraction reagent ISOPLANT was obtained from the ground material.
Genomic DNA was extracted using (Nippon Gene) according to the attached manual. Using the obtained genomic DNA as a template, PCR was performed using, as primers, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2,
A nucleotide sequence encoding a chloroplast transit peptide (CTP) sequence of Petunia hybrida EPSPS and Agrobacterium sp. Str.
ain CP4) and a DNA fragment containing the EPSPS gene (hereinafter, this CTP-C
Described as P4 EPSPS gene. ) Was amplified. The oligonucleotide was synthesized using a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RNA Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). In addition, PCR was performed once at 94 ° C. for 5 minutes, then at 55 ° C. for 2 minutes, and further at 72 ° C. for 3 minutes.
This was carried out 38 times as one heat retention cycle of 1 minute at 55 ° C., 2 minutes at 55 ° C., and 3 minutes at 72 ° C., and finally 94 ° C.
For 1 minute, then at 55 ° C. for 2 minutes and further at 72 ° C. for 10 minutes. The amplified DNA fragment is transferred to plasmid pCR
Plasmid pCREPSPS (FIG. 1) was obtained by ligation to the cloning site of the PCR product of 2.1 (Invitrogen). Next, the plasmid was used as a competent cell of Escherichia coli JM109 strain.
(Manufactured by Takara Shuzo) and selected for ampicillin-resistant strains. Furthermore, the nucleotide sequence of the plasmid contained in the selected ampicillin-resistant strain was determined by Thermo Sequence II Dye Termi
The determination was performed using a nator kit (Amersham Pharmacia Biotech) and a DNA sequencer 373S (PE Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 3 became clear, and the plasmid pCREPSPS
Was confirmed to contain this CTP-CP4 EPSPS gene.

Reference Example 2 Acquisition of a Gene Encoding a Modified Protein of the Protoporphyrin IX Binding Subunit of Tobacco Magnesium Keratase Using an RNeasy Plant Kit (manufactured by QIAGEN) from the leaf tissue of tobacco (Nicotiana tabacum cv. SR1) The operation was performed according to the attached manual to prepare total RNA. In addition, perform the operation using RNA LA PCR Kit (AMV) Ver 1.1 (Takara Shuzo) according to the attached manual,
A DNA fragment containing a gene encoding a protoporphyrin IX binding subunit protein of tobacco magnesium keratase lacking a chloroplast translocation signal (hereinafter, referred to as the present modified tobacco keratase subunit) was obtained. First, Oligo dT-Adap included in the kit as a primer
Using tor Primer, using tobacco total RNA as a template,
Add the reverse transcriptase included in the kit and add 1st strand cD
NA was synthesized. Subsequently, PCR was performed using the 1st strand cDNA as a template and LA Taq polymerase contained in the kit.
Was performed to amplify a DNA fragment containing the gene encoding the modified tobacco keratase subunit. Here, as a primer for PCR, an oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 4 and an oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 5 were used. The oligonucleotide is a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RN)
A Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). In addition, PCR
Incubation was performed once at 94 ° C for 2 minutes, followed by 30 cycles at 94 ° C for 30 seconds, then at 50 ° C for 30 seconds, and further at 72 ° C for 7 minutes as one cycle. The PC
TA Cloning Kit for DNA fragment amplified by R reaction
(Invitrogen) according to the attached manual, and the DNA fragment was cloned into the cloning site of the PCR product of the pCR2.1 plasmid. After the DNA of the obtained plasmid was digested with the restriction enzyme KpnI, it was analyzed by agarose gel electrophoresis, and the plasmid in which the 8.0 kb DNA fragment was detected was named pTCHLH. The plasmid contains the gene encoding the modified tobacco keratase subunit
It has a structure inserted such that it can be expressed in the sense direction under the control of the c promoter. Furthermore, this plasmid pT
After digesting CHLH with the restriction enzyme KpnI, self-cyclization was performed, and about 60
A plasmid pTCHLH1 (FIG. 2) having a structure in which a base DNA fragment was removed from the plasmid pTCHLH was obtained.

Reference Example 3 Introduction of the Present CTP-CP4 EPSPS Gene into Tobacco A plasmid for introducing the present CTP-CP4 EPSPS gene into plants by the Agrobacterium method was constructed. First, pNG01
[Shiota et al. (1994) Plant Physiol 106: 17-23] (Fig. 3)
Was digested with the restriction enzyme HindIII, and DNA polymerase was used to add nucleotides to the gaps in the double-stranded DNA, thereby
After blunting the end of A, self-cyclization was performed using T4 DNA ligase to obtain pNG04 (FIG. 4). Digest pNG04 with XbaI to obtain a DNA fragment containing the nopaline synthase gene terminator and the cauliflower mosaic virus 35S promoter located downstream thereof, and insert this into the XbaI cleavage site of plasmid pUC19 (Takara Shuzo). Thus, pNT35S (FIG. 5) was obtained. Next, the plasmid pCREPSPS prepared in Reference Example 1 was digested with HindIII and SalI, and the resulting CTP was obtained.
A plasmid pCENS (FIG. 6) was obtained by inserting a DNA fragment containing the -CP4 EPSPS gene between the HindIII cleavage site and the SalI cleavage site of pNT35S. After digesting this plasmid pCENS with the restriction enzyme HindIII,
Nucleotide was added to the gap of the main-strand DNA to blunt the DNA end, treated with calf intestine-derived alkaline phosphatase to dephosphorylate the 5 ′ end of the DNA, and a phosphorylated KpnI linker (Takara Shuzo 4668A) was inserted. And the plasmid pCE
NSK (FIG. 7) was obtained. On the other hand, the binary vector pBI121 (Cl
ontech) by digesting with the restriction enzyme SmaI and inserting a KpnI linker (manufactured by Takara Shuzo) into the plasmid to remove the SmaI recognition site of pBI121 and add a KpnI recognition site.
pBI121K was produced. After digesting the DNA of the plasmid pBI121K with the restriction enzyme SacI,
The 3 'protruding end of the DNA was digested to blunt the end of the DNA. Next, the 5 ′ end of the DNA was dephosphorylated with an alkaline phosphatase derived from calf small intestine, and a phosphorylated SalI linker (Takara Shuzo 4680P) was inserted to cyclize the plasmid.
121KS was built. By digesting the binary vector pBI121KS with restriction enzymes KpnI and SalI, β-glucuroni
The dase gene was removed and replaced with the plasmid pC
This CTP-CP obtained by digesting ENSK with restriction enzymes KpnI and SalI
4 Insert the DNA fragment containing the EPSPS gene and insert this CTP-CP4 EPS downstream of the cauliflower mosaic virus 35S promoter.
A plasmid pBICE to which the PS gene was linked (FIG. 8) was prepared. Further, by digesting the binary vector pBI121 (manufactured by Clontech) with the restriction enzymes BamHI and SacI, β-glu
After removing the curonidase gene and blunting the end of the obtained DNA fragment using T4 DNA polymerase, T4 DNA
Self-cyclization was performed using gase to construct a plasmid pNO (FIG. 9). This plasmid was used as a vector control for the present CTP-CP4 EPSPS gene expression plasmid pBICE. The plasmids pBICE and pNO were each transferred to Agrobacterium
tumefaciens LBA4404 (manufactured by Clontech), and this was introduced into an LB medium (0.5% yeast) containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin.
Agrobacterium strain having pBICE and Agrobacterium strain having pNO were isolated by culturing with extract, 1.0% Bacto tryptone, 0.5% NaCl) and selecting transformants. Next, the gene was introduced into tobacco according to the method described in a plant gene manipulation manual (Hirofumi Uchimiya, Kodansha Scientific, 1992). L. Agrobacterium strain with plasmid pBICE
B medium (0.5% Yeast extract, 1.0% Bacto tryptone, 0.5%
% NaCl) at 28 ° C. overnight, and leaf pieces of tobacco (Nicotiana tabacum cv. SR1) aseptically cultured were immersed in the culture solution. The tobacco leaf pieces were placed in Murashige-Skoog medium [Murashig
e T. and Skoog F., Physiol. Plant. (1962) 15, p473
Described in. Hereinafter, it is referred to as MS medium. ] On a medium supplemented with 0.8% agar, 0.1 mg / l naphthaleneacetic acid and 1.0 mg / l benzylaminopurine at room temperature for 2 days. Next, after washing the tobacco leaf pieces with sterilized water, 0.8% agar, 0.1 mg / l naphthalene acetic acid, 1.0 mg / l benzylaminopurine, and 5%
The cells were cultured on an MS medium supplemented with 00 μg / ml cefotaxime for 7 days. Then, the tobacco leaf pieces were 0.8% agar, 0.1 mg / l naphthalene acetic acid, 1.0 mg / l benzylaminopurine, 500 μg /
The cells were transplanted and cultured on an MS medium supplemented with ml cefotaxime and 100 μg / ml kanamycin (hereinafter referred to as a selection MS medium). The culturing was continuously performed for 4 months while transplanting the tobacco leaf pieces into a fresh MS medium for selection every one month. During this time, shoots differentiated from foliage emerged from the tobacco leaf pieces were transplanted to an MS medium supplemented with 0.8% agar, 300 μg / ml cefotaxime and 50 μg / ml kanamycin, and rooted to obtain regenerated individuals. The regenerated individual was transplanted and cultured in an MS medium supplemented with 0.8% agar and 50 μg / ml kanamycin to obtain a tobacco individual introduced with the present CTP-CP4 EPSPS gene. Similarly, an Agrobacterium strain having pNO was transmitted to tobacco leaf pieces, and a regenerated individual was obtained from the tobacco leaf pieces to obtain tobacco individuals (hereinafter referred to as control recombinant tobacco).

Example 1 Introduction of the present CTP-CP4 EPSPS gene and the gene encoding the present modified tobacco keratase subunit into tobacco The present CTP-CP4 EPSPS gene and the gene encoding the present modified tobacco keratase subunit were transformed into Agrobacterium Plasmids for introduction into plants were constructed by the bacterium method. First, the plasmid pTCHLH1 prepared in Reference Example 2 was digested with restriction enzymes KpnI and SalI to obtain a DNA containing the gene encoding the modified tobacco keratase subunit.
Fragments were prepared. On the other hand, the β-glucuronidase gene was removed by digesting the binary vector pBI121KS prepared in Reference Example 3 with the restriction enzymes KpnI and SalI, and in place of this, the gene encoding the present modified tobacco keratase subunit described above. A DNA fragment containing
Plasmid pBI linked downstream of the 35S promoter
TCHLH (FIG. 10) was prepared. Next, the plasmid pCENSK prepared in Reference Example 3 was digested with a restriction enzyme KpnI,
The present CTP-CP4 EPSPS gene, a terminator of a gene encoding nopaline synthase located downstream of the gene, and a DNA fragment containing a cauliflower mosaic virus 35S promoter located further downstream thereof were obtained. Plasmid pBICETCH (FIG. 11) in which the present CTP-CP4 EPSPS gene and the gene encoding the modified tobacco keratase subunit are respectively linked downstream of the cauliflower mosaic virus 35S promoter by insertion into the KpnI cleavage site of plasmid pBITCHLH. ) Was prepared. The plasmid pBICETCH was replaced with Agrobacterium tumefaci
Agrobacterium strain having pBICETCH was isolated by introducing into ens LBA4404, culturing it in LB medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin, and selecting transformants. . The Agrobacterium strain was used to infect aseptically cultured tobacco leaf pieces.
Tobacco into which the gene encoding the CTP-CP4 EPSPS gene and the present modified tobacco keratase subunit was introduced was obtained.

Test Example 1 Herbicide resistance test of tobacco into which the present CTP-CP4 EPSPS gene was introduced and tobacco into which the gene encoding the present CTP-CP4 EPSPS gene and the modified tobacco keratase subunit were introduced Reference Example 3 Of the present CTP-CP4 EPSPS gene-introduced two tobacco (individual names; CE-1, CE-2) and control recombinant tobacco leaves, and the present CTP-CP4 obtained in Example 1. Tobacco 2 into which the CP4 EPSPS gene and the gene encoding the modified tobacco keratase subunit have been introduced
The leaves of an individual (individual name; CETCH-1, CETCH-2) are collected, each of which is equally divided into two along the main vein, and one of the leaf pieces is a PPO-inhibited herbicide represented by the above structural formula 8. An aqueous solution containing the active compound at a concentration of 0.3 ppm was applied to the entire surface. The other leaf piece was not coated with the herbicidal compound. These leaf pieces were placed on an MS medium containing 0.8% agar and allowed to stand in the light at room temperature for 7 days. Next, each leaf piece was ground in 5 ml of an 80% acetone aqueous solution using a mortar and a pestle to extract chlorophyll. After diluting the extract 10 times with 80% acetone aqueous solution, 750 nm, 663n
m, absorbance at 645 nm and measured by MacknneyG., J. Bio.
The total chlorophyll content was calculated by the method described in l. Chem. (1941) 140, p315. Table 1 shows the results. In the table, P
The degree of resistance to the PO-inhibiting herbicidal compound was determined by measuring the total chlorophyll content of the leaf pieces treated with the PPO-inhibiting herbicidal compound,
Expressed as a percentage of the total chlorophyll content of untreated leaf pieces.

[0040]

[Table 1] In addition, two tobacco individuals (individual names; CE-1, CE-2) into which the present CTP-CP4 EPSPS gene has been introduced, the present CTP-CP4 EPSPS gene and a gene encoding the present modified tobacco keratase subunit have been introduced. 2 tobacco individuals (individual names; CETCH-1, CET
CH-2) and control recombinant tobacco were similarly treated with an aqueous solution containing glyphosate (isopropylammonium N- (phosphonomethyl) glycinate) at a concentration of 100 ppm, and the degree of resistance to the glyphosate compound was measured. Table 2 shows the results. In the table, the degree of resistance to the glyphosate compound was expressed as a percentage of the total chlorophyll content of the leaf pieces treated with the glyphosate compound relative to the total chlorophyll content of the untreated leaf pieces.

[0041]

[Table 2]

Reference Example 4 Expression of soybean-derived PPO gene in E. coli lacking the hemG gene After sowing soybean (Glycine max var. Williams 82),
For 20 days, and green leaves were collected. 5 g of the collected green leaves were frozen with liquid nitrogen, ground with a mortar and a pestle, and RNA was extracted from the ground using an RNA extraction reagent ISOGEN (Nippon Gene) according to the attached manual. Total RNA was collected from the obtained RNA extract by ethanol precipitation,
This is poly (A) RNA fractionation kit BIOMAG mRNA Purification
Using a Kit (manufactured by Perceptive Biosystems), fractionation was performed according to the attached manual, and a poly (A) RNA fraction was collected. Using 1 μg of this poly (A) RNA fraction as a template, Marathon c
Using the cDNA synthesis reagent included in the DNAamplification kit (Clontech), cDNA was synthesized according to the attached manual. Using the obtained cDNA as a template, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7
PCR was performed using an oligonucleotide having the nucleotide sequence represented by the above as a primer to amplify a DNA fragment containing the chloroplast PPO gene. The oligonucleotide is D
NA synthesizer (PE Applied Biosystems: Model
394 DNA / RNA Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). Also, P
The CR is kept at 94 ° C for 1 minute and then at 65 ° C for 5 minutes.
This was performed 29 times with one cycle of keeping the temperature at 94 ° C. for 15 seconds and then at 65 ° C. for 5 minutes. After the PCR reaction, the amplified DNA fragment was purified by filtering the reaction solution through MicroSpin S-400HR (manufactured by Pharmacia Biotech), and the DNA fragment was subjected to plasmid pCR2.1 (Invitrogen) digested with the restriction enzyme SalI. (Made by the company)
The plasmid pSPPO-P was obtained. Next, the plasmid was introduced into competent cells of Escherichia coli INVαF '(manufactured by Invitrogen), and ampicillin-resistant strains were selected. Further, the nucleotide sequence of the plasmid contained in the selected ampicillin-resistant strain was analyzed using a Dye terminator cycle sequencing kit (manufactured by PE Applied Biosystems) and a DNA sequencer 37.
It was determined using 3S (manufactured by PE Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 8 was clarified, and the plasmid pSPPO-P was found to be a soybean chloroplast PPO.
It was confirmed to contain the gene. This plasmid pSPPO-
P was digested with the restriction enzyme PshBI, and the end of the obtained DNA fragment was blunted using T4 DNA polymerase.
After digestion with I, a DNA fragment containing the soybean chloroplast PPO gene and the lac promoter was isolated. Next, the plasmid p
ACYC184 (Nippon Gene) is restricted to restriction enzymes NruI and SphI
To remove the 410 bp fragment and replace it with the DNA
Plasmid pACYCSP (Fig. 1)
2) was obtained. Next, the plasmid pACYCSP was
o, F. et al., Japanese J. Genet., 63; p237 (1988) described in Pana gene (hemG locus) deletion mutant strain E. coli BT3 strain Hanahan, DJ, Mol. 166;
p557 (1983), and introduced at 15 μg /
ml chloramphenicol, contains 10μg / ml kanamycin
By culturing in YPT medium (5 g / l yeast extract, 5 g / l tryptone, 5 g / l peptone, 10 g / l NaCl, pH 7.0), it is resistant to chloramphenicol and kanamycin, and hemG
An Escherichia coli BT3 / pACYCSP strain whose gene deletion was complemented with a soybean-derived PPO gene was selected.

Reference Example 5 Acquisition of a gene encoding a protein that is a modified protein of soybean PPO and has no ability to oxidize protoporphyrinogen IX and specifically binds to protoporphyrinogen IX SEQ ID NO: 9 PCR was performed using the oligonucleotide consisting of the sequence and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10 as primers, and using the plasmid pSPPO-P prepared in Reference Example 4 as a template, to bind to the chloroplast translocation signal and FAD binding. Soybean P lacking sequence
A DNA fragment encoding PO (hereinafter referred to as the present modified soybean PPO) was amplified. The oligonucleotide is a DNA synthesizer (PE Applied Biosystems: M
odel 394 DNA / RNA Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). In addition, the PCR was carried out for one cycle at 94 ° C. for 1 minute, then at 55 ° C. for 2 minutes, and further at 72 ° C. for 3 minutes.
Times. The amplified DNA fragment was digested with restriction enzymes NcoI and Sa
The plasmid pTVGMP (FIG. 13) was constructed by digesting with lI and inserting between the NcoI cleavage site and the SalI cleavage site of plasmid pTV118N (Takara Shuzo). The plasmid pTVG
MP was replaced with PPO gene deletion mutant strain Escherichia coli BT3 by Hanah.
an, DJ, Mol. Biol., 166; p557 (1983), and introduced at 100 μg / ml ampicillin, 10 μg / m
When the cells were cultured in a YPT medium containing lkanamycin (5 g / l yeast extract, 5 g / l tryptone, 5 g / l peptone, 10 g / l NaCl, pH 7.0), no growth-complemented clones were obtained.

Example 2 Introduction of the present CTP-CP4 EPSPS gene and the gene encoding the present modified soybean PPO into tobacco The present CTP-CP4 EPSPS gene and the gene encoding the present modified soybean PPO were introduced into plants by the Agrobacterium method. A plasmid for constructing the plasmid was constructed. Similarly to the method described in Reference Example 5, the plasmid pSPPO prepared in Reference Example 4 was prepared using an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 12. By performing PCR using -P as a template, the modified soybean PP
A DNA fragment containing the gene encoding O was amplified. Subsequently, the plasmid pBI121K prepared in Reference Example 3 was digested with restriction enzymes KpnI and SacI to obtain β-glucuronida.
The se gene is removed, and the DNA fragment containing the gene encoding the present modified soybean PPO is replaced with a restriction enzyme.
Insert a DNA fragment obtained by digestion with KpnI and SacI,
A plasmid pBIGMP (FIG. 14) in which the gene was linked downstream of the 35S promoter was prepared. Next, Reference Example 3
Digest the plasmid pCENSK prepared in the above with the restriction enzyme KpnI, the present CTP-CP4 EPSPS gene, the terminator of the gene encoding nopaline synthase located downstream thereof, and further the cauliflower mosaic virus 35S promoter located downstream thereof Obtain a DNA fragment containing
By inserting this into the KpnI cleavage site of the above plasmid pBIGMP, the plasmid pBICEGMP in which the present CTP-CP4 EPSPS gene and the gene encoding the modified soybean PPO are respectively linked downstream of the cauliflower mosaic virus 35S promoter (FIG. 15) ). The plasmid
pBICEGMP, introduced into Agrobacterium tumefaciens LBA4404, 300 μg / ml streptomycin, 100 μg / ml
By culturing on LB medium containing rifampicin and 25 μg / ml kanamycin and selecting transformants, pBICEGMP
Agrobacterium strain with The Agrobacterium strain was infected aseptically into leaf pieces of tobacco, and tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present modified soybean PPO were introduced was obtained in the same manner as described in Reference Example 3. I do.

Test Example 2 Tobacco herbicide tolerance test of tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present modified soybean PPO were introduced The present CTP-CP4 EPSPS gene obtained in Example 2 and the present modified soybean PPO were coded Tobacco into which the gene to be introduced has been introduced,
By testing the control recombinant tobacco in the same manner as in Test Example 1, the degree of resistance to the PPO-inhibiting herbicidal compound represented by Structural Formula 8 is quantitatively confirmed. In addition, by testing the tobacco and the control recombinant tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present modified soybean PPO were introduced by the same operation as in Test Example 1, the degree of resistance to the glyphosate compound was determined. Confirm quantitatively.

Reference Example 6 Acquisition of PPO gene of Paramecium bursaria Chlamydomonas reinhardtii CC407
Transfer the strain to the Chlamydomonas Genetics Center (address: DCM
B Group, Department of Botany, Box 91000, Duke Uni
versity, Durham, NC 27708-1000, USA)
7 μM NH ₄ Cl, 0.4 mM MgS under 0 μE / m ² / s photosynthesis active illumination
_{O 4 · 7H 2 O, 0.34mM} CaCl 2 · 2H 2 O, potassium 25mM phosphate,
TAP liquid medium (EH Harr) consisting of 0.5 mM Tris (pH 7.5), 1 ml / L Hatner trace elements, and 1 ml / L glacial acetic acid
is, The Chlamydomonas Sourcebook, Academic Press,
(San Diego, 1989, pp. 576-577) for 5 days, and 200 ml (1.0 × 10 ⁶ cells) of a culture solution containing cells in the initial stationary growth phase.
s / ml). From these cells, total RNA was prepared by using ISOGEN (manufactured by Nippon Gene) according to the attached manual. In addition, BioMag mRNA Purific
Perform the operation using the ation Kit (Perceptive Biosystems) in accordance with the attached manual to obtain poly (A) RNA.
Was fractionated. From the obtained poly (A) RNA, Marathon cD
Using the NAAmplification Kit (manufactured by Clontech), perform the operation according to the attached manual to synthesize cDNA,
It was used as a CR template. As a PCR primer, an oligonucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 13 and an oligonucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 14 were prepared. These oligonucleotides are used in a DNA synthesizer (PE Applied Biosystems: Mod
The DNA was synthesized using an El 394 DNA / RNA Synthesizer, and purified using an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). PCR
Prepare a reaction mixture using the Advantage cDNA PCR Kit (Clontech) according to the attached manual,
After 1 minute, then keep at 65 ° C once for 5 minutes, then 94 ° C
This was carried out 29 times with one cycle of keeping the temperature for 15 seconds and then at 65 ° C. for 5 minutes. After the PCR reaction, mix the reaction solution with Micr
The amplified DNA fragment was purified by filtration through oSpin S-400HR (Pharmacia Biotech). TA
Using a Cloning Kit (manufactured by Invitrogen), the operation was performed according to the attached manual, and the amplified DNA fragment was cloned into pCR2.1
The plasmid was cloned into the cloning site of the PCR product to construct a plasmid pCPPO. Obtained recombinant plasmid p
Dye terminator c
The determination was performed using a ycle sequencing kit (manufactured by PE Applied Biosystems) and a DNA sequencer 373S (manufactured by PE Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 15 became clear, and it was revealed that pCPPO was a plasmid containing the full-length cDNA of PPO of Paramecium bursaria.

Reference Example 7 Acquisition of a Gene Encoding a Protein That Is a Modified Protein of Paramecium bursaria PPO and Does Not Have Oxidizing Activity on Protoporphyrinogen IX and Specificly Binds to Protoporphyrinogen IX Using the oligonucleotide consisting of the base sequence and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 17 as primers, PCR was performed using the plasmid pCPPO prepared in Reference Example 6 as a template,
PPO of Paramecium bursaria lacking a chloroplast translocation signal and an FAD binding sequence (hereinafter referred to as the present modified Paramecium bursaria PPO)
It is written. ) Was amplified. In addition,
Oligonucleotides were synthesized using a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RNA Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). The PCR was carried out 30 times, with one cycle of 94 ° C. for 1 minute, followed by 55 ° C. for 2 minutes and further 72 ° C. for 3 minutes as one cycle. The amplified DNA fragment was digested with restriction enzymes BamHI and SacI.
Plasmid pTVCRP (FIG. 16) was inserted between the BamHI and SacI cleavage sites of TV119N (Takara Shuzo).
Was built. The plasmid pTVCRP was transformed into a PPO gene deletion mutant strain E. coli BT3 strain by Hanahan, DJ, Mol. Biol., 1
66; p557 (1983).
YPT containing 0 μg / ml ampicillin, 10 μg / ml kanamycin
Medium (5 g / l yeast extract, 5 g / l tryptone, 5 g / l peptone,
When the cells were cultured at 10 g / l NaCl (pH 7.0), no clones with growth complementation were obtained at all.

Example 3 Introduction of the present CTP-CP4 EPSPS gene and the gene encoding the present modified Paramecium bursaria PPO into tobacco The present CTP-CP4 EPSPS gene and the present modified Paramecium bursaria PPO
A plasmid was constructed for introducing the gene encoding the above into a plant by the Agrobacterium method. The plasmid pTVCRP prepared in Reference Example 7 was digested with the restriction enzymes BamHI and SacI to prepare a DNA fragment containing the gene encoding the modified P. lactis PPO. Binary vector pBI121 (Clontech) was digested with restriction enzymes BamHI and SacI.
The β-glucuronidase gene is removed by digestion with
A DNA fragment containing a gene encoding was inserted to prepare a plasmid pBICRP (FIG. 17) in which the gene was ligated downstream of the 35S promoter. Then, the plasmid
After digesting pBICRP with the restriction enzyme BamHI, DNA Polymerase I
To add a nucleotide to the gap between the double-stranded DNA
Alkaline phosphatas derived from calf small intestine
e) to dephosphorylate the 5 ′ end of the DNA, and phosphorylate Kp
An nI linker (Takara Shuzo 4668A) is inserted and cyclized to obtain plasmid pBICRPK. Next, the plasmid pCENSK prepared in Reference Example 3 was digested with the restriction enzyme KpnI, and the CTP-
The CP4 EPSPS gene, a terminator of the gene encoding nopaline synthase located downstream thereof, and the cauliflower mosaic virus 35 located further downstream thereof
By obtaining a DNA fragment containing the S promoter and inserting it into the KpnI cleavage site of the plasmid pBICRPK, the CTP
-CP4 EPSPS gene and the gene encoding this modified soybean PPO are cauliflower mosaic virus 35S, respectively.
Plasmid pBICEC linked downstream of the promoter
RP (FIG. 18) is prepared. The plasmid pBICECRP was
bacterium tumefaciens LBA4404,
g / ml streptomycin, 100 μg / ml rifampicin, 2
An Agrobacterium strain having pBICECRP is isolated by culturing in an LB medium containing 5 μg / ml kanamycin and selecting transformants. The Agrobacterium strain was infected aseptically into leaf pieces of tobacco, and the tobacco into which the gene encoding the present CTP-CP4 EPSPS gene and the gene encoding the present modified E. paramecium PPO were introduced in the same manner as described in Reference Example 3. get.

Test Example 3 Tobacco herbicide resistance test of tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present modified E. parasitoid PPO were introduced The present CTP-CP4 EPSPS gene obtained in Example 3 and the present modified E. parasitoid PPO And a control recombinant tobacco, into which the gene encoding the PPO-inhibiting herbicidal compound represented by the above structural formula 8 has been quantitatively determined. To confirm. Further, by testing the tobacco and the control recombinant tobacco in which the present CTP-CP4 EPSPS gene and the gene encoding the present modified chlamydomonas PPO were introduced by the same operation as in Test Example 1, the resistance to the glyphosate compound was determined. Is quantitatively confirmed.

Reference Example 8 Obtaining Gene Encoding Arabidopsis Chloroplast-Localized Ferrochelatase An operation was performed from the leaf tissue of Arabidopsis thaliana (Arabidopsis thaliana WS) using RNeasy Plant Kit (manufactured by QIAGEN) according to the attached manual. And total RNA was prepared. In addition, perform the operation using RNA LA PCR Kit (AMV) Ver 1.1 (Takara Shuzo) according to the attached manual,
A DNA fragment containing a gene encoding chloroplast-localized ferrochelatase (hereinafter, referred to as the present Arabidopsis ferrochelatase) was obtained. First, the oligo dT-Adaptor Primer included in the kit was used as a primer, and the reverse transcriptase included in the kit was added using Arabidopsis total RNA as a template to synthesize 1st strand cDNA. Subsequently, PCR was performed using the 1st strand cDNA as a template and LA Taq polymerase included in the kit to amplify a DNA fragment containing the gene encoding the present Arabidopsis thaliana ferrochelatase. Here, as a primer for PCR, an oligonucleotide primer having a base sequence represented by SEQ ID NO: 18 and an oligonucleotide primer having a base sequence represented by SEQ ID NO: 19 were used. The oligonucleotide was synthesized using a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RNA Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). PCR was performed once at 94 ° C. for 2 minutes, followed by 94 ° C. for 30 seconds and 55 ° C. for 3 seconds.
This was repeated 30 times with one cycle of keeping the temperature at 72 ° C. for 3 minutes for 0 second. After the PCR reaction, the PC
TA Cloning Kit for DNA fragment amplified by R reaction
(Invitrogen) according to the attached manual, and the DNA fragment was subjected to PCR of pCR2.1 plasmid.
The product is cloned into the cloning site and plasmid pCRATF
(FIG. 19). The plasmid has a structure in which a gene encoding the present Arabidopsis thaliana ferrochelatase is inserted in a form capable of being expressed in the sense direction under the control of the lac promoter. Next, the plasmid was introduced into competent cells of Escherichia coli JM109 strain (Takara Shuzo), and ampicillin-resistant strains were selected. Furthermore, the nucleotide sequence of the plasmid contained in the selected ampicillin-resistant strain was
nce II Dye Terminator kit (Amersham Pharmacia Biotech) and DNA sequencer 373S (PE Applied Biosystems). As a result, the base sequence represented by SEQ ID NO: 20 became apparent,
Plasmid pCRATF was confirmed to contain the gene encoding the present Arabidopsis thaliana ferrochelatase.

Example 4 Introduction of the present CTP-CP4 EPSPS gene and the gene encoding the present Arabidopsis thaliana ferrochelatase into tobacco The present CTP-CP4 EPSPS gene and the gene encoding the present Arabidopsis thaliana ferrochelatase were subjected to the Agrobacterium method. A plasmid was constructed for introduction into plants. First, a DNA fragment containing the gene encoding the present Arabidopsis thaliana ferrochelatase was prepared by digesting the plasmid pCRATF prepared in Reference Example 8 with the restriction enzyme SacI. The plasmid pBIATFGUS was obtained by inserting this DNA fragment into the restriction enzyme SacI site of the binary vector pBI121 (Clontech). This plasmid was digested with the restriction enzyme BamHI to remove the β-glucuronidase gene,
By self-cyclization, a plasmid pBIATF (FIG. 20) in which the gene was linked downstream of the 35S promoter was prepared. Next, the plasmid pBIATF was replaced with the restriction enzyme BamHI.
After digestion with DNA polymerase I, nucleotides were added to the gaps of the double-stranded DNA using DNA polymerase I to blunt the DNA ends, and treated with alkaline phosphatase derived from calf small intestine.
The 5 'end is dephosphorylated, and a phosphorylated KpnI linker (Takara Shuzo 4668A) is inserted for cyclization to obtain plasmid pBIATFK. Next, the plasmid pCEN prepared in Reference Example 3 was used.
SK is digested with the restriction enzyme KpnI, and the CTP-CP4 EPSPS gene,
A DNA fragment containing the 35S promoter of the cauliflower mosaic virus located further downstream of the terminator of the gene encoding nopaline synthase located downstream thereof and the Kp of the plasmid pBIATFK was obtained.
Plasmid pBICEATF in which the present CTP-CP4 EPSPS gene and the gene encoding the present Arabidopsis thaliana ferrochelatase are respectively connected downstream of the cauliflower mosaic virus 35S promoter by inserting into the nI cleavage site.
(FIG. 21) is produced. The plasmid pBICEATF was
cterium tumefaciens introduced into LBA4404, 300 μg /
ml streptomycin, 100 μg / ml rifampicin, 25
An Agrobacterium strain having pBICEATF is isolated by culturing in LB medium containing μg / ml kanamycin and selecting transformants. The Agrobacterium strain was infected into leaf pieces of tobacco that had been aseptically cultured, and tobacco into which the gene encoding the present CTP-CP4 EPSPS gene and the gene encoding the present Arabidopsis thaliana ferrochelatase had been introduced in the same manner as in the method described in Reference Example 3. get.

Test Example 4 Tobacco herbicide tolerance test of tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present Arabidopsis thaliana ferrochelatase were introduced The present CTP-CP4 EPSPS gene obtained in Example 4 and the present Arabidopsis thaliana ferrochelatase And a control recombinant tobacco, to which the gene encoding the gene of the present invention has been introduced, is tested in the same manner as in Test Example 1 to quantitatively determine the degree of resistance to the PPO-inhibiting herbicidal compound represented by the structural formula 8. To confirm. Also, this CTP-CP4 EPSPS
By testing the tobacco and the control recombinant tobacco into which the gene and the gene encoding the present Arabidopsis thaliana ferrochelatase have been introduced by the same operation as in Test Example 1, the degree of resistance to the glyphosate compound is quantitatively confirmed.

Reference Example 9 Isolation of Gene Encoding Soybean Coproporphyrinogen III Oxidase Soybean (Glycine max cv. Jack) was sown, cultivated at 25 ° C. for 20 days, and green leaves were collected. 5 g of the collected green leaves were frozen with liquid nitrogen, and this was ground with a mortar and pestle, from the ground material,
Total RNA was prepared using an RNA extraction reagent ISOGEN (Nippon Gene) according to the attached manual. Further, using RNA LA PCR Kit (AMV) Ver 1.1 (manufactured by Takara Shuzo Co., Ltd.), the operation was performed according to the attached manual, and coproporphyrinogen III oxidase (hereinafter, this soybean CPOX
It is written. ) Was obtained. First, Oligo d contained in the kit as a primer
Using T-Adaptor Primer, using the soybean total RNA as a template, add the reverse transcriptase included in the kit and add 1st s
trand cDNA was synthesized. Subsequently, PCR was performed using the 1st strand cDNA as a template and LA Taq polymerase included in the kit to amplify a DNA fragment containing the gene encoding the present soybean CPOX. Here, as a primer for PCR, an oligonucleotide primer having the base sequence of SEQ ID NO: 21 and an oligonucleotide primer having the base sequence of SEQ ID NO: 22 were used. The oligonucleotide is a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RNA Synthesi).
and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). In addition, PCR was performed once at 94 ° C. for 2 minutes, then at 94 ° C. for 30 seconds and then 55 ° C.
This was carried out for 30 cycles at a temperature of 30 ° C. for 30 seconds and at a temperature of 72 ° C. for 3 minutes. After the PCR reaction, the DNA fragment amplified by the PCR reaction is subjected to TA C
Using a loning Kit (manufactured by Invitrogen), the operation was performed according to the attached manual, and the DNA fragment was cloned into the cloning site of the PCR product of pCR2.1 plasmid, and named plasmid pCRSCPOX (FIG. 22). The plasmid has a structure in which the gene encoding the present soybean CPOX is inserted downstream of the lac promoter in the antisense direction. Next, the plasmid was used as a competent cell of Escherichia coli JM109 strain.
(Manufactured by Takara Shuzo) and selected for ampicillin-resistant strains. Furthermore, the nucleotide sequence of the plasmid contained in the selected ampicillin-resistant strain was determined by Thermo Sequence II Dye Termi
The determination was performed using a nator kit (Amersham Pharmacia Biotech) and a DNA sequencer 373S (PE Applied Biosystems). As a result, SEQ ID NO: 23
The nucleotide sequence indicated by is clarified, and the plasmid pCRSCP
OX was confirmed to contain this soybean CPOX gene.

Example 5 Introduction of the present CTP-CP4 EPSPS gene and the gene encoding the present soybean CPOX into tobacco To introduce the present CTP-CP4 EPSPS gene and the gene encoding the present soybean CPOX into plants by the Agrobacterium method Was constructed. First, a DNA fragment containing a gene encoding the present soybean CPOX was prepared by digesting the plasmid pCRSCPOX prepared in Reference Example 9 with the restriction enzyme BamHI. This DNA fragment was inserted into the BamHI restriction site of the binary vector pBI121KS prepared in Reference Example 3 to obtain a plasmid pBISCPOXGUS. This plasmid is digested with the restriction enzyme SalI to give β-glu
The curonidase gene was removed and self-cyclization was performed to prepare a plasmid pBISCPOX (FIG. 23) in which the gene was linked downstream of the 35S promoter. Next, after digesting the plasmid pBISCPOX with the restriction enzyme BamHI, the DNA
Using ymerase I, nucleotides were added to the gaps in the double-stranded DNA and the DNA ends were blunted.
Dephosphorylate the 5 'end of the DNA by treating with phosphatase,
A phosphorylated KpnI linker (Takara Shuzo 4668A) is inserted and cyclized to obtain plasmid pBISCPOXK. Next, the plasmid pCENSK prepared in Reference Example 3 was digested with a restriction enzyme KpnI, and the present CTP-CP4 EPSPS gene, a terminator of a gene encoding nopaline synthase located downstream thereof,
And a DNA fragment containing the cauliflower mosaic virus 35S promoter located further downstream thereof, and inserting this into the KpnI cleavage site of the plasmid pBISCPOXK, the present CTP-CP4 EPSPS gene and the present soybean CPOX
The plasmid pBICESCPOX (FIG. 24), in which the genes encoding are linked downstream of the cauliflower mosaic virus 35S promoter, is prepared. The plasmid pBIC
ESCPOX was introduced into Agrobacterium tumefaciens LBA4404, and this was cultured in an LB medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin to select transformants, thereby obtaining pBICESCPOX.
Agrobacterium strain with The Agrobacterium strain was infected aseptically into leaf pieces of tobacco, and tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present soybean CPOX were introduced was obtained in the same manner as in the method described in Reference Example 3. I do.

Test Example 5 Herbicide resistance test of tobacco into which the present CTP-CP4 EPSPS gene and the gene encoding the present soybean CPOX were introduced The present CTP-CP4 EPSPS gene obtained in Example 5 and the present soybean C
By testing the tobacco transfected with the gene encoding POX and the control recombinant tobacco in the same manner as in Test Example 1, the degree of resistance to the PPO-inhibiting herbicidal compound represented by the above structural formula 8 was determined. To confirm. In addition, this CTP-CP4 EPSPS gene and this soybean CPOX
By testing the tobacco and the control recombinant tobacco into which the gene encoding is introduced by the same operation as in Test Example 1, the degree of resistance to the glyphosate compound is quantitatively confirmed.

[0056]

Industrial Applicability According to the present invention, it is possible to provide a plant having resistance to a glyphosate compound and resistance to a PPO-inhibiting herbicidal compound.

[Sequence List Free Text] SEQ ID NO: 1 A DNA fragment having a chimeric gene in which an Agrobacterium-derived EPSPS gene is bound downstream of a base sequence encoding a chloroplast transit peptide of a petunia-derived EPSPS gene SEQ ID NO: 2 Amplifies a DNA fragment having a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of a base sequence encoding a chloroplast transit peptide of a petunia-derived EPSPS gene SEQ ID NO: 4 DNA having partial nucleotide sequence of tobacco-derived chlH gene
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 5 DNA having partial base sequence of tobacco-derived chlH gene
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 6 Oligonucleotide primer designed to amplify PPO gene from soybean SEQ ID NO: 7 Oligonucleotide primer designed to amplify PPO gene from soybean SEQ ID NO: 9 DNA having partial base sequence of PPO gene derived from soybean
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 10 DNA having partial base sequence of PPO gene derived from soybean
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 11 DNA having partial base sequence of PPO gene derived from soybean
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 12 DNA having partial base sequence of PPO gene derived from soybean
Oligonucleotide primer designed to amplify the fragment SEQ ID NO: 13 Oligonucleotide primer designed to amplify the PPO gene derived from E. paramecium SEQ ID NO. 14: Oligo designed to amplify the PPO gene derived from E. paramecium Nucleotide primer SEQ ID NO: 16 Oligonucleotide primer designed to amplify a DNA fragment having a partial base sequence of PPO gene derived from Paramecium bursaria SEQ ID No. 17: Amplify a DNA fragment having a partial base sequence of PPO gene derived from Paramecium bursaria SEQ ID NO: 18 Oligonucleotide primer designed for amplifying chloroplast-localized ferrochelatase gene derived from Arabidopsis thaliana SEQ ID NO: 19 Chloroplast derived from Arabidopsis thaliana Oligonucleotide primer designed to amplify type ferrochelatase gene SEQ ID NO: 21 Oligonucleotide primer designed to amplify soybean-derived coproporphyrinogen III oxidase gene SEQ ID NO: 22 Soybean-derived coproporphyrinogen III oxidase Oligonucleotide primers designed to amplify genes

[0058]

[Sequence List] SEQUENCE LISTING <110> Sumitomo Chemical Company Limited <120> Herbicide resistant plants <130> P152117 <150> JP 11/310244 <151> 1999-10-29 <160> 23 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having nuc leotide sequence encoding the Petunia hybrida EPSPS chloroplast transit peptide and the Agrobacterium sp.strain CP4 EPSPS gene <400> 1 ggaagcttca agaatggcac aaattaacaa catggc 36 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having nucl eotide sequence encoding the Petunia hybrida EPSPS chloroplast transit p eptide and the Agrobacterium sp. strain CP4 EPSPS gene <400> 2 gagtcgacgg tcatcaggca gccttcgtat cg 32 <210> 3 <211> 1587 <212> DNA <213> Petunia hybrida EPSPS chloroplast transit peptide and Agrobacteriu m sp.strain CP4 EPSPS <220> <221> CDS < 222> (1) ... (1584) <400> 3 atg gca caa att aac aac atg gct caa ggg ata caa acc ctt aat ccc 48 Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1 5 10 15 aat tcc aat ttc cat aaa ccc caa gtt cct aaa tct tca agt ttt ctt 96 Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 20 25 30 gtt ttt gga tct aaa aaa ctg aaa aat tca gca aat tct atg ttg gtt 144 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu Val 35 40 45 ttg aaa aaa gat tca att ttt atg caa aag ttt tgt tcc ttt agg att 192 Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50 55 60 tca gca tca gtg gct aca gcc tgc atg ctt cac ggt gca agc agcgg 240 Ser Ala Ser Val Ala Thr Ala Cys Met Leu His Gly Ala Ser Ser Arg 65 70 75 ccc gca acc gcc cgc aaa tcc tct ggc ctt tcc gga acc gtc cgc att 288 Pro Ala Thr Ala Arg Lys Ser Ser Gly Leu Ser Gly Thr Val Arg Ile 80 85 90 95 ccc ggc gac aag tcg atc tcc cac cgg tcc ttc atg ttc ggc ggt ctc 336 Pro Gly Asp Lys Ser Ile Ser His Arg Ser Phe Met Phe Gly Gly Leu 100 105 110 gcg agc ggt gaa acg cgc atc acc ggc ctt ctg gaa ggc gag gac gtc 384 Ala Ser Gly Glu Thr Arg Ile Thr Gly Leu Leu Glu Gly Glu Asp Val 115 120 125 atc aat acg ggc aag gcc atg cag gcc atg ggc gcc agg atc cgt aag 432 Ile Asn Thr Gly Lys Ala Met Gln Ala Met Gly Ala Arg Ile Arg Lys 130 135 140 gaa ggc gac acc tgg atc atc gat ggc gtc ggc aat ggc ggc ctc ctg 480 Glu Gly Asp Thr Trp Ile Ile Asp Gly Val Gly Asn Gly Gly Leu Leu 145 150 155 gcg cct gag gat ttc ggc aat gcc gcc acg ggc tgc cgc 528 Ala Pro Glu Ala Pro Leu Asp Phe Gly Asn Ala Ala Thr Gly Cys Arg 160 165 170 175 ctg acc atg ggc ctc gtc ggg gtc tac gat ttc gac atgc accc ttc Lec Met Gly Leu Val Gly Val Tyr Asp Phe Asp Ser Thr Ple Ile 180 185 190 ggc gac gcc tcg ctc aca aag cgc ccg atg ggc cgc gtg ttg aac ccg 624 Gly sp Ala Ser Leu Thr Lys Arg Pro Met Gly Arg Val Leu Asn 195 200 205 ctg cgc gaa atg ggc gtg cag gtg aaa tcg gaa gac ggt gac cgt ctt 672 Leu Arg Glu Met Gly Val Gln Val Lys Ser Glu Asp Gly Asp Arg Leu 210 215 220 ccc gtt acc ttg cgc gg ccg acg acg c cg atc acc tac cgc 720 Pro Val Thr Leu Arg Gly Pro Lys Thr Pro Thr Pro Ile Thr Tyr Arg 225 230 235 gtg ccg atg gcc tcc gca cag gtg aag tcc gcc gtg ctg ctc gcc ggc 768 Val Pro Met Ala Ser Ala Gln Val Lys Ser Ala Val Leu Leu Ala Gly 240 245 250 255 ctc aac acg ccc ggc atc acg acg gtc atc gag ccg atc atg acg cgc 816 Leu Asn Thr Pro Gly Ile Thr Thr Val Ile Glu Pro Ile Met Thr Arg 260 265 270 gat cat acg gaa aag atg ctg cag ggc ttt ggc gcc aac ctt acc gtc 864 Asp His Thr Glu Lys Met Leu Gln Gly Phe Gly Ala Asn Leu Thr Val 275 280 285 gag acg gat gcg gac ggc gtg cgc acc atc cgc ccg 912 Glu Thr Asp Ala Asp Gly Val Arg Thr Ile Arg Leu Glu Gly Arg Gly 290 295 300 aag ctc acc ggc caa gtc atc gac gtg ccg ggc gac ccg tcc tcg acg 960 Lys Leu Thr Gly Gln Val Ile Asp Val Pro Gly Asp Ser Ser Thr 305 310 315 gcc ttc ccg ctg gtt gcg gcc ctg ctt gtt ccg ggc tcc gac gtc acc 1008 Ala Phe Pro Leu Val Ala Ala Leu Leu Val Pro Gly Ser Asp Val Thr 320 325 330 335 atc ctc aac gtg ctg atg aac ccc acc cgc acc ggc ctc atc ctg acg 1056 Ile Leu Asn Val Leu Met Asn Pro Thr Arg Thr Gly Leu Ile Leu Thr 340 345 350 ctg cag gaa atg ggc gcc gac atc gaa gtc atc aac ccg cgc ctt gcc G104 Lec Gly Ala Asp Ile Glu Val Ile Asn Pro Arg Leu Ala 355 360 365 ggc ggc gaa gac gtg gcg gac ctg cgc gtt cgc tcc tcc acg ctg aag 1152 Gly Gly Glu Asp Val Ala Asp Leu Arg Val Arg Ser Ser Thr Leu Lys 370 380 ggc gtc acg gtg ccg gaa gac cgc gcg cct tcg atg atc gac gaa tat 1200 Gly Val Thr Val Pro Glu Asp Arg Ala Pro Ser Met Ile Asp Glu Tyr 385 390 395 ccg att ctc ctc gct gtc gcc gcc gcc gcc gcc gcc gcc gcc gcc gcc gcc gcc acc gtg atg 1248 Pro Ile Leu Ala Val Ala Ala Ala Phe Ala Glu Gly Ala Thr Val Met 400 405 410 415 aac ggt ctg gaa gaa ctc cgc gtc aag gaa agc gac cgc ctc tcg gcc 1296 Asn Gly Leu Glu Lys Glu Ser Asp Arg Leu Ser Ala 420 425 430 gtc gcc aat ggc ctc aag ctc aat ggc gtg gat tgc gat gag ggc gag 1344 Val Ala Asn Gly Leu Lys Leu Asn Gly Val Asp Cys Asp Glu Gly Glu 435 440g 445g ctc gtc gtg cgc ggc cgc cct gac ggc aag ggg ctc ggc aac 1392 Thr Ser Leu Val Val Arg Gly Arg Pro Asp Gly Lys Gly Leu Gly Asn 450 455 460 gcc tcg ggc gcc gcc gtc gcc acc cat atcc gat ccc 1440 Ala Ser Gly Ala Ala Val Ala Thr His Leu Asp His Arg Ile Ala Met 465 470 475 agc ttc ctc gtc atg ggc ctc gtg tcg gaa aac cct gtc acg gtg gac 1488 Ser Phe Leu Val Met Gly Leu Val Ser Glun Thr Val Asp 480 485 490 495 gat gcc acg atg atc gcc acg agc ttc ccg gag ttc atg gac ctg atg 1536 Asp Ala Thr Met Ile Ala Thr Ser Phe Pro Glu Phe Met Asp Leu Met 500 505 510 gcc ggg ctg aggc gg gaa ctc tcc gat acg aag gct gcc tga 1584 Ala Gly Leu Gly Ala Lys Ile Glu Leu Ser Asp Thr Lys Ala Ala 515 520 525 tga 1587 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of tobacco chlH gene <400> 4 ccaatgtaat gctatggtac ctatgttatt cactc 35 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220 > <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of tobacco chlH gene <400> 5 gagatcattc tttttgctgt cgacttatcg atcg 34 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223 > Designed oligonucleotide primer to amplify soybean PPO gene <400> 6 tcgagctcca tggtttccgt cttcaacgag atcctattc 39 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify soybean PPO gene < 400> 7 ttgtcgacaa ctgctactat ttgtacactc tatttg 36 <210> 8 <211> 1632 <212> DNA <213> Glycine max var. Williams82 <220> <221> CDS <222> (1) ... (1632) <400> 8 atg gtt tcc gtc ttc aac gag atc cta ttc ccg ccg aac caa acc ctt 48 Met Val Ser Val Phe Asn Glu Ile Leu Phe Pro Pro Asn Gln Thr Leu 1 5 10 15 ctt cgc ccc tcc ctc cat tcc cca acc tct ttc ttc acc tct ccc act 96 Leu Arg Pro Ser Leu His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr 20 25 30 cga aaa ttc cct cgc tct cgc cct aac cct att cta cgc tgc tcc att 144 Arg Lys Phe Pro Arg Sers Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile 35 40 45 gcg gag gaa tcc acc gcg tct ccg ccc aaa acc aga gac tcc gcc ccc 192 Ala Glu Glu Ser Thr Ala Ser Pro Pro Lys Thr Arg Asp Ser Ala Pro 50 55 60 gtg gac tgc gtc gtc gtc ggc gga ggc gtc agc ggc ctc tgc atc gcc 240 Val Asp Cys Val Val Val Gly Gly Gly Val Ser Gly Leu Cys Ile Ala 65 70 75 80 cag gcc ctc gcc acc aaa cac gcc aat gcc aac gtc gcgt 288 Gln Ala Leu Ala Thr Lys His Ala Asn Ala Asn Val Val Val Thr Glu 85 90 95 gcc cga gac cgc gtc ggc ggc aac atc acc acg atg gag agg gac gga 336 Ala Arg Asp Arg Val Gly Gly Asn Ile Thr Thr Met Glu Arg Asp Gly 100 105 110 tac ctc tgg gaa gaa ggc ccc aac agc ttc cag cct tct gat cca atg 384 Tyr Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp Pro Met 115 120 125 ctc acc atg gtg gtg gac agtt tta aag gat gag ctt gtt ttg ggg 432 Leu Thr Met Val Val Asp Ser Gly Leu Lys Asp Glu Leu Val Leu Gly 130 135 140 gat cct gat gca cct cgg ttt gtg ttg tgg aac agg aag ttg agg ccg 480 Asp Pro Asp Ala Pro Arg Phe Val Leu Trp Asn Arg Lys Leu Arg Pro 145 150 155 160 gtg ccc ggg aag ctg act gat ttg cct ttc ttt gac ttg atg agc att 528 Val Pro Gly Lys Leu Thr Asp Leu Pro Phe Phe Asp Leu Met Ser Ile 165 170 175 ggt ggc aaa atc agg gct ggc ttt ggt gcg ctt gga att cgg cct cct 576 Gly Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu Gly Ile Arg Pro Pro 180 185 190 cct cca ggt cat gag gaa tcg gtt gaa gag ttt gtt cgt cgg ac 624 Pro Pro Gly His Glu Glu Ser Val Glu Glu Phe Val Arg Arg Asn Leu 195 200 205 ggt gat gag gtt ttt gaa cgg ttg ata gag cct ttt tgt tca ggg gtc 672 Gly Asp Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser Gly Val 210 215 220 tat gca ggc gat cct tca aaa tta agt atg aaa gca gca ttc ggg aaa 720 Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe Gly Lys 225 230 235 240 gtt tgg aag ctg gaa aaa aat ggt ggt agc att att ggt gga act ttc 768 Val Trp Lys Leu Glu Lys Asn Gly Gly Ser Ile Ile Gly Gly Thr Phe 245 250 255 aaa gca ata caa gag aga aat gga gct tca aaa cca cct cga gat ccg 816 Lys Ala Ile GlnGlu Arg Asn Gly Ala Ser Lys Pro Pro Arg Asp Pro 260 265 270 cgt ctg cca aaa cca aaa ggt cag act gtt gga tct ttc cgg aag gga 864 Arg Leu Pro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe Arg Lys Gly 275 280 285 ctt acc atg ttg cct gat gca att tct gcc aga cta ggc aac aaa gta 912 Leu Thr Met Leu Pro Asp Ala Ile Ser Ala Arg Leu Gly Asn Lys Val 290 295 300 aag tta tct tgg aag ctt tca agt att agt aaa ctg gat agt gga gag 960 Lys Leu Ser Trp Lys Leu Ser Ser Ile Ser Lys Leu Asp Ser Gly Glu 305 310 315 320 tac agt ttg aca tat gaa aca cca gaa gga gtg gtt tct ttg cag tgc 1008 Tyr Ser Leu Thr Tyr Glu Thr Pro Glu Gly Val Val Ser Leu Gln Cys 325 330 335 aaa act gtt gtc ctg acc att cct tcc tat gtt gct agt aca ttg ctg 1056 Lys Thr Val Val Leu Thr Ile Pro Ser Tyr Val Ala Ser Thr Leu Leu 340 345 350 cgt cct ctg tct gct gct gct gca gat gca ctt tca aag ttt tat tac 1104 Arg Pro Leu Ser Ala Ala Ala Ala Asp Ala Leu Ser Lys Phe Tyr Tyr 355 360 365 cct cca gtt gct gca gtt tcc ata tcc tat cca aaa gaa gct att aga 1152 Pro Pro Val Ala Ala Val Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg 370 375 380 tca gaa tgc ttg ata gat ggt gag ttg aag ggg ttt ggt caa ttg cat 1200 Ser Glu Cys Leu Ile Asp Gly Glu Leu Lys Gly Phe Gly Gln Leu His 385 390 395 400 cca cgt agc caa gga gtg gaa aca tta gga act ata tac agc tca tca 1248 Pro Arg Ser Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser 405 410 415 cta ttc ccc aac cga gca cca cct gga agg gtt cta ctc ttg aat tac 1296 Leu Phe Pro Asn Arg Ala Pro Pro Gly Arg Val Leu Leu Leu Asn Tyr 420 425 430 att gga gga gca act aat act gga att tta tcg aag acg gac agt gaa 1344 Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys Thr Asp Ser Glu 435 440 445 ctt gtg gaa aca gtt gat cga gat ttg agg aaa atc ctt ata aac cca 1392 Leu Val Glu Thr Val Asp Arg Asp Leu Arg Lys Ile Leu Ile Asn Pro 450 455 460 460 aat gcc cag gat cca ttt gta gtg ggg gtg aga ctg tgg cct caa gct 1440 Asn Ala Gln Asp Pro Phe Val Val Gly Val Arg Leu Trp Pro Gln Ala 465 470 475 475 480 att cca cag ttc tta gtt ggc cat ctt gat ctt ct a gat gtt gct aaa 1488 Ile Pro Gln Phe Leu Val Gly His Leu Asp Leu Leu Asp Val Ala Lys 485 490 495 gct tct atc aga aat act ggg ttt gaa ggg ctc ttc ctt ggg ggt aat 1536 Ala Ser Ile Arg Asn Thr Gly Phe Glu Gly Leu Phe Leu Gly Gly Asn 500 505 510 tat gtg tct ggt gtt gcc ttg gga cga tgc gtt gag gga gcc tat gag 1584 Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu 515 520 525 gta gca g gaa gta aac gat ttt ctc aca aat aga gtg tac aaa tag 1632 Val Ala Ala Glu Val Asn Asp Phe Leu Thr Asn Arg Val Tyr Lys 530 535 540 543 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 9 ggcggaggcg tcaccatggt ctgcatcgcc caggcc 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 10 gcctgcaggt cgacaactgc tactatttgt acactc 36 <210> 11 <211> 33 <212> DNA <2 13> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 11 cacaggaaag gtaccatggt ctgcatcgcc cag 33 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 12 cctgcagctc gagagctcct actatttgta cac 33 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220 > <223> Designed oligonucleotide primer to amplify Chlamydomonas PPO gene <400> 13 aatgatgttg acccagactc ctgggacc 28 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify Chlamydomonas PPO gene <400> 14 tactacacat cccagcaagc gccaatg 27 <210> 15 <211> 1838 <212> DNA <213> Chlamydomonas reinhardtii CC407 <220> <221> CDS <222> (2) ... (1693) <400> 15 a atg atg ttg acc cag act cct ggg acc gcc acg gct tct agc cgg 46 Met Met Leu Thr Gln Thr Pro Gly Thr Ala Thr Ala Ser Ser Arg 1 5 10 15 c gg tcg cag atc cgc tcg gct gcg cac gtc tcc gcc aag gtc gcg cct 94 Arg Ser Gln Ile Arg Ser Ala Ala His Val Ser Ala Lys Val Ala Pro 20 25 30 cgg ccc acg cca ttc tcg gtc gcg agc ccc gg acc agc ccc 142 Arg Pro Thr Pro Phe Ser Val Ala Ser Pro Ala Thr Ala Ala Ser Pro 35 40 45 gcg acc gcg gcg gcc cgc cgc aca ctc cac cgc act gct gcg gcg gcc 190 Ala Thr Ala Ala Ala Ala Arg Arg Thr Leu His Arg Thr Ala Ala Ala Ala 50 55 60 act ggt gct ccc acg gcg tcc gga gcc ggc gtc gcc aag acg ctc gac 238 Thr Gly Ala Pro Thr Ala Ser Gly Ala Gly Val Ala Lys Thr Leu Asp 65 70 75 aat gtg tat gac gtg atc gtg gtc ggt gga ggt ctc tcg ggc ctg gtg 286 Asn Val Tyr Asp Val Ile Val Val Gly Gly Gly Leu Ser Gly Leu Val 80 85 90 95 acc ggc cag gcc ctg gcg gct cag cac aaa att cag aac ttc ctt gtt 334 Thr Gln Ala Leu Ala Ala Gln His Lys Ile Gln Asn Phe Leu Val 100 105 110 acg gag gct cgc gag cgc gtc ggc ggc aac att acg tcc atg tcg ggc 382 Thr Glu Ala Arg Glu Arg Val Gly Gly Asn Ile Thr Ser Met Ser Gly 115 120 125 gat gg c tac gtg tgg gag gag ggc ccg aac agc ttc cag ccc aac gat 430 Asp Gly Tyr Val Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Asn Asp 130 135 140 agc atg ctg cag att gcg gtg gac tct ggc tgc gag agg gtg 478 Ser Met Leu Gln Ile Ala Val Asp Ser Gly Cys Glu Lys Asp Leu Val 145 150 155 ttc ggt gac ccc acg gct ccc cgc ttc gtg tgg tgg gag ggc aag ctg 526 Phe Gly Asp Pro Thr Ala Pro Arg Phe Val Trp Glu Gly Lys Leu 160 165 170 175 cgc ccc gtg ccc tcg ggc ctg gac gcc ttc acc ttc gac ctc atg tcc 574 Arg Pro Val Pro Ser Gly Leu Asp Ala Phe Thr Phe Asp Leu Met Ser 180 185 190 atc ccc ggc aag atcg gcc ggg ctg ggc gcc atc ggc ctc atc aac 622 Ile Pro Gly Lys Ile Arg Ala Gly Leu Gly Ala Ile Gly Leu Ile Asn195 200 205 Pro Ser Phe Glu Glu Ser Val Glu Gln Phe Ile Arg Arg 210 215 220 aac ctg ggc gat gag gtg ttc ttc cgc ctg atc gag ccc ttc tgc tcc 718 Asn Leu Gly Asp Glu Val Phe Phe Arg Leu Ile Glu Pro Phe Cy twenty three 0 235 ggc gtg tac gcg ggc gac ccc tcc aag ctg tcc atg aag gcg gcc ttc 766 Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe 240 245 250 255 aac agg atc tgg att ctg gag agg aac gg agc ctg gtg gga ggt 814 Asn Arg Ile Trp Ile Leu Glu Lys Asn Gly Gly Ser Leu Val Gly Gly 260 265 270 gcc atc aag ctg ttc cag gaa cgc cag tcc aac ccg gcc ccg ccg cgg 862 Ala Ile Lys Gln Ser Asn Pro Ala Pro Pro Arg 275 280 285 gac ccg cgc ctg ccg ccc aag ccc aag ggc cag acg gtg ggc tcg ttc 910 Asp Pro Arg Leu Pro Pro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe 290 295 300cgc aag ctg aag atg ctg ccg gac gcc att gag cgc aac atc ccc 958 Arg Lys Gly Leu Lys Met Leu Pro Asp Ala Ile Glu Arg Asn Ile Pro 305 310 315 gac aag atc cgc gtg aac tgg aag ctg gtg cct gc ggg Asp Lys Ile Arg Val Asn Trp Lys Leu Val Ser Leu Gly Arg Glu Ala 320 325 330 335 gac ggg cgg tac ggg ctg gtg tac gac acg ccc gag ggc cgt gtc aag 1054 Asp Gly Arg Tyr Gly Leu Val Tyr Asp Thr Pro Glu GlyArg Val Lys 340 345 350 350 gtg ttt gcc cgc gcc gtg gct ctg acc gcg ccc agc tac gtg gtg gcg 1102 Val Phe Ala Arg Ala Val Ala Leu Thr Ala Pro Ser Tyr Val Val Ala 355 360 365 gac ctg gtc aag gag cag gc gcc gcc gcc gag gcc ctg ggc tcc 1150 Asp Leu Val Lys Glu Gln Ala Pro Ala Ala Ala Glu Ala Leu Gly Ser 370 375 380 ttc gac tac ccg ccg gtg ggc gcc gtg acg ctg tcg tac ccg Prog Asg Pro Val Gly Ala Val Thr Leu Ser Tyr Pro Leu Ser 385 390 395 gcc gtg cgg gag gag cgc aag gcc tcg gac ggg tcc gtg ccg ggc ttc 1246 Ala Val Arg Glu Glu Arg Lys Ala Ser Asp Gly Ser Val Pro Gly Phe 400 405 410 415 ggt cag ctg cac ccg cgc acg cag ggc atc acc act ctg ggc acc atc 1294 Gly Gln Leu His Pro Arg Thr Gln Gly Ile Thr Thr Leu Gly Thr Ile 420 425 430 tac agc tcc agc ctg ttc ccc ggc cgggggggg cac atg ctg 1342 Tyr Ser Ser Ser Leu Phe Pro Gly Arg Ala Pro Glu Gly His Met Leu 435 440 445 ctg ctc aac tac atc ggc ggc acc acc aac cgc ggc atc gtc aac cag 1390 Leu Leu Asn Tyr Ile Gly Gly Thr T hr Asn Arg Gly Ile Val Asn Gln 450 455 460 acc acc gag cag ctg gtg gag cag gtg gac aag gac ctg cgc aac atg 1438 Thr Thr Glu Gln Leu Val Glu Gln Val Asp Lys Asp Leu Arg Asn Met 465 470 470 475 gtc atc aag ccc gac gcg ccc aag ccc cgt gtg gtg ggc gtg cgc gtg 1486 Val Ile Lys Pro Asp Ala Pro Lys Pro Arg Val Val Gly Val Arg Val 480 485 490 495 tgg ccg cgc gcc atc ccg cag ttc aac ctg ggcac gc 1534 Trp Pro Arg Ala Ile Pro Gln Phe Asn Leu Gly His Leu Glu Gln Leu 500 505 510 gac aag gcg cgc aag gcg ctg gac gcg gcg ggg ctg cag ggc gtg cac 1582 Asp Lys Ala Arg Lys Ala Leu Asp Ala Ala Gla Gly Val His 515 520 525 ctg ggg ggc aac tac gtc agc ggt gtg gcc ctg ggc aag gtg gtg gag 1630 Leu Gly Gly Asn Tyr Val Ser Gly Val Ala Leu Gly Lys Val Val Glu 530 535 540 cac ggc tac gcc tcc ctg gcc aag agc gtg tcc aag gcc 1678 His Gly Tyr Glu Ser Ala Ala Asn Leu Ala Lys Ser Val Ser Lys Ala 545 550 555 gca gtc aag gcc taa gcggctgcag cagtagcagc agcagcatcg ggctgtagsct 1733 Ala Vals a 560 563 ggtaaatgcc gcagtggcac cggcagcagc aattggcaag cacttggggc aagcggagtg 1793 gaggcgaggg gggggctacc attggcgctt gctgggatgt gtagt 1838 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence Identified Sequencer <220> of Chlamydomonas PPO gene <400> 16 ggtcggtgga ggggatccga tgctggtgac cg 32 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA fragment having par tial sequence of Chlamydomonas PPO gene <400> 17 gctactgctg cgagctctta ggccttgact gc 32 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify Arabidopsis ferrochelat ase gene <400> 18 gatcggttct gaaatttgga tccatgcagg c 31 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify Arabidopsis ferrochelatase gene <400> 19 cacaaaacca acgagctcct ataggttccg g 31 <210> 20 <211> 1401 <212> DNA <213> Arabidop sis thaliana WS <220> <221> CDS <222> (1) ... (1401) <400> 20 atg cag gca acg gct tta tca tct ggg ttc aat cct cta acg aaa cgt 48 Met Gln Ala Thr Ala Leu Ser Ser Gly Phe Asn Pro Leu Thr Lys Arg 1 5 10 15 aaa gat cac aga ttt ccc agg tca tgc tct cag aga aat tct ctg tct 96 Lys Asp His Arg Phe Pro Arg Ser Cys Ser Gln Arg Asn Ser Leu Ser 20 25 30 ttg att caa tgc gat ata aaa gag aga tct ttc gga gag tct atg acg 144 Leu Ile Gln Cys Asp Ile Lys Glu Arg Ser Phe Gly Glu Ser Met Thr 35 40 45 atc acg aat cgt gga ttg agt ttt aag acg aat gtg ttt gag caa gct 192 Ile Thr Asn Arg Gly Leu Ser Phe Lys Thr Asn Val Phe Glu Gln Ala 50 55 60 cgt tct gtg act gga gac tgt tct tat gat gaa act tca gca aaa gca 240 Arg Ser Val Thr Gly Asp Cys Ser Tyr Asp Glu Thr Ser Ala Lys Ala 65 70 75 80 cgt tct cat gtt gtt gca gaa gat aag att ggt gtc ttg ctt ttg aat 288 Arg Ser His Val Val Ala Glu Asp Lys Ile Gly Val Leu Leu Leu Asn 85 90 95 tta ggt ggt cct gaa act ctt aac gat gtt caa cct ttc ttg tat aat 336 Leu Gly Gly Pro Glu Thr Leu Asn Asp Val Gln Pro Phe Leu Tyr Asn 100 105 110 ctc ttt gct gat ccg gat att ata agg ctt cct aga cca ttt cag ttt 384 Leu Phe Ala Asp Pro Asp Ile Ile Arg Leu Pro Arg Pro Phe Gln Phe 115 120 125 ctt caa ggg act ata gct aag ttt ata tct gtt gtt cgt gct ccg aaa 432 Leu Gln Gly Thr Ile Ala Lys Phe Ile Ser Val Val Arg Ala Pro Lys 130 135 140 tct aaa gaa ggg tat gct gct att ggt ggt ggc tct cct ttg cgt aaa 480 Ser Lys Glu Gly Tyr Ala Ala Ile Gly Gly Gly Ser Pro Leu Arg Lys 145 150 155 160 ata act gat gag caa gcg gat gct att aag atg tct ttg caa gcg aag 528 Ile Thr Asp Glu Gln Ala Asp Ala Ile Lys Met Ser Leu Gln Ala Lys 165 170 175 aac att gct gct aat gtc tat gtt ggt atg cgg tat tgg tat ccg ttc 576 Asn Ile Ala Ala Asn Val Tyr Val Gly Met Arg Tyr Trp Tyr Pro Phe 180 185 190 act gag gag gct gtt cag cag ata aag aag gac aaa att act aga ctt 624 Thr Glu Glu Ala Val Gln Gln Ile Lys Lys Asp Lys Ile Thr Arg Leu 195 200 205 gtt gta ctg cca ttg tat cct cag tat tct atc tct aca acg ggt tca 672 Val Val Leu Pro Leu Tyr Pro Gln Tyr Ser Ile Ser Thr Thr Gly Ser 210 215 220 agc ata cgc gtt ctc caa gat tta ttc agg aaa gat ccg tac cta gct 720 Ser Ile Arg Val Leu Gln Asp Leu Phe Arg Lys Asp Pro Tyr Leu Ala 225 230 235 240 gga gtg ccg gta gct att ata ata aag tcc tgg tac caa agg cga ggc tat 768 Gly Val Pro Val Ala Ile Ile Lys Ser Trp Tyr Gln Arg Arg Gly Tyr 245 250 255 gtc aat tct atg gct gac ctc att gag aag gag ctt caa act ttc tct 816 Val Asn Ser Met Ala Asp Leu Ile Glu Lys Glu Leu Gln Thr Phe Ser 260 265 270 gat cct aag gag gtt atg ata ttc ttc agt gcc cat ggt gtt ccg gtc 864 Asp Pro Lys Glu Val Met Ile Phe Phe Ser Ala His Gly Val Pro Val 275 280 285 agc tac gta gag aat gct gga gat ccg tac cag aag cag atg gaa gag 912 Ser Tyr Val Glu Asn Ala Gly Asp Pro Tyr Gln Lys Gln Met Glu Glu 290 295 300 tgt att gac ttg ata atg gaa gag cta aaa gcc aga ggg gtt ctt aac 960 Cys Ile Asp Leu Ile Met Glu Glu Leu Lys Ala Arg Gly Val Leu Asn 305 310 315 320 gac cat aaa ttg gca tac cag agt cgt gtt ggc cct gtt caa 1008 Asp His Lys Leu Ala Tyr Gln Ser Arg Val Gly Pro Val Gln Trp Leu 325 330 335 aag cca tac acc gat gag gtt ctt gtc gac ctt ggt aag agt ggt gtt 1056 Lys Pro Tyr Thr Asp Glu Val Leu Val Asp Leu Gly Lys Ser Gly Val 340 345 350 aag agt cta cta gcc gtt cca gtc agt ttc gtg agt gag cac att gag 1104 Lys Ser Leu Leu Ala Val Pro Val Ser Phe Val Ser Glu His Ile Glu 355 360 365 aca ctt gag gag ata gac atg gag tat agg gaa tta gct ctt gag tca 1152 Thr Leu Glu Glu Ile Asp Met Glu Tyr Arg Glu Leu Ala Leu Glu Ser 370 375 380 ggg gta gag aac tgg gga cgg gta ccc gcg cta ggt ctc aca cca tcc 1200 Gly Val Glu Asn Gly Arg Val Pro Ala Leu Gly Leu Thr Pro Ser 385 390 395 400 ttc atc acc gac tta gct gat gca gtg ata gaa tca ctt cct tca gca 1248 Phe Ile Thr Asp Leu Ala Asp Ala Val Ile Glu Ser Leu Pro Ser Ala 405 410 415 gaa gca atg tca aac cca aat gca gtg gtt gac tca gaa gat agc gag 1296 Glu Ala Met Ser Asn Pro Asn Ala Val Val Asp Ser Glu Asp Ser Glu 420 425 430 tcg tca gat gct ttc agt tac att gtc aag atg ttc ttc ggt tcg att 1344 Ser Ser Asp Ala Phe Ser Tyr Ile Val Lys Met Phe Phe Gly Ser Ile 435 440 445 ctg gct ttc gtc cta ctt ctc tcc cca aag atg ttc cat gcg ttc cgg 1392 Leu Ala Lehe Leu Leu Pro Lys Met Phe His Ala Phe Arg 450 455 460 aac cta tag 1401 Asn Leu 465 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify Soybean coproporphyrino gen III oxidase gene <400> 21 gaatcggatc cgaagcatga tgcattgtgc 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify Soybean coproporphyrino gen III oxidase gene <400> 22 gggggtcgac tgatgaatta gatccattcc 30 <210> 23 <211> 1158 <212> DNA <213> Glycine max cv. Jack <220> <221> CDS <222> (1) ... (1158) <400> 23 atg atg cat tgt gcg agc att gtc tcg gct ccg tcc tac gcg ttc cct 48 Met Met His Cys Ala Ser Ile Val Ser Ala Pro Ser Tyr Ala Phe Pro 1 5 10 15 ttt ctc tct ggc tcc gct tcc act act cca act gcg atc tcg ctc act 96 Phe Arg Ser Gly S er Ala Ser Thr Thr Pro Thr Ala Ile Ser Leu Thr 20 25 30 aag cgc agt tgg aag cca cct ccg agc atg gca aaa ggc cca gtc aga 144 Lys Arg Ser Trp Lys Pro Pro Pro Ser Met Ala Lys Gly Pro Val Arg 35 40 45 gcc acc gtt tct ata gag aaa gag acc ccg gag gcc aat cgt ccc gaa 192 Ala Thr Val Ser Ile Glu Lys Glu Thr Pro Glu Ala Asn Arg Pro Glu 50 55 60 acg ttt ctc aga gga gtg gac gag gcc cag tct tcc act tcg gtt cgg 240 Thr Phe Leu Arg Gly Val Asp Glu Ala Gln Ser Ser Thr Ser Val Arg 65 70 75 80 gcc cgc ttc gag aag atg ata agg gag gcc cag gac acc gtg tgc agt 288 Ala Arg Phe Glu Lys Met Ile Arg Glu Ala Gln Asp Thr Val Cys Ser 85 90 95 gcc ctc gag gcc gct gat ggt ggg gcc cag ttc aag gag gac gtt tgg 336 Ala Leu Glu Ala Ala Asp Gly Gly Ala Gln Phe Lys Glu Asp Val Trp 100 105 110 tcc agg ccc ggt ggc ggc ggt ggc att agc agg gtc ctt caa gac ggt 384 Ser Arg Pro Gly Gly Gly Gly Gly Ile Ser Arg Val Leu Gln Asp Gly 115 120 125 gcc gtt tgg gag aag gct ggg gtt aat gtc tct gtt gtt tac ggt gtc 432 Val Trp Glu Lys A la Gly Val Asn Val Ser Val Val Tyr Gly Val 130 135 140 atg cca cct gac gca tat cgt gct gcc aag ggt gtt cct acc gat caa 480 Met Pro Pro Asp Ala Tyr Arg Ala Ala Lys Gly Val Pro Thr Asp Gln 145 150 155 160 aaa cct ggt cct gtg cct ttc ttc gct gct gga atc agc tcc gtt tta 528 Lys Pro Gly Pro Val Pro Phe Phe Ala Ala Gly Ile Ser Ser Val Leu 165 170 175 cac cca aag aac ccg ttt gcc cct acc cta cat ttc aac tat cgc tat 576 His Pro Lys Asn Pro Phe Ala Pro Thr Leu His Phe Asn Tyr Arg Tyr 180 185 190 ttt gaa acc gat gct cct aaa ggt gct cct gga gca cct aga caa tgg 624 Phe Glu Thr Asp Ala Pro Lys Gly Ala Pro Gly Ala Pro Arg Gln Trp 195 200 205 tgg ttt ggt ggg gga act gac ttg aca cca gct tac att ttt gag gag 672 Trp Phe Gly Gly Gly Gly Thr Asp Leu Thr Pro Ala Tyr Ile Phe Glu Glu 210 215 220 gat gtt aag cac ttc cat tca att caa aaa caa gcc tgt gac aag ttt 720 Asp Val Lys His Phe His Ser Ile Gln Lys Gln Ala Cys Asp Lys Phe 225 230 235 240 gaa cct act ttt tac ccc cga ttc aag aaa tgg tgt gat gat tac ttt 768 Glu P ro Thr Phe Tyr Pro Arg Phe Lys Lys Trp Cys Asp Asp Tyr Phe 245 250 255 tat atc aag cat cga ggt gaa agg aga ggg ctt gga gga ata ttt ttt 816 Tyr Ile Lys His Arg Gly Glu Arg Arg Gly Leu Gly Gly Ile Phe Phe 260 265 270 gat gat ctt aat gac tat gat cag gag atg ctc ctt tcc ttt gct act 864 Asp Asp Leu Asn Asp Tyr Asp Gln Glu Met Leu Leu Ser Phe Ala Thr 275 280 285 ggt tgt gca aat tct gtt att cct gct tat tta cct atc ata gag aaa 912 Gly Cys Ala Asn Ser Val Ile Pro Ala Tyr Leu Pro Ile Ile Glu Lys 290 295 300 agg aag gat ttg ccc ttc aat gat cat cag aaa gca tgg caa caa ttg 960 Arg Lys Asp Leu Pro Phe Asn Asp His Gln Lys Ala Trp Gln Gln Leu 305 310 315 320 cga agg gga cga tat gtt gaa ttc aat ttg gta tat gat agg ggt aca 1008 Arg Arg Gly Arg Tyr Val Glu Phe Asn Leu Val Tyr Asp Arg Gly Thr 325 330 335 aca ttt gga ctg aaa act gga ggg aga ata gag agt ata ctt gtt tct 1056 Thr Phe Gly Leu Lys Thr Gly Gly Arg Ile Glu Ser Ile Leu Val Ser 340 345 350 ctc cca ctg act gct cgg tgg gaa tac gat cat aaa ccg gaaga gga 1104 Leu Pro Leu Thr Ala Arg Trp Glu Tyr Asp His Lys Pro Glu Glu Gly 355 360 365 agc gaa gaa tgg aaa ctc ttg gac gca tgc atc aac ccc aag gaa tgg 1152 Ser Glu Glu Trp Lys Leu Leu Asp Ala Cys Pro Lys Glu Trp 370 375 380 atc taa 1158 Ile 385

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pCREPSPS. CT
P-EPSPS is a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of a nucleotide sequence encoding a chloroplast transit peptide of petunia-derived EPSPS, and lac pro means a lactose operon promoter sequence. Amp ^r is an ampicillin resistance gene, Km
^r represents the kanamycin resistance gene, and ori represents the origin of replication.

FIG. 2 shows a restriction map of plasmid pTCHLH1. TCH
LH is a protoporphyrin IX binding subunit gene of tobacco magnesium keratase lacking a chloroplast translocation signal, and lac pro means a promoter sequence of a lactose operon. Amp ^r is an ampicillin resistance gene, Km ^r is a kanamycin resistance gene, and ori is a replication origin.

FIG. 3 shows a restriction map of plasmid pNG01. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII represents the kanamycin resistance gene, GUS represents the β-glucuronidase gene, and RB and LB represent the right and left border sequences of T-DNA.

FIG. 4 shows a restriction map of plasmid pNG04. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII represents the kanamycin resistance gene, GUS represents the β-glucuronidase gene, and RB and LB represent the right and left border sequences of T-DNA.

FIG. 5 shows a restriction map of plasmid pNT35S. NT is the nopaline synthase gene terminator sequence, 35S
Represents the cauliflower mosaic virus 35S promoter, and lac pro represents the promoter sequence of the lactose operon. Amp ^r is an ampicillin resistance gene, or
i represents the duplication start point.

FIG. 6 shows a restriction map of plasmid pCENS. CTP-E
PSPS is a chimeric gene in which the Agrobacterium-derived EPSPS gene is linked downstream of the base sequence encoding the chloroplast transit peptide of petunia-derived EPSPS, NT is the terminator sequence of the nopaline synthase gene, and 35S is the cauliflower. The mosaic virus 35S promoter and lac pro mean the lactose operon promoter sequence. Amp ^r represents an ampicillin resistance gene, and ori represents a replication origin.

FIG. 7 shows a restriction map of plasmid pCENSK. CTP-
EPSPS is a chimeric gene in which the EPSPS gene derived from Agrobacterium is linked downstream of the base sequence encoding the chloroplast transit peptide derived from Petunia, NT is the terminator sequence of the nopaline synthase gene, and 35S is the cauliflower. The mosaic virus 35S promoter and lac pro mean the promoter sequence of the lactose operon. Amp ^r represents an ampicillin resistance gene, and ori represents a replication origin.

FIG. 8 shows a restriction map of plasmid pBICE. CTP-E
PSPS is a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of the base sequence encoding the chloroplast transit peptide of Petunia-derived EPSPS, NP is the promoter sequence of the nopaline synthase gene, and NT is the nopaline 35S means the 35S promoter of the cauliflower mosaic virus. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 9 shows a restriction map of plasmid pNO. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycin resistance gene, RB and L
B represents the right and left border sequence of T-DNA.

FIG. 10 shows a restriction map of plasmid pBITCHLH.
TCHLH is a protoporphyrin IX binding subunit gene of tobacco magnesium keratase lacking chloroplast translocation signal, NP is the promoter sequence of nopaline synthase gene, NT is the terminator sequence of nopaline synthase gene, 35S is Cauliflower mosaic virus 3
Means 5S promoter. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 11 shows a restriction map of plasmid pBICETCH.
CTP-EPSPS is a chimeric gene in which the Agrobacterium-derived EPSPS gene is linked downstream of the nucleotide sequence encoding the chloroplast transit peptide of Petunia-derived EPSPS, and TCHLH is a tobacco that lacks the chloroplast translocation signal. Protoporphyrin IX binding subunit gene of magnesium keratase. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 12 shows a restriction map of plasmid pACYCSP. P
PO is the soybean protoporphyrinogen IX oxidase gene, meaning the promoter sequence of the lac pro lactose operon. Cm ^r represents a chloramphenicol resistance gene, and ori represents a replication origin.

FIG. 13 shows a restriction map of plasmid pTVGMP. GM
P is a soybean PPO gene lacking a chloroplast translocation signal and a FAD binding sequence, and lac pro means a promoter sequence of a lactose operon. Amp ^r represents an ampicillin resistance gene, and ori represents a replication origin.

FIG. 14 shows a restriction map of plasmid pBIGMP. GM
P is the soybean PPO gene lacking the chloroplast translocation signal and the FAD binding sequence, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, and 35S is the cauliflower mosaic virus. Means 35S promoter. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 15 shows a restriction map of plasmid pBICEGMP.
CTP-EPSPS is a chimeric gene in which the Agrobacterium-derived EPSPS gene is linked downstream of the nucleotide sequence encoding the chloroplast transit peptide derived from Petunia, and GMP is a chloroplast translocation signal and FAD binding sequence. This is a soybean PPO gene lacking. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycin resistance gene, RB and LB are T-DNA
Represents the left and right border array.

FIG. 16 shows a restriction map of plasmid pTVCRP. CR
P is the Chlamydomonas reinhardtii PPO gene lacking the chloroplast translocation signal and the FAD binding sequence, and lac pro means the promoter sequence of the lactose operon. Also, Am
p ^r is the ampicillin resistance gene, ori represents an origin of replication.

FIG. 17 shows a restriction map of plasmid pBICRP. CR
P is the Chlamydomonas reinhardtii PPO gene lacking the chloroplast translocation signal and the FAD binding sequence, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, and 35S is the cauliflower mosaic virus. Means 35S promoter. NPTII is a kanamycin resistance gene, RB and LB are T-DNA
Represents the left and right border array.

FIG. 18 shows a restriction map of plasmid pBICECRP.
CTP-EPSPS is a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of a nucleotide sequence encoding a chloroplast transit peptide derived from Petunia, and CRP is a chloroplast translocation signal and a FAD binding sequence. Is the P. chrysalis PPO gene lacking. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycin resistance gene, RB and LB
Represents the right and left border sequence of T-DNA.

FIG. 19 shows a restriction map of plasmid pCRATF. AT
F is a chloroplast-localized Arabidopsis ferrochelatase gene, and lac pro means a lactose operon promoter sequence. Amp ^r is an ampicillin resistance gene, Km ^r is a kanamycin resistance gene, and ori is a replication origin.

FIG. 20 shows a restriction map of plasmid pBIATF. AT
F is the chloroplast-localized Arabidopsis ferrochelatase gene, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, and 35S is the cauliflower mosaic virus 35S promoter . NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 21 shows a restriction map of plasmid pBICEATF.
CTP-EPSPS is a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of a nucleotide sequence encoding a chloroplast transit peptide derived from Petunia, and ATF is a chloroplast-localized Arabidopsis ferrokera It is a Tase gene. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 22 shows a restriction map of plasmid pCRSCPOX.
SCPOX is the soybean coproporphyrinogen III oxidase gene, and lac pro means the promoter sequence of the lactose operon. Amp ^r is an ampicillin resistance gene, Km ^r is a kanamycin resistance gene, and ori is a replication origin.

FIG. 23 shows a restriction map of plasmid pBISCPOX.
SCPOX is the soybean coproporphyrinogen III oxidase gene, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, and 35S is the 35S promoter of cauliflower mosaic virus. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 24 shows a restriction map of plasmid pBICESCPOX. CTP-EPSPS is a chimeric gene in which an Agrobacterium-derived EPSPS gene is linked downstream of a partial nucleotide sequence encoding a chloroplast transit peptide derived from petunia, and SCPOX is a soybean coproporphyrinogen III oxidase gene. It is. NP means the promoter sequence of the nopaline synthase gene, NT means the terminator sequence of the nopaline synthase gene, and 35S means the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycin resistance gene, RB and LB are T-DNA
Represents the left and right border array.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12N 5/00 C // A01N 57/20 15/00 ZNAA

Claims (56)

[Claims] 1. A plant having natural 5-enolpyruvylshikimate-3-phosphate synthase having at least one enzyme activity selected from the following (1) and (2): A plant that exhibits tolerance to an activity-inhibiting amount of a herbicide, comprising: (1) a 5-enol substantially different from the plant's natural 5-enol pyruvylshikimate-3-phosphate synthase activity Pyruvylshikimate-3-phosphate synthase activity. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. In addition, a plant which has a gene encoding a protein having the following properties (3), (4) and (5) introduced therein and expresses the gene. (3) It specifically binds to a substance related to the herbicidal activity of a protoporphyrinogen IX oxidase-inhibiting herbicide. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 2. The plant according to claim 1, wherein the gene is a gene operably linked to a promoter operable in plant cells and a terminator operable in plant cells. 3. The method according to claim 1, wherein the protein is protoporphyrinogen.
3. The plant according to claim 1, which is a protein having a property of specifically binding to a substance contained as an active ingredient in an IX oxidase-inhibiting herbicide. 4. A protein having a property of specifically binding to a plant cell endogenous substance involved in the expression of protoporphyrinogen IX oxidase inhibitory activity of a protoporphyrinogen IX oxidase-inhibiting herbicide in a plant cell. The plant according to claim 1 or 2. 5. A protoporphyrinogen IX oxidase.
Inhibition type herbicide is a compound of any of the following (1) to (3)
Protoporphy containing a compound selected from among the active ingredients
Claim 1 which is a linogen IX oxidase inhibitory herbicide.
5. The plant according to any one of to 4 above. (1) Chlormethoxynil, bifenox, chlorni
Trophen (CNP), acifluorfen [5- [2-chloro-4
-(Trifluoromethyl) phenoxy] -2-nitrobenzoate
Acid] and its ethyl ester, acifluorfen
Sodium, oxyfluorfen [2-chloro-1- (3-E
(Toxi-4-nitrophenoxy) -4-trifluoromethylbe
Benzene), oxadiazone (3- [2,4-dichloro-5- (1-
Tylethoxy) phenyl] -5- (1,1-dimethylethyl) -1,3,
4-oxadiazole-2 (3H) -one], S-23142 [2- [4-
Loro-2-fluoro-5- (prop-2-iniloxy) phenyl]
-2,3,4,5,6,7-Hexahydro-1H-isoindole-1,3-di
On), chlorophthalim (N- (4-chlorophenyl) -3,4,
5,6-tetrahydrophthalimide), TNPP-ethyl (ethyl
2- [1- (2,3,4-trichlorophenyl) -4-nitropyrazoli
-5-oxy] propionate], LS82-556 [N3- (1-Fe
Nylethyl) -2,6-dimethyl-5-propionylnicotinia
Mido] (2) Compound JG represented by general formula I (general formula I) wherein G is a group represented by any of the following G-1 to G-9,
J is a group represented by any of the following J-1 to 30. Where the dashed lines in equations J-5, J-6, J-12 and J-24
Indicates that the ring on the left contains only a single bond or
Represents that one bond is a double bond between carbon atoms,
X represents an oxygen atom or a sulfur atom; Y represents an oxygen atom or
Represents a sulfur atom; R¹ Is a hydrogen atom or a halogen atom
And R^Two Is a hydrogen atom, C₁-C₈ Alkyl group, C₁-C₈ C
Roalkyl group, halogen atom, hydroxyl group, -OR²⁷ Group, -SH
Group, -S (O) pR²⁷ Group, -COR²⁷ Group, -CO_TwoR²⁷ Group, -C (O) SR²⁷
Group, -C (O) NR²⁹R³⁰ Group, -CHO group, -CR²⁷= NOR³⁶ Group, -CH =
CR³⁷CO_TwoR²⁷ Group, -CH_TwoCHR ³⁷CO_TwoR²⁷ Group, -CO_TwoN = CR³¹R³² 
Group, nitro group, cyano group, -NHSO_TwoR³³ Group, -NHSO_TwoNHR³³ 
Group, -NR²⁷R³⁸ Group, -NH_Two Group or one or more of the same species
Or different C1-C4 alkyl groups
Represents a phenyl group, p represents 0, 1 or 2;^Three Is
 C₁-C_Two Alkyl group, C₁-C_Two Haloalkyl group, -OCH_Three 
Group, -SCH_Three Group, -OCHF_TwoGroup, halogen atom, cyano group,
Or a nitro group, R^Four Is a hydrogen atom, C₁-C_Three Alkyl
Group, C₁-C_Three Shows haloalkyl group or halogen atom
Then R^Five Is a hydrogen atom, C₁-C_Three Alkyl group, halogen source
Child, C₁-C_Three Haloalkyl group, cyclopropyl group, vinyl
Group, C_Two Alkynyl group, cyano group, -C (O) R³⁸ Group, -CO_TwoR^Three
8 Group, -C (O) NR³⁸R³⁹ Group, -CR³⁴R³⁵CN group, -CR³⁴R³⁵C (O)
R³⁸ Group, -CR³⁴R³⁵CO_TwoR³⁸Group, -CR³⁴R³⁵C (O) NR³⁸R³⁹ Group,
-CHR³⁴OH group, -CHR³⁴OC (O) R³⁸ Group, or -OCHR³⁴OC
(O) NR³⁸R³⁹ Represents a group, or G is G-2 or
 R for G-6^FourAnd R^FiveIs the carbon source to which they are attached
And a child may represent a C = O group;⁶ Is C₁-C₆ Al
Kill group, C₁-C₆ Haloalkyl group, C_Two-C₆ Alkoxyal
Kill group, C_Three-C₆ Alkenyl group, or C_Three-C₆ Alkini
X¹ Is a direct bond, an oxygen atom, a sulfur atom, -NH
Group, -N (C₁-C_Three Alkyl) group, -N (C₁-C_ThreeHaloalkyl)
Group, or -N (allyl) group, R⁷ Is a hydrogen atom, C₁-C
₆ Alkyl group, C₁-C₆ Haloalkyl group, halogen atom,
-S (O)_Two(C₁-C₆Alkyl) group, or -C (= O) R⁴⁰ Table
Then R⁸ Is a hydrogen atom, C₁-C₈ Alkyl group, C_Three-C₈ Cyclo
Alkyl group, C_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl
Group, C₁-C₈ Haloalkyl group, C_Two-C₈ Alkoxyalkyl
Group, C_Three-C₈ Alkoxyalkoxyalkyl group, C_Three-C₈ C
Loalkynyl group, C_Three-C₈Haloalkenyl group, C₁-C₈ Al
Killsulfonyl group, C₁-C₈ Haloalkylsulfonyl group,
C_Three-C₈ Alkoxycarbonylalkyl group, -S (O)_TwoNH (C₁-
C₈ Alkyl) group, -C (O) R⁴¹ Group, or on the phenyl ring
R⁴² Represents a benzyl group which may be substituted with n and n and
And m are each independently 0, 1, 2 or 3;
And m + n represents 2 or 3, and Z is -CR⁹R^Ten Group, oxygen
Atom, sulfur atom, -S (O) group, -S (O)_Two Group, or -N (C₁-
C _Four Alkyl) group, each R⁹ Is independently hydrogen
Atom, C₁-C_Three Alkyl group, halogen atom, hydroxy
Group, C₁-C₆ Alkoxy group, C₁-C₆ Haloalkyl group, C₁-C
₆ Haloalkoxy group, C_Two-C₆ Alkylcarbonyloxy
Group or C_Two-C₆ A haloalkylcarbonyloxy group
Represent each R^Ten Is independently a hydrogen atom, C₁-C_Three A
Represents a alkyl group, a hydroxy group, or a halogen atom,
R¹¹ And R¹² Are each independently a hydrogen atom, halogen
Atom, C₁-C₆ Alkyl group, C_Three-C₆ Alkenyl groups, also
Is C₁-C₆ Represents a haloalkyl group, R¹³ Is a hydrogen atom, C₁-
C₆ Alkyl group, C₁-C₆ Haloalkyl group, C_Three-C₆ Arche
Nyl group, C_Three-C₆ Haloalkenyl group, C_Three-C₆ Alkynyl
Group, C_Three-C₆ Haloalkynyl group, HC (= 0) group, (C₁-C_Four Al
Kill) C (= O) group, or -NH_Two Represents a group, R¹⁴ Is C₁-C₆ 
Alkyl group, C₁-C₆ Alkylthio group, C₁-C₆ Haloalk
Or -N (CH_Three)_Two W represents a nitrogen atom or
 -CR^Fifteen Represents a group, R^Fifteen Is a hydrogen atom, C₁-C₆ Alkyl
Group, halogen atom, or C₁-C₆ No alkyl group, 1
And two halogen atoms, C₁-C₆ An alkoxy group or
-CF_Three Represents a phenyl group which may be substituted with
Each Q independently represents an oxygen or sulfur atom
Then Q¹ Represents an oxygen atom or a sulfur atom; Z¹ Is -CR¹⁶
R¹⁷ Group, oxygen atom, sulfur atom, -S (O) group, -S (O)_Two Group,
Or -N (C₁-C_Four Alkyl) group, each R¹⁶ 
Is independently a hydrogen atom, a halogen atom, a hydroxy group, C₁
-C₆ Alkoxy group, C₁-C₆ Haloalkyl group, C₁-C₆ Halo
Alkoxy group, C_Two-C₆ An alkylcarbonyloxy group,
Or C_Two-C₆ Represents a haloalkylcarbonyloxy group,
Each R¹⁷ Is independently a hydrogen atom, a hydroxy group,
Or represents a halogen atom, R¹⁸ Is C₁-C₆ Alkyl
Group, halogen atom, or C₁-C₆ Table showing haloalkyl groups
Then R¹⁹ And R²⁰ Are each independently a hydrogen atom, C₁-
C₆ Alkyl group, or C₁-C ₆ Represents a haloalkyl group,
Z^Two Is an oxygen atom, a sulfur atom, -NR⁹ Group, or -CR⁹R^Ten 
Represents a group, R^{twenty one} And R^{twenty two} Are each independently C₁-C₆ 
Alkyl group, C₁-C₆ Haloalkyl group, C_Three-C₆ Alkenyl
Group, C_Three-C₆ Haloalkenyl group, C_Three-C₆ Alkynyl group
Or C_Three-C₆ Represents a haloalkynyl group, R^{twenty three} Is a hydrogen atom,
Represents a halogen atom or a cyano group, R^{twenty four} Is C₁-C₆ 
Alkylsulfonyl group, C₁-C₆ Alkyl group, C₁-C₆ C
Loalkyl group, C_Three-C₆ Alkenyl group, C_Three-C₆ Alkynyl
Group, C₁-C₆ Alkoxy group, C₁-C₆Haloalkoxy groups
Or a halogen atom, R^{twenty five} Is C₁-C₆ Alkyl group, C
₁-C₆ Haloalkyl group, C_Three-C₆ Alkenyl group, or C_Three
-C₆ Represents an alkynyl group, R²⁶ Is C₁-C₆ Alkyl group, C
₁-C₆ Haloalkyl group or C on the ring₁-C₆ Alkyl
Group, one or two halogen atoms, one or two
Toro group, C₁-C₆ Alkoxy group, and CF_Three Consisting of
Even if substituted with a substituent selected from the group
Represents a good phenyl group, W¹ Represents a nitrogen atom or a CH group
And T is any of the following general formulas T-1, T-2, or T-3
Represents a group of(Where E¹, E^Two, E^Three, E^Four, E^Five, E⁶, E⁷, E⁸, E⁹, E^Ten, E
¹¹, And E¹² Is a hydrogen atom or C₁-C_Three Al
Represents a kill group. ) R²⁷ Is C₁-C₈ Alkyl group, C_Three-C₈ Cycloalkyl group, C
_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl group, C₁-C₈ Halo
Alkyl group, C_Two-C₈ Alkoxyalkyl group, C_Two-C₈Al
Quilthioalkyl group, C_Two-C₈ Alkylsulfinyl al
Kill group, C_Two-C₈ Alkylsulfonylalkyl group, C₁-C
₈ Alkyl sulfonyl group, halogen source on phenyl ring
Child and C₁-C_Four From the group consisting of alkyl groups
Substituted with at least one selected substituent
Phenylsulfonyl group, C_Four-C₈ Alkoxyal
Coxyalkyl group, C_Four-C₈ Cycloalkylalkyl group,
C₆-C₈ Cycloalkoxyalkyl group, C_Four-C₈ Alkenyl
Oxyalkyl group, C_Four-C₈Alkynyloxyalkyl
Group, C_Three-C₈ Haloalkoxyalkyl group, C_Four-C₈ Haloal
Kenyloxyalkyl group, C_Four-C₈ Haloalkynyloxy
Alkyl group, C₆-C₈ Cycloalkylthioalkyl group, C_Four
-C₈ Alkenylthioalkyl group, C_Four-C₈ Alkynylthio
Alkyl group, halogen atom on the ring, C₁-C_Three Alkyl group
And C₁-C_Three From the group consisting of haloalkyl groups
Substituted with at least one selected substituent
Substituted with a good phenoxy group₁-C_Four Alkyl group,
Halogen atom on the ring, C₁-C_Three Alkyl group and C₁-C_Three 
Selected from the group consisting of haloalkyl groups
Benzy optionally substituted with at least one substituent
C substituted with a ruoxy group₁-C_Four Alkyl group, C_Four-C₈bird
Alkylsilylalkyl group, C_Three-C₈ Cyanoalkyl group,
C_Three-C₈ Halocycloalkyl group, C_Three-C₈ Haloalkenyl
Group, C_Five-C₈ Alkoxyalkynyl group, C_Five-C₈ Halo alco
Xylalkynyl group, C_Five-C₈ Alkylthioalkenyl group,
C_Three-C₈ Haloalkynyl group, C_Five-C₈ Alkoxyalkynyl
Group, C_Five-C₈ Haloalkoxyalkynyl group, C_Five-C₈ Archi
Luthioalkynyl group, C_Two-C₈ Alkylcarbonyl group, ring
A halogen atom, C₁-C_Three Alkyl group and C₁-C_Three C
Selected from the group consisting of
Benzyl optionally substituted with at least one substituent
Group, -CHR³⁴COR²⁸ Group, -CHR³⁴COOR²⁸ Group, -CHR³⁴P (O) (OR
²⁸)_Two Group, -CHR³⁴P (S) (OR²⁸)_TwoGroup, -CHR³⁴C (O) NR²⁹R³⁰ 
Group, or -CHR³⁴C (O) NH_Two Represents a group, R²⁸ Is C₁-C₆ A
Lucyl group, C_Two-C₆ Alkenyl group, C_Three-C₆ Alkynyl group,
Or a tetrahydrofuranyl group, R²⁹ And R³¹
 Is independently a hydrogen atom, or C₁-C_Four Table showing alkyl groups
Then R³⁰ And R³² Is independently C₁-C_Four Alkyl group
Or a halogen atom on the ring, C₁-C_Three Alkyl group and C₁-
C_Three Selected from the group consisting of haloalkyl groups
A group optionally substituted with at least one substituent
Represents a phenyl group, or R²⁹And R³⁰And in-(CH_Two)_Five-,
-(CH_Two)_Four-Or -CH_TwoCH_TwoOCH_TwoCH_Two-May be represented
In each ring thus formed, C₁-
C_Three Consists of alkyl, phenyl and benzyl groups
Even if substituted with a substituent selected from the group
Good or R³¹And R³²And these are combined
C with carbon atoms_Three-C₈ Even if it represents a cycloalkyl group
Well, R³³ Is C₁-C_Four Alkyl group, C₁-C_Four Haloalkyl
Group or C_Three-C₆ Represents an alkenyl group, R³⁴ And R
³⁵ Is independently a hydrogen atom or C₁-C_Four Table showing alkyl groups
Then R³⁶ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Arche
Nyl group, or C_Three-C₆ Represents an alkynyl group, R³⁷ Is water
Elementary atom, C₁-C_Four Displays an alkyl group or halogen atom
Then R³⁸ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Cyclo
Alkyl group, C_Three-C₆ Alkenyl group, C_Three-C₆ Alkynyl
Group, C_Two-C₆ Alkoxyalkyl group, C₁-C₆ Haloalkyl
Group, halogen atom on the ring, C₁-C_Four Alkyl group and C₁-
C_Four Selected from the group consisting of alkoxy groups
Fe which may be substituted with at least one substituent
Nyl group, -CH_TwoCO_Two(C₁-C_Four Alkyl) group, or -CH (CH_Three)
CO_Two(C₁-C_Four Alkyl) group, R³⁹ Is a hydrogen atom, C₁-C_Two
 Alkyl group or C (O) O (C₁-C_Four Alkyl) group
Then R⁴⁰ Is a hydrogen atom, C₁-C₆ Alkyl group, C₁-C₆ Arco
Xyl group, or NH (C₁-C₆ Alkyl) group, R⁴¹ Is
C₁-C₆ Alkyl group, C₁-C₆ Haloalkyl group, C₁-C₆ Al
Coxy group, NH (C₁-C₆ Alkyl) group, R⁴² Substituted with a group
Optionally phenyl, benzyl, or C_Two-C₈ The
Represents an alkylamino group, R⁴² Is C₁-C₆ Alkyl group, 1
 Or two halogen atoms, C₁-C₆ An alkoxy group
Or CF_Three Represents a group. (3) a compound represented by the general formula II and nipyraclofen general formula (II)Where R⁴³ Is C₁-C_Four Represents an alkyl group, R⁴⁴ Is C₁-C
_Four Alkyl group, C₁-C_Four Alkylthio group, C₁-C_Four Arco
Xyl group, C₁-C_FourHaloalkyl group, C₁-C_Four Haloalkyl
Thio group, or C₁-C_Four Represents a haloalkoxy group;
Well, R⁴³ And R⁴⁴And in-(CH_Two)_Three-Or- (CH_Two)_Four-Represents
May be R⁴⁵ Represents a hydrogen atom or a halogen atom
Then R⁴⁶ Is a hydrogen atom or C₁-C_Four Represents an alkyl group, R
⁴⁷ Is hydrogen atom, nitro group, cyano group, -COOR⁴⁹ Group, -C
(= X) NR⁵⁰R⁵¹ Group, or -C (= X^Two) R⁵² Represents a group, R⁴⁸ Is
Hydrogen atom, halogen atom, cyano group, halogen atom and
At least selected from the group consisting of
May also be substituted with one substituent₁-C_Four Archi
Group, C₁-C_Four An alkoxy group, a halogen atom on the ring,
Toro, cyano, C₁-C_Four Alkyl group, C₁-C_Four Arco
Xy group and halo-C₁-C_Four Group consisting of alkyl groups
Substituted with at least one substituent selected from
Optionally substituted phenyl, pyrrolyl, C_Two-C₈ A
Ruquil group, C_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl
Group, C_Three-C₈ An alkoxy group, (the C_Two-C₈ An alkyl group,
C _Three-C₈ An alkenyl group, the C_Three-C₈ An alkynyl group and
C_Three-C₈ An alkoxy group contains at least one oxygen source
Or a group shown below.
AndR⁴⁹ R⁵⁰ And R⁵¹ May be the same or different,
Hydrogen atom or C₁-C_FourRepresents an alkyl group, or R
⁵⁰ And R⁵¹Is a 5-member or a nitrogen atom to which these bind
May form a 6-membered saturated ring, R⁵² Is hydrogen
Atom, C₁-C_Four An alkyl group, or at least one or more
C substituted with a halogen atom₁-C_Four Represents an alkyl group, R
⁵³ Is a hydrogen atom, C₁-C_Four Alkyl group, C_Two-C₆ Alkene
Group, C_Three-C₆ Alkynyl group, phenyl group (C₁-C_Four A
Alkyl group, the C_Two-C₆ Alkenyl group, the C_Three-C₆ Alkini
And the phenyl group is substituted with a halogen atom.
May be present), C _Three-C₈ Cycloalkyl group, cyanomethy
Or R⁶³Represents a CO- group, R⁵⁴ Is a hydrogen atom, halo
C optionally substituted with a gen atom₁-C₆ Alkyl group,
C optionally substituted with a halogen atom_Two-C₆ Alkene
C, which may be substituted with_Three-C₆ A
Alkynyl group,
Nyl group, C_Three-C₈ Cycloalkyl group, cyanomethyl group,
C₁-C_Four Alkoxy C ₁-C₆ Alkyl group, di C₁-C_Four Al
Killamino C₁-C_Four Alkyl group, tetrahydrofurfury
Methyl group, C_Three-C₆ Alkynyloxy C₁-C_Four Alkyl
Group, benzyl group, halogen atom on the ring, nitro group, cyano
No group, C₁-C_Four Alkyl group, C₁-C_Four An alkoxy group and
Halo C₁-C_Four Select from groups consisting of alkyl groups
Benzyl group which may be substituted with a substituent represented by -C (=
X^Two) R⁶³Group,-(CH_Two)_a-(O)_d-R⁷⁰ Group,-(CH_Two)_a-O- (CH_Two)_b-R
⁷⁰ Group, or-(CH_Two)_a-X^Two-R⁷⁶Represents a group, or R⁵³
 And R⁵⁴Is the nitrogen atom to which they are attached, 3 members, 5 members
6 or 6 membered saturated ring or 5 or 6 membered ring
Aromatic ring (in the saturated ring and the aromatic ring, one carbon
Element atom may be replaced by one oxygen atom)
May be formed, R⁵⁵ Is a hydrogen atom, C₁-C_Four Alkyl
Group, C_Two-C₆ Alkenyl group, or C_Three-C₆ Alkynyl group
Or R⁵⁵ And R⁵⁶ And in-(CH_Two)_e-Form a group
May be, R⁵⁶ And R⁵⁷ Is C₁-C_Four Alkyl
Group, C_Two-C₆ Alkenyl group, C_Three-C₆ Alkynyl group or
Is a phenyl group (the C₁-C_Four An alkyl group, the C_Two-C₆ Arche
Nyl group, the C_Three-C₆ An alkynyl group and the phenyl group are
Hydrogen atom, C_Three
-C₆ Cycloalkyl group, -X^TwoR⁶⁰ Group, or -NR⁶¹R⁶²Base
And R⁵⁸ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Two-C₆ 
Alkenyl group, C_Three-C₆ Alkynyl group, C₁-C_Four Alkyl
Carbonyl group, cyano C₁-C_Three Alkyl group, C₁-C_Four Al
Coxycarbonyl C₁-C_Four Alkyl group, di C₁-C_Four Arco
Xycarbonyl C₁-C_FourAlkyl group, benzyl group, C₁-C_Four
Alkoxy-C₁-C_FourAlkynyl group,-(CH_Two)_a-R⁷⁵Group,-(CH
_Two)_a-X^Two-R⁷²Group,-(CH_Two)_a-X^Two-(CH_Two)_b-R⁷²Group, or-(C
H_Two)_a-X^Two-(CH_Two)_b-X ^Two-(CH_Two)_c-R⁷²Represents a group, R⁵⁹ Is hydrogen field
Child, C₁-C_Four Alkyl group, C_Two-C₆ Alkenyl group, C_Three-C₆
 Alkynyl group, cyano C₁-C_Three Alkyl group, C₁-C_Four A
Lucylcarbonyl C₁-C_Three Alkyl group or phenyl
Represents a group, R⁶⁰ Is replaced by at least one halogen atom
May be C₁-C_Four Represents an alkyl group, R⁶¹ And R⁶²
 May be the same or different and represent a hydrogen atom, or
C₁-C_Four Represents an alkyl group, R⁶³ Is at least one
C optionally substituted with a halogen atom₁-C_Four Alkyl
Group, C₁-C_Four Alkoxy C₁-C_Four Alkyl group, C₁-C_Four A
Lucircio C₁-C_Four Alkyl group, C_Three-C₆ Cycloalkyl
Group, halogen atom on the ring, nitro group, cyano group, C₁-C_Four
 Alkyl group, C₁-C_Four Alkoxy group and halo C₁-C_Four 
One selected from the group consisting of alkyl groups
A phenyl group optionally substituted with a substituent of -NR⁷³R⁷⁴
Group, or-(CH_Two)_a-(O)_d-R⁷⁵Represents a group, R⁶⁴ Is C₁-C_Four
Displays an alkoxycarbonyl group or carboxyl group
Then R⁶⁵ Is a chloromethyl group, a cyanomethyl group, at least
May also have one or more oxygen atoms inserted C_Three-C₆ Shi
Chloroalkyl group, or C₁-C_Four Alkoxycarbonyl
C₁-C_Four Represents an alkyl group, R⁶⁶ Is a hydroxyl group, or -NR⁶⁷R
⁶⁸And A is -NR⁶⁷R⁶⁸, Or -S (O)_f-R⁶⁹Table
Then R⁶⁷ And R⁶⁸ May be the same or different, and hydrogen
Atom, or C₁-C_Four Represents an alkyl group, R⁶⁹ Is C₁-C_Four 
Alkyl group, or C₁-C_Four Represents a haloalkyl group, R⁷⁰
 Is a hydrogen atom, hydroxyl group, halogen atom, at least one
C on₁-C_Four C optionally substituted with an alkoxy group₁-C
_Four Alkyl group, at least one oxygen atom is inserted
May be C_Three-C₆1 or 2 cycloalkyl groups
C optionally substituted with a methyl group_Three-C₆Cycloalkyl
Group, furyl group, thienyl group, or -C (= O) R⁷¹Table
Then R⁷¹ And R⁷² May be the same or different and C₁-
C_Four Alkyl group, or C₁-C _Four Represents an alkoxy group, R
⁷³ And R⁷⁴ May be the same or different and C₁-C_Four A
Represents a alkyl group or a phenyl group;⁷⁵ Is at least
One or more oxygen atoms may be inserted C_Three-C₆Shiku
Substituted with 1 or 2 methyl groups
May be C_Three-C₆Cycloalkyl group, furyl group, thienyl
Group, or -C (= O) R⁷¹Represents a group, R⁷⁶ Is C₁-C_Four Archi
A, b, and c are each independently 1, 2,
Or 3; d represents 0 or 1; e represents 2 or 3
Represents, f represents 1 or 2, X^Two Is an oxygen atom or sulfur
Represents an atom. 6. The plant according to claim 1, wherein the protein has a property of specifically binding to protoporphyrin IX. 7. The protein according to claim 1, wherein the protein is a protoporphyrin IX-binding subunit protein of magnesium keratase, or a modified protein of said protein, which has a property of specifically binding to protoporphyrin IX. The plant according to 2, 4, 5, or 6. 8. The protein is a protoporphyrin IX-binding subunit protein of magnesium keratase derived from a plant, or a modified protein of the protein, which has a property of specifically binding to protoporphyrin IX. The plant according to claim 1, 2, 4, 5, 6, or 7. 9. The protein is a protoporphyrin IX binding subunit protein of tobacco-derived magnesium keratase, or a modified protein of the protein, which has a property of specifically binding to protoporphyrin IX. Claims 1, 2, 4, 5,
The plant according to 6, 7 or 8. 10. The plant according to claim 1, wherein the protein is a protein having a property of specifically binding to protoporphyrinogen IX. 11. The protein is a modified protein of protoporphyrinogen IX oxidase, which does not have the ability to oxidize protoporphyrinogen IX and has the property of specifically binding to protoporphyrinogen IX. The plant according to claim 1, 2, 4, 5, or 10. 12. A protein which is a modified protein of protoporphyrinogen IX oxidase, does not have the ability to oxidize protoporphyrinogen IX, and is contained as an active ingredient in a protoporphyrinogen IX oxidase-inhibiting herbicide. The plant according to claim 1, 2, 3, 5, or 10, which is a protein having a property of specifically binding to a plant. 13. The plant according to claim 11, wherein the protoporphyrinogen IX oxidase is plant-derived protoporphyrinogen IX oxidase. 14. The plant according to claim 11, 12 or 13, wherein the protoporphyrinogen IX oxidase is soybean-derived protoporphyrinogen IX oxidase. 15. The plant according to claim 11, wherein the protoporphyrinogen IX oxidase is algae-derived protoporphyrinogen IX oxidase. 16. The protoporphyrinogen IX oxidase is a protoporphyrinogen derived from E. coli.
The plant according to claim 11, 12 or 15, which is IX oxidase. 17. A member selected from the following (1) and (2):
A plant that has the above enzyme activity and is resistant to a herbicide in an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant by the enzyme activity, (1) A 5-enolpyruvylshikimate-3-phosphate synthase activity that is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. In addition, a plant which has a gene encoding a protein having the following properties (3), (4) and (5) introduced therein and expresses the gene. (3) Specific binding to protoporphyrin IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 18. The plant according to claim 17, wherein the gene is a gene operably linked to a promoter operable in plant cells and a terminator operable in plant cells. 19. The plant according to claim 17, wherein the protein is ferrochelatase or a modified protein of the protein, which is a protein having a property of specifically binding to protoporphyrin IX. 20. The plant according to claim 19, wherein the ferrochelatase is a plant-derived ferrochelatase. 21. The plant according to claim 19, wherein the ferrochelatase is a ferrochelatase derived from Arabidopsis thaliana. 22. One selected from the following (1) and (2):
A plant that has the above enzyme activity and is resistant to a herbicide in an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant by the enzyme activity, (1) A 5-enolpyruvylshikimate-3-phosphate synthase activity that is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. In addition, a plant which has a gene encoding a protein having the following properties (3), (4) and (5) introduced therein and expresses the gene. (3) Specific binding to protoporphyrinogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 23. The plant according to claim 22, wherein the gene is a gene operably linked to a promoter operable in plant cells and a terminator operable in plant cells. 24. The protein according to claim 2, wherein the protein is coproporphyrinogen III oxidase or a modified protein of the protein, which has a property of specifically binding to protoporphyrinogen IX.
24. The plant according to 2 or 23. 25. The plant according to claim 24, wherein the coproporphyrinogen III oxidase is plant-derived coproporphyrinogen III oxidase. 26. The plant according to claim 24, wherein the coproporphyrinogen III oxidase is soybean-derived coproporphyrinogen III oxidase. 27. A method for producing a herbicide-tolerant plant, comprising growing the plant according to any one of claims 1 to 26. 28. A method for controlling weeds, which comprises applying a herbicide to a plant cultivation area according to any one of claims 1 to 26. 29. A method for selecting a herbicide-tolerant plant, which comprises spraying a herbicide on a cultivation area of the plant according to any one of claims 1 to 26. 30. A method for selecting a herbicide-tolerant plant cell, comprising adding a herbicide to a culture area of the plant cell according to any one of claims 1 to 26. 31. One selected from the following (1) and (2):
A step of introducing a gene encoding the above protein into a plant cell and expressing the gene; (1) a protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (2) A protein having substantially glyphosate oxidoreductase activity. Introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene in a plant cell. (3) It specifically binds to a substance related to the herbicidal activity of a protoporphyrinogen IX oxidase-inhibiting herbicide. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 32. A protein having the properties of (3), (4) and (5), which is a plant cell endogenous substance involved in the expression of herbicidal activity of a protoporphyrinogen IX oxidase-inhibiting herbicide in a plant cell. The method according to claim 31, which is a protein having a property of specifically binding. 33. The method according to claim 31, wherein the protein having the properties of (3), (4) and (5) is a protein having a property of specifically binding to protoporphyrin IX. 34. The protein having the properties of (3), (4) and (5) is a protoporphyrin IX binding subunit protein of magnesium keratase, or a modified protein of said protein,
34. The method according to claim 31, 32 or 33, which is a protein having a property of specifically binding to IX. 35. A protein having the properties of (3), (4) and (5), which is a protoporphyrin IX-binding subunit protein of magnesium keratase derived from a plant,
35. The method according to claim 31, wherein the modified protein is a protein having a property of specifically binding to protoporphyrin IX. 36. The protein having the properties of (3), (4) and (5) is a protoporphyrin IX binding subunit protein of tobacco-derived magnesium keratase, or a modified protein thereof. 37. A protein having the property of specifically binding to protoporphyrin IX.
The described method. 37. The protein having the properties of (3), (4) and (5) is a protein having a property of specifically binding to protoporphyrinogen IX.
2. The method according to 2. 38. The protein having the properties of (3), (4) and (5), which is a modified protein of protoporphyrinogen IX oxidase, wherein the protein is protoporphyrinogen IX.
IX, which does not have the ability to oxidize protoporphyrinogen
38. The method according to claim 31, 32 or 37, which is a protein having a property of specifically binding to. 39. The method according to claim 38, wherein the protoporphyrinogen IX oxidase is plant-derived protoporphyrinogen IX oxidase. 40. The method according to claim 38, wherein the protoporphyrinogen IX oxidase is soybean-derived protoporphyrinogen IX oxidase. 41. The method according to claim 38, wherein the protoporphyrinogen IX oxidase is algae-derived protoporphyrinogen IX oxidase. 42. The protoporphyrinogen IX oxidase is a protoporphyrinogen derived from E. coli.
42. The method according to claim 38 or 41, which is IX oxidase. 43. One selected from the following (1) and (2):
Having the above enzymatic activity, by the enzymatic activity, to the plant cells showing resistance to herbicides in an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant, (1) A 5-enolpyruvylshikimate-3-phosphate synthase activity that is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A method for producing a herbicide-tolerant plant, comprising a step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. (3) It specifically binds to a substance related to the herbicidal activity of a protoporphyrinogen IX oxidase-inhibiting herbicide. (4) The protein has substantially no ability to alter the substance to which it specifically binds. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 44. A plant cell into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene: (1) Protoporphyrinogen IX oxidase inhibition It specifically binds to substances related to the herbicidal activity of herbicides. (2) the protein has substantially no ability to alter the substance to which it specifically binds; (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) A protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity. 45. One selected from the following (1) and (2):
A step of introducing a gene encoding the above protein into a plant cell and expressing the gene; (1) a protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (2) A protein having substantially glyphosate oxidoreductase activity. Introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene in a plant cell. (3) Specific binding to protoporphyrin IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 46. The protein having the properties of (3), (4) and (5) is ferrochelatase, or a modified protein thereof, which has the property of specifically binding to protoporphyrin IX 46. The method of claim 45, wherein 47. The method according to claim 46, wherein the ferrochelatase is a plant-derived ferrochelatase. 48. The method according to claim 46, wherein the ferrochelatase is derived from Arabidopsis thaliana. 49. One selected from the following (1) and (2):
Having the above enzymatic activity, by the enzymatic activity, to the plant cells showing resistance to herbicides in an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant, (1) A 5-enolpyruvylshikimate-3-phosphate synthase activity that is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A method for producing a herbicide-tolerant plant, comprising a step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. (3) Specific binding to protoporphyrin IX. (4) It does not have the ability to transform protoporphyrinogen IX. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 50. A plant cell into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene: (1) specific for protoporphyrin IX Join. (2) It does not have the ability to transform protoporphyrinogen IX. (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) A protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity. 51. One selected from the following (1) and (2):
A step of introducing a gene encoding the above protein into a plant cell and expressing the gene; (1) a protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (2) A protein having substantially glyphosate oxidoreductase activity. Introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene in a plant cell. (3) Specific binding to protoporphyrinogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 52. The protein having the properties of (3), (4) and (5) is coproporphyrinogen III oxidase or a modified protein thereof which is specific for protoporphyrinogen IX. 52. The method according to claim 51, which is a protein having a property of binding to. 53. The method according to claim 52, wherein the coproporphyrinogen III oxidase is plant-derived coproporphyrinogen III oxidase. 54. The method according to claim 52 or 53, wherein the coproporphyrinogen III oxidase is soybean-derived coproporphyrinogen III oxidase. 55. One selected from the following (1) and (2):
Having the above enzymatic activity, by the enzymatic activity, to the plant cells showing resistance to herbicides in an amount that inhibits the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of the plant, (1) A 5-enolpyruvylshikimate-3-phosphate synthase activity that is substantially different from the natural 5-enolpyruvylshikimate-3-phosphate synthase activity of a plant. (2) Glyphosate oxidoreductase activity substantially different from the natural glyphosate oxidoreductase activity of the plant. A method for producing a herbicide-tolerant plant, comprising a step of introducing a gene encoding a protein having the following properties (3), (4) and (5) into a plant cell and expressing the gene. (3) Specific binding to protoporphyrinogen IX. (4) It has the ability to transform coproporphyrinogen III. (5) It does not substantially contain a framework region of an immunoglobulin variable region. 56. A plant cell into which a gene encoding a protein having the following properties (1), (2) and (3) has been introduced and expressing the gene: (1) Specific to protoporphyrinogen IX To combine. (2) It has the ability to transform coproporphyrinogen III. (3) It does not substantially contain a framework region of an immunoglobulin variable region. A method for producing a herbicide-tolerant plant, comprising a step of introducing and expressing a gene encoding one or more proteins selected from the following (4) and (5): (4) A protein having substantially 5-enolpyruvylshikimate-3-phosphate synthase activity. (5) A protein having substantially glyphosate oxidoreductase activity.