JP2000312586A - Imparting of resistance against weed-controlling agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for importing resistance against a weed- controlling agent to a plant by directly transducing a gene into a plant cell which codes for a protein which specifically binds to a substance involved in a weed-controlling action of a weed-controlling agent, does not have an ability of denaturing it, and does not contain an immunoglobulin variable region. SOLUTION: This is a method for importing resistance to a plant against a weed-controlling agent such as protoporphyrinogen IX oxidase inhibiting herbicides which inhibit or suppress porphyrin synthesis of the plant by linking to a promotor and a terminator which are functionable in a plant cell in a funcitonable form, a gene coding for the protein which specifically binds to a substance involved in the weed-controlling action of the weed-controlling agent, does not have an ability of denaturing it to the specifically binding substance, and does not substantially contains a frame work region of an immunoglobulin variable region, followed by transducing the obtained gene into a plant cell for expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for imparting resistance to a weed control agent to a plant.

[0002]

2. Description of the Related Art Weed control is an important operation for improving crop yield and quality, and herbicides are mainly used for this purpose. However, in the use of weed control agents such as herbicides, it is often difficult to clearly distinguish crops from closely related weeds and selectively control only weeds. Attempts have been made to produce highly resistant crops, and some of them have been put to practical use. In recent years, genetic engineering techniques have been used to produce such crops resistant to weed control agents. Examples of such a technique include, for example, 5-enolpyruvylshikimate-3-phosphate synthase (5-enolpyruvyls), which is a glyphosate target enzyme.
hikimate-3-phosphate synthase, hereinafter referred to as EPSPS. ) Is known to produce a glyphosate-tolerant plant by introducing the gene into a cultivated plant [Hinchee, MAW et al., BIO / TECH].
NOLOGY, 6: p915 (1988)]. Further, for example, a method of producing a glyphosinate-resistant plant by introducing a gene for phosphinothricin acetyltransferase, which is an enzyme for acetylating glyphosinate, into a cultivated plant is also known [Block, MD et al. , EMBO J., 6;
p2513 (1987)].

[0003]

However, the production of plants resistant to a weed-controlling agent by such a technique is not yet sufficient, and it is desired to develop a new method for imparting plant-resistant weed-controlling agents. ing.

[0004]

Under such circumstances, the present inventors have conducted intensive studies, and as a result, by imparting a certain type of protein to plant cells, the plant is imparted with a weed controlling agent resistance. The inventors have found that this is possible and have led to the present invention. That is, the present invention provides 1) a method for imparting resistance to a weed control agent to a plant, which comprises introducing a gene encoding a protein having the following properties (1), (2) and (3) into plant cells: (Hereinafter referred to as Method 1 of the present invention)
It is written. (1) It specifically binds to a substance related to the weed controlling action of the weed controlling agent. (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. 2) the method according to the above 1), wherein the protein is a protein having a property of specifically binding to a substance contained as an active ingredient in the weed controlling agent; 3) the weed controlling activity of the weed controlling agent in a plant cell; 1) The method according to the above 1), which is a protein having a property of specifically binding to an endogenous plant cell involved in expression; 4) The weed controlling agent is a weed controlling agent that inhibits or suppresses porphyrin biosynthesis in plants. 1) to 3), 5) the method according to 1) to 4), wherein the weed control agent is a protoporphyrinogen IX oxidase-inhibiting herbicide, 6) the protein specifically binds to protoporphyrin IX 1), 3), which are proteins having the property of
4) or the method according to 5), 7) the protein is a protoporphyrin IX binding subunit protein of magnesium keratase, or is a modified protein of the protein and has a property of specifically binding to protoporphyrin IX. 1), 3), 4), 5) or 6), wherein the protein comprises the amino acid sequence represented by SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, or SEQ ID NO: 57 The method according to the above 1), 3), 4), 5) or 6), wherein the protein is a protein having a property of specifically binding to protoporphyrinogen IX;
3) The method according to 4) or 5), 10) the protein is a modified protein of protoporphyrinogen IX oxidase, and has no ability to oxidize protoporphyrinogen IX and specifically to protoporphyrinogen IX 1), 3), 4), 5) or 9), wherein the protein is a protein having the property of binding, 11) the protein is a modified protein of protoporphyrinogen IX oxidase, and the protein is protoporphyrinogen IX. (1), (2), (4), (5), or (9), which is a protein that has no property of oxidizing the protein and specifically binds to a substance contained as an active ingredient in a protoporphyrinogen IX oxidase-inhibiting herbicide 12) The protein is a modified protein of coproporphyrinogen III oxidase. 1), 3), 4), 5) or 9 which is a protein that does not have the ability to oxidize protoporphyrinogen IX and coproporphyrinogen III and has the property of specifically binding to protoporphyrinogen IX. 13) A method for imparting resistance to a weed control agent to a plant, which comprises introducing a gene encoding a protein having the following properties (1), (2) and (3) into plant cells: A method characterized by including a step of causing expression (hereinafter, referred to as method 2 of the present invention). (1) Specific binding to protoporphyrin IX. (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. 14) The method according to the above 13), wherein the weed controlling agent is a weed controlling agent that inhibits or suppresses porphyrin biosynthesis of a plant; ) Or the method of 14), 16) whether the protein is magnesium keratase,
Or 13) to 15), wherein the protein is a modified protein of the protein and has a property of specifically binding to protoporphyrin IX; 17) the protein is ferrochelatase, or a modified protein of the protein; The method according to the above 13) to 15), which is a protein having a property of specifically binding to protoporphyrin IX, and 18) the method according to the above 13) to 15), wherein the protein is a protein consisting of 4 to 100 amino acids. Method 19) A method for imparting resistance to a weed control agent to a plant, comprising the step of introducing a gene encoding a protein having the following properties (1), (2) and (3) into a plant cell and expressing the gene: (Hereinafter referred to as Method 3 of the present invention). (1) Specific binding to protoporphyrinogen IX To. (2) It has the ability to transform coproporphyrinogen III. (3) It does not substantially contain a framework region of an immunoglobulin variable region. 20) The method according to the above item 19), wherein the weed controlling agent is a weed controlling agent that inhibits or suppresses plant porphyrin biosynthesis; 22) The method according to 19), 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. ) To 21)
23) A plant to which weed control agent resistance has been imparted by the method according to any one of 1) to 22), 24) A method for producing a plant resistant to weed control, which comprises growing the plant according to 23). 25) a method for controlling weeds, which comprises spraying a weed controlling agent on the cultivation area of the plant according to the above item 23); 27) A method for selecting a plant cell resistant to a weed controlling agent, which comprises adding a weed controlling agent to the culture area of the plant cell according to the above item 23).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. In the present invention, examples of the weed control agent include a herbicide, a plant growth regulator and the like. Such herbicides include:
As active ingredients, compounds that inhibit electron transfer in photosynthesis, compounds that inhibit carotenoid biosynthesis, compounds that inhibit porphyrin biosynthesis, compounds that inhibit amino acid biosynthesis, compounds that inhibit lipid biosynthesis, and cell wall biosynthesis Examples include compounds that inhibit compounds, proteins, nucleic acids, or compounds that affect cell division, or compositions containing compounds that have auxin antagonism. More specifically, compounds that inhibit porphyrin biosynthesis include protoporphyrinogen IX oxidase (protopo
rphyrinogen IX oxidase; EC 1.3.3.4. Hereinafter, it is described as PPO. )) (Hereinafter referred to as PPO-inhibiting herbicidal compounds). Compounds that inhibit electron transfer in photosynthesis include compounds that inhibit photosystem I or II, and 4-hydroxypyruvate dioxygenase (4-hydroxyphenyl pyruvate dioxygase) located upstream of the plastoquinone biosynthesis system.
enase; EC 1.13.11.27. Hereinafter, it is referred to as 4-HPPD. ) Can be exemplified. Examples of the compound that inhibits carotenoid biosynthesis include phytoene desaturase (hereinafter, referred to as PDS).
And the like, which inhibit the activity. Compounds that inhibit amino acid biosynthesis include compounds that inhibit EPSPS, acetolactates synthase (EC 4.
1.3.18. Hereinafter, it is referred to as ALS. ), A compound that inhibits glutamine synthetase (EC 6.3.1.2; hereinafter referred to as GS), or a compound that inhibits dihydropteroate synthase (EC).
2.5.1.15. Hereinafter, it is referred to as DHP. ) Can be exemplified. Compounds that inhibit lipid biosynthesis include, for example, acetyl-CoA carboxylase (acetylCoA
carboxylase; EC 6.4.1.2. Hereinafter, it is referred to as ACC. ) Can be exemplified. Specific examples of compounds that affect cell division include compounds that inhibit microtubule formation, and specific examples of compounds that inhibit cell wall biosynthesis include cellulose synthesis. And the like. As a plant growth regulator, as an active ingredient, a compound having an action of antagonizing a plant hormone that promotes cell elongation or differentiation, specifically, for example, 2,4-D, phenoxyalkanecarboxylic acid, benzoic acid or picoline A composition containing a derivative such as an acid can be used.

In the present invention, the substance related to the weed controlling action of the weed controlling agent (hereinafter, referred to as the present substance) is a compound contained as an active ingredient in the weed controlling agent and a compound which is applied to a plant. Means a plant cell endogenous substance involved in the expression of the weed controlling action of the weed controlling agent in the plant cell. Examples of the compound contained as an active ingredient in the weed control agent include the above compounds contained in herbicides, plant growth regulators and the like. Specifically, for example, P
PO inhibiting herbicidal compounds include Duke, SO, Rebei
z, CA ACS Symposium Series 559, Porphyric Pestic
ides, Chemistry, Toxicology, and Pharmaceutical Ap
plications. American Chemical Society, Washington
DC (1994) and the like. PPO-inhibiting herbicidal compounds encompass many different structural molecular species [D
uke et al., Weed Sci. 39: p465 (1991); Nandihalli e
t al., Pesticide Biochem. Physiol. 43: p193 (1992);
Matringe et al., FEBS Lett. 245: p35 (1989); Yanas
e, Andoh, Pesticide Biochem.Physiol. 35: p70 (198
9)], specifically, diphenyl ethers such as chlormethoxynil, bifenox, chloronitrophen (C
NP), 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-oxadiazol-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 [eg LS8
2-556 [N3- (1-phenylethyl) -2,6-dimethyl-5-propionylnicotinamide]], phenopyrate, an O-phenylpyrrolidino carbamate analog of phenopyrate,
And piperidino carbamate analogs of phenopyrate.

Particularly important diphenyl ethers include:
Compounds represented by the following structural formulas 1 to 7 may be mentioned [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)].

Embedded image

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.

Embedded image

Embedded image

Embedded image

Embedded image

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 -CR¹⁵ Represents a group, R¹⁵ 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, E¹² Is a hydrogen atom or C₁-C_Three Alkyl group
Represents ) R²⁷ Is C₁-C₈ Alkyl group, C_Three-C₈ Cycloa
Lucyl group, C_Three-C₈ Alkenyl group, C_Three-C₈ Alkynyl group,
C₁-C₈ Haloalkyl group, C_Two-C₈ Alkoxyalkyl group,
C_Two-C₈Alkylthioalkyl group, C_Two-C₈ Alkylsulf
Ynylalkyl group, C_Two-C₈ Alkylsulfonylalkyl
Group, C₁-C₈ Alkylsulfonyl group on the phenyl ring
Halogen atom and C₁-C_Four Glue composed of alkyl groups
Substituted with at least one substituent selected from
An optionally substituted phenylsulfonyl group, C_Four-C₈ Al
Coxyalkoxyalkyl group, C_Four-C₈ Cycloalkylua
Lucyl group, C₆-C₈ Cycloalkoxyalkyl group, C_Four-C₈ 
Alkenyloxyalkyl group, C_Four-C₈Alkynyloxy
Alkyl group, C_Three-C₈ Haloalkoxyalkyl group, C_Four-C₈ 
Haloalkenyloxyalkyl group, C_Four-C₈ Halo alkini
Roxyalkyl group, C₆-C₈ Cycloalkylthioalkyl
Group, C_Four-C₈ Alkenylthioalkyl group, C_Four-C₈ Archi
Nylthioalkyl group, halogen atom on the ring, C₁-C_Three Al
Kill group and C₁-C_Three Group consisting of haloalkyl groups
Substituted with at least one substituent selected from
Substituted with an optionally substituted phenoxy group₁-C_Four Al
Kill group, halogen atom on the ring, C₁-C_Three Alkyl group and
 C₁-C_Three Select from a group consisting of haloalkyl groups
May be substituted with at least one substituent
Substituted with a benzyloxy group₁-C_Four Alkyl group, C_Four
-C₈Trialkylsilylalkyl group, C_Three-C₈ Cyanoal
Kill group, C_Three-C₈ Halocycloalkyl group, C_Three-C₈ Haloal
Kenyl group, C_Five-C₈ Alkoxyalkynyl group, C_Five-C₈ Halo
Alkoxyalkynyl group, C_Five-C₈ Alkylthioalkenyl
Group, C_Three-C₈ Haloalkynyl group, C_Five-C₈ Alkoxyal
Quinyl group, C_Five-C₈ Haloalkoxyalkynyl group, C_Five-C₈ 
Alkylthioalkynyl group, C_Two-C₈ Alkylcarbonyl
Group, halogen atom on the ring, C₁-C_Three Alkyl group and C₁-
C_Three Selected from the group consisting of haloalkyl groups
Be substituted with at least one substituent.
Benzyl group, -CHR³⁴COR²⁸ Group, -CHR³⁴COOR²⁸ Group, -CHR³⁴P
(O) (OR²⁸)_Two Group, -CHR³⁴P (S) (OR²⁸)_TwoGroup, -CHR³⁴C (O) NR²⁹
R³⁰ Group, or -CHR³⁴C (O) NH_Two Represents a group, R²⁸ Is C₁-C
₆ Alkyl group, C_Two-C₆ Alkenyl group, C_Three-C₆ Alkynyl
Group, or tetrahydrofuranyl group, R²⁹ and
 R³¹ Is independently a hydrogen atom, or C₁-C_Four An alkyl group
Represents, R³⁰ And R³² Is independently C₁-C_Four Alkyl group,
Or a halogen atom on the ring, C₁-C_Three Alkyl group and C
₁-C_Three Selected from the group consisting of haloalkyl groups
May be 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-Represents
In each ring thus formed,
 C₁-C_Three From alkyl, phenyl and benzyl groups
Substituted with a substituent selected from the group consisting of
Or R³¹And R³²And these are combined
Carbon atoms and C_Three-C₈ Represents a cycloalkyl group
May be, R³³ Is C₁-C_Four Alkyl group, C₁-C_Four Haloal
Kill group, or C_Three-C₆ Represents an alkenyl group, R³⁴ And
And R³⁵ Is independently a hydrogen atom or C₁-C_Four An alkyl group
Represents, R³⁶ Is a hydrogen atom, C₁-C₆ Alkyl group, C_Three-C₆ Al
Kenyl group, or C_Three-C₆ Represents an alkynyl group, R³⁷ Is
Hydrogen 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]

Embedded image

Embedded image

Embedded image

Embedded image

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
Group optionally substituted with a halogen group or a halogen atom_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 charcoal
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 Alkoshki 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)}.

Embedded image

[0013] The compound contained as an active ingredient of a weed controlling agent that inhibits electron transfer in photosynthesis includes:
The following compounds may be mentioned. For example, compounds that inhibit electron transfer of photosystem I include paraquat (paraq
uat) or diquat, and the compound that inhibits electron transfer of photosystem II is atrazine
(atrazine) and other triazine (triazine) compounds, diuron (diuron) and other urea (urea) compounds, or bromoxynil (bromoxynil) or ioxynil (ioxyni
l) and the like, and nitrile compounds such as 4-H
Compounds that inhibit PPD include isoxaflutol (is
oxaflutole) and other isoxazole compounds.
Pyrazole compounds or triketones
tone) -based compounds. Compounds included as active ingredients of the weed control agent that inhibits carotenoid biosynthesis include compounds that inhibit PDS, such as norfurazone
(norflurazon), flurochloridone,
Examples include fluridone, flutamone, and diflufenican.
Examples of the compound contained as an active ingredient of the weed control agent that inhibits amino acid biosynthesis include glyphosate (glyphosate) as an example of a compound that inhibits EPSPS.
Examples of compounds that inhibit S include sulfonylurea (su
lfonylurea) -based compound, imidazolinone
Compound, pyrimidinyl thiobenzoate (pyrimidinin
ylthiobenzoate) compound or triazolopyrimidine
(triazolopyrimidine) compounds and the like.Examples of compounds that inhibit GS include bialaphos or glufosinate, and DHP
Examples of the compound that inhibits the activity include aslan (asulam). Examples of the compound contained as an active ingredient of the weed control agent that inhibits lipid biosynthesis include cyclohexanedione (cyclohexanedione) -based compound or allyloxyphenoxypropionate (ary) as an example of a compound that inhibits ACC.
loxyphenoxypropionate) compounds. Examples of the compound contained as an active ingredient of the weed control agent that inhibits cell wall biosynthesis include dichlobenil as an example of a compound that inhibits cellulose biosynthesis.

The plant cell endogenous substance involved in the expression of the weed controlling action in the plant cells when the weed controlling agent is applied to the plant includes, for example, a substrate of the target enzyme on which the active ingredient of the weed controlling agent acts or the target enzyme. A substance that is a precursor or metabolite of a substrate that, when accumulated in plant cells, causes cell dysfunction, or that is produced in plant cells by such a substance and causes cell dysfunction. Substances. More specifically, when a plant is treated with a PPO-inhibiting herbicidal compound, protoporphyrinogen IX, a substrate of PPO, accumulates in the cells of the plant and is metabolized in the cell to produce protoporphyrin IX. In addition, it has been known that active oxygen is generated in cells in the presence of protoporphyrin IX and light, resulting in impaired cell function (Miyamoto, Jun., Eds., 1993; Chapter 3, section 3.3, p106, Hirokawa Shoten Tokyo), protoporphyrinogen IX, protoporphyrin IX in this system
And active oxygen.

The gene used in the method 1 of the present invention is a gene encoding a protein having the following properties (1) to (3). (1) Specific binding to the substance. (2) The protein does not substantially have the ability to alter the substance to which it specifically binds. (3) 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 metamorphosis" 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 variable region of the immunoglobulin" refers to the heavy chain and the light chain constituting the 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 amino acid sequence is relatively highly conserved and serves to maintain the highly conserved three-dimensional structure of the variable region. By taking the above-mentioned 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 region of the immunoglobulin variable region” can be selected, for example, based on the amino acid sequence of the protein. Proteins that do not contain an amino acid sequence consisting of about 30 amino acids or more that 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 may be determined, for example, by using a gene encoding the protein as a template and immunoglobulin.
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, 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 protein encoded by the gene used in the method 1 of the present invention may be a naturally occurring protein, as long as it has the above-mentioned properties (1) to (3). Even proteins that have amino acid substitutions, additions, deletions, etc. introduced into them, are proteins that have been selected based on the binding to the substance from among artificially created proteins having random amino acid sequences. There may be. In the present invention, the term “protein” refers to a substance in which two or more amino acids are bound by a peptide bond, and includes, for example, a substance in which about 4 to 100 amino acids are bound by a peptide bond. Specific examples of proteins having the above properties (1) to (3) include the following proteins.
Proteins that specifically bind to protoporphyrin IX include, for example, magnesium keratase (magnesium pro
toporphyrin IX chelatase), or a modified protein of the protein, which specifically binds to protoporphyrin IX, and more specifically, an organelle from the subunit protein. Examples include proteins from which the translocation signal sequence has been removed. In addition, ferrokeratase (protoheme ferrolyase; EC 4.9.
9.1) the modified protein of protoporphyrin IX,
And proteins that specifically bind to protoporphyrin IX without influencing the ability to bind to protoporphyrin IX.More specifically, a region in ferrochelatase that is presumed to be a binding site for iron ions is modified to produce protoporphyrin.
Proteins that do not have the ability to alter IX can be mentioned. Furthermore, cobalt chelatase (cobaltochelat)
ase) substrate-binding subunit protein, or a modified protein of said protein, comprising protoporphyrin IX
And proteins that specifically bind to.
Examples of the protein that specifically binds to protoporphyrin IX include a protein that specifically includes the amino acid sequence represented by SEQ ID NO: 51 or 53 and a protein that specifically binds to protoporphyrin IX, a porphyrin compound that includes the amino acid sequence represented by SEQ ID NO: 55 or 57 And proteins that specifically bind to E. coli. Specific examples of such proteins include SEQ ID NOs: 51, 53, and 55.
Or a protein containing the amino acid sequence represented by 57 once or a protein containing the amino acid sequence plural times, and the number of amino acids constituting the protein is, for example, about 4 to about 100. Can be. As more specific examples, SEQ ID NOs: 52, 54, 5
A protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 58, or a protein comprising the amino acid sequence represented by SEQ ID NO: 51, 53, 55 or 57 repeated 4 or 8 times, for example, represented by SEQ ID NO: 59, 60, 61 or 62 And the like consisting of the amino acid sequence of the present invention. Proteins that specifically bind to protoporphyrinogen IX include modified proteins of PPO that do not have the ability to oxidize protoporphyrinogen IX and specifically bind to protoporphyrinogen IX. And more specifically, PP
Region predicted to be the FAD binding site in O (amino acid sequence
A region having GXGXXG, where X represents any amino acid. For example, Arabidopsis chloroplast localized PPO
And a protein comprising the amino acids 63-68 from the amino terminus and lacking the amino acid sequence GGGISG). In addition, proteins that bind to protoporphyrinogen IX include coproporphyrinogen III oxidase.
dase; EC 1.3.3.3), comprising protoporphyrinogen IX and coproporphyrinogen.
Protoporphyrinogen I without the ability to oxidize III
Proteins that specifically bind to X can also be mentioned. By binding of the protein as described above to the substance in the cell, cell dysfunction caused by the substance is prevented, and resistance to the weed control agent 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 the 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
cDNA is synthesized, and this is 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 subunit protein gene , Deletion, addition, etc., and the gene was transferred to Gibson, LCD et al., Proc.
d. Sci. USA, 92; introduced into E. coli BL21 (DE3) strain according to the method described in p1941 (1995) to obtain a transformant, and transforming the transformant under conditions in which the introduced gene is highly expressed. Incubate. By selecting strains in which the culture cells turn red and exhibit fluorescence absorption (excitation wavelength 405 nm, fluorescence wavelength 630 nm) indicating accumulation of protoporphyrin IX, a modified protein of the subunit protein, protoporphyrin IX
To obtain a gene encoding a protein that specifically binds to.

[0018] 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 based on 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. Protoporphyrin, a modified protein of ferrochelatase, that has no altering ability for protoporphyrin IX
To obtain a gene encoding a protein that specifically binds to IX, for example, a protein in which a region presumed to be a binding site for iron ions in ferrochelatase is modified and has no metamorphic property to protoporphyrin IX, Based on the nucleotide sequence encoding the amino acid sequence in the vicinity of the region, a primer for introducing a mutation into the region is prepared, and a commercially available site-directed mutagenesis kit (Mutan-Super Express Km, By performing PCR using Takara Shuzo, a gene encoding a mutant in the above region can be prepared. Specifically, the wild-type ferrochelatase gene was inserted into the cloning site of the plasmid vector pKF19k, and the above-mentioned mutagenesis primer and pKF
PCR is performed using a selection primer for reversing the amber mutation on the 19k kanamycin resistance gene.
By introducing the gene amplified by the PCR into Escherichia coli MV1184 strain (suppressor-free) and selecting the transgenic 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. By analyzing the base sequence of the plasmid DNA of the Escherichia coli, it can be confirmed that the isolated gene is a gene encoding the desired modified protein.

A gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 51, 53, 55 or 57 repeated four or eight times may be, for example, the start codon AT
After G, a base sequence encoding the amino acid sequence is selected a base sequence repeated a predetermined number of times, an oligonucleotide consisting of the base sequence and an oligonucleotide consisting of a base sequence complementary to the base sequence, DNA It can be manufactured by synthesizing each using a synthesizer and annealing them. For a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 52, 54, 56 or 58, a nucleotide sequence encoding the amino acid sequence is selected, and an oligonucleotide consisting of the nucleotide sequence and complementary to the nucleotide sequence are selected. Oligonucleotides consisting of a specific base sequence can be synthesized by using a DNA synthesizer and annealed. Here, when selecting a base sequence encoding a predetermined amino acid sequence, for example, a codon frequently used in a plant-derived gene may be selected.

The PPO gene has been used in 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 protein that is a modified protein of PPO and specifically binds to protoporphyrinogen IX without urinary ability to oxidize protoporphyrinogen IX, for example, substitution of a base into the PPO gene, Mutations such as deletion and addition are introduced, and the modified gene is introduced into Escherichia coli described above 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 selected in this manner is expressed in a host cell such as Escherichia coli to prepare a protein encoded by the gene. , 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. More specifically, the modified gene is inserted into an expression vector for Escherichia coli, Yamamoto, F. et al., Japanese J. Genet., 63;
such as the E. coli BT3 strain described in p237 (1988), etc.
A PPO gene (hemG locus) -deficient mutant strain Escherichia coli was introduced, and the Escherichia coli was cultured in a medium containing hemin and aminolevulinic acid in addition to a cell growth inhibitor corresponding to the selectable marker on the vector introduced into the Escherichia coli. A transformant may be obtained, and a protein encoded by the modified gene may be prepared from the transformant. 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 for selecting a gene encoding a protein having no ability to oxidize protoporphyrinogen IX. Also, PPO
In order to obtain a gene encoding a protein in which a region presumed to be a binding site to FAD (a region having the amino acid sequence GXGXXG, where X represents an arbitrary amino acid), Based on the nucleotide sequence encoding the amino acid sequence near the region, a primer for introducing a deletion mutation in the region is prepared. Using this mutagenesis primer, as described above, using a commercially available site-directed mutagenesis kit (Mutan-Super Express Km, Takara Shuzo)
By performing R, a gene encoding a deletion mutant of 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 to 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 a 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. In order 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, base substitution, deletion, in coproporphyrinogen III oxidase gene,
A mutation such as addition is introduced, and the modified gene is introduced into Escherichia coli described above 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, for example, in a host cell such as Escherichia coli, to prepare a protein encoded by the gene. , NJand Jacobs, JM (1982)
By measuring according to the method described in Enzyme, 28, 206-219, etc., a gene of a protein having no ability to oxidize protoporphyrinogen IX can be selected. Further, for the protein prepared from E. coli as described above, the ability to convert coproporphyrinogen III to protoporphyrinogen IX (coproporphyrinogen III oxidase activity), for example, Yosh
inaga, T., Sano, S., J. Biol. Chem., 255; p4722 (198
By performing the measurement according to the method described in (0) and the like, a gene of a protein having no oxidizing ability to coproporphyrinogen III can be selected.

The genes of proteins capable of specifically binding 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. When the phage library described above is screened on a plate coated with this substance, a phage displaying a target protein capable of binding to the substance can be isolated. Gene can be obtained.

The gene used in the method 2 of the present invention is a gene encoding a protein having the following properties (1) to (3). (1) Specific binding to protoporphyrin IX. (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. 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. Further, `` 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. As the protein encoded by the gene used in the method 2 of the present invention, even if it has the above-mentioned properties (1) to (3), even if it is a naturally-occurring protein, it can be substituted with a natural protein by amino acid substitution. , Addition, deletion, etc., even if the protein is selected from the artificially created protein having a random amino acid sequence based on the binding to the substance as an index Good. Specific examples of such proteins include:
For example, magnesium keratase and modified proteins of magnesium keratase which specifically bind to protoporphyrin IX and do not have the ability to denature protoporphyrinogen IX can be mentioned. 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. Further, a protein containing the amino acid sequence represented by SEQ ID NO: 51 or 53 and specifically binding to protoporphyrin IX, a protein containing the amino acid sequence represented by SEQ ID NO: 55 or 57 and specifically binding to a porphyrin compound, and the like are mentioned. You can also. Specific examples of such a protein include a protein containing the amino acid sequence represented by SEQ ID NO: 51, 53, 55, or 57 once, a protein containing the amino acid sequence multiple times, and the like. The number of amino acids
For example, about 4 to about 100 pieces can be given.
As a more specific example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 52, 54, 56 or 58 or a protein comprising the amino acid sequence represented by SEQ ID NO: 51, 53, 55 or 57 repeated 4 or 8 times For example, a protein comprising the amino acid sequence represented by SEQ ID NO: 59, 60, 61 or 62 can 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, a gene encoding the D subunit protein of magnesium keratase is used as a photosynthetic bacterium Rhodobacte.
r sphaerides (Genebank accession AJ001690), photosynthetic bacterium Rhodobacter capsulatus (Genebank accession Z11
165), pea (Genebank accession AF014399), tobacco (Genebank accession Y10022), Synechocystis PC
Genes derived from C.6803 (Genebank accession X96599) and the like are known. 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, L.
CD et al., Proc.Acad.Sci. USA, 92; p1941 (1995)
And the like. The gene encoding ferrochelatase can be obtained by the method described above. The detection of the activity of ferrochelatase can be carried out, for example, according to the method described in Porra, RJ, Anal. Biochem., 68; p289 (1975) and the like.

The gene used in the method 3 of the present invention is a gene encoding a protein having the following properties (1) to (3). (1) Specific binding to protoporphyrinogen IX. (2) It has the ability to transform coproporphyrinogen III. (3) It does not substantially contain a framework region of an immunoglobulin variable region. "Specifically binds to protoporphyrinogen IX" refers to, as described above, for example, an enzyme and a substrate, or an enzymatic chemical bond between an enzyme and an inhibitor or activity modulator, 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. As the protein encoded by the gene used in the method 3 of the present invention, even if it has the above-mentioned properties (1) to (3), even if it is a naturally-occurring protein, it can be substituted with a naturally-occurring protein by amino acid substitution. , Addition, deletion, etc., even if the protein is selected from the artificially created protein having a random amino acid sequence based on the binding to the substance as an index Good. Specific examples of such proteins include coproporphyrinogen
III oxidase and coproporphyrinogen I
A modified protein of II oxidase that oxidizes coproporphyrinogen III to oxidize protoporphyrinogen I
Examples include proteins capable of producing X.
The gene encoding coproporphyrinogen III oxidase can be obtained, for example, by the method described above.

In the methods 1 to 3 of the present invention, a gene encoding the above-described protein having the properties required in each method (hereinafter collectively referred to as the present protein) is replaced with a target plant cell. And expressed. One kind of the gene encoding the present protein may be introduced into a cell, or a plurality of kinds of genes encoding different present proteins may be introduced. Methods for introducing genes into plant cells include the Agrobacterium infection method (Japanese Patent Publication No.
-58917 and JP-A-60-70080), an electroporation method for protoplasts (JP-A-60-251887 and JP-A-5-68575), or a particle gun method (Tokuhyohei 5-508)
316 and JP-A-63-258525) 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 encoding this protein in plant cells, homologous recombination [Fraley, RT et al., Proc. Natl.
cad. Sci. USA, 80; p4803 (1983)], the gene may be introduced into the chromosome of a plant, and a cell expressing the gene may be selected. It may be operably linked to a terminator operable in a plant cell and introduced into the cell. Here, `` in a operable form '' means that the gene encoding the present protein is linked to these promoter and terminator such that the gene is expressed under the control of the promoter and terminator in a plant cell to be introduced. Means in state. Examples of a promoter operable in a plant cell include, for example, a nopaline synthase gene promoter, a constitutive promoter derived from T-DNA such as an octopine synthase gene promoter, a plant virus such as a cauliflower mosaic virus-derived 19S promoter or 35S promoter. Inducible promoters such as a promoter, a phenylalanine ammonia lyase gene promoter, a chalcone synthase gene promoter, and a Pathogenesis-related protein gene promoter can be exemplified.
Further, other plant promoters not limited to these may be used. Examples of the terminator that can function in a plant cell include a T-DNA-derived terminator such as a nopaline synthase gene terminator, and a plant virus-derived terminator such as a garlic virus GV1 or GV2 terminator. 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 weed-controlling agent to which resistance is to be imparted or a medium containing the 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 19)
1990), pp. 27-55, and the like, and 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 rice plants
Arabidopsis edited by Isao Shimamoto and Kiyotaka Okada, Shujunsha 1996
By the method described in Chapter 4, rice and Arabidopsis thaliana expressing the gene encoding the present protein can be obtained. Further, a soybean expressing a gene encoding the present protein can be obtained by introducing the gene encoding the present protein into a soybean somatic embryo using a particle gun by a method described in JP-A-3-291501. Similarly, Fromm, ME, et al. Bio /
Technology, 8; In accordance with the method described in p838 (1990), transfected into a maize somatic embryo using a particle gun, it is possible to obtain corn expressing a gene encoding the present protein. According to the usual method described in the Journal of the Japanese Society of Breeding, 1995, Vol. 44, Separate Volume 1, page 57, introduced into a wheat immature scutellum aseptically cultured using a particle gun and encodes the protein Wheat expressing the gene can be obtained. Similarly, Hagio et al., Journal of Japanese Society of Breeding, 1995, Vol. 44, separate volume 1
No., p. 67, and introduced into a barley immature scutellum aseptically cultured using a particle gun to obtain a barley expressing a gene encoding the present protein. To confirm the resistance of a plant expressing the gene encoding the present protein to a weed control agent,
It is preferable to spray a weed controlling agent for imparting resistance to the cultivation area of the plant and measure the growth of the plant. To confirm more quantitatively, for example, in the case of a plant resistant 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 Sprayed with an aqueous solution of a herbicide and allowed to stand on an agar medium at room temperature in the light, and several days later, Macknney, G., J. Bol. Chem.,
140; p315 (1941), chlorophyll may be extracted and the chlorophyll content measured according to the method described in p315 (1941).

The weed controlling agent-tolerant plant obtained according to the present invention is resistant to the weed controlling agent, and therefore can grow well even when the weed controlling agent is sprayed. Therefore, by imparting the weed control agent resistance to the weed control agent to the target cultivated plant according to the present invention, by cultivating the plant and spraying the weed control agent on the cultivation area, weeds other than the plant can be efficiently treated. The cultivated plant can be removed well, so that the yield of the cultivated plant can be improved, the quality can be improved, the amount of the herbicide to be used can be reduced, and labor can be saved.

[0029]

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 Protoporphyrin IX Binding Subunit Protein Gene of Magnesium Keratase Genomic DNA of the photosynthetic bacterium Rhodobacter sphaeroides ATCC17023 was prepared using a genomic DNA preparation kit ISOPLANT (Nippon Gene). Next, Gibson, LCD e
Acad. Sci. USA, 92; p1941 (1995), and using about 1 μg of the genomic DNA as a template, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 PCR was performed using 10 pmol of each oligonucleotide consisting of the nucleotide sequence as a primer to amplify a DNA fragment containing the protoporphyrin IX binding subunit protein gene bchH of magnesium keratase.
The oligonucleotide was used in 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 carried out at 94 ° C. for 2 minutes, then at 96 ° C. for 40 seconds, and further at 68 ° C. for 7 minutes.
This cycle was repeated 28 times, with the heat retention at 96 ° C for 40 seconds and then at 68 ° C for 7 minutes as one cycle.
For 40 seconds and then at 68 ° C. for 7 minutes and at 72 ° C. for 10 minutes once.

Reference Example 2 Expression of Protoporphyrin IX Binding Subunit Protein Gene of Magnesium Keratase in Escherichia coli Gibson, LCD et al., Proc. Acad. Sci. USA, 92; p.
According to the description of 1941 (1995), the DNA fragment containing the bchH gene prepared in Reference Example 1 was digested with the restriction enzymes NdeI and BglII.
The resulting DNA fragment was digested with the expression vector pET11a (S
Plasmid pETBCH (FIG. 1) was prepared by insertion between the NdeI cleavage site and BamHI cleavage site of tratagene. This plasmid pETBCH was introduced into competent cells of Escherichia coli BL21 (DE3) strain (manufactured by Stratagene) as described in the instruction manual attached to the competent cells.
BL21 (DE3) / pETBCH strain was obtained. The strain was placed in a test tube (14 × 10 m
LB liquid medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) containing 100 μg / ml ampicillin
The test tubes were covered with aluminum foil (hereinafter referred to as dark conditions) and cultured at 37 ° C. with shaking under fluorescent light (about 8000 lux). The absorbance of the culture at 600 nm is about 0.6
Reached, isopropyl β-D-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 0.4 mM, and the culture was further continued for about 20 hours.
CD et al., Proc.Acad.Sci. USA, 92; p1941 (1995)
As described in the above, the Escherichia coli turns red, and fluorescence absorption indicating the accumulation of protoporphyrin IX in the Escherichia coli (excitation wavelength
405 nm, fluorescence wavelength 630 nm) were observed. In addition, E. coli BL
When the 21 (DE3) / pETBCH strain was cultured in the same manner as described above without adding IPTG, red coloration of Escherichia coli was not observed, and the fluorescence absorption was not detected. On the other hand, E. coli BL21 (DE3) / pETB
When the CH strain was cultured in the same manner as described above with the addition of IPTG, but without covering the test tube with aluminum foil (hereinafter referred to as light conditions), the E. coli strain grew and became red as described above. did.

Reference Example 3 Expression of PPO Gene Derived from Soybean in Escherichia coli Deleting the hemG Gene Soybean (Glycine max var. Williams 82) was inoculated at 25 ° C.
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 pestle, and RNA was extracted from the ground with 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 having the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4
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 as one heat retention cycle at 94 ° C. for 15 seconds and then at 65 ° C. for 5 minutes. After the PCR reaction,
The reaction solution was filtered through MicroSpin S-400HR (Pharmacia Biotech) to purify the amplified DNA fragment, and the DNA fragment was treated with plasmid pCR2.1 (Invitrogen) cut with restriction enzyme SalI. By ligation, plasmid pSPPO-P was obtained. The plasmid was then transformed into E. coli I
The strain was introduced into competent cells of NVαF ′ strain (manufactured by Invitrogen), and ampicillin-resistant strains were selected. Furthermore, 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 373S (P
E Applied Biosystems).
As a result, the nucleotide sequence represented by SEQ ID NO: 5 was clarified, and it was confirmed that the plasmid pSPPO-P contained the soybean chloroplast PPO gene. This plasmid pSPPO-P was digested with the restriction enzyme PshBI, and the end of the obtained DNA fragment was
4 After blunting using DNA polymerase, the fragment was further digested with SphI to isolate a DNA fragment containing the soybean chloroplast PPO gene and the lac promoter. Next, the plasmid pAC
YC184 (manufactured by Nippon Gene) was digested with restriction enzymes NruI and SphI, the 410 bp fragment was removed, and the DNA fragment was inserted in place of the fragment to obtain plasmid pACYCSP (FIG. 2).
I got Then, the plasmid pACYCSP, Yamamoto, F.
et al., Japanese J. Genet., 63; p237 (1988) and the like.Hanahan, DJ, Mol. Biol., 166; p557 ( 1
983) and introduced into a YPT medium containing 5 μg / ml chloramphenicol and 10 μg / ml kanamycin (5 g / l yeast extract, 5 g / l tryptone, 5 g / l peptone, 1 g
0g / l NaCl, pH 7.0), the cells were resistant to chloramphenicol and kanamycin, and the hemG gene deletion was complemented with the soybean-derived PPO gene.
The BT3 / pACYCSP strain was selected.

Example 1 Test of the ability of magnesium keratase to impart herbicide resistance to protoporphyrin IX binding subunit protein The E. coli BT3 / pACYCSP strain prepared in Reference Example 3 was
Inoculating a YPT medium containing 10 or 1 ppm of a PPO-inhibiting herbicidal compound represented by Structural Formula 8 and containing 10 μg / ml hemin, 50 μg / ml aminolevulinic acid, 15 μg / ml chloramphenicol and 10 μg / ml kanamycin, In the same manner as in Example 2, the cells were cultured under dark or light conditions. As a control, Escherichia coli BT3 / pACYCSP strain was similarly cultured using the above-mentioned medium containing no herbicidal compound. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 1 shows the results.

[0034]

[Table 1]

An oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 were used as primers in the same manner as in Reference Example 1 to derive from the photosynthetic bacterium Rhodobacter sphaeroides.
The DNA fragment containing the bchH gene was amplified and the resulting DNA
The fragment was digested with restriction enzymes NcoI and BglII, and inserted into the plasmid pTV118N (Takara Shuzo) between the NcoI cleavage site and the BamHI cleavage site to obtain the plasmid pTVBCH (FIG. 3).
Was built. The plasmids pTVBCH and pTV118N were each added to the Escherichia coli BT3 / pACYCSP strain prepared in Reference Example 3 by Hanana.
han, DJ, Mol. Biol., 166; p557 (1983), and these were introduced at 100 μg / ml ampicillin, 15 μg / ml.
By culturing in YPT medium containing μg / ml chloramphenicol and 10 μg / ml kanamycin, plasmid pACYCSP
Escherichia coli BT3 / pACYCSP + with pTVBCH and E. coli BT3 / pACYCSP + with plasmids pACYCSP and pTV118N
pTV118N strain was obtained. These E. coli strains were converted to
Containing 10 or 1 ppm of a PPO-inhibiting herbicidal compound represented by the formula: 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml hemin and
YPT medium containing 50 μg / ml aminolevulinic acid was inoculated, and cultured under dark or light conditions in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 2 shows the results.

[0036]

[Table 2]

These Escherichia coli strains were prepared by the above-mentioned structural formula 1, structural formula 14, structural formula 15, structural formulas 18 to 22, structural formula 29, structural formula 32, structural formula 33, structural formula 34 or structural formula 36. And inoculated in a YPT medium containing 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml hemin and 50 μg / ml aminolevulinic acid, containing In the same manner as in Reference Example 2, the cells were cultured under dark or bright conditions. 18 hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was defined as 1. I asked. Table 3 shows the results.

[0038]

[Table 3]

Example 2 Introduction of Gene Encoding Protoporphyrin IX Binding Subunit Protein of Magnesium Keratase into Tobacco A plasmid for introducing the bchH gene into plants by the Agrobacterium method was constructed. First, the binary vector
pBI121 (manufactured by Clontech) was digested with a restriction enzyme SacI, a KpnI linker (manufactured by Takara Shuzo) was inserted, the SacI recognition site of pBI121 was removed, and a plasmid pBIK having a KpnI recognition site added was prepared. On the other hand, in the same manner as in the method described in Reference Example 1, the photosynthetic bacterium Rhodobac
A DNA fragment containing the bchH gene was amplified by performing PCR using the genomic DNA of ter sphaeroides as a template. Next, the plasmid pBIK was replaced with the restriction enzymes XbaI and KpnI.
The β-glucuronidase gene was removed by digestion with a DNA fragment obtained by digesting the DNA fragment containing the bchH gene with the restriction enzymes XbaI and KpnI in place of this, and bchH downstream of the 35S promoter. A plasmid pBIBCH (FIG. 4) to which the gene was linked was prepared. In addition, the binary vector pBI121 (Clontech) was replaced with the restriction enzyme BamHI.
The β-glucuronidase gene was removed by digestion with SacI and the end of the obtained DNA fragment was T4 DNA polymeras
After blunting using e, self-cyclization was performed using T4 DNA ligase to construct a plasmid pNO (FIG. 5). This plasmid was used as a vector control for the bchH expression plasmid pBIBCH. Each of the plasmids pBIBCH and pNO was introduced into Agrobacterium tumefaciens LBA4404, and these were cultured in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin, and a transformant was selected. Bacterium strains and Agrobacterium strains with pNO were isolated. Next, the gene was introduced into tobacco according to the method described in a plant gene manipulation manual (Hirofumi Uchimiya, Kodansha Scientific, 1992). LB Agrobacterium strain carrying plasmid pBIBCH
Medium (0.5% Yeast extract, 1.0% Bacto tryptone, 0.5%
(NaCl) at 28 ° C. overnight, and aseptically cultured tobacco (Nicotiana tabacum cv. SR1) leaf pieces were immersed in the culture solution. Murashige containing 0.8% agar, 0.1 mg / l naphthalene acetic acid and 1.0 mg / l benzylaminopurine
It was cultured at room temperature for 2 days on a Murashige-Skoog medium (MS medium) described in T. and Skoog F., Physiol. Plant. (1962) 15, p473. Next, after washing the tobacco leaf pieces with sterile water,
M containing 0.8% agar, 0.1 mg / l naphthalene acetic acid, 1.0 mg / l benzylaminopurine, and 500 μg / ml cefotaxime
The cells were cultured on S medium for 7 days. Then 0.8% of the tobacco leaf pieces
Agar, 0.1 mg / l naphthalene acetic acid, 1.0 mg / l benzylaminopurine, 500 μg / ml cefotaxime, and 100 μg / ml
The cells were transplanted and cultured on an MS medium containing kanamycin (hereinafter, referred to as an MS medium for selection). 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 in foliage emerged from the tobacco leaf pieces were 0.8% agar, 300 μg / ml
Contains cefotaxime and 50 μg / ml kanamycin
Transplanted into MS medium and rooted to obtain regenerated individuals. The regenerated individual was transplanted and cultured in an MS medium containing 0.8% agar and 50 μg / ml kanamycin to obtain a tobacco individual into which the bchH gene had been introduced. 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 3 Tobacco herbicide tolerance test of tobacco into which a gene encoding a protoporphyrin IX binding subunit protein of magnesium keratase has been introduced Tobacco leaf into which the bchH gene obtained in Example 2 has been introduced and a control group Replaced tobacco leaves are collected and divided equally into two along the main vein, and an aqueous solution containing the PPO-inhibiting herbicidal compound represented by the above structural formula 8 at a concentration of 0.3 ppm in one leaf piece. It 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 a 5 ml 80% aqueous acetone solution with a mortar and pestle to extract chlorophyll. After diluting the extract 10-fold with 80% acetone aqueous solution, the absorbance at 750 nm, 663 nm, and 645 nm was measured, and Macknney G., J. Biol. Chem.
(1941) 140, p315 The total chlorophyll content was calculated by the method described. Table 4 shows the results obtained for four tobacco clones (BCH1 to 4) into which the bchH gene was introduced and control recombinant tobacco. In the table, the degree of resistance to the herbicidal compound was expressed as a percentage of the total chlorophyll content of leaf pieces treated with the herbicidal compound, relative to the total chlorophyll content of untreated leaf pieces.

[0041]

[Table 4]

Further, the tobacco into which the bchH gene was introduced and the control recombinant tobacco were subjected to the structural formulas 3, 7, 7, 10, 11, 13, and 1 respectively.
7, structural formula 23, structural formula 24, structural formula 25, structural formula 2
7, treated similarly with an aqueous solution containing any of the PPO-inhibiting herbicidal compounds represented by Structural Formula 28, Structural Formula 30, or Structural Formula 35, and the degree of resistance to each herbicidal compound was measured. Table 5 shows the results. In the table, the degree of resistance to the herbicidal compound was expressed as a percentage of the total chlorophyll content of leaf pieces treated with the herbicidal compound, relative to the total chlorophyll content of untreated leaf pieces.

[0043]

[Table 5]

Reference Example 4 Acquisition of Gene Encoding a Modified Protein of Protoporphyrin IX-Binding Subunit of Tobacco Magnesium Keratase Using RNeasy Plant Kit (QIAGEN) from 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, the reverse transcriptase contained in the kit was added to the tobacco 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 modified tobacco keratase subunit. Here, as a primer for PCR, an oligonucleotide primer consisting of the base sequence of SEQ ID NO: 8 and an oligonucleotide primer consisting of the base sequence of SEQ ID NO: 9 were used. The oligonucleotide is a DNA synthesizer (PE
Applied Biosystems: Model 394 DNA / RNA Sy
nthesizer) and a cartridge for oligonucleotide purification (PE Applied Biosystems: OP
C cartridge). The PCR was performed once at 94 ° C. for 2 minutes, followed by 94 ° C. for 30 seconds, then at 50 ° C. for 30 seconds, and further at 72 ° C. for 7 minutes.
This was carried out for 30 cycles. The PC mentioned above
After the R reaction, operation was performed using a TA Cloning Kit (manufactured by Invitrogen) according to the attached manual, and the DNA fragment amplified by the PCR reaction was cloned into pCR2.1 plasmid. The resulting plasmid DNA was digested with the restriction enzyme KpnI.
After digestion with agarose gel electrophoresis,
The plasmid in which the DNA fragment of b was detected was named pTCHLH. The plasmid has a structure in which a gene encoding the modified tobacco keratase subunit is inserted in a direction that allows expression under the control of a lac promoter. further,
After digesting this plasmid pTCHLH with the restriction enzyme KpnI, self-cyclization was performed, and a DNA fragment consisting of about 60 bases was converted into plasmid pTCHLH.
A plasmid pTCHLH1 (FIG. 6) having a structure removed from HLH was obtained.

Example 4 Test of the ability of the modified tobacco keratase subunit to impart herbicide tolerance The plasmid pTCHLH1 or pCR2.1 prepared in Reference Example 4
Were introduced into the E. coli BT3 / pACYCSP strain prepared in Reference Example 3 according to the method described in Hanahan, DJ, Mol.Biol., 166; p557 (1983). Chloranphenicol, Escherichia coli BT3 / pACYCSP + pTCH having plasmids pACYCSP and pTCHLH1 by culturing in YPT medium containing 50 μg / ml kanamycin
The LH1 strain and the E. coli BT3 / pACYCSP + pCR2.1 strain having the plasmids pACYCSP and pCR2.1 were obtained. These Escherichia coli strains contain 10 or 1 ppm of the PPO-inhibiting herbicidal compound represented by the above structural formula 8, and 100 μg / ml ampicillin, 15 μg
/ ml chloramphenicol, 50 μg / ml kanamycin, 10
YP with μg / ml hemin and 50 μg / ml aminolevulinic acid
The medium was inoculated into a T medium, and cultured in the dark or in the light in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 6 shows the results.

[0046]

[Table 6]

Reference Example 5 Introduction of Gene Encoding the Modified Tobacco Keratase Subunit into Tobacco A plasmid for introducing the gene encoding the modified tobacco keratase subunit into plants by the Agrobacterium method was constructed. First, the plasmid pTCHLH1 prepared in Reference Example 4 was digested with restriction enzymes KpnI and SalI to prepare a DNA fragment containing a gene encoding the present modified tobacco keratase subunit. On the other hand, by digesting the binary vector pBI121 (manufactured by Clontech) with the restriction enzyme SmaI and inserting a KpnI linker (manufactured by Takara Shuzo), the plasmid pBI121K to which the SmaI recognition site of pBI121 was removed and the KpnI recognition site was added was added. Produced. The plasmid p
After digesting the DNA of BI121K with the restriction enzyme SacI, the DNA poly
Using merase I, nucleotides were added to the gaps of the double-stranded DNA to blunt the ends of the DNA. Next, the 5 ′ end of the DNA was dephosphorylated with an alkaline phosphatase derived from calf small intestine, a phosphorylated SalI linker (4680P, manufactured by Takara Shuzo) was inserted and cyclized to construct plasmid pBI121KS. The β-glucuronidase gene was removed by digesting the binary vector pBI121KS with restriction enzymes KpnI and SalI,
Instead, a DNA fragment containing the gene encoding the present modified tobacco keratase subunit was inserted, and a plasmid pBITCHLH (FIG. 7) in which the gene was ligated downstream of the 35S promoter was prepared. The plasmid pBIT
CHLH was introduced into Agrobacterium tumefaciens LBA4404,
This was cultured in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin, and a transformant was selected to isolate an Agrobacterium strain having pBITCHLH. The Agrobacterium strain is infected into aseptically cultured tobacco leaf pieces, and tobacco into which the gene encoding the modified tobacco keratase subunit has been introduced is obtained in the same manner as in Example 2.

Reference Example 6 Confirmation of herbicide resistance of tobacco into which the gene encoding the present modified tobacco keratase subunit was introduced The gene encoding the present modified tobacco keratase subunit produced in Reference Example 5 was introduced. By testing tobacco in the same manner as in Example 3, the degree of resistance of the tobacco to the herbicidal compound is quantitatively confirmed.

Reference Example 7 Acquisition of a gene encoding a protein which is a modified protein of soybean PPO and has no ability to oxidize protoporphyrinogen IX and specifically binds to protoporphyrinogen IX SEQ ID NO: 10 Using the oligonucleotide consisting of the sequence and the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 11 as primers, PCR was performed using the plasmid pSPPO-P prepared in Reference Example 3 as a template, and a chloroplast translocation signal and FAD binding A DNA fragment encoding a soybean PPO having a deleted sequence (hereinafter referred to as the present modified soybean PPO) 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). PCR was performed at 94 ° C for 1 minute and then at 55 ° C for 2 minutes.
This was carried out 30 times with one cycle of keeping the temperature at 72 ° C. for 3 minutes. The amplified DNA fragment was digested with the restriction enzyme Nc.
digested with oI and SalI, and the plasmid pTV118N (Takara Shuzo)
Plasmid pTVGMP (FIG. 8) was constructed by inserting between the coI and SalI cleavage sites. The plasmid pTVGMP was introduced into the PPO gene deletion mutant strain Escherichia coli BT3 according to the method described in Hanahan, DJ, Mol. Biol., 166; p557 (1983).
YPT medium containing 0 μg / ml kanamycin (5 g / l yeast extract, 5
g / l tryptone, 5 g / l peptone, 10 g / l NaCl, pH 7.0), no clones with growth complementation were obtained.

Example 5 Test of ability of the modified soybean PPO to impart herbicide tolerance Plasmids pTVGMP and pTV118N prepared in Reference Example 7
Were introduced into the E. coli BT3 / pACYCSP strain prepared in Reference Example 3 according to the method described in Hanahan, DJ, Mol. Biol., 166; By culturing in YPT medium containing lanfenicol and 10 μg / ml kanamycin, plasmid p
E. coli BT3 / pACYCSP + pTVGMP strain with ACYCSP and pTVGMP,
And Escherichia coli BT having plasmids pACYCSP and pTV118N
3 / pACYCSP + pTV118N strain was obtained. These Escherichia coli strains were isolated from the PPO-inhibiting herbicidal compound represented by the above structural formula 8 by 10
Or 1 ppm, 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml
YPT medium containing hemin and 50 μg / ml aminolevulinic acid was inoculated and cultured in the dark or in the light in the same manner as in Reference Example 2. 18 hours after the start of culture, 600 n
The absorbance at m was measured, and the absorbance when cultured in a medium containing no herbicidal compound was defined as 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was determined.
Table 7 shows the results.

[0051]

[Table 7]

Example 6 Introduction of the Gene Encoding the Modified Soy PPO into Tobacco A plasmid for introducing the gene encoding the modified soy PPO into plants by the Agrobacterium method was constructed. Similarly to the method described in Reference Example 7, using the oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 12 and the oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 13, the plasmid pSPPO prepared in Reference Example 3 By performing PCR using -P as a template, a DNA fragment containing the gene encoding the present modified soybean PPO was amplified. Next, the plasmid pBI121K prepared in Reference Example 5 was digested with restriction enzymes KpnI and SacI to remove the β-glucuronidase gene, and instead, a DNA fragment containing the gene encoding the present modified soybean PPO was replaced with the DNA fragment containing the gene encoding the modified soybean PPO. A DNA fragment obtained by digestion with the restriction enzymes KpnI and SacI was inserted to prepare a plasmid pBIGMP (FIG. 9) in which the gene was ligated downstream of the 35S promoter. The plasmid pBIGMP was replaced with Agrobacterium tumefaciens.
The Agrobacterium strain having pBIGMP was isolated by introducing the gene into LBA4404, culturing it in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin, and selecting transformants. The Agrobacterium strain was infected to aseptically cultured tobacco leaf pieces, and tobacco into which the gene encoding the modified soybean PPO was introduced was obtained in the same manner as in Example 2.

Example 7 Confirmation of herbicide resistance of tobacco into which the gene encoding the present modified soybean PPO was introduced Example 3 was carried out on the tobacco introduced with the gene encoding the modified soybean PPO produced in Example 6 By the same operation as described above, the degree of resistance to the PPO-inhibiting herbicidal compound represented by Structural Formula 8 was quantitatively measured. Tobacco into which the gene encoding the modified soybean PPO has been introduced
Table 8 shows the results obtained for the four clones (GMP1 to 4) and the control recombinant tobacco. In the table, the degree of resistance to the herbicidal compound was expressed as a percentage of the total chlorophyll content of leaf pieces treated with the herbicidal compound, relative to the total chlorophyll content of untreated leaf pieces.

[0054]

[Table 8]

Reference Example 8 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 Hartner 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 a 200 ml (1.0 × 10 ⁶ cell) 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: 14 and an oligonucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 15 were prepared. The oligonucleotide is a DNA synthesizer (PE Applied Biosystems: Model 394)
The DNA was synthesized using a DNA / RNA Synthesizer, and purified using an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). PCR
A reaction solution was prepared using a vantage cDNA PCR Kit (manufactured by Clontech) in accordance with the attached manual.
This was carried out 29 times with one cycle of keeping the temperature at 65 ° C. for 5 minutes for one second. After the PCR reaction, the reaction solution is
The amplified DNA fragment was purified by filtration through S-400HR (Pharmacia Biotech). TA Cloni
Using ng Kit (manufactured by Invitrogen), the operation was performed according to the attached manual, and the amplified DNA fragment was cloned into pCR2.1 plasmid to construct plasmid pCPPO.
The nucleotide sequence of the DNA fragment contained in the obtained recombinant plasmid pCPPO was analyzed using a dye terminator cycle sequencing kit (PE
It was determined using a DNA sequencer 373S (manufactured by Applied Biosystems) and a DNA sequencer 373S (manufactured by PE Applied Biosystems). As a result, the nucleotide sequence represented by SEQ ID NO: 16 became clear, and it was revealed that pCPPO was a plasmid containing the full-length cDNA of PPO of Paramecium bursaria.

Reference Example 9 Acquisition of a gene encoding a protein which is a modified protein of P. chrysalis PPO and has no ability to oxidize protoporphyrinogen IX and specifically binds to protoporphyrinogen IX SEQ ID NO: 17 Using the oligonucleotide consisting of the nucleotide sequence and the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 18 as primers, PCR was performed using the plasmid pCPPO prepared in Reference Example 8 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, and
Plasmid pTVCRP (FIG. 10) 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 8 Test of the ability of the modified P. paramecium PPO to impart herbicide resistance The plasmids pTVCRP and pTV118N prepared in Reference Example 9
Were introduced into the E. coli BT3 / pACYCSP strain prepared in Reference Example 3 according to the method described in Hanahan, DJ, Mol. Biol., 166; By culturing in YPT medium containing lanfenicol and 10 μg / ml kanamycin, plasmid p
E. coli BT3 / pACYCSP + pTVCRP strain with ACYCSP and pTVCRP,
And Escherichia coli BT having plasmids pACYCSP and pTV118N
3 / pACYCSP + pTV118N strain was obtained. These Escherichia coli strains were isolated from the PPO-inhibiting herbicidal compound represented by the above structural formula 8 by 10
Or 1 ppm, 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml
YPT medium containing hemin and 50 μg / ml aminolevulinic acid was inoculated and cultured in the dark or in the light in the same manner as in Reference Example 2. 18 hours after the start of culture, 600 n
The absorbance at m was measured, and the absorbance when cultured in a medium containing no herbicidal compound was defined as 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was determined.
Table 9 shows the results.

[0058]

[Table 9]

Example 9 Introduction of the Gene Encoding the Modified P. paramecium PPO into Tobacco A plasmid for introducing the gene encoding the modified P. amphida PPO into plants by the Agrobacterium method was constructed. The plasmid pTVCRP prepared in Reference Example 9 was digested with the restriction enzymes BamHI and SacI to prepare a DNA fragment containing the gene encoding the modified P. lactis PPO. Β-g by digesting the binary vector pBI121 (Clontech) with restriction enzymes BamHI and SacI
The lucuronidase gene was removed and replaced with a D-containing gene encoding the present modified chlamydomonas PPO.
The plasmid pBICRP (FIG. 11) was prepared by inserting the NA fragment and ligating the gene downstream of the 35S promoter. The plasmid pBICRP was replaced with Agrobacterium tumefaciens.
The Agrobacterium strain having pBICRP was isolated by introducing it into LBA4404, culturing it in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin, and selecting transformants. The Agrobacterium strain was infected into aseptically cultured leaf pieces of tobacco, and tobacco into which the gene encoding the modified P. lactis PPO was introduced was obtained in the same manner as in Example 2.

Example 10 This modified P. amphida PPO
Confirmation of herbicide resistance of tobacco into which the gene encoding the gene of the present invention has been introduced.
By the same operation as described above, the degree of resistance to the herbicidal compound represented by Structural Formula 8 was quantitatively measured. 4 of the tobacco into which the gene encoding the modified P.
Table 10 shows the obtained results of the clones (CRP1 to 4) and the control recombinant tobacco. In the table, the degree of resistance to the herbicidal compound was expressed as a percentage of the total chlorophyll content of leaf pieces treated with the herbicidal compound, relative to the total chlorophyll content of untreated leaf pieces.

[0061]

[Table 10]

Example 11 Test of ability to impart herbicide resistance to a modified protein of barley ferrochelatase, which specifically binds to protoporphyrinogen IX Miyamoto, K. et al., Plant Physiol. 105; p769 (1994)
The plasmid having the barley ferrochelatase gene obtained by the method described in (1) is digested with restriction enzymes NspI and EcoRI to delete the signal sequence. A DNA fragment containing the gene encoding was obtained. This DNA fragment was converted to plasmid pTV119N (Takara Shuzo).
Plasmid pTVHVF1 (FIG. 12) was constructed by insertion between the SphI cleavage site and the EcoRI cleavage site. The plasmids pTVHVF1 and pTV118N were used in Reference Example 3 respectively.
E. coli BT3 / pACYCSP strain prepared in
l. Biol., 166; p557 (1983), and these were cultured in a YPT medium containing 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, and 10 μg / ml kanamycin to obtain the plasmid E. coli BT3 / pACYCSP + pTVHVF1 strain having pTVHVF1 and pTVHVF1, and E. coli BT3 / pACYCSP + pTV1 having plasmids pACYCSP and pTV118N
18N strains were obtained. These Escherichia coli strains contain 10 or 1 ppm of the PPO-inhibiting herbicidal compound represented by the structural formula 8,
100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml hemin and 50 μ
A YPT medium containing g / ml aminolevulinic acid was inoculated, and cultured under dark or light conditions in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 11 shows the results.

[0063]

[Table 11]

Example 12 Introduction of a Gene Encoding the Modified Barley Ferrokeratase into Tobacco A plasmid was constructed for introducing the ferrochelatase gene of the modified barley into plants by the Agrobacterium method. The plasmid pTVHVF1 described in Example 11 was replaced with the restriction enzyme NcoI.
After digestion with double-stranded DNA using DNA polymerase I
Nucleotides were added to the gaps to make the ends of the DNA blunt. Next, the DNA was treated with alkaline phosphatase derived from calf small intestine to dephosphorylate the 5 ′ end of the DNA, and phosphorylated BamH
An I linker (Takara Shuzo 4610P) was inserted and cyclized to construct a plasmid pTVHVF2. Then, pTVHVF2 was replaced with the restriction enzyme Ec.
After digestion with oRI, double-stranded DN
A nucleotide was added to the gap of A to blunt the end of the DNA. In addition, Alkaline phospha from calf small intestine
The DNA was treated with tase to dephosphorylate the 5'-end of the DNA, and a phosphorylated SalI linker (4680P, manufactured by Takara Shuzo) was inserted for cyclization to construct a plasmid pTVHVF3. The β-glucuronidase gene was removed by digesting the plasmid pBI121KS prepared in Reference Example 5 with restriction enzymes BamHI and SalI, while the above-mentioned pTVHVF3 was digested with restriction enzymes BamHI and SalI to obtain the modified barley ferrochela A DNA fragment containing the gene encoding the enzyme is prepared, and the DNA fragment is
By inserting the gene encoding the modified barley ferrochelatase downstream of the 35S promoter by inserting the plasmid pBI121KS in place of the curonidase gene, a plasmid pBIHVF (FIG. 13) was prepared. The plasmid pBIHVF was introduced into Agrobacterium tumefaciens LBA4404, and this was cultured in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin to select an Agrobacterium strain having pBIHVF by transformant selection. Isolated. The Agrobacterium strain was infected aseptically into leaf pieces of tobacco, and tobacco into which the gene encoding the modified barley ferrochelatase was introduced was obtained in the same manner as in Example 2.

Example 13 Confirmation of Herbicide Tolerance of Tobacco Introduced with Gene Encoding the Modified Barley Ferrokeratase The tobacco transfected with the gene encoding the modified barley ferrochelatase prepared in Example 12 was obtained. Example 3
By the same operation as described above, the degree of resistance to the herbicidal compound represented by Structural Formula 8 was quantitatively measured. Table 12 shows the results obtained for four tobacco clones (HVF1-4) into which the gene encoding the modified barley ferrochelatase was introduced (HVF1-4) and control recombinant tobacco. In the table, the degree of resistance to the herbicidal compound was expressed as a percentage of the total chlorophyll content of leaf pieces treated with the herbicidal compound, relative to the total chlorophyll content of untreated leaf pieces.

[0066]

[Table 12]

Example 14 Test of ability to impart herbicide resistance to a modified protein of cucumber ferrochelatase which specifically binds to protoporphyrinogen IX Oligonucleotide and sequence consisting of base sequence represented by SEQ ID NO: 19 Using an oligonucleotide consisting of the nucleotide sequence represented by No. 20 as a primer, Miyamoto, K. et al.
et al., Plant Physiol., 105; p769 (1994), PCR was performed using a cucumber ferrochelatase cDNA clone as a template, and a signal sequence-deleted cucumber ferrochelatase (hereinafter referred to as this modified product). A DNA fragment encoding cucumber ferrochelatase) was amplified. Oligonucleotides were synthesized using 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
This was carried out 30 times with one cycle of holding at 94 ° C. for 1 minute, then at 55 ° C. for 2 minutes, and further at 72 ° C. for 3 minutes. The amplified DNA fragment is digested with restriction enzymes BamHI and SacI,
BamHI cleavage site of plasmid pTV119N (Takara Shuzo) and SacI
Plasmid pTVC is inserted by inserting into the cleavage site.
The SF (Fig. 14) was constructed. The plasmids pTVCSF and pTV1
E. coli BT3 / pACYC prepared in Reference Example 3
The SP strain was introduced according to the method described in Hanahan, DJ, Mol. Biol., 166; p557 (1983), and these were introduced in a YPT medium containing 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, and 10 μg / ml kanamycin. By culturing, E. coli BT3 / pACYCSP + pTVCS with plasmids pACYCSP and pTVCSF
F strain and E. coli BT3 / pACYCSP + pTV118N strain having plasmids pACYCSP and pTV118N were obtained. These Escherichia coli strains contain 10 or 1 ppm of the PPO-inhibiting herbicidal compound represented by the above structural formula 8, and 100 μg / ml ampicillin, 15 μg
/ ml chloramphenicol, 10 μg / ml kanamycin, 10
YP with μg / ml hemin and 50 μg / ml aminolevulinic acid
The medium was inoculated into a T medium, and cultured in the dark or in the light in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 13 shows the results.

[0068]

[Table 13]

Example 15 Introduction of a Gene Encoding the Modified Cucumber Ferrochelatase into Tobacco A plasmid for introducing the modified cucumber ferrochelatase gene into plants by the Agrobacterium method was constructed. Plasmid pBI121 (Clontech) with restriction enzyme BamHI
The β-glucuronidase gene was removed by digestion with SacI and SacI, while the plasmid pTVCSF described in Example 14 was digested with the restriction enzymes BamHI and SacI, and the DNA fragment containing the gene encoding the modified cucumber ferrochelatase. And by inserting the DNA fragment into the plasmid pBI121 instead of the β-glucuronidase gene, the plasmid pBICSF in which the gene encoding the modified cucumber ferrochelatase is linked downstream of the 35S promoter.
(FIG. 15) was produced. The plasmid pBICSF was replaced with Agrobacter
ium tumefaciens LBA4404, 300 μg / ml streptomycin, 100 μg / ml rifampicin, 25 μg / m
An Agrobacterium strain having pBICSF was isolated by culturing in a medium containing lkanamycin and selecting transformants. The Agrobacterium strain was infected into aseptically cultured tobacco leaf pieces, and tobacco into which the modified cucumber ferrochelatase gene had been introduced was obtained in the same manner as in Example 2.

Reference Example 10 Confirmation of herbicide resistance of tobacco into which the gene encoding the present modified cucumber ferrochelatase was introduced The tobacco introduced with the gene encoding the present modified cucumber ferrochelatase produced in Example 15 was Example 3
And the degree of resistance of the tobacco to the herbicidal compound is quantitatively confirmed.

Reference Example 11 Isolation of Escherichia coli coproporphyrinogen III oxidase (hemF) gene A genomic DNA is prepared from Escherichia coli LE392 using a genomic DNA preparation kit ISOPLANT (manufactured by Nippon Gene). On the other hand, according to the Escherichia coli hemF gene registered in GenBank (Accession X75413) and the nucleotide sequence of the 5 ′ and '3 ′ regions thereof, it consists of an oligonucleotide primer consisting of the nucleotide sequence of SEQ ID NO: 21 and the nucleotide sequence of 22 Synthesize oligonucleotide primers. The oligonucleotide is 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). Using about 1 μg of genomic DNA of E. coli LE392 strain as a template, PCR is performed using 10 pmol of each of the above oligonucleotides as primers to amplify a DNA fragment containing the hemF gene of E. coli. PCR was performed at 96 ° C for 1 minute and at 55 ° C for 2 minutes.
30 minutes at 72 ° C for 3 minutes.
Perform cycle.

Example 16 Test of Escherichia coli hemF protein for imparting herbicide resistance The hemF gene contains the hemF gene amplified by the method described in Reference Example 11.
The DNA fragment was digested with restriction enzymes FbaI and PstI, and inserted between the BamHI cleavage site and the PstI cleavage site of the commercially available plasmid pUC118 (manufactured by Takara Shuzo Co., Ltd.).
16) was constructed. The plasmids pHEMF and pTV118N were added to E. coli BT3 / pACYCSP prepared in Reference Example 3, respectively.
ahan, DJ, Mol. Biol. 166; p557 (1983), and introduced these at 100 μg / ml ampicillin, 15 μg.
/ ml chloramphenicol and 10 μg / ml kanamycin in a YPT medium containing plasmid pACYCSP.
E. coli BT3 / pACYCSP + with pHEMF and E. coli BT3 / pACYCSP + with plasmids pACYCSP and pTV118N
pTV118N strain was obtained. These E. coli strains were converted to
Containing 10 or 1 ppm of a PPO-inhibiting herbicidal compound represented by the formula: 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin, 10 μg / ml hemin and
YPT medium containing 50 μg / ml aminolevulinic acid was inoculated, and cultured under dark or light conditions in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 14 shows the results.
Shown in

[0073]

[Table 14]

Reference Example 12 Introduction of E. coli hemF Gene into Tobacco A plasmid for introducing the E. coli hemF gene into plants by the Agrobacterium method was constructed. First, in order to obtain the E. coli hemF gene, an oligonucleotide primer having the nucleotide sequence represented by SEQ ID NO: 23 and an oligonucleotide primer having the nucleotide sequence represented by SEQ ID NO: 24 were synthesized. The oligonucleotide is a DNA synthesizer (PE Applied Biosystems: Model 394).
The DNA was synthesized using a DNA / RNA Synthesizer, and purified using an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). Using this oligonucleotide primer, PCR was performed in the same manner as in the method described in Reference Example 11, and DNA containing the E. coli hemF gene was obtained.
The fragment was amplified. Β-glucuron by digesting plasmid pBI121 (Clontech) with restriction enzymes BamHI and SacI
The enzyme is removed from the PCR amplified DNA
The fragment was digested with restriction enzymes BamHI and SacI to prepare an E. coli hemF gene fragment, and the gene fragment was treated with the β-glucuroni
By inserting the plasmid pBI121 in place of the dase gene, a plasmid pBIHEMF (FIG. 17) in which the E. coli hemF gene was linked downstream of the 35S promoter was prepared.
The plasmid pBIHEMF was transformed into Agrobacterium tumefaciens LBA
4404, 300 μg / ml streptomycin, 1
PBIH was obtained by culturing in a medium containing 00 μg / ml rifampicin and 25 μg / ml kanamycin and selecting transformants.
Agrobacterium strain with EMF was isolated. The Agrobacterium strain is infected aseptically into leaf pieces of tobacco, and tobacco into which the E. coli hemF gene has been introduced is obtained in the same manner as described in Example 2.

Reference Example 13 Confirmation of herbicide resistance of tobacco introduced with E. coli hemF gene The tobacco introduced with E. coli hemF gene prepared in Reference Example 12 was tested in the same manner as in Example 3, and To quantitatively confirm the degree of tolerance to herbicidal compounds.

Reference Example 14 Binding of porphyrin compound binding protein to protoporphyrin IX Kitano et al., Proceedings of the 74th Annual Meeting of the Chemical Society of Japan in 1998,
According to the method described on page 1353 4G511, a phage library displaying a protein containing an amino acid sequence consisting of five random amino acids, and a porphyrin compound 5,10,15,20-tetrakis (N-methylpyridinium-4-yl) -twenty one
Amino acid sequence HASYS or RASSL capable of specifically binding to H, 23H-porphine (H ₂ TMpyP) (H represents histidine, A represents alanine, S represents serine, Y represents tyrosine, R represents arginine, and L represents leucine.) A phage clone displaying a protein containing was prepared. First, a phage library displaying a protein containing an amino acid sequence consisting of five random amino acids was prepared as follows. SEQ ID NO: 2
A mixed oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5 and a mixed oligonucleotide consisting of the base sequence represented by SEQ ID NO: 26 were synthesized. The mixed 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). T4 DNA kin to each 50 pmol of the mixed oligonucleotide
After reacting ase to phosphorylate the 5 ′ end of the oligonucleotide, mix them, and heat them at 70 ° C. for 10 minutes,
Annealing was performed by slow cooling to room temperature at a rate of 0.5 ° C. per minute. By digesting plasmid pCANTAB5E (manufactured by Pharmacia Biotech) with restriction enzymes SfiI and NotI, a recombinant antibody gene was obtained from the plasmid.
By removing the ScFv and instead inserting the phosphorylated and annealed oligonucleotide pair, the amino acid sequence of the random five amino acids is followed by the amino acid sequence of the surface protein of M13 phage. A plasmid containing a nucleotide sequence encoding a protein consisting of the amino acid sequence was prepared. The plasmid was transformed into E. coli TG-1 strain by Hanahan, DJ, Mol. Biol. 166; p.
557 (1983).
Recombinant Escherichia coli TG-1 strain was obtained by culturing in 2 × YT medium (10 g / l yeast extract, 15 g / l tryptone, 5 g / l NaCl, pH 7.2) containing 00 μg / ml ampicillin. The recombinant E. coli
The TG-1 strain was inoculated into 2xYT medium containing 100 μg / ml ampicillin, and shake-cultured at 37 ° C. 1 hour after the start of culture, 6x10 ¹⁰ p
fu helper phage M13KO7 (Pharmacia Biotech)
And cultured with shaking for another 18 hours. Next, the culture solution was centrifuged at 1000 × g for 20 minutes, and a phage library displaying a protein containing an amino acid sequence consisting of five random amino acids was recovered. Also, the amino acid sequence HASY
In order to prepare a phage clone displaying a protein containing S (SEQ ID NO: 51), an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 27 and SEQ ID NO: 2
An oligonucleotide having the base sequence represented by No. 8 was synthesized. Furthermore, the amino acid sequence RASSL (SEQ ID NO: 53)
In order to prepare a phage clone displaying a protein containing, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 29 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 30 were synthesized. These 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 same operation as in the case of preparing the phage library displaying the protein containing the amino acid sequence consisting of five random amino acids was performed to obtain a phage clone displaying the protein containing the amino acid sequence HASYS or RASSL. Phage clone displaying protein containing amino acid sequence HASYS, amino acid sequence RASSL
Phage clones presenting, or phage suspension containing the phage library displaying a protein consisting of five random amino acids (titer 10 ⁵ pfu) respectively nitrocellulose filters (Schleicher & Schuell, Inc.)
After spotting the nitrocellulose filter,
% Bovine serum albumin in PBT buffer (137 mM NaCl, 8.10
mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , 0.05% Tween
20, pH 7.2) for 1 hour for blocking. After washing the nitrocellulose filter with PBT buffer, 10
2xSSC buffer containing 0.3M protoporphyrin IX (0.3M NaCl,
(0.03M sodium citrate) for 18 hours. Further, the nitrocellulose filter was washed with 2 × SSC buffer and dried, and then the fluorescence derived from protoporphyrin IX was detected under irradiation with ultraviolet light (365 nm). The spots in the phage library did not show fluorescence, but the amino acid sequence H
The spot of the phage clone displaying the protein containing ASYS and the spot of the phage clone displaying the protein containing the amino acid sequence RASSL showed clear fluorescence.

Example 17 Test of ability of protoporphyrin IX-binding protein to impart herbicide resistance First, amino acid sequence HASYS (SEQ ID NO: 51) or
A plasmid capable of expressing a gene encoding a protein containing the amino acid sequence RASSL (SEQ ID NO: 53) was prepared. First, in order to prepare a plasmid capable of expressing a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 52 (hereinafter, referred to as protein MGHASYS), an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 31, And an oligonucleotide having a base sequence represented by SEQ ID NO: 32 was synthesized. 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). Each of the oligonucleotides 50 pmol T4 DNA k
After phosphorylation of the 5 'end of the oligonucleotide by the action of inase, these were mixed, heated at 70 ° C for 10 minutes, and then annealed by slowly cooling to room temperature at a rate of 0.5 ° C per minute. By digesting the plasmid pTV118N with the restriction enzymes NcoI and EcoRI, a 16-bp gene fragment was removed from the plasmid, and the above-described phosphorylated and annealed oligonucleotide pair was inserted instead. Protein MGHA
PHASYS, a plasmid capable of expressing the gene encoding SYS
Was prepared. Next, in order to prepare a plasmid capable of expressing a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 54 (hereinafter referred to as protein MGRASSL), an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 33 , And an oligonucleotide having the base sequence of SEQ ID NO: 34 was synthesized. 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). The same operation as in the case of preparing the plasmid pHASYS was performed to prepare a plasmid pRASSL capable of expressing a gene encoding the protein MGRASSL. Next, the amino acid sequence YAG capable of binding to the porphyrin compound H ₂ TMPyP
A plasmid capable of expressing a gene encoding a protein containing Y or YAGF (Y represents tyrosine, A represents alanine, G represents glycine, and F represents phenylalanine) was prepared. In order to prepare a plasmid capable of expressing a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 56 (hereinafter, referred to as protein MGYAGY), an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 35, and a sequence An oligonucleotide having the base sequence represented by No. 36 was synthesized. In addition, in order to prepare a plasmid capable of expressing a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 58 (hereinafter, referred to as protein MGYAGF), an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 37, And an oligonucleotide having the nucleotide sequence of SEQ ID NO: 38 was synthesized. These oligonucleotides were used on a DNA synthesizer (PE Applied Biosystems: Model 394 DNA / RN).
A Synthesizer) and purified with an oligonucleotide purification cartridge (PE Applied Biosystems: OPC cartridge). Plasmid pHASYS
Perform the same operation as in the case of preparing the protein MGYAGY
A plasmid pYAGY capable of expressing a gene encoding the gene and a plasmid pYAGF capable of expressing a gene encoding the protein MGYAGF were prepared. The above plasmid pHASY
S, pRASSL, pYAGY, pYAGF or pTV118N, respectively, were added to the E. coli BT3 / pACYCSP strain prepared in Reference Example 3 by Hanahan, D .;
J., Mol. Biol., 166; p557 (1983), and these were introduced into a YPT containing 100 μg / ml ampicillin, 15 μg / ml chloramphenicol, 10 μg / ml kanamycin.
By culturing in a medium, plasmids pACYCSP and pHASY
E. coli BT3 / pACYCSP + pHASYS strain with S, plasmid pAC
E. coli BT3 / pACYCSP + pRASSL strain with YCSP and pRASSL, E. coli BT3 / pACYCSP + pY with plasmids pACYCSP and pYAGY
AGY strain, E. coli BT3 / p with plasmids pACYCSP and pYAGF
ACYCSP + pYAGF strain, and plasmids pACYCSP and pTV118N
Escherichia coli BT3 / pACYCSP + pTV118N strain was obtained. These Escherichia coli strains contained 1 ppm of the PPO-inhibiting herbicidal compound represented by the structural formula 8, and contained 100 μg / ml ampicillin,
μg / ml chloramphenicol, 10 μg / ml kanamycin,
Contains 10 μg / ml hemin and 50 μg / ml aminolevulinic acid
The cells were inoculated into a YPT medium and cultured under dark or light conditions in the same manner as in Reference Example 2. Eighteen hours after the start of the culture, the absorbance of the culture solution at 600 nm was measured, and the absorbance when cultured in a medium containing no herbicidal compound was set to 1, and the relative value of the absorbance when cultured in a medium containing the herbicidal compound was set to 1. I asked. Table 15 shows the results.

[0078]

[Table 15]

Furthermore, the amino acid sequences HASYS and RASSL
Proteins containing amino acid sequences connected consecutively
A plasmid capable of expressing the encoding gene was prepared.
A protein consisting of the amino acid sequence represented by SEQ ID NO: 59
Quality (hereinafter, protein MG (HASYS)_FourIt is written. )[here,
(HASYS)_nIs a sequence consisting of the peptide HASYS repeated n times
Means That can express the gene encoding
SEQ ID NO: 39, SEQ ID NO: 40,
The nucleotide sequence represented by SEQ ID NO: 41 or 42
Was synthesized. These ori
The oligonucleotide is a DNA synthesizer (PE Applied Biosystems)
Stems: Model 394 DNA / RNA Synthesizer)
And oligonucleotide purification cartridge (PE
Applied Biosystems: OPC cartridge)
Purified. First, from the base sequence represented by SEQ ID NO: 40
And a base represented by SEQ ID NO: 41
Create T4 DNA kinase on sequence oligonucleotide
The 5 'end of each was phosphorylated. After that,
Oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 39
Base sequence represented by SEQ ID NO: 40 phosphorylated with tide
An oligonucleotide consisting of, or phosphorylated
An oligonucleotide comprising the base sequence represented by SEQ ID NO: 41
An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 42
And 300 pmol each at 70 ° C
For 5 minutes and slowly reach room temperature at a rate of 0.5 ° C / min.
Annealed by cooling. The ani
The two pairs of oligonucleotides were mixed and T4
 After ligating these using DNA ligase, the resulting
The 5 'end of the DNA fragment is phosphorylated using T4 DNA kinase.
Oxidized. On the other hand, the vector pTV118N was replaced with the restriction enzymes NcoI and Eco.
Digestion with RI to remove the 16 bp DNA fragment
Instead of inserting the phosphorylated DNA fragment
Therefore, protein MG (HASYS)_FourGene that encodes
The resulting plasmid, pHASYS4, was obtained. Furthermore, the array number
No. 60 (hereinafter referred to as a protein)
Bottom, protein MG (HASYS)₈It is written. Genetics that encode)
SEQ ID NO:
An oligonucleotide having a base sequence represented by 43,
And an oligo consisting of the base sequence represented by SEQ ID NO: 44
Nucleotides were synthesized. The oligonucleotide is DNA
Synthesizer (PE Applied Biosystems: Model 39)
4 DNA / RNA Synthesizer)
Reotide Purification Cartridge (PE Applied Biosis
(Thames: OPC cartridge). First, T4 D
The 5 'end of the oligonucleotide is
Phosphorylated. Thereafter, the base sequence shown in SEQ ID NO: 39
Sequence oligonucleotides and phosphorylated sequence numbers
Oligonucleotide consisting of the base sequence represented by No. 40
Are mixed at 300 pmol each and phosphorylated SEQ ID NO: 41
Oligonucleotide consisting of the indicated base sequence and sequence number
Oligonucleotide consisting of the base sequence represented by No. 42
Are mixed at 300 pmol each, and the phosphorylated sequence
Oligonucleotide consisting of the base sequence represented by No. 43
From the base sequence represented by SEQ ID NO: 44
Oligonucleotides of 600 pmol each
After heating it at 70 ° C for 5 minutes, slowly heat it at a rate of 0.5 ° C / min.
Annealed by cooling to room temperature. Said
Mixes three pairs of annealed oligonucleotide pairs
And after ligation using T4 DNA ligase, the resulting D
Phosphate the 5 'end of the NA fragment using T4 DNA kinase
It has become. Then, the above-described plasmid pHASYS4 was prepared.
Perform the same operation as in the above procedure to obtain protein MG (HASYS).₈The
Obtain the plasmid pHASYS8 that can express the gene to be
Was. Next, from the amino acid sequence represented by SEQ ID NO: 61,
Protein (hereinafter referred to as protein MG (RASSL)_FourIt is written. )
[Where (RASSL)_nIs the peptide RASSL repeated n times
Means a crooked array. ] Can be expressed
SEQ ID NO: 45, to produce a functional plasmid
SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48
Oligonucleotides consisting of different base sequences were synthesized. Ma
A protein consisting of the amino acid sequence represented by SEQ ID NO: 62
Protein (hereinafter referred to as protein MG (RASSL)₈It is written. )
A plasmid capable of expressing the gene to be expressed
An oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 49
The nucleotide sequence represented by SEQ ID NO: 50
Oligonucleotides were synthesized. The oligonucleotide
Otide is a DNA synthesizer (PE Applied Biosystems)
(Synthesis using Model 394 DNA / RNA Synthesizer)
And an oligonucleotide purification cartridge (PE
Id Biosystems: OPC cartridge)
Was. Perform the same operation as when plasmid pHASYS4 was prepared.
Do, protein MG (RASSL) _FourPlasmid pR capable of expressing
ASSL4 was prepared. In addition, plasmid pHASYS8 was prepared.
Perform the same operation as in the above procedure to obtain protein MG (RASSL).₈Express
A possible plasmid, pRASSL8, was created. Plus above
Mid pHASYS4, pHASYS8, pRASSL4, pRASSL8 or pTV118
N represents E. coli BT3 / pACYCSP prepared in Reference Example 3, respectively.
Described in Hanahan, D.J., Mol. Biol., 166; p557 (1983)
And introduce them at 100 μg / ml ampicillin
, 15 μg / ml chloramphenicol, 10 μg / ml kanamai
By culturing in YPT medium containing syn, plasmid p
Escherichia coli BT3 / pACYCSP + pHASYS4 with ACYCSP and pHASYS4
Strain, Escherichia coli BT3 / pA with plasmids pACYCSP and pHASYS8
CYCSP + pHASYS8 strain, carrying plasmids pACYCSP and pRASSL4
E. coli BT3 / pACYCSP + pRASSL4 strain, plasmid pACYCSP
E. coli BT3 / pACYCSP + pRASSL8 strain with pRASSL8, and
E. coli BT3 / pA with plasmids pACYCSP and pTV118N
CYCSP + pTV118N strain was obtained. These E. coli strains were
Contains 1 ppm of a PPO-inhibiting herbicidal compound represented by Structural Formula 8
100 μg / ml ampicillin, 15 μg / ml chlorampheni
Cole, 10 μg / ml kanamycin, 10 μg / ml hemin and
Inoculate YPT medium containing 50 μg / ml aminolevulinic acid,
Culture under dark or light conditions as in Example 2.
did. 18 hours after the start of the culture, the absorption of the culture solution at 600 nm was performed.
Measure the light intensity and culture in a medium without the herbicidal compound.
The absorbance at the time of exposure was set to 1, and the medium containing the herbicidal compound was used.
The relative value of the absorbance when cultured was determined. Table 16 shows the results.
Shown in

[0080]

[Table 16]

REFERENCE EXAMPLE 15 Introduction of Gene Encoding Protoporphyrin IX-Binding Protein into Tobacco First, protoporphyrin IX-binding protein MG (HASY
S) A plasmid was constructed for introducing the gene encoding ₈ into plants by the Agrobacterium method. After the plasmid pHASYS8 prepared in Example 17 was digested with the restriction enzyme NcoI, nucleotides were added to the gaps of the double-stranded DNA using DNA polymerase I to blunt the ends of the DNA.
Next, the DNA was treated with Alkaline phosphatase derived from calf small intestine to dephosphorylate its 5 'end, and a phosphorylated BamHI linker (4610P, manufactured by Takara Shuzo) was inserted and cyclized to construct plasmid pHASYS8B. In addition, plasmid pBI121 (Clont
ech) was digested with restriction enzymes BamHI and SacI to remove the β-glucuronidase gene. On the other hand, plasmid pHASYS8B is digested with restriction enzymes BamHI and SacI to prepare a DNA fragment containing a gene encoding protein MG (HASYS) ₈ , and the DNA fragment is inserted into plasmid pBI121 instead of the β-glucuronidase gene described above. As a result, a plasmid pBIHASYS8 (FIG. 18) in which the gene encoding the protoporphyrin IX-binding protein MG (HASYS) ₈ was linked downstream of the 35S promoter. Next, a plasmid for introducing the gene encoding the protoporphyrin IX-binding protein MG (RASSL) ₈ into plants by the Agrobacterium method was constructed. The plasmid pRASSL8 prepared in Example 17 was constructed according to the method for preparing pBIHASYS8 described above, and the protoporphyrin IX binding protein MG was constructed.
A plasmid pBIRASSL8 (FIG. 19) in which the gene encoding (RASSL) ₈ was linked downstream of the 35S promoter was prepared. Plasmid pBIHASYS8 or pBIRASSL8 above,
Agrobacterium strain having pBIHASYS8, and pBIRASSL8 were introduced by introducing each into Agrobacterium tumefaciens LBA4404, culturing them in a medium containing 300 μg / ml streptomycin, 100 μg / ml rifampicin, and 25 μg / ml kanamycin and selecting transformants. Agrobacterium strains were isolated. The Agrobacterium strain was infected into aseptically cultured tobacco leaf pieces, and tobacco into which the gene encoding the protoporphyrin IX binding protein MG (HASYS) ₈ was introduced in the same manner as described in Example 2, and Porphyrin IX binding protein MG (R
ASSL) Tobacco into which the gene encoding ₈ has been introduced is obtained.

Reference Example 16 Confirmation of herbicide resistance of tobacco into which a gene encoding a protoporphyrin IX-binding peptide was introduced The tobacco having a gene encoding a protoporphyrin IX-binding protein produced in Reference Example 15 was introduced. A test was conducted in the same manner as in Example 3 to quantitatively confirm the degree of resistance of the tobacco to the herbicidal compound.

[0083]

Industrial Applicability According to the present invention, it is possible to produce a plant having resistance to a weed control agent.

[Sequence Listing Free Text] SEQ ID NO: 1 Oligonucleotide primer designed to amplify bchH gene SEQ ID NO: 2 Oligonucleotide primer designed to amplify bchH gene SEQ ID NO: 3 Soybean-derived PPO gene Oligonucleotide primer designed to amplify SEQ ID NO: 4 Oligonucleotide primer designed to amplify soybean-derived PPO gene SEQ ID NO: 6 Oligonucleotide primer designed to amplify bchH gene SEQ ID NO: 7 bchH gene SEQ ID NO: 8 DNA having partial nucleotide sequence of tobacco-derived chlH gene
Oligonucleotide primer designed to amplify fragment SEQ ID NO: 9 DNA having partial base sequence of tobacco-derived chlH gene
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 fragment SEQ ID NO: 13 DNA having partial base sequence of PPO gene derived from soybean
Oligonucleotide primer designed to amplify the fragment SEQ ID NO: 14 Oligonucleotide primer designed to amplify the PPO gene derived from Euglena sequence SEQ ID NO: 15 Oligo designed to amplify the PPO gene derived from Euglena Nucleotide primer SEQ ID NO: 17 Oligonucleotide primer designed to amplify a DNA fragment having a partial base sequence of PPO gene derived from Paramecium bursaria SEQ ID No. 18: Amplify DNA fragment having a partial base sequence of PPO gene derived from Paramecium bursaria Oligonucleotide primer designed for amplifying a DNA fragment having a partial base sequence of cucumber-derived ferrochelatase gene SEQ ID NO: 20 Oligonucleotide primer designed to amplify DNA fragment having partial base sequence of erokeratase gene SEQ ID NO: 21 Oligonucleotide primer designed to amplify hemF gene derived from Escherichia coli SEQ ID NO: 22 Amplify hemF gene derived from Escherichia coli SEQ ID NO: 23 Oligonucleotide primer designed to amplify hemF gene derived from E. coli SEQ ID NO: 24 Oligonucleotide primer designed to amplify hemF gene derived from E. coli SEQ ID NO: 25 Oligonucleotide designed to prepare a gene encoding a protein containing an amino acid sequence consisting of five random amino acids SEQ ID NO: 26 Protein containing an amino acid sequence consisting of five random amino acids Oligonucleotide designed to produce gene encoding SEQ ID NO: 27 Oligonucleotide designed to produce gene encoding protein containing amino acid sequence HASYS SEQ ID NO: 28 Gene encoding protein containing amino acid sequence HASYS SEQ ID NO: 29 designed to produce a gene encoding a protein comprising the amino acid sequence RSSL SEQ ID NO: 30 Designed to produce a gene encoding a protein comprising the amino acid sequence RASS SEQ ID NO: 31 designed to produce a gene encoding protein MGHASYS SEQ ID NO: 32 designed to produce a gene encoding protein MGHASYS Oligonucleotides designed to make genes SEQ ID NO: 33 Oligonucleotides designed to make a gene encoding protein MGRASSSL SEQ ID NO: 34 Oligonucleotides designed to make a gene encoding protein MGRASSSL SEQ ID NO: 35 Oligonucleotide designed to produce gene encoding protein MGYAAGY SEQ ID NO: 36 Oligonucleotide designed to produce gene encoding protein MGYAAGY SEQ ID NO: 37 Gene producing gene encoding protein MGYAGF Oligonucleotide designed for generating a gene encoding protein MGYAGF SEQ ID NO: 38 ASYS) ₄ is designed to produce a gene encoding the oligonucleotide SEQ ID NO: 40 protein MG (hasys) ₄ is designed to produce a gene encoding the oligonucleotide SEQ ID NO: 41 protein MG a (hasys) ₄ Oligonucleotide designed to produce gene encoding SEQ ID NO: 42 Oligonucleotide designed to produce gene encoding protein MG (HASYS) ₄ SEQ ID NO: 43 Gene encoding protein MG (HASYS) ₈ Oligonucleotides designed to produce SEQ ID NO: 44 Oligonucleotides designed to produce a gene encoding protein MG (HASYS) ₈ SEQ ID NO: 45 To produce a gene encoding protein MG (RASSSL) ₄ Designed Oligonucleotides SEQ ID NO: designed to produce a gene encoding the oligonucleotide SEQ ID NO: 47 protein MG (RASSL) _4, which is designed to produce a gene encoding the oligo nucleotide SEQ ID NO: 46 protein MG (RASSL) ₄ 48 Oligonucleotide designed to create a gene encoding protein MG (RASSSL) ₄ SEQ ID NO: 49 Oligonucleotide designed to create a gene encoding protein MG (RASSSL) ₈ SEQ ID NO: 50 Protein MG ( oligonucleotide designed SEQ ID NO: 51 protoporphyrin IX binding protein HASYS SEQ ID NO: 52 protoporphyrin IX binding protein MGHASYS SEQ ID NO: 53 protoporphyrin IX binding to produce a gene encoding RASSL) ₈ Protein RASSL SEQ ID NO: 54 protoporphyrin IX binding protein MGRASSL SEQ ID NO: 55 H ₂ TMPyP binding protein YAGY SEQ ID NO: 56 H ₂ TMPyP binding protein MGYAGY SEQ ID NO: 57 H ₂ TMPyP binding protein YAGF SEQ ID NO: 58 H ₂ TMPyP binding protein MGYAGF SEQ ID NO: 59 Protoporphyrin IX binding protein MG (HASYS) ₄ SEQ ID NO: 60 Protoporphyrin IX binding protein MG (HASYS) ₈ SEQ ID NO: 61 Protoporphyrin IX binding protein MG (RASSL) ₄ SEQ ID NO: 62 Protoporphyrin IX binding protein MG (RASSL) ₈

[0085]

[Sequence List] SEQUENCE LISTING <110> Sumitomo Chemical Company Limited <120> Method for producing herbicide resistant plants <130> P150318 <150> JP 10/120553 <151> 1998-04-30 <150> JP 10/281127 <151 > 1998-10-02 <150> JP 10/330981 <151> 1998-11-20 <150> JP 11/054730 <151> 1999-03-02 <160> 62 <210> 1 <211> 39 <212 > DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify bchH gene <400> 1 gacatctaga ggagacgacc atatgcacgg tgaagtctc 39 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify bchH gene <400> 2 acggaagctt agatcttcac tcggcggcaa t 31 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify soybean PPO gene <400> 3 tcgagctcca tggtttccgt cttcaacgag atcctattc 39 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify soybean PPO gene <400> 4 ttgtcgacaa ctgctactat ttgtacactc tatttg <210 > 5 <211> 1 632 <212> DNA <213> Glycine max var. Williams82 <220><221> CDS <222> (1) ... (1632) <400> 5 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 Ser 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 atp gcc Valc 240 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 gtc gtc acg gag 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 g ga 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 agt ggt 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 ggt 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 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 180 185 190 cct cca ggt cat gag gaa tcg gtt gaa gag ttt gtt cgt cgg aac ctt 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 t gt 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 Gln Glu 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 gga864 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 g aa 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 ca 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 gc a 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 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 465 470 475 480 att cca cag ttc tta gtt ggc cat ctt gat ctt cta 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 Seryr Ala Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu 515 520 525 gta gca gct gaa gta aac gat ttt ctc aca aat aga gtg tac aaa tag 1632 Val Ala Ala Gla Val Glu Val Asn Asp Phe Leu Thr Asn Arg Val Tyr Lys 530 535 540 543 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220><213> Designed oligonucleotide primer to amplify bchH gene <400> 6 gacatctagt ctagacgacc atatgcacgg tgaagtctc 39 <210> 7 <211> 31 <212 > DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify bchH gene <400> 7 acggaagctt ggtacctcac tcggcggcaa t 31 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of tobacco chlH gene <400> 8 ccaatgtaat gctatggtac ctatgttatt cactc 35 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of tobacco chlH gene <400> 9 gagatcattc tttttgctgt cgacttatcg atcg 34 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 10 ggcggaggcg tcaccatggt ctgcatcgcc caggcc 36 <210> 1 1 <211> 36 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 11 gcctgcaggt cgacaactgc tactatttgt acactc 36 <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 cacaggaaag gtaccatggt ctgcatcgcc cag 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of soybean PPO gene <400> 13 cctgcagctc gagagctcct actatttgta cac 33 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Chlamydomonas PPO gene <400> 14 aatgatgttg acccagactc ctgggacc 28 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Chlamydomonas PPO gene <400> 15 tactacacat cccagcaagc gccaatg 27 <210> 1 6 <211> 1838 <212> DNA <213> Chlamydomonas reinhardtii CC407 <220><221> CDS <222> (2) ... (1693) <400> 16 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 cgg 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 gcg acc gct gcg 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 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 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 t tc ctt gtt 334 Thr Gly 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 Ile Thr Ser Met Ser Gly 115 120 125 gat ggc 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 aag gac ctt 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 agg Asp Pro Thr Ala Pro Arg Phe Val Trp 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 atc cgc 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 Asn 195 200 205 gga gcc atg ccc tcc ttc gag gag g ag cag ttc atc cgc cgc 670 Gly Ala Met 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 Phe Arg Leu Ile Glu Pro Phe Cys Ser 225 230 235 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 aag aac ggc ggc 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 gcc ccc gcc cgg 862 Ala Ile Lys Leu Phe Gln Glu Arg Gln Ser Asn Pro Ala Pro Pro Arg 275 280 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 Val Gly Ser Phe 290 295 300 cgc aag ggc 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 gt a ag ctg gtg tct ctg ggc cgc gag gcg 1006 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 Ggt gag Arg Tyr Gly Leu Val Tyr Asp Thr Pro Glu Gly Arg Val Lys 340 345 350 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 Ala 355 360 365 gac ctg gtc aag gag cag gcg ccc 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 gg gcc gcc gcc tac ccg ctg agc 1198 Phe Asp Tyr Pro 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 A 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 ag c tcc agc ctg ttc ccc ggc cgc gcg ccc gag ggc 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 ac cgc acc ac cgc cag 1390 Leu Leu Asn Tyr Ile Gly Gly Thr Thr 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 465 470 475 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 480 485 490 490 495 tgg ccg cgg gcc cag ttc aac ctg ggc cac ctg gag cag ctg 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 ggg ggg ctg cag ggg gg Arg Lys Ala Leu Asp Ala Ala Gly Leu Gln 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 530 535 540 cac ggc tac gag tcc gca gcc aac 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 545 550 555 555 gca gtc aag gcc taa gcggctgcag cag cag cag cag cag tagg Val Lys Ala 560 563 ggtaaatgcc gcagtggcac cggcagcagc aattggcaag cacttggggc aagcggagtg 1793 gaggcgaggg gggggctacc attggcgctt gctgggatgt gtagt 1838 <210> 17 <211> 32 <212> DNA <213> fragment Artificial Sequencer tial sequence of Chlamydomonas PPO gene <400> 17 ggtcggtgga ggggatccga tgctggtgac cg 32 <210> 18 <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> 18 gctactgctg cgagctctta ggccttgact gc 32 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having par tial sequence of cucumber ferrochelatase gene <4 00> 19 gctttagaat cggatcctat ggcagtggat gac 33 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify DNA fragment having partial sequence of cucumber ferrochelatase gene <400> 20 ggtgaacttc tatttgagct ctcaggtaaa tataag 36 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Esherichia coli hemF gene ne <400> 21 gctgaaggcg tgatcagtta tttcc 25 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Esherichia coli hemF gene <400> 22 catcagcctg cagtgcgaaa agtg 24 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Esherichia coli hemF gene <400> 23 cgaaaaaggg atccgttatg aaaccc 26 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide primer to amplify Esherichia coli hemF gene <400> 24 gctgttttcc gagctcccgt cac 23 <210> 25 <211> 22 <2 12> DNA <213> Artificial Sequence <220><223> Designed oligonucleotides to synthesize genes coding proteins whic h contain amino acid sequences comprised with random 5 amino acids <400> 25 tggccnnknn knnknnknnk gc 22 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotides to synthesize genes coding proteins whic h contain amino acid sequences comprised with random 5 amino acids <400> 26 ggccgcmnnm nnmnnmnnmn nggccagct 29 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein including amino acid sequence HASYS <400> 27 tggcccatgc tagttagtcg gc 22 <210> 28 <211> 29 <212> DNA <213 > Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein including amino acid squence HASYS <400> 28 tggcgccgac taactagcat gggccagct 29 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220 ><223> Designed oligonucleotide to synthesize the gene coding the protein including amino acid sequence RASSL <400> 29 tggcccgggc gtcgtcgttg gc 22 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein including amino acid sequence RASSL <400> 30 ggccgccaac gacgacgccc gggccagct 29 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGHASYS <400> 31 catgggtcac gcttcttact cctaag 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGHASYS <400> 32 aattcttagg agtaagaagc gtgacc 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGRASSL <400> 33 catgggtcgt gcttcttccc tgtaag 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGRASSL <400> 34 aattct taca gggaagaagc acgacc 26 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGYAGY <400> 35 catgggttac gctggctact aag 23 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGYAGY <400> 36 aattcttagt agccagcgta acc 23 <210> 37 <211> 23 <212> DNA <213 > Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGYAGF <400> 37 catgggttac gctggcttct aag 23 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MGYAGF <400> 38 aattcttaga agccagcgta acc 23 <210> 39 <211> 34 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (HASYS) ₄ <400> 39 catgggtcac gcttcttact cccatgcatc ttac 34 <210> 40 <211> 34 <212> DNA <213> Artificial Sequence <220><223> D esigned oligonucleotide to synthesize the gene coding the protein MG (HASYS) ₄ <400> 40 gtgggagtaa gatgcatggg agtaagaagc gtgacc 36 <210> 41 <211> 37 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (HASYS) ₄ <400> 41 tcccacgctt cttactccca tgcatcttac tcctaag 37 <210> 42 <211> 35 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (HASYS) ₄ <400> 42 aattcttagg agtaagatgc atgggagtaa gaagc 35 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (HASYS ) ₈ <400> 43 tcccacgctt cttactccca tgcatcttac 30 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (HASYS) ₈ <400> 44 gtgggagtaa gatgcatggg agtaagaagc 30 <210> 45 <211> 34 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to sy nthesize the gene coding the protein MG (RASSL) ₄ <400> 45 catgggtcgt gcttcttccc tgcgcgcatc ttcc 34 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (RASSL) ₄ <400> 46 acgcagggaa gatgcgcgca gggaagaagc acgacc 36 <210> 47 <211> 37 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG ( RASSL) ₄ <400> 47 ctgcgtgctt cttccctgcg cgcatcttcc ctgtaag 37 <210> 48 <211> 35 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (RASSL) ₄ <400> 48 aattcttaca gggaagatgc gcgcagggaa gaagc 35 <210> 49 <211> 30 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (RASSL) ₈ <400> 49 ctgcgtgctt cttccctgcg cgcatcttcc 30 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide to synthesize the gene coding the protein MG (RASSL) ₈ <400> 50 acgcagggaa gatgcgcgca gggaagaagc 30 <210> 51 <211> 5 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein HASYS <400> 51 His Ala Ser Tyr Ser 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein MGHASYS <400> 52 Met Gly His Ala Ser Tyr Ser 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein RASSL <400> 53 Arg Ala Ser Ser Leu 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein MGRASSL <400> 54 Met Gly Arg Ala Ser Ser Leu 1 5 <210> 55 <211> 4 <212> PRT <213> Artificial Sequence <220><223> H ₂ TMpyP binding protin YAGY <400> 55 Tyr Ala Gly Tyr 1 <210> 56 <211> 6 <212> PRT <213> Artificial Sequence <220><223> H ₂ TMpyP binding protin MGYAGY <400> 56 Met Gly Tyr Ala Gly Tyr 1 5 <210> 57 <211> 4 <212> PRT <213> Artificial Sequence <220><223> H ₂ TMpyP binding protin YAGF <400> 5 7 Tyr Ala Gly Phe 1 <210> 58 <211> 6 <212> PRT <213> Artificial Sequence <220><223> H ₂ TMpyP binding protin MGYAGF <400> 58 Met Gly Tyr Ala Gly Phe 1 5 <210> 59 <211> 22 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein MG (HASYS) ₄ <400> 59 Met Gly His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr 1 5 10 15 Ser His Ala Ser Tyr Ser 20 <210> 60 <211> 42 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein MG (HASYS) ₈ <400> 60 Met Gly His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr 1 5 10 15 Ser His Ala Ser Tyr Ser His Ala Ser Tyr Ser His Ala Ser Tyr Ser 20 25 30 His Ala Ser Tyr Ser His Ala Ser Tyr Ser 35 40 <210> 61 <211> 22 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX binding protein MG (RASSL) ₄ <400> 61 Met Gly Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu Arg Ala Ser Ser 1 5 10 15 Leu Arg Ala Ser Ser Leu 20 <210> 62 <211> 42 <212> PRT <213> Artificial Sequence <220><223> Protoporphyrin IX bind ining protein MG (RASSL) ₈ <400> 62 Met Gly Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu Arg Ala Ser Ser 1 5 10 15 Leu Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu 20 25 30 Arg Ala Ser Ser Leu Arg Ala Ser Ser Leu 35 40

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pETBCH. bchH
Is a magnesium keratase subunit gene of photosynthetic bacterium Rhodobacter sphaeroides, T7 pro means a promoter sequence of T7 phage, and T7 ter means a terminator sequence of T7 phage. Amp ^r is an ampicillin resistance gene, lacI ^q is a lactose operon repressor protein gene, and ori is a replication origin.

FIG. 2 shows a restriction map of plasmid pACYCSP. PPO
Is the soybean protoporphyrinogen IX oxidase gene, which means the promoter sequence of the lac pro lactose operon. Cm ^r represents a chloramphenicol resistance gene, and ori represents a replication origin.

FIG. 3 shows a restriction map of plasmid pTVBCH. bchH
Is the magnesium keratase subunit gene of the photosynthetic bacterium Rhodobacter sphaeroides, and lac pro means the promoter sequence of the lactose operon. Also,
Amp ^r represents the ampicillin resistance gene, and ori represents the replication origin.

FIG. 4 shows a restriction map of plasmid pBIBCH. bchH
Is the magnesium keratase subunit gene of the photosynthetic bacterium Rhodobacter sphaeroides, 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 is a kanamycin resistance gene, RB and LB are T-DNA
Represents the left and right boundary array.

FIG. 5 shows a restriction map of plasmid pNO. NP indicates the promoter sequence of the nopaline synthase gene, NT indicates the terminator sequence of the nopaline synthase gene, and 35S indicates 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. 6 shows a restriction map of plasmid pTCHLH. TCHL
H 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. 7 shows a restriction map of plasmid pBITCHLH. TC
HLH is a protoporphyrin IX binding subunit gene of tobacco magnesium keratase lacking the chloroplast translocation signal, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, 35S is the Cauliflower mosaic virus 35S
Means promoter. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 8 shows a restriction map of plasmid pTVGMP. GMP
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. 9 shows a restriction map of plasmid pBIGMP. GMP
Is the soybean PPO gene lacking the chloroplast translocation signal and 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 35S promoter of cauliflower mosaic virus. Means NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 10 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, Amp ^r
Represents an ampicillin resistance gene, and ori represents an origin of replication.

FIG. 11 shows a restriction map of plasmid pBICRP. CR
P is the Chlamydomonas reinhardtii PPO gene lacking the chloroplast translocation signal and 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. Also, N
PTII represents the kanamycin resistance gene, and RB and LB represent the left and right border sequences of T-DNA.

FIG. 12 shows a restriction map of plasmid pTVHVF1. H
VF is a barley ferrochelatase gene lacking a signal 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. 13 shows a restriction map of plasmid pBIHVF. HV
F is the barley ferrochelatase gene with the signal sequence deleted, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, 35S is the cauliflower mosaic virus 35S
Means promoter. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 14 shows a restriction map of plasmid pTVCSF. CS
F is a cucumber ferrochelatase gene lacking a signal 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. 15 shows a restriction map of plasmid pBICSF. CS
F is the cucumber ferrochelatase gene lacking the signal sequence, NP is the promoter sequence of the nopaline synthase gene, NT is the terminator sequence of the nopaline synthase gene, 35S is the cauliflower mosaic virus 35S
Means promoter. NPTII represents a kanamycin resistance gene, and RB and LB represent left and right border sequences of T-DNA.

FIG. 16 shows a restriction map of plasmid pHEMF. HEM
F is E. coli coproporphyrinogen III oxidase gene hemF, 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. 17 shows a restriction map of plasmid pBIHEMF. H
EMF is Escherichia coli coproporphyrinogen III oxidase gene hemF, 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. 18 shows a restriction map of plasmid pBIHASYS8. HASYS8 is a gene encoding the protein MG (HASYS) ₈ , 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. 19 shows a restriction map of plasmid pBIRASSL8. RASSL8 is a gene encoding the protein MG (RASSL) ₈ , 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.

 ──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number Japanese Patent Application No. 11-54730 (32) Priority date March 2, 1999 (1999.3.2) (33) Priority claim country Japan (JP)

Claims (56)

[Claims] 1. A method for imparting resistance to a weed controlling agent to a plant, wherein a gene encoding a protein having the following properties (1), (2) and (3) is introduced into a plant cell and expressed. A method comprising the steps of: (1) It specifically binds to a substance related to the weed controlling action of the weed controlling agent. (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. 2. The method 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 a protein having a property of specifically binding to a substance contained as an active ingredient in a weed controlling agent. 4. The method according to claim 1, wherein the protein has a property of specifically binding to a plant cell endogenous substance involved in the expression of the weed controlling activity of the weed controlling agent in the plant cell. 5. The weed controlling agent according to claim 1, which is a weed controlling agent that inhibits or suppresses porphyrin biosynthesis in plants.
The described method. 6. The method for controlling weeds according to claim 6, wherein the weed control agent is protoporphyrinogen.
The method according to claims 1 to 5, which is an IX oxidase-inhibiting herbicide. 7. The weed controlling agent according to any one of the following (1) to (3):
A professional whose active ingredient is a compound selected from the group of compounds
Topolphyrinogen IX oxidase inhibitory herbicide
The method according to claim 1, wherein (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
Senzen), 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 the following G-1 to 9;
Groups represented by 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
 -CR¹⁵ Represents a group, R¹⁵ 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, E¹² Is a hydrogen atom or C₁-C_Three Alkyl group
Represents ) 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
Benzen 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 an 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
Group optionally substituted with a halogen group or a halogen atom_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 charcoal
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 Alkoshki 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. 8. The protein according to claim 1, wherein the protein has a property of specifically binding to protoporphyrin IX.
The method according to 2, 4, 5, 6 or 7. 9. 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. 2,4,5,6,7 or 8
The described method. 10. The protein is a protoporphyrin IX-binding subunit protein of magnesium keratase derived from a photosynthetic microorganism, or a modified protein of the protein having a property of specifically binding to protoporphyrin IX. Certain claims 1, 2,
The method according to 4, 5, 6, 7, 8 or 9. 11. 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. Claims 1, 2, 4, 5,
The method according to 6, 7, 8 or 9. 12. 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 method according to 6, 7, 8, 9 or 11. 13. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 51.
The method according to 4, 5, 6, 7 or 8. 14. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 52.
The method according to 4, 5, 6, 7, 8 or 13. 15. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 53.
The method according to 4, 5, 6, 7 or 8. 16. The protein according to claim 1, wherein the protein comprises the amino acid sequence represented by SEQ ID NO: 54.
The method according to 4, 5, 6, 7, 8 or 15. 17. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 55.
The method according to 4, 5, 6, 7 or 8. 18. The protein according to claim 1, wherein the protein comprises the amino acid sequence represented by SEQ ID NO: 56.
The method according to 4, 5, 6, 7, 8 or 17. 19. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 57.
The method according to 4, 5, 6, 7 or 8. 20. The protein according to claim 1, wherein the protein comprises an amino acid sequence represented by SEQ ID NO: 58.
The method according to 4, 5, 6, 7, 8 or 19. 21. The protein according to claim 1, wherein the protein is a protein consisting of 4 to 100 amino acids.
5, 6, 7, 8, 13, 14, 15, 16, 17, 1
The method according to 8, 19 or 20. (22) the protein is protoporphyrinogen;
The method according to claim 1, 2, 4, 5, 6, or 7, which is a protein having a property of specifically binding to IX. 23. The protein is a modified protein of protoporphyrinogen IX oxidase, which has no ability to oxidize protoporphyrinogen IX and has a property of specifically binding to protoporphyrinogen IX. Claim 1, 2, 4, 5, 6, 7 or 2
2. The method according to 2. 24. A protein which is a modified protein of protoporphyrinogen IX oxidase, does not have an ability to oxidize protoporphyrinogen IX, and is contained as an active ingredient in a protoporphyrinogen IX oxidase-inhibiting herbicide. 23. The method according to claim 1, 2, 3, 3, 5, 6, 7 or 22, which is a protein having a property of specifically binding to. 25. The method according to claim 23, wherein the protoporphyrinogen IX oxidase is plant-derived protoporphyrinogen IX oxidase. 26. The method according to claim 23, 24 or 25, wherein the protoporphyrinogen IX oxidase is soybean-derived protoporphyrinogen IX oxidase. 27. The method according to claim 23, wherein the protoporphyrinogen IX oxidase is algae-derived protoporphyrinogen IX oxidase. (28) the protoporphyrinogen IX oxidase is a protoporphyrinogen derived from Paramecium bursaria
28. The method according to claim 23, 24 or 27, which is IX oxidase. 29. The modified protein of coproporphyrinogen III oxidase, wherein the protein is protoporphyrinogen IX and coproporphyrinogen.
23. The method according to claim 1, 2, 4, 5, 6, 7, or 22, which is a protein having no ability to oxidize III and having a property of specifically binding to protoporphyrinogen IX. 30. A method for imparting resistance to a weed control agent to a plant, comprising introducing a gene encoding a protein having the following properties (1), (2) and (3) into a plant cell and expressing the gene: A method comprising the steps of: (1) It specifically binds to protoporphyrin IX. (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. 31. The method according to claim 30, wherein the gene is a gene operably linked to a promoter operable in plant cells and a terminator operable in plant cells. 32. The weed controlling agent which is a weed controlling agent which inhibits or suppresses porphyrin biosynthesis in plants.
Or the method of 31. 33. The weed control agent is a protoporphyrinogen IX oxidase-inhibiting herbicide.
The described method. 34. The method according to any one of claims 30 to 33, wherein the protein is magnesium keratase, or a modified protein of the protein, which has a property of specifically binding to protoporphyrin IX. 35. The method according to any one of claims 30 to 33, wherein the protein is ferrochelatase, or a modified protein of the protein, which has a property of specifically binding to protoporphyrin IX. 36. The method according to claim 30, 31, 32, 33 or 35, wherein the protein is a plant-derived ferrochelatase. 37. The method according to claim 30, 31, 32, 33, 35 or 36, wherein the protein is ferrochelatase derived from barley. 38. The method according to claim 30, 31, 32, 33, 35 or 36, wherein the protein is cucumber-derived ferrochelatase. 39. The method according to claim 30, wherein the protein is a protein consisting of 4 to 100 amino acids. 40. A method for imparting resistance to a weed control agent to a plant, comprising introducing a gene encoding a protein having the following properties (1), (2) and (3) into a plant cell and expressing the gene: A method comprising the steps of: (1) It specifically binds to protoporphyrinogen IX. (2) It has the ability to transform coproporphyrinogen III. (3) It does not substantially contain a framework region of an immunoglobulin variable region. 41. The method according to claim 40, wherein the gene is a gene operably linked to a promoter operable in plant cells and a terminator operable in plant cells. 42. The weed controlling agent which is a weed controlling agent which inhibits or suppresses porphyrin biosynthesis in plants.
Or the method of 41. 43. The weed control agent is a protoporphyrinogen IX oxidase-inhibiting herbicide.
The described method. (44) the protein is coproporphyrinogen;
41. A protein which is III oxidase or a modified protein of the protein, which has a property of specifically binding to protoporphyrinogen IX.
-43. (45) the protein is coproporphyrinogen III oxidase derived from a microorganism;
The described method. 46. The protein according to claim 40, wherein the protein is coproporphyrinogen III oxidase derived from Escherichia coli.
The described method. 47. A plant which has been rendered tolerant to a weed control agent according to the method of any one of claims 1 to 39. 48. A plant to which weed control agent resistance has been imparted by the method according to claim 40. 49. A method for producing a plant resistant to a weed controlling agent, which comprises growing the plant according to claim 47. 50. A method for producing a weed-controlling agent-resistant plant, comprising growing the plant according to claim 48. 51. A method for controlling weeds, which comprises spraying a weed controlling agent on a plant cultivation area according to claim 47. 52. A method for controlling weeds, which comprises spraying a weed controlling agent on the plant cultivation area according to claim 48. 53. A method for selecting a weed-controlling agent-resistant plant, which comprises spraying the weed controlling agent on the cultivation area of the plant according to claim 47. 54. A method for selecting a weed controlling agent-resistant plant, which comprises spraying the weed controlling agent on the cultivation area of the plant according to claim 48. 55. A method for selecting a plant cell resistant to a weed controlling agent, which comprises adding a weed controlling agent to the culture area of the plant cell according to claim 47. 56. A method for selecting a plant cell resistant to a weed controlling agent, comprising adding a weed controlling agent to the culture area of the plant cell according to claim 48.