# Patent

## Title
Herbicidal compound resistant plant

## Abstract

The present invention provides a plant on which resistance to at least one of protoporphyrinogen IX oxidize inhibitory-type herbicidal compounds is conferred, wherein to said plant (1) a DNA encoding a protein showing protoporphyrinogen IX oxidize activity and (2) a DNA encoding cytochrome P450 showing activity of metabolizing said herbicidal compound, have been introduced and expressed, and the like.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a herbicidal compound resistant plant.
2. Description of the Related Art
Protoporphyrinogen IX oxidize inhibitory-type herbicidal compound is contained as an active ingredient in a weed control agent. As a plant on which resistance to the protoporphyrinogen IX oxidize inhibitory-type herbicidal compound has been conferred, for instance, (1) a plant overexpressing protoporphyrinogen IX oxidize in the plant body, (2) a plant expressing a variant protoporphyrinogen IX oxidize in the plant body, wherein mutation has been introduced to the amino acid sequence of said protoporphyrinogen IX oxidize such that the sensitivity to a protoporphyrinogen IX oxidize inhibitory-type herbicidal compound is reduced (for example, described in International Patent Publication 97/32011 and the like), (3) a plant expressing cytochrome P-450 derived from actinomyces such that a protoporphyrinogen IX oxidize inhibitory-type herbicidal compound is metabolized for inactivation in the plant body (for example, described in International Patent Publication 03/40370) and the like are known.
SUMMARY OF THE INVENTION
As to the above herbicidal compound resistant plant, phytotoxicity from the herbicidal compound tends to occur, since herbicidal action of the herbicidal compound is generally immediate. Therefore, it has been desired to develop a plant in which phytotoxicity from the herbicidal compound is further reduced.
The present invention provides a plant on which a synergistic resistance to the herbicidal compound has been conferred by introducing in to the plant combination of two kinds of proteins with different functions. Namely, the present invention provides,
1. a plant on which resistance to at least one of protoporphyrinogen IX oxidize inhibitory-type herbicidal compounds is conferred, wherein to said plant both of the following DNAs have been introduced and expressed (hereinafter, may be referred to as the plant of the present invention):

    (1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein showing protoporphyrinogen IX oxidize activity, and
    (2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 showing activity of metabolizing said herbicidal compound;

2. the plant of the above 1, wherein said protein showing protoporphyrinogen IX oxidize activity and said cytochrome P450 showing activity of metabolizing said herbicidal compound in combination in said plant results in synergistic resistance to said herbicidal compound;

3. the plant of the above 1 or 2, wherein said cytochrome P450 is cytochrome P450 derived from actinomyces: 

4. the plant of the above 1 or 2, wherein said cytochrome P450 is selected from the group consisting of:
    
        (1) cytochrome P450 derived from actinomyces belonging to Streptomyces, 
        (2) cytochrome P450 comprising an amino acid sequence having 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or 2,
        (3) cytochrom P450 comprising the amino acid sequence of SEQ ID NO: 1, and
        (4) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 2;

5. the plant of any one of the above 1 to 4, wherein said protein showing protoporphyrinogen IX oxidize activity is a protein derived from a plant and showing protoporphyrinogen IX oxidize activity;

6. the plant of any one of the above 1 to 4, wherein said protein showing protoporphyrinogen IX oxidize activity is

    
    
    (1a) a protein derived from a plant and showing protoporphyrinogen IX oxidize activity that is inhibited by said herbicidal compound, or
    (1b) a protein derived from a plant and showing protoporphyrinogen IX oxidize activity that is not inhibited by said herbicidal compound;

7. a method for producing a herbicidal compound resistant plant comprising a step of propagating the plant of any one of the above 1 to 6;

8. a method for controlling weeds comprising a step of applying to a cultivation area of the plant of any one of the above 1 to 6, a compound to which said plant exhibit resistance, (hereinafter, may be referred to as the method for controlling weeds of the present invention);

9. a method for selecting a herbicidal compound resistant plant, said method comprising:
    
        1) a step of applying or adding to a cultivation area of the plant of any one of the above 1 to 6, a compound to which said plant exhibit resistance, and
        2) a step of selecting a plant which has survived the weed control effect of said applied or added compound; and

10. a method for conferring on a plant, resistance to at least one of protoporphyrinogen IX oxidize inhibitory-type herbicidal compound, said method comprising a step of introducing to and expressing in said plant both of the following DNAs:

    
    
    (1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein showing protoporphyrinogen IX oxidize activity, and
    (2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 showing activity of metabolizing said herbicidal compound;

and the like.






BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the restriction map of the plasmid pBI-NdG6-rSt-1609soy.
FIG. 2 shows the restriction map of the plasmid pBI-NdG6-rSt-1584soy.
FIG. 3 shows the restriction map of the plasmid pKFGMP03.
FIG. 4 shows the restriction map of the plasmid pSUM-NdG6-sPPOav.
FIG. 5 shows the restriction map of the plasmid pBI-NdG6-sPPOav.
FIG. 6 shows the restriction map of the plasmid pBI-35S-sPPOav.
FIG. 7 shows the photos of wild-type line SR-1, the plants of comparative examples (lines 6-S-1 and S-P-1) and the present invention (line 6-P-1) grown for 14 days in a greenhouse after spraying the compound (II) (applied amount 1.78 mg/20 mL spray liquid).
FIG. 8 is a figure indicating the results of that, before the spraying of the compound (II), as to the recombinant tobacco of lines 6-S-1, S-6-1, P-S-1, S-P-1, P-6-1, 6-P-1, 3-6-1, 6-3-1, 3-S-1 and S-3-1, expression of cytochrome P450 was assayed by using Western blotting. The protein was extracted from the leaf of each recombinant tobacco to investigate the accumulation of the protein having the amino acid sequence of SEQ ID NO: 1. The letters at bottom of the lanes in the figure indicate the line of the recombinant tobacco. Also, “Positive” indicates the results of the Western blotting followed by the electrophoresis of the 10 ng of the protein (the protein having the amino acid sequence of SEQ ID NO: 1) produced by the expression in E. coli, wherein said protein was the antigen to the antibody used in the above Western blotting method.
FIG. 9 is a figure indicating the results of that, as to wild-type line SR-1, the recombinant tobacco lines 6-S-1, S-P-1, 6-P-1, 5-S-1 and 5-P-1, DNAs of introduced genes were amplified from the chromosomal DNA solution by PCR, and the amplified DNAs were detected after the electrophoresis in agarose gel. “Marker” is 0.5 μg of the λ/Hind III marker (manufactured by TAKARA BIO INC.) applied to the electrophoresis.



The abbreviations described in the above figures are explained below.

    P4501609soyDNA: the DNA encoding cytochrom P450 comprising the amino acid sequence of SEQ ID NO: 1.
    P4501584soyDNA: the DNA encoding cytochrom P450 comprising the amino acid sequence of SEQ ID NO: 2.
    RuBPCOssCTP: the nucleotide sequence encoding the chloroplast transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase of soybean (cv. Jack).
    CR16G6p: DNA in which the nucleotide sequence upstream of restriction site of the restriction enzyme NdeI is removed from the CR16G6 promoter.
    CR16t: DNA in which the nucleotide sequence downstream of restriction site of the restriction enzyme ScaI is removed from the CR16 terminator.
    NOSp: promoter of the nopaline synthase gene.
    NPTII: kanamycin resistance gene.
    NOSt: terminator of nopaline synthase gene.
    35Sp: 35S promoter of cauliflower mosaic virus.
    GUS: β-glucuronidase gene.
    RB: the right border sequence of T-DNA.
    LB: the left border sequence of T-DNA.
    ColE1 ori: the replication origin of plasmid ColE1
    Kmr: kanamycin resistance gene.
    DNA (sPPOav): the DNA having a nucleotide sequence encoding an amino acid sequence of a variant soybean PPO (sPPOav).
    HindIII, EcoRI, EcoT221, SacI, NotI, SalI: the cleavage sites of the respective restriction enzyme.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to a plant on which resistance to at least one of protoporphyrinogen IX oxidize inhibitory-type herbicidal compounds is conferred, wherein to said plant both of the following DNAs have been introduced and expressed (hereinafter, may be referred to as the plant of the present invention): (1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein showing protoporphyrinogen LX oxidize activity, and (2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 showing activity of metabolizing said herbicidal compound.
The present invention relates to a plant, wherein the protein showing protoporphyrinogen IX oxidize activity and the cytochrome P450 showing activity of metabolizing said herbicidal compound in combination in said plant results in synergistic resistance to said herbicidal compound.
The cytochrome P450 can be cytochrome P450 derived from actinomyces. For example, the cytochrome P450 can be (1) cytochrome P450 derived from actinomyces belonging to Streptomyces, (2) cytochrome P450 comprising an amino acid sequence having 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or 2, (3) cytochrom P450 comprising the amino acid sequence of SEQ ID NO: 1, and (4) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 2.
The protein showing protoporphyrinogen IX oxidize activity can be a protein derived from a plant and showing protoporphyrinogen LX oxidize activity. For example, the protein showing protoporphyrinogen IX oxidize activity can be (1a) a protein derived from a plant and showing protoporphyrinogen IX oxidize activity that is inhibited by said herbicidal compound, or (1b) a protein derived from a plant and showing protoporphyrinogen IX oxidize activity that is not inhibited by said herbicidal compound.
In another embodiment, the present invention relates to a method for producing a herbicidal compound resistant plant comprising a step of propagating the plant of the present invention.
In another embodiment, the present invention relates to a method for controlling weeds comprising a step of applying to a cultivation area of the plant of the present invention, a compound to which said plant exhibit resistance, (hereinafter, may be referred to as the method for controlling weeds of the present invention).
In another embodiment, the present invention relates to a method for selecting a herbicidal compound resistant plant, by 1) a step of applying or adding to a cultivation area of the plant of the present invention, a compound to which said plant exhibit resistance, and 2) a step of selecting a plant which has survived the weed control effect of said applied or added compound; and
In another embodiment, the present invention relates to a method for conferring on a plant, resistance to at least one of protoporphyrinogen IX oxidize inhibitory-type herbicidal compound, the method comprising a step of introducing to and expressing in the plant both of the following DNAs: (1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein showing protoporphyrinogen IX oxidize activity, and (2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 showing activity of metabolizing said herbicidal compound.
Hereinafter, the present invention will be described in detail.
The weed control agent in the present invention is a composition for weed control containing a herbicidal compound as an active ingredient. The composition may contain as needed insecticidal compounds, fungicidal compounds, plant growth regulatory compounds, fertilizer ingredients and the like along with the herbicidal compound. As such weed control agents, for example, there are compositions containing as an active ingredient a compound inhibiting porphyrin biosynthesis and the like. As the compounds inhibiting porphyrin biosynthesis, for example, there are compounds inhibiting activity of protoporphyrinogen IX oxidize (EC 1.13.11.27, hereinafter may be referred to as PPO) and the like. Specifically for example, as the compounds inhibiting activity of PPO (hereinafter, may be referred to as the present herbicidal compounds), there are the compounds disclosed in Duke, S. O., Rebeiz, C. A. ACS Symposium Series 559, Porphyric Pesticides, Chemistry, Toxicology, and Pharmaceutical Applications. American Chemical Society, Washington D.C. (1994) and the like. In the present herbicidal compounds, various molecular species having different structures are contained (Duke et al., Weed Sci. 39: p 465 (1991); Nandihalli et al., Pesticide Biochem. Physiol. 43: p 193 (1992); Matringe et al., FEBS Lett. 245: p 35 (1989); Yanase, Andoh, Pesticide Biochem. Physiol. 35: p 70 (1989)), there may be, for example,
diphenylether:
for instance, chloromethoxynil, biphenox, chloronitrophene (CNP), acifluorfen (i.e. 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid and the like) and it's ethyl ester, acifluorfen-sodium, oxyfluorfen (i.e. 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethylbenzene), or oxadiazol (for instance, oxadiazon (i.e. 3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2(3H)-one) and the like);
cyclic imide:
for instance, S-23142 (i.e. N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophtalimide and the like), or chlorphthalim (i.e. N-(4-chlorophenyl)-3,4,5,6-tetrahydrophtalimide); phenylpyrazole:
for instance, TNPP-ethyl (i.e. ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate) and the like;
pyridine derivatives
for instance, LS82-556 (i.e. N3-(1-phenylethyl)-2,6-dimethyl-5-propyonylnicotinamide) and the like;
phenopylate, o-phenylpyrrolidinocarbamate analog of phenopylate, or piperidinocarbamate analog of phenopylate and the like.
As the present herbicidal compounds, there may be, specifically for example, a compound of the following formula (I):




wherein in formula (I) G represents a group shown in any one of the following G-1 to G-9;








wherein in G-1 to G-9,

X represents an oxygen atom or sulfur atom;
Y represents an oxygen atom or sulfur atom;
R1 represents a hydrogen atom or halogen atom;
R2 represents a hydrogen atom, C1-C8 alkyl group, C1-C8 haloalkyl group, halogen atom, hydroxyl group, —OR9 group, —SH group, —S(O)pR9 group, —COR9 group, —CO2R9 group, —C(O)SR9 group, —C(O)NR11R12 group, —CONH2 group, —CHO group, —CR9═NOR18 group, —CH═CR19CO2R9 group, —CH2CHR19CO2R9 group, —CO2N═CR13R14 group, nitro group, cyano group, —NHSO2R15 group, —NHSO2NHR15 group, —NR9R20 group, —NH2 group or phenyl group that may be substituted with one or more C1-C4 alkyl groups which may be the same or different;
p represents 0, 1 or 2;
R3 represents C1-C2 alkyl group, C1-C2 haloalkyl group, —OCH3 group, —SCH3 group, —OCHF2 group, halogen atom, cyano group, nitro group or C1-C3 alkoxy group substituted with a phenyl group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C3 alkyl group, C1-C3 haloalkyl group, OR28 group, NR11R28 group, SR28 group, cyano group, CO2R28 group and nitro group;
R4 represents a hydrogen atom, C1-C3 alkyl group or C1-C3 haloalkyl group;
R5 represents a hydrogen atom, C1-C3 alkyl group, C1-C3 haloalkyl group, cyclopropyl group, vinyl group, C2 alkynyl group, cyano group, —C(O)R20 group, —CO2R20 group, —C(O)NR20R21 group, —CHR16R17CN group, —CR16R17C(O)R20 group, —C16R17CO2R20 group, —CR16R17C(O)NR2OR21 group, —CHR16OH group, —CHR16OC(O)R20 group or —OCHR16OC(O)NR20R21 group, or, when G represents G-2 or G-6, R4 and R5 may represent C═O group together with the carbon atom to which they are attached;
R6 represents C1-C6 alkyl group, C1-C6 haloalkyl group, C2-C6 alkoxyalkyl group, C3-C6 alkenyl group or C3-C6 alkynyl group;
R7 represents a hydrogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, halogen atom, —S(O)2(C1-C6 alkyl) group or —C(═O)R22 group;
R8 represents a hydrogen atom, C1-C8 alkyl group, C3-C8 cycloalkyl group, C3-C8 alkenyl group, C3-C8 alkynyl group, C1-C8 haloalkyl group, C2-C8 alkoxyalkyl group, C3-C8 alkoxyalkoxyalkyl group, C3-C8 haloalkynyl group, C3-C8 haloalkenyl group, C1-C8 alkylsulfonyl group, C1-C8 haloalkylsulfonyl group, C3-C8 alkoxycarbonylalkyl group, —S(O)2NH(C1-C8 alkyl) group, —C(O)R23 group or benzyl group which may be substituted with R24 on the phenyl ring;
R9 represents C1-C8 alkyl group, C3-C8 cycloalkyl group, C3-C8 alkenyl group, C3-C8 alkynyl group, C1-C8 haloalkyl group, C2-C8 alkoxyalkyl group, C2-C8 alkylthioalkyl group, C2-C8 alkylsulfinylalkyl group, C2-C8 alkylsulfonylalkyl group, C4-C8 alkoxyalkoxyalkyl group, C4-C8 cycloalkylalkyl group, C4-C8 cycloalkoxyalkyl group, C4-C8 alkenyloxyalkyl group, C4-C8 alkynyloxyalkyl group, C3-C8 haloalkoxyalkyl group, C4-C8 haloalkenyloxyalkyl group, C4-C8 haloalkynyloxyalkyl group, C4-C8 cycloalkylthioalkyl group, C4-C8 alkenylthioalkyl group, C4-C8 alkynylthioalkyl group, C1-C4 alkyl group substituted with a phenoxy group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C3 alkyl group and C1-C3 haloalkyl group, C1-C4 alkyl group substituted with a benzyloxy group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C3 alkyl group and C1-C3 haloalkyl group, C4-C8 trialkylsilylalkyl group, C2-C8 cyanoalkyl group, C3-C8 halocycloalkyl group, C3-C8 haloalkenyl group, C5-C8 alkoxyalkenyl group, C3-C8 haloalkoxyalkenyl group, C5-C8 alkylthioalkenyl group, C3-C8 haloalkynyl group, C5-C8 alkoxyalkynyl group, C5-C8 haloalkoxyalkynyl group, C5-C8 alkylthioalkynyl group, C2-C8 alkylcarbonyl group, benzyl group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C3 alkyl group, C1-C3 haloalkyl group, —OR28 group, —NR11R28 group, —SR26 group, cyano group, —CO2R28 group and nitro group, —CR16R17COR10 group, —CR16R17CO2R20 group, —CR16R17P(O)(OR10)2 group, —CR16R17P(S)(OR10)2 group, —CR16R17C(O)NR11R12 group, —CR16R17C(O)NH2 group, —C(═CR26R27)COR10 group, —C(═CR26R27)CO2R20 group, C(═CR26R27)P(O)(OR10)2 group, —C(═CR26R27)P(S)(OR10)2 group, —C(═CR26R27)C(O)NR11R12 group, —C(═CR26R27)C(O)NH2 group, or any one of rings shown in Q-1 to Q-7:




which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C2-C6 alkenyl group, C2-C6 haloalkenyl group, C2-C6 alkynyl group, C3-C6 haloalkynyl group, C2-C8 alkoxyalkyl group, —OR28 group, —SR28 group, —NR11R28 group, C3-C8 alkoxycarbonylalkyl group, C2-C4 carboxyalkyl group, —CO2R28 group and cyano group;

R10 represents a C1-C6 alkyl group, C2-C6 alkenyl group, C3-C6 alkynyl group or tetrahydrofuranyl group;
R11 and R13 independently represent a hydrogen atom or C1-C4 alkyl group:
R12 represents C1-C6 alkyl group, C3-C6 cycloalkyl group, C3-C6 alkenyl group, C3-C6 alkynyl group, C2-C6 alkoxyalkyl group, C1-C6 haloalkyl group, C3-C6 haloalkenyl group, C3-C6 haloalkynyl group, phenyl group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C4 alkyl group and C1-C4 alkoxy group or —CR16R17CO2R25 group; or,
R11 and R12 together may represent —(CH2)5—, —(CH2)4— or —CH2CH2OCH2CH2—, or in that case the resulting ring may be substituted with a substituent selected from a C1-C3 alkyl group, a phenyl group and benzyl group;
R14 represents a C1-C4 alkyl group or phenyl group which may be substituted on the ring with a substituent selected from a halogen atom, C1-C3 alkyl group and C1-C3 haloalkyl group; or,
R13 and R14 may represent C3-C8 cycloalkyl group together with the carbon atom to which they are attached;
R15 represents C1-C4 alkyl group, C1-C4 haloalkyl group or C3-C6 alkenyl group;
R16 and R17 independently represent a hydrogen atom or C1-C4 alkyl group, C1-C4 haloalkyl group, C2-C4 alkenyl group, C2-C4 haloalkenyl group, C2-C4 alkynyl group, C3-C4 haloalkynyl group; or,
R16 and R17 may represent C3-C6 cycloalkyl group with the carbon atom to which they are attached, or the ring thus formed may be substituted with at least one substituent selected from a halogen atom, a C1-C3 alkyl group and C1-C3 haloalkyl group;
R18 represents a hydrogen atom, C1-C6 alkyl group, C3-C6 alkenyl group or C3-C6 alkynyl group;
R19 represents a hydrogen atom, C1-C4 alkyl group or halogen atom,
R20 represents a hydrogen atom, C1-C6 alkyl group, C3-C6 cycloalkyl group, C3-C6 alkenyl group, C3-C6 alkynyl group, C2-C6 alkoxyalkyl group, C1-C6 haloalkyl group, C3-C6 haloalkenyl group, C3-C6 haloalkynyl group, phenyl group which may be substituted on the ring with at least one substituent selected from a halogen atom, C1-C4 alkyl group and —OR28 group, or —CR16R17CO2R25 group;
R21 represents a hydrogen atom, C1-C2 alkyl group or —CO2(C1-C4 alkyl) group;
R22 represents a hydrogen atom, C1-C6 alkyl group, C1-C6 alkoxy group or NH(C1-C6 alkyl) group;
R23 represents C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group, NH(C1-C6 alkyl) group, benzyl group, C2-C8 dialkylamino group or phenyl group which may be substituted with R24;
R24 represents C1-C6 alkyl group, 1 to 2 halogen atoms, C1-C6 alkoxy group or CF3 group;
R25 represents C1-C6 alkyl group, C1-C6 haloalkyl group, C3-C6 alkenyl group, C3-C6 haloalkenyl group, C3-C6 alkynyl group or C3-C6 haloalkynyl group;
R26 and R27 each represent independently a hydrogen atom, C1-C4 alkyl group, C1-C4 haloalkyl group, C2-C4 alkenyl group, C2-C4 haloalkenyl group, C2-C4 alkynyl group, C3-C4 haloalkynyl group, —OR28 group, —NHR28 group, or —SR28 group; or,
R26 and R27 may represent C3-C8 cycloalkyl group with the carbon atom to which they are attached, or each of the ring thus formed may be substituted with at least one substituent selected from a halogen atom, C1-C3 alkyl group and C1-C3 haloalkyl group; and,
R28 represents a hydrogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C3-C6 alkenyl group, C3-C6 haloalkenyl group, C3-C6 alkynyl group, C3-C6 haloalkynyl group, C2-C4 carboxyalkyl group, C3-C8 alkoxycarbonylalkyl group, C3-C8 haloalkoxycarbonylalkyl group, C5-C9 alkenyloxycabonylalkyl group, C5-C9 haloalkenyloxycabonylalkyl group, C5-C9 alkynyloxycabonylalkyl group, C5-C9 haloalkynyloxycabonylalkyl group, C5-C9 cycloalkoxycabonylalkyl group or C5-C9 halocycloalkoxycabonylalkyl group.
And further, there may be, specifically for example, a compound having the following formula (hereinafter, may be referred to as the compound (II)):




butafenacil, flufenpyr-ethyl, methyl 2-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1(2H)-pyrimidinyl]-4-fluorophenoxy]phenoxyacetate, ethyl [3-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1(2H)-2-pyrimizinyl]-4-fluorophenoxy]-2-pyridazinyl]oxyacetate, carfentrazone-ethyl, sulfentrazone and the like.

Here, “butafenacil” is the compound of CAS registry no. 134605-64-4, “flufenpyr-ethyl” is the compound of CAS registry no. 188489-07-8, “carfentrazone-ethyl” is the compound of CAS registry no. 128639-02-1, “sulfentrazone” is the compound of CAS registry no. 122836-35-5. Hereinafter, methyl 2-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluorom ethyl)-1(2H)-pyrimidinyl]-4-fluorophenoxy]phenoxyacetate (CAS registry no. 344419-98-3) may be referred to as the compound (III), and ethyl [3-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoro methyl)-1(2H)-2-pyrimizinyl]-4-fluorophenoxy]-2-pyridazinyl]oxyacetate (CAS registry no. 353292-31-6) may be referred to as the compound (IV).
In the present invention, “sequence homology” refers to the homology between two nucleotide sequences or two amino acid sequences. Such “sequence homology” may be determined by comparing the two sequences, each aligned in an optimal state, over the whole region of the test sequences. As such, additions or deletions (for example, gaps) can be utilized in the optimal alignment of the test nucleic acid sequences or amino acid sequences. Such sequence homology can be calculated through the step of producing the alignment conducted by a homology analysis using a program such as FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)), BLAST (Altschul et al., Journal of Molecular Biology, 215, 403-410 (1990)), CLUSTAL W (Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)) and the like. Such programs, for example, can be typically utilized on the webpage of the DNA Data Bank of Japan (the international databank operated within the Center for Information Biology and DNA Data Bank of Japan). Further, the sequence homology may be determined by utilizing a commercially available sequence analysis software. Specifically for example, it can be calculated by producing an alignment conducted by a homology analysis by the Lipman-Pearson method (Lipman, D. J. and Pearson, W. R., Science, 227, 1435-1441, (1985)) utilizing GENETYX-WIN Ver. 6 (manufactured by GENETYX Corporation). For example, as the results of alignment at homology analysis of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 by using the method, it is calculated 90% of sequence homology.
As the gene (DNA) having a nucleotide sequence encoding an amino acid sequence of “the protein showing protoporphyrinogen IX oxidize activity” used in the present invention, for example, there are a gene having a nucleotide sequence encoding an amino acid sequence of a wild-type PPO (for instance, refer to U.S. Pat. No. 5,767,373 and the like), a gene having a nucleotide sequence encoding an amino acid sequence of a variant PPO having PPO activity that is not inhibited by the present herbicidal compound (for instance, refer to U.S. Pat. No. 5,939,602, WO 9704089 and the like), a gene having a nucleotide sequence encoding an amino acid sequence of PPO derived from bacterium having PPO activity that is not inhibited by the present herbicidal compound (for instance, refer to EPO 0770682, WO 9833927 and the like) and the like. These genes may be a gene having a nucleotide sequence encoding an amino acid sequence of naturally-occurring PPO, a gene having a nucleotide sequence encoding an amino acid sequence of a protein having PPO activity wherein substitution, addition or deletion and the like of 1 or more amino acids has been introduced to the amino acid sequence of naturally-occurring PPO, or a gene having a nucleotide sequence encoding an amino acid sequence of a protein selected based on PPO activity. Also, these genes may be a gene having a nucleotide sequence encoding an amino acid sequence of a protein having PPO activity that is inhibited by the present herbicidal compound in an amount to inhibit natural PPO activity of plant or a gene having a nucleotide sequence encoding an amino acid sequence of a protein having PPO activity that is not inhibited by the present herbicidal compound. Concretely, as the gene having a nucleotide sequence encoding an amino acid sequence of naturally-occurring PPO, there are, for example, genes derived from Esherichia coli (Genbank accession X68660), Bacillus subtilis (Genbank accession X97208), Haemophilus influnzae (Genbank accession L42023), mouse (Genbank accession D45185), human (Genbank accession D38537), Arabidopsis (Genbank accession D83139) or tobacco (Genbank accession Y13465, Y13466) and the like. Such gene used in the present invention has a nucleotide sequence encoding an amino acid sequence of a protein which shows PPO activity and can confer on a plant resistance to the present herbicidal compound when said gene and a cytochrome P450 gene (DNA) showing herbicidal compound metabolizing activity described hereinafter were introduced to and expressed in the plant to utilize said gene in combination with the cytochrome P450 gene.
Cytochrome P450 used in the present invention is a family of proteins containing protoheme and named for its spectroscopic property indicating Soret near 450 nm when it binds with carbon monoxide at a reduction state. The cytochrome P450 exists in various animal tissues, plant tissues, fungi, yeast and bacteria. The cytochrome p450 has abilities to catalyze, with free oxygen and 2 electrons generally derived from NADPH, or rarely derived from NADH, monooxygenation of the present herbicidal compounds and the like and subsequent elimination of functional groups. The cytochrome p450 may be (1) a type to be provided electron from the electron transfer system with both of ferredoxin and NADPH-ferredoxin reductase, or (2) a type to be provided electron directly from NADPH-cytochrome P450 reductase. Preferably, it may be the former. The cytochrome P450 in the former may be located in any one of subcellular organelles of a host cell or in the cytoplasm. The ferredoxin may be an endogenous ferredoxin in the host cell or a foreign ferredoxin produced in the host cell wherein a foreign ferredoxin gene has been introduced into the host cell.
Preferable cytochrome P450 used in the present invention may be cytochrome P450 derived from actinomyces. Here, “actinomyces” is a family of prokaryote belonging to the Actinomycetales, which are a family of Gram-positive bacteria to be grouped 8 genera of Streptomyces. Actinomyces, Mycobacterium, Frankia, Nocardia and the like. More preferable cytochrome P450 may be cytochrome P450 derived from actinomyces belonging to Streptomyces, concretely, for example, cytochrome P450 derived from Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomyces achromogenes, Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus, Streptomyces roseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis, Saccharopolyspora taberi and the like. Concrete examples of cytochrome P450 derived from actinomyces belonging to Streptomyces may be cytochrome P450 having the amino acid sequence of SEQ ID NO: 1, cytochrome P450 having the amino acid sequence of SEQ ID NO: 2, cytochrome P450 having an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 1 or 2, and the like.
The gene (DNA) having a nucleotide sequence encoding an amino acid sequence of cytochrome P450 may be a cytochrome P450 gene having naturally-occurring nucleotide sequence, and a gene having a nucleotide sequence encoding cytochrome P450 in which the codon usage has been optimized for its expression in the host cell. Also, it may be a gene encoding a protein having cytochrome P450 activity wherein substitution, addition, deletion or the like of amino acid has been introduced to an amino acid sequence of a naturally-occurring cytochrome P450, and, a gene encoding a protein selected based on the cytochrome P450 activity. Concretely, the gene encoding cytochrome P450 may be a gene encoding cytochrome P450 described in International Patent Publication WO 03040370.
The gene having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 introduced to the host cell may be located in any one of subcellular organelles in the cell or in chromosome in nuclear. Also, the cytochrome P450 may be located in any one of subcellular organelles, cytoplasm or extracellular space, preferably subcellular organelles, more preferably plastid.
For the transition of cytochrome P450 to subcellular organelles in the cell, it may be introduced to the host cell a chimeric DNA in which a DNA having a nucleotide sequence encoding organelles transit peptide sequence is linked in frame upstream of a DNA having a nucleotide sequence encoding an amino acid sequence of cytochrome P450. Here, “linked in frame” means that the reading frame of the nucleotide sequence encoding the organelles transit peptide sequence and the reading frame of the nucleotide sequence encoding the amino acid sequence of the cytochrome P450 are linked to form one continuous reading frame. The transit peptide sequence to transit and localize the protein to subcellular organelles in the host cell is, for example, a transit peptide sequence of a cytoplasmic precursor of a chloroplast protein of a plant described in U.S. Pat. No. 5,717,084, and a chimeric sequence comprising plural kinds of transit peptide sequences described in U.S. RE36449 etc. Concretely, it is, for example, chloroplast transit peptide sequence derived from ribulose-1,5-bisphosphate carboxylase (hereinafter, may be referred to as RuBPCO) small subunit of soybean which is obtainable by a method described in International Patent Publication WO03040370.
As the method of artificially causing deletions, additions or substitutions of amino acid residues in an amino acid sequence of the protein showing PPO activity or an amino acid sequence of the cytochrome P450 described above, for example, there is a method comprising a steps of carrying out site-directed mutagenesis on a DNA having a nucleotide sequence encoding the amino acid sequence, and then allowing the expression of such DNA by a conventional method. Concretely, for example, it may be a method utilizes amber mutations (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a method by PCR utilizing primers for introducing a mutation and the like. Also, for example, it may be a method comprising the steps of carrying out random mutagenesis on a DNA having a nucleotide sequence encoding the amino acid sequence and the like. Concretely, for example, it may be a method of conducting PCR by utilizing the DNA having a nucleotide sequence encoding the amino acid sequence as a template and a primer pair which can amplify the full length of the DNA under the conditions in which concentration of each of dATP, dTTP, dGTP and dCTP utilized as a substrate are changed, or, under the conditions in which the concentration of Mg2+ is made increase more to promote the polymerase reaction, and the like. Such methods of PCR may be, for example, the conventional methods described in Method in Molecular Biology, (31), 1994, 97-112 and the like.
In the method for confirming PPO activity of the protein to which mutation has been artificially introduced as described above, for example, the DNA having a nucleotide sequence encoding the amino acid sequence of the protein to which the mutation has been artificially introduced is inserted to a vector plasmid firstly, as described in WO9732011 and the like. Next, by introducing the vector plasmid and expressing the DNA in a PPO-deficient host cell, it may be confirmed that the introduced DNA compensates PPO deficiency and the PPO-deficient host cell can be grown heme-independently. Also, in the method for confirming cytochrome P450 activity of the protein to which mutation has been introduced as described above, for example, the DNA having a nucleotide sequence encoding the amino acid sequence of the protein to which the mutation has been introduced is inserted to a vector plasmid firstly. The vector plasmid is introduced in a host cell to express the DNA, as described in WO03040370 and the like. Thus, such activity may be confirmed by adding and reacting the present herbicidal compound as a substrate, ferredoxin and NADPH-ferredoxin reductase to an extract of the host cell in which mutated cytochrome P450 was produced, and measuring the decreased substrate in the reaction mixture after the reaction.
The DNA having a nucleotide sequence encoding an amino acid sequence wherein substitution, addition, deletion or the like of amino acid has been introduced to the particular amino acid sequence of the protein showing PPO activity or the particular amino acid sequence of cytochrome P450 can be obtained from various DNA libraries, by utilizing a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence encoding the particular amino acid sequences described above as a probe and carrying out hybridization under stringent conditions according to the conventional genetic engineering methods. Also, the DNA having a nucleotide sequence encoding an amino acid sequence having 90% or more sequence homology to the particular amino acid sequences, concretely, for example, a DNA having a nucleotide sequence encoding cytochrome P450 having an amino acid sequence having 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or 2 can be obtained from various DNA libraries, by utilizing a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence encoding the particular amino acid sequence as a probe and carrying out hybridization under a stringent condition according to the conventional genetic engineering methods. As the “stringent conditions”, there can be mentioned, for example, the conditions under which hybridization is performed at 68° C. in a solution containing 6×SSC (let the solution containing 3 N NaCl and 0.3 M trisodium citrate be 20×SSC) and then the hybridized membrane is washed at 68° C. with 0.1×SSC and 0.5% SDS in a hybridization conducted according to the conventional method described in such as Sambrook, J., Frisch, E. F., and Maniatis, T.; chapter 9.53, Molecular Cloning 2nd edition, Cold Spring Harbor Press (1989). The salt concentration in the washing step can be selected, for example, from the conditions of 2×SSC (low stringency condition) to the conditions of 0.1×SSC (high stringency conditions). A temperature in the washing step can be selected, for example, from room temperature (low stringency condition) to 68° C. (high stringency condition). Alternatively, both of the salt concentration and temperature may be changed.
To introduce to plant and express the gene to be introduced in the present invention, generally, a DNA having a nucleotide sequence encoding the amino acid sequence of the protein to be expressed, a DNA in which aforementioned DNA and a promoter functional in the plant cell is operably linked or the like, may be inserted to a vector plasmid functional in the plant cell, and introduced to the plant cell. When the vector plasmid already possessing the promoter functional in the plant cell is utilized, aforementioned DNA may be inserted downstream of the promoter present in the vector plasmid so that the promoter and the DNA having a nucleotide sequence encoding the amino acid sequence of the protein to be expressed are operably linked.
Here, the promoter functional in the host cell such as plant cell is a nucleotide sequence which is connected 5′ upstream of a nucleotide sequence of a gene having a nucleotide sequence encoding an amino acid sequence of a protein (the structural gene) and has a function to control initiation of the transcription of the gene in the host cell such as plant cell. As the functional promoter in the plant cell, for example, there is mentioned T-DNA derived constitutive promoters such as nopaline synthase gene promoter and octopine synthase gene promoter; plant virus-derived promoters such as cauliflower mosaic virus derived 19S and 35S promoters; inducible promoters such as phenylalanine ammonia-lyase gene promoter, chalcone synthase gene promoter and pathogenesis-related protein gene promoter; the plant promoter described in International Patent Publication WO2000020613, and the like. Also, a terminator functional in the host cell such as plant cell may be linked downstream of a DNA in which the promoter functional in the host cell such as plant cell described above and a DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity or the cytochrome P450 are operably linked.
The terminator functional in the host cell such as plant cell is a nucleotide sequence which is connected 3′ downstream of a nucleotide sequence of a gene having a nucleotide sequence encoding an amino acid sequence of a protein (the structural gene) and has a function to add polyadenine sequence for stabilization of the transcription of the gene. As the functional terminator in the plant cell, for example, there is mentioned T-DNA derived constitutive terminators such as nopaline synthase gene (NOS) terminator; plant virus derived terminators such as terminators of garlic virus GV1 or GV2; the plant terminator described in International Patent Publication WO2000020613; and the like.
As the plant cell to be utilized as the host cell, there are, for example, plant cells derived from dicotyledonous plant, including solanaceous plant such as eggplant, potato and tomato; cruciferous plant such as rape, canola, lettuce, sugar beet and arabidopsis; leguminous plant such as soybean, pea and alfalfa; rosaceous plant such as apple, pear and almond; citrus such as orange and lemon; cotton, linum, sunflower, grape, almond, poplar, and plant cells derived from monocotyledonous plant including poaceous plant such as corn, rice, wheat, barley, rye, oat and sorghum and the like.
As the plant cell to be utilized as the host cell, there are various plant cells such as plant tissues, plant bodies, cultured cells, seeds and the like.
As the method for introducing into a host cell such as a plant cell DNA having the structural gene to which a promoter and a terminator functional in the host cell such as the plant cell are linked, there are, for example, a method by infection with agrobacterium (Japanese examined patent publication No. Hei2-58917 and Japanese unexamined patent publication No. Syo-60-70080), electroporation into protoplast (Japanese unexamined patent publication No. Syo-60-251887 and Japanese unexamined patent publication No. Hei5-68575), particle gun methods (Japanese unexamined patent publication No. Hei5-508316 and Japanese unexamined patent publication No. Syo-63-258525) or the like.
In such case, the transformant to which the DNA has been introduced can be selected based on phenotype of a selective marker gene, by simultaneously introducing a selective marker gene selected from hygromycin phosphotransferase gene, neomycin phosphotransferase gene, chloramphenicol acetyltransferase gene and the like, and a DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity or a DNA having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 showing the present herbicidal compound metabolizing activity. The selective marker gene, and the DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity or the DNA having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 showing the present herbicidal compound metabolizing activity may be tandemly inserted into the same vector and introduced. A vector plasmid comprising the selective marker gene, and the DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity or the DNA having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 showing the present herbicidal compound metabolizing activity may be introduced simultaneously. A transformant to which the objective gene has been introduced may also be selected by culturing with a medium containing the present herbicidal compound, plant cells to which a vector comprising the objective gene has been introduced and isolating grown clones.
As a method for introducing into a plant a DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity and a DNA having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 showing the present herbicidal compound metabolizing activity, there may be (1) a method of introducing both of the DNAs simultaneously to the same plant cell, or (2) a method of introducing to the same plant cell a DNA in which both of the DNAs are tandemly connected.
The presence of both of the DNAs in the transformant may be confirmed by analyzing DNA prepared from the transformant using genetic engineering analysis methods (such as confirming restriction enzyme sites, analysis of nucleotide sequence, southern hybridization, PCR and the like) described in, for example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press and the like.
Specifically, for example, rice or Arabidopsis having introduced therein both of the DNAs can be obtained according to the method described in Model-Shokubutu-No-Jikken-Protocol: Ine, Shiroinunazuna-Hen (Supervisors: Koh SHIMAMOTO and Kiyotaka OKADA, Shujun-sha, 1996), Fourth chapter. Further, there can be obtained a soybean having introduced therein both of the DNAs by an introduction into a soybean somatic embryo with a particle gun according to the method described in Japanese Unexamined Patent Publication No. 3-291501. Likewise, a maize having introduced therein both of the DNAs can be obtained by an introduction into maize somatic embryo with a particle gun according to the method described by Fromm, M. E., et al., Bio/Technology, 8; p 838 (1990). Wheat having introduced therein both of the DNAs can be obtained by introducing the DNAs into aseptically-cultured wheat immature embryo with a particle gun according to a conventional method described by TAKUMI et al., Journal of Breeding Society (1995), 44: Extra Vol. 1, p 57. Likewise, barley having introduced therein both of the DNAs can be obtained by an introduction into aseptically-cultured barley immature embryo with a particle gun according to a conventional method described by HAGIO, et al., Journal of Breeding Society (1995), 44; Extra Vol. 1, p 67.
From thus produced transformant, a transgenic plant to which both of the DNAs have been introduced can be obtained, by regenerating a plant body according to the method for culturing the plant cell described in, for example, “Shokubutu-Saibou-Soshiki-Baiyo, Jissai, Ouyou, Tenbou”, Harada, Komamine Ed., Rikogakusha (1979), p 65-118 and the like.
Further, by crossing of the transgenic plant having introduced and expressed therein both of the DNAs and a plant of the targeted variety, both of the DNAs can be introduced to a chromosome of the plant of the targeted variety, and the plant of the targeted variety to which both of the DNAs has been introduced can be obtained.
Also, by introducing separately to different plant cells, a DNA having a nucleotide sequence encoding an amino acid sequence of the protein showing PPO activity and a DNA having a nucleotide sequence encoding an amino acid sequence of the cytochrome P450 showing the present herbicidal compound metabolizing activity, and selecting and regenerating the each individual, and then crossing the progeny line of the separately regenerated transformants, the plant of the present invention can be obtained.
Specifically, for example, to produce a recombinant soybean line to which a DNA having a nucleotide sequence encoding an amino acid sequence of a variant soybean PPO (sPPOav) has been introduced and a recombinant soybean line to which a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, both of the DNAs are separately introduced to somatic embryos of soybean by using a particle gun according to the method described in Japanese unexamined patent publication No. Hei3-291501. Next, a crossed line is obtained by crossing the produced recombinant soybean lines. For investigating the resistance of the crossed line to the present herbicidal compound, scoring evaluation may be carried out as to sensitivity in a spray test of the present herbicidal compound according to the method described below in Example 9 and the like (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying).
Also, concretely, for example, to produce a recombinant corn line to which a DNA having a nucleotide sequence encoding an amino acid sequence of a variant corn PPO has been introduced and a recombinant corn line to which a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, both of the DNAs are separately introduced to somatic embryos of corn by using a particle gun according to the method described in Fromm, M. E., et al., Bio/Technology, 8; p 838 (1990). Next, a crossed line is obtained by crossing the produced recombinant corn lines. For investigating the resistance of the crossed line to the present herbicidal compound, scoring evaluation may be carried out as to sensitivity in a spray test of the present herbicidal compound according to the method described below in Example 9 and the like (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying).
Also, concretely, for example, to produce a recombinant cotton line to which a DNA having a nucleotide sequence encoding an amino acid sequence of a variant cotton PPO has been introduced and a recombinant cotton line to which a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, both of the DNAs are separately introduced to cotton according to Agrobacterium infection method. Next, the crossed line is obtained by crossing the produced recombinant cotton lines. For investigating the resistance of the crossed line to the present herbicidal compound, scoring evaluation may be carried out as to sensitivity in a spray test of the present herbicidal compound according to the method described below in Example 9 and the like (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying).
Also, concretely, for example, to produce a recombinant rape line to which a DNA having a nucleotide sequence encoding an amino acid sequence of a variant rape PPO has been introduced and a recombinant rape line to which a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, both of the DNAs are separately introduced to rape according to Agrobacterium infection method. Next, a crossed line is obtained by crossing produced recombinant rape lines. For investigating the resistance of the crossed line to the present herbicidal compound, scoring evaluation may be carried out as to sensitivity in a spray test of the present herbicidal compound according to the method described below in Example 9 and the like (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying).
Also, concretely, for example, to produce a recombinant wheat line to which a DNA having a nucleotide sequence encoding an amino acid sequence of a variant wheat PPO has been introduced and a recombinant wheat line to which a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, both of the DNAs are separately introduced to calli derived from immature embryo of wheat by using a particle gun according to the method described in TAKUMI et al., Journal of Breeding Society (1995), 44: Extra Vol. 1, p 57. Next, a crossed line is obtained by crossing produced recombinant wheat lines. For investigating the resistance of the crossed line to the present herbicidal compound, scoring evaluation may be carried out as to sensitivity in a spray test of the present herbicidal compound according to the method described below in Example 9 and the like (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying).
In the method for controlling weeds of the present invention, an effective amount of the present herbicidal compound is applied to the cultivation area of the plant of the present invention. The amount of the application of the present herbicidal compound may be appropriately decided according to season of the application, variety of the weed, kind of the present herbicidal compound and the like.
The plant of the present invention may express each one kind of both of the protein showing PPO activity and the cytochrome P450 showing the present herbicidal compound metabolizing activity simultaneously, one kind of the one protein and plural kinds of the other protein simultaneously, or plural kinds of both of the proteins simultaneously. Such expression of the proteins in combination may confer synergistic resistance to the present herbicidal compound on the plant. Because degree of phytotoxicity of the plant of the present invention is highly reduced in applying the present herbicidal compound, the plant of the present invention can be grown well when the present herbicidal compound is sprayed or added to the area growing or culturing the plant of the present invention. By cultivating the plant of the present invention and applying a weed controlling agent containing the present herbicidal compound as an active ingredient to the cultivation area of the plant, it can be to remove efficiently plants such as weeds except for the plant of the present invention, then to improve the yield and quality of the plant of the present invention, to reduce the amount of application of weed controlling agents, to save the labor and the like.
EXAMPLES
Hereinafter, the present invention is further explained with Examples in detail, but not limited thereto.
Example 1
Production of Recombinant Tobacco Plants into which a Cytochrome P450 Gene has been Introduced
Plasmid pBI-NdG6-rSt-1609soy (FIG. 1) (described in International Patent Publication WO0304370) is a binary vector plasmid to express the chimeric protein comprising the chloroplast transit peptide sequence of soybean (cv. Jack) RuBPCO small subunit and cytochrome P450 having the amino acid sequence of SEQ ID NO: 1 under the control of CR16G6 promoter (described in International Patent Publication WO00020613). Plasmid pBI-NdG6-rSt-1584 (FIG. 2) (described in International Patent Publication WO0304370) is a binary vector plasmid to express the chimeric protein comprising the chloroplast transit peptide sequence of soybean (cv. Jack) RuBPCO small subunit and cytochrome P450 having the amino acid sequence of SEQ ID NO: 2 under the control of CR16G6 promoter (described in International Patent Publication WO00020613).
These plasmids pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt-1584soy were introduced separately to Agrobacterium tumefaciens LBA4404 strain (manufactured by Clontech). The resultant transformants were cultured on LB agar medium (0.5% Yeast extract, 1.0% Bacto tryptone, 0.5% NaCl) containing 300 μg/L streptomycin, 100 μ/L rifampicin and 25 μ/L kanamycin, followed by selection of drug resistant colonies to isolate a recombinant Agrobacterium strain having plasmid pBI-NdG6-rSt-1609soy and a recombinant Agrobacterium strain having plasmid pBI-NdG6-rSt-1584soy respectively.
Then, according to the method described in Manual for Gene Manipulation of Plant (by Hirofumi Uchimiya, Kodan-sha Scientific (1992)), gene introduction to tobacco was carried out. The above recombinant Agrobacterium strains were each cultured at 28° C. overnight in LB liquid medium containing 300 μg/L streptomycin, 100 μ/L rifampicin and 25 μ/L kanamycin. To the obtained liquid culture medium, leaf disks sampled from aseptically-cultured tobacco (Nicotinia tabacum strain SR-1) were dipped. The leaf disks were planted on MS agar medium (MS inorganic salts, MS vitamins, 3% sucrose, and 0.8% agar; Murashige T. and Skoog F., Physiol. Plant. (1962) 15, p473) containing 0.1 mg/L naphthalene acetic acid and 1.0 mg/L benzyl aminopurine, and cultured in the light at room temperature for 2 days. Then, the leaf disks were washed with sterilized water, and cultured for 7 days on MS agar medium containing 0.1 mg/L naphthalene acetic acid, 1.0 mg/L benzyl aminopurine and 500 mg/L cefotaxime. Next, the leaf disks were transplanted to and cultured on MS agar medium containing 0.1 mg/L naphthalene acetic acid, 1.0 mg/L benzylaminopurine, 500 mg/L cefotaxime and 100 mg/L kanamycin. The culture was conducted continuously for 2 months while transplanting the leaf disks to fresh medium of the same composition at intervals of 2 weeks. During that time, the adventitious buds developed from the leaf disks were transplanted to and rooted on MS agar medium containing 100 mg/L kanamycin to obtain regenerated plants. Then, the regenerated plants were transplanted to and cultured on MS agar medium containing 100 mg/L kanamycin dispensed to culture pots (Technopot manufactured by SUMITOMO BAKELITE Co., Ltd) to obtain a recombinant tobacco individual into which the T-DNA region of plasmid pBI-NdG6-rSt-1609soy has been introduced and a recombinant tobacco individual into which the T-DNA region of plasmid pBI-NdG6-rSt-1584soy has been introduced. The obtained individuals were transplanted to growing pots containing horicultural soil (Kureha Engei Baido manufactured by Kureha Chemical Industry Co., Ltd.) from the culture pots, acclimated to the external environment in a growth chamber, then grown in a greenhouse. Flowers were covered with paper bags during flowering period to avoid crossing with other individuals, and seeds were harvested from them.
Example 2
Selection of the Recombinant Tobacco Individual with an Accumulation of the Cytochrome P450
From the recombinant tobacco individuals obtained in Example 1, recombinant tobacco individuals in which the protein having the amino acid sequence of SEQ ID NO: 1 or 2 has been accumulated in their leaves were selected by using Western blotting method. Firstly, about 1 cm square of a leaf piece of the recombinant tobacco to be assayed was sampled, and put into 2 mL sampling tube. It was added one zirconia bead of 5 mm diameter (YTZ ball manufactured by NIKKATO CORPORATION) therein and put the lid thereon, and then quickly frozen in liquid nitrogen. Using a cell disruption apparatus (Mixer Mill MM300 manufactured by QIAGEN), it was shaken twice at the rate of 30 times/second for 15 seconds to homogenize the sample. By adding 0.1 mL of sample buffer (PBS buffer (137 mM sodium chloride, 8.1 mM disodium hydrogenphosphate, 2.68 mM potassium chloride, and 1.47 mM potassium dihydrogenphosphate) containing 1 mM phenylmethylsulfonyl fluoride) and shaking, proteins were extracted. Sampling one part of the extract, the concentration of the protein in the extract was measured by using BIO-RAD Protein Assay Kit (manufactured by BIO-RAD) and measuring absorbance at 595 nm according to the protocol attached the kit using bovine serum albumin as a standard. To the extract thus prepared from the recombinant tobacco individual, it was mixed the same volume of 2×SDS sample buffer (manufactured by Nakalai tesque), heated 100° C. for 3 minutes, and then cooled on ice. It was added into the well of SDS-PAGE gel (PAG mini “Daiich” manufactured by Daiichi Pure Chemicals Co., Ltd) such that 10 μg protein was applied per 1 well. Electrophoresis was carried out in the SDS-PAGE electrophoresis buffer (tris(hydroxymethyl)aminomethane 15 g, glycine 72 g, and SDS 5 g/L) at 40 mA per gel for 1 hour. From this gel, the proteins after the electrophoresis were transferred onto PVDF membrane (Immobilon-P manufactured by Millipore) for 30 minutes at 10 V in transfer buffer of Bjerrum and Schafer-Nielsen (48 mm tris(hydroxymethyl)amino methane, 39 mM glycine, and 20% methanol) by using a semi-dry blotting device (Transblot SD cell manufactured by BIO-RAD) according to the attached instruction manual. The membrane was treated by using Immune blot kit (manufactured by BIO-RAD) and anti-rabbit IgG antibody labeled with alkaline phosphatase, and then the coloring reaction was carried out by using NBT/BCIP coloring system to detect a band of the protein extracted from the leaf of the recombinant tobacco individual. Firstly, the blocking treatment was carried out by shaking the membrane gently in TBS buffer (20 mM Tris-HCl (pH7.5) and 0.5 mM sodium chloride) containing 3% gelatin for 30 minutes at room temperature. Then, the membrane was washed with TBS buffer for 5 minutes, and gently shaken for 1 hour at room temperature with antiserum of the primary antibody diluted 3,000 fold with TBS buffer containing 0.05% Tween 20 to carry out the primary antibody reaction. As the primary antibody, the rabbit antiserum was used which was obtained by immunization of rabbit with the protein having the amino acid sequence of SEQ ID NO: 1 (described in International Patent Publication WO03040370). Next, the membrane was washed twice with TBS buffer containing 0.05% Tween 20 for 5 minutes, and then shaken gently for 1 hour at room temperature with anti-rabbit IgG antibody labeled with alkaline phosphatase (manufactured by Bio-Rad) diluted 3,000 fold with TBS buffer containing 0.05% Tween 20 to carry out the second antibody reaction. Then, the membrane was washed twice with TBS buffer containing 0.05% Tween 20 for 5 minutes and then the coloring reaction was carried out by using AP coloring kit (manufactured by Bio-Rad). The membrane after coloring was kept after drying. By the above Western blotting method, in 26 of 40 recombinant tobacco individuals to which the plasmid pBI-NdG6-rSt-1609soy containing DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 has been introduced, it was detected relatively high amounts of the protein having molecular weight of 44 kDa. In 6 of 20 recombinant tobacco individuals to which the plasmid pBI-NdG6-rSt-1584soy containing DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 has been introduced, it was detected relatively high amounts of the protein having molecular weight of 44 kDa.
These recombinant tobacco individuals in which relatively high amounts of the protein of 44 kDa was detected were selected and seeds were harvested from the selected individuals to assay copy number of the introduced gene.
Example 3
Selection of a Recombinant Tobacco Line to which 1 Copy of the Cytochrome P450 Gene has been Introduced
Based on the principle that a kanamycin resistant gene and a gene connected tandem to the resistant gene are linked when these genes are introduced into a plant by Agrobacterium method using a binary vector, from the recombinant tobacco individuals selected in Example 2, tobacco lines in which 1 copy of the introduced gene is located on one of homologous chromosomes were further selected.
The seeds were harvested from T0 generation of the recombinant tobacco individuals selected in Example 2, and dipped in a 5-fold dilution of sodium hypochlorite solution (manufactured by Nacalai tesque) for 15 minutes for sterilization. About 50 seeds treated for sterilization were aseptically seeded on MS agar medium containing 100 mg/L kanamycin, and cultured at 25° C. in the light to germinate aseptically. After about 2 weeks, germinated seedlings were observed to select tobacco lines which indicate segregation rate of 3:1 at 5% significance level by chi-square test. As the results, of 26 lines selected in Example 2 from the recombinant tobacco individuals to which the plasmid pBI-NdG6-rSt-1609soy has been introduced, 9 lines were identified and selected as lines in which 1 copy of the introduced gene is located on one of homologous chromosomes. Of 6 lines selected in Example 2 from the recombinant tobacco individuals to which the plasmid pBI-NdG6-rSt-1584soy has been introduced, 2 lines were identified and selected as lines in which 1 copy of the introduced gene is located on one of homologous chromosomes.
Next, T1-generation individuals of the above selected lines were transplanted to growing pots containing horicultural soil (Kureha Engei Baido manufactured by Kureha Chemical Industry Co., Ltd.), acclimated to the external environment in a growth chamber, then grown in a greenhouse. Flowers were covered with paper bags during flowering period to avoid crossing with other individuals, and seeds were harvested from them.
Example 4
Selection of Homozygote of the Recombinant Tobacco Line to which the Cytochrome P450 Gene has been Introduced
Seeds were harvested from the recombinant tobacco lines #17, #19, #22, #23, #25, #29, #30, #34 and #40 which were selected in Example 3 from among the lines to which the plasmid pBI-NdG6-rSt-1609soy has been introduced. Also, seeds were harvested from the recombinant tobacco lines #5 and #16 which were selected in Example 3 from among the lines to which the plasmid pBI-NdG6-rSt-1584soy has been introduced. Seeds of each 4 individuals of the lines #17 and #25 of the recombinant tobacco line into which the plasmid pBI-NdG6-rSt-1609soy has been introduced and the line #16 of the recombinant tobacco line to which the plasmid pBI-NdG6-rSt-1584soy has been introduced were seeded aseptically on MS agar medium containing 100 mg/L kanamycin according the method described in Example 3. An individual of which all seedlings show kanamycin resistance was selected as a homozygote.
Example 5
Production of Recombinant Tobacco Plants to which a Variant of Soybean Protoporphyrinogen IX Oxidize Gene has been Introduced
DNA of a variant soybean PPO gene having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 was operably linked with a promoter and a terminator which are functional in plant cells to construct an expression plasmid for expression of the variant soybean PPO gene in plant. The DNA of the variant soybean PPO gene having the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 was obtained by substitution of cytosine at the 677th nucleotide of a nucleotide sequence encoding a PPO derived from soybean (Glycine max cv. Williams82) with thymine, whereby alanine at the 226th amino acid residue of the PPO derived from soybean was substituted with valine.
Firstly, the plasmid pSPPO-E described in Japanese patent application publication No. Hei11-18775 was digested with restriction enzymes Sac I and Sal I to obtain a DNA having the nucleotide sequence encoding the amino acid sequence of the PPO derived from soybean between Sac I cleavage site and Sal I cleavage site (Sac I-Sal I fragment). The obtained DNA was inserted by ligation between Sac I cleavage site and Sal I cleavage site in the multicloning site of plasmid pKF18k-2 (manufactured by TAKARA BIO INC.) to subclone the DNA having the nucleotide sequence encoding the amino acid sequence of the PPO derived from soybean.
The plasmid to which the above DNA was thus subcloned was treated by using Mutan-Express Km Kit (manufactured by TAKARA BIO INC.) according to the protocol attached to the kit to substitute cytosine at the 677th nucleotide of the nucleotide sequence encoding the amino acid sequence of the PPO derived from soybean with thymine by the Oligonucleotide-directed Dual Amber method, whereby plasmid pKFGMP03 (FIG. 3) containing a DNA having the nucleotide sequence of SEQ ID NO: 4 between Sac I cleavage site and Sal I cleavage site in the multicloning site was obtained.
After digesting plasmid pNdG6-AT described in Example 16 of WO03040370 with restriction enzymes Bgl II and Sac I, plasmid pNdG6-#89 was constructed by inserting between the Bgl II cleavage site and the Sac I cleavage site of plasmid pNdG6-ΔT a synthetic oligonucleotide adapter containing a Sac I recognition sequence and a Sal I recognition sequence. Plasmid pNdG6-#89 contains downstream of CR16G6 promoter a Sac I recognition sequence and a Sal I recognition sequence, and thereby an expression unit to express a DNA in plant can be constructed when the DNA is inserted between cleavage sites in each of the recognition sequences. The adapter inserted in plasmid pNdG6-#89 was produced by mixing in equal volume of each 100 μM/L solution of a synthetic oligonucleotide having the nucleotide sequence 5′-GATCTGAGCTCCATGGATCCGTCGACAGCT-3′ (SEQ ID NO: 13) and a synthetic oligonucleotide having the nucleotide sequence 5′-GTCGACGGATCCATGGAGCTCA-3′ (SEQ ID NO: 14), heating at 65° C. for 10 minutes, and then cooling at room temperature. Then, by digesting the above plasmid pKFGMP03 with Sac I and Sal I, it was obtained a DNA having between Sac I cleavage site and Sal I cleavage site (Sac I-Sal I fragment) the nucleotide sequence (SEQ ID NO: 4) encoding the amino acid sequence of the variant soybean PPO (SEQ ID NO: 3) in which alanine at 226th amino acid residue of the PPO derived from soy bean was substituted with valine (hereinafter, the variant PPO may be referred to as sPPOav). The obtained DNA was inserted by ligation between Sac I cleavage site and Sal I cleavage site of plasmid pNdG6-#89 to connect the DNA having the nucleotide sequence encoding the amino acid sequence of the variant soybean PPO (sPPOav) downstream of CR16G6 promoter. Thus, plasmid pSUM-NdG6-sPPOav (FIG. 4) containing an expression unit to express the variant soybean PPO (sPPOav) was constructed.
Next, after digesting the binary vector plasmid pBI121 (manufactured by Clontech) with restriction enzyme Not I, the DNA termini were blunted by adding nucleotides complementary to the single strand part of the resultant Not I cleavage sites, using TaKaRa BKL Kit (manufactured by TAKARA BIO INC.) according to the protocol attached to the kit. Then, the resultant DNA was self-ligated to form a plasmid.
The obtained plasmid was digested with restriction enzymes Hid III and Eco RI. Between Hind III cleavage site and Eco RI cleavage site of the digested plasmid, a DNA containing CR16G6 promoter obtained by digestion of plasmid pNdG6-ΔT with Hind III and EcoRI (Hind III II-EcoRI fragment) was inserted to construct plasmid pBI-NdG6.
By inserting a DNA containing the expression unit obtained by digestion of plasmid pSUM-NdG6-sPPOav with Not I, to the Not I cleavage site of plasmid pBI-NdG6, plasmid pBI-NdG6-sPPOav (FIG. 5) was constructed.
By analyzing the nucleotide sequence of plasmid pBI-NdG6-sPPOav, it was confirmed that the nucleotide sequence of SEQ ID NO: 5 was contained between Hind III cleavage site and ECo RI cleavage site of the plasmid.
Next, plasmid pBI121 (manufactured by Clontech), which contains an expression unit to express an introduced gene under the control of 35S promoter derived from cauliflower mosaic virus in plant cells, was digested with restriction enzymes Bgl II and Sac I. Between Bgl II cleavage site and Sac I cleavage site of the digested plasmid pBI121, the adapter containing a Sac I recognition sequence and a Sal I recognition sequence was inserted to construct plasmid pBI121#89. Plasmid pBI121#89 contains downstream of 35S promoter a Sac I recognition sequence and a Sal I recognition sequence, and thereby an expression unit to express a DNA in plant can be constructed when the DNA is inserted between cleavage sites in each of the recognition sequences. The adapter inserted in plasmid pBI121#89 was produced by mixing in equal volume of each 100 μmol/L solution of a synthetic oligonucleotide having the nucleotide sequence 5′-GATCTGAGCTCCATGGATCCGTCGACAGCT-3′ (SEQ ID NO: 13) and a synthetic oligonucleotide having the nucleotide sequence 5′-GTCGACGGATCCATGGAGCTCA-3′ (SEQ ID NO: 14), heating at 65° C. for 10 minutes, and then cooling at room temperature. Then, by digesting the above plasmid pKFGMP03 with Sac I and Sal I, it was obtained DNA having the nucleotide sequence encoding the amino acid sequence of the variant soybean PPO (sPPOav) between Sac I cleavage site and Sal I cleavage site (Sac I-Sal I fragment). The obtained DNA was inserted by ligation between Sac I cleavage site and Sal I cleavage site of plasmid pBI121#89 to connect the DNA having the nucleotide sequence encoding the amino acid sequence of the variant soybean PPO (sPPOav) downstream of 35S promoter. Thus, plasmidpBI-35S-sPPOav (FIG. 6) containing an expression unit to express the variant soybean PPO (sPPOav) was constructed.
By analyzing the nucleotide sequence of plasmid pBI-35S-sPPOav, it was confirmed that the nucleotide sequence of SEQ ID NO: 6 was contained between Hind III cleavage site and Eco RI cleavage site of the plasmid.
Thus constructed plasmid pBI-NdG6-sPPOav and plasmid pBI-35S-sPPOav were introduced separately to Agrobacterium tumefaciens LBA4404 strain (manufactured by Clontech) according to the method described in Example 1 such that recombinant tobacco individuals were produced by using the obtained recombinant Agrobacterium strains. Seeds were individually harvested from the produced recombinants.
Example 6
Selection of a Recombinant Tobacco Line to which 1 Copy of the Variant Soybean PPO (sPPOav) Gene has been Introduced
From tobacco lines derived from the recombinant tobacco individuals produced in Example 5, according to the method described in Example 3, tobacco lines in which 1 copy of the introduced gene is located on one of homologous chromosomes were selected. As the results, of 27 lines of the recombinant tobacco individuals to which the plasmid pBI-NdG6-sPPOav has been introduced, 17 lines were identified and selected as lines in which 1 copy of the introduced gene is located on one of homologous chromosomes. Of 4 lines of the recombinant tobacco individuals to which the plasmid pBI-35S-sPPOav has been introduced, 3 lines were identified and selected as lines to which 1 copy of the introduced gene is located on one of homologous chromosomes.
Next, T1-generation individuals of the above selected lines were transplanted to growing pots containing horicultural soil (Kureha Engei Baido manufactured by Kureha Chemical Industry Co., Ltd.), acclimated to the external environment in a growth chamber, then grown in a greenhouse. Flowers were covered with paper bags during flowering time to avoid crossing with other individuals, and seeds were from them. Using the obtained seeds, homozygotes were selected according to the method described in Example 4.
Example 7
Analysis of the Recombinant Tobacco Lines to which the Variant Soybean PPO (sPPOav) Gene has been Introduced
Among the homozygotes selected in Example 6, as to the recombinant tobacco line PO23 to which plasmid pBI-NdG6-sPPOav has been introduced and the recombinant tobacco line 35S-2 to which plasmid pBI-35S-sPPOav has been introduced, it was assayed production of the variant PPO soybean (sPPOav) by using Western blotting method according to the operations described in Example 2. As the primary antibody, it was used rat anti-soybean PPO antiserum (manufactured by ASAHI TECHNOGLASS CORPORATION), which was produced by fusing a nucleotide sequence encoding a partial peptide of the variant soybean PPO (sPPOav) downstream of dehydrofolic acid reductase gene derived from mouse (plasmid pQE-40 manufactured by QIAGEN) and expressing the fused gene to produce a fused protein, immunizing rat with the fused protein as an antigen.
By the Western blotting method, it was confirmed that the band corresponding to molecular weight of the variant soybean PPO (sPPOav) was detected from each leaf of the recombinant tobacco line P023 to which plasmid pBI-NdG6-sPPOav has been introduced and the recombinant tobacco line 35S-2 to which plasmid pBI-35S-sPPOav has been introduced.
Example 8
Crossing the Recombinant Tobacco Line in which the Variant Soybean PPO (sPPOav) Gene has been Introduced with the Recombinant Tobacco Line to which the Cytochrome P450 Gene Encoding the Amino Acid Sequence of SEQ ID NO: 1 has been Introduced
It was carried out crossing the homozygote line 1609soy #25 which was selected in Example 4 from among the recombinant tobacco lines to which DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 (cytochrome P450) has been introduced, with the line P023 to which plasmid pBI-NdG6-sPPOav has been introduced or the line 35S-2 to which plasmid pBI-35S-sPPOav has been introduced which are homozygote lines selected in Example 6 from among the recombinant tobacco lines to which the variant soybean PPO (sPPOav) gene has been introduced. Also, it was carried out crossing the 1609soy #25 line, the P023 line or the 35S-2 line, with the wild-type tobacco strain SR-1, to produce the individuals having each introduced gene heterogeneously.
Seeds harvested from the T1 generation of the recombinant tobacco individuals were aseptically seeded on MS agar medium containing 100 mg/L kanamycin. Seeds of the wild-type tobacco were aseptically seeded on MS agar medium that contains no kanamycin. Next, germinated plants were transplanted to growing pots containing horicultural soil (Kureha Engei Baido manufactured by Kureha Chemical Industry Co., Ltd.), acclimated to the external environment in a growth chamber, then grown at 23° C., 23 hours of day length in a growth chamber.
From a blossom bud of the tobacco line used as the female line, the petals were removed and the anthers before splitting were removed for emasculation. The stigma in the resultant blossom bud of the female plant was contacted with the splitting anther of the line used as the male line. Reciprocal crossing was conducted for each line. The flower after the pollination was covered with a paper bag to avoid pollination with the pollens of other lines. After the crossing, the plant was grown for about 2 months at 23° C., 23 hours of day length in a growth chamber and the seeds were collected from the flower. Seeds collected from the same pod derived from the same female plant were used as 1 line for analysis of the next generation. Name of the crossed lines thus produced, the male lines and female lines are shown in Table 1.











TABLE 1







crossed





line
female line
male line









P-S-1
SR-1
P023



S-P-1
P023
SR-1



3-S-1
SR-1
35S-2



S-3-1
35S-2
SR-1



6-S-1
SR-1
1609soy#25



S-6-1
1609soy#25
SR-1



P-6-1
1609soy#25
P023



6-P-1
P023
1609soy#25



3-6-1
1609soy#25
35S-2



6-3-1
35S-2
1609soy#25










Example 9
Spray Test of the Present Herbicidal Compound to the Hybrid Line Between the Recombinant Tobacco Line to which the Variant Soybean PPO (sPPOav) Gene has been Introduced and the Recombinant Tobacco Line to which the Gene of the Cytochrome P450 Having the Amino Acid Sequence of SEQ ID NO: 1 has been Introduced
With respect to each recombinant tobacco line produced by crossing in Example 8, seeds were aseptically seeded on MS agar medium containing 100 mg/L kanamycin. After about 2 weeks, germinated seedlings were observed. All of the crossed lines obtained by crossing using the wild-type line SR-1 as female plant showed resistance to kanamycin and grew normally.
Germinated individuals were transplanted to growing pots containing horicultural soil (Kureha Engei Baido manufactured by Kureha Chemical Industry Co., Ltd.), acclimated to the external environment in a growth chamber, then grown for about 2 weeks at 23° C., 23 hours of day length in a growth chamber. Thus obtained plants of the present invention were applied to the spray test of the present herbicidal compound.
The compound (II) of the present herbicidal compound was dissolved with Solvesso cocktail mixed Solvesso 200 (manufactured by Valent) and Sorpol 3816 (manufactured by Sumitomo Chemical Co., Ltd.) at 87.5:10 such that the compound (II) was respectively contained 0.22 mg, 0.45 mg, 0.89 mg or 1.78 mg in 0.5 mL Solvesso cocktail. The spray liquid of the present herbicidal compound was prepared as the aqueous solution containing the Solvesso cocktail of 2.5% concentration in which the compound (II) was dissolved and Agri dex of 1% concentration (manufactured by Valent) as adjuvant.
Spray of the spray liquid to the plant of the present invention above described was carried out, by using running automatic spray machine (manufactured by Nambasekkei), such that splay liquid 20 mL was sprayed to the recombinant tobacco seedlings put at 0.9 square meter of the sprayed area in a uniform way. After about 2 weeks, the sensitivity of the applied recombinant tobacco line to the compound (II) was compared to the sensitivity of wild-type tobacco line SR-1 to the compound (II). In the above spray test, 4 individuals of the recombinant tobacco were used with respect to each applied amount of the compound (II). The sensitivity of the recombinant tobacco to the compound (II) was decided by scoring it based on the later index, and calculating the average of the score of 4 individuals with respect to each recombinant tobacco 1 line and applied amount for it. And, the same spray test of wild-type line SR-1 was carried out as negative control. The results are shown in Table 2.
<Scoring Index Based on the Degree of Phytotoxicity as Dying of the Individual and Browning or Whitening of the Leaves or Stems Caused by Compound Spraying>


    “0”: the case in that the individual was died;
    “1”: the case in that phytotoxicity as browning or whitening of the leaves or stems was caused, and the phytotoxicity seriously affect the individual's growth, but it was not died;
    “2”: the case in that phytotoxicity as browning or whitening of the leaves or stems was caused, but the phytotoxicity did not seriously affect the individual's growth, but it was not died; and
    “3”: the case in that phytotoxicity as browning or whitening of the leaves or stems was small, or almost not observed.











TABLE 2









Applied amount of the compound (II)



(mg/20 mL spray liquid)











Applied tobacco line
0.22
0.45
0.89
1.78





wild type SR-1
1.5
1.0
0.0
0.0


(negative control)


6-S-1 (Comparative
2.0
1.0
0.3
0.3


Example)


S-P-1 (Comparative
3.0
2.0
2.0
1.5


Example)


6-P-1 (the plant of
3.0
3.0
2.5
2.5


the present invention)









And further, the photos of wild-type line SR-1, the plants of comparative examples (lines 6-S-1 and S-P-1) and the present invention (line 6-P-1) grown for 14 days in a greenhouse after spraying the compound (II) (applied amount 1.78 mg/20 mL spray liquid) are shown in FIG. 7.
Example 10
Expression Analysis of the Crossed Line Between the Recombinant Tobacco Line to which the Variant Soybean PPO (sPPOav) Gene has been Introduced and the Recombinant Tobacco Line to which the Gene of the Cytochrome P450 having the Amino Acid Sequence of SEQ ID NO: 1 has been Introduced
Before the spraying of the compound (II), as to the recombinant tobacco of lines 6-S-1, S-6-1, P-S-1, S-P-1, P-6-1, 6-P-1, 3-6-1, 6-3-1, 3-S-1 and S-3-1, expression of cytochrome P450 was assayed by using Western blotting method according to the operations described in Example 2. The protein was extracted from the leaf of each recombinant tobacco to investigate the accumulation of the protein having the amino acid sequence of SEQ ID NO: 1. The results are shown in FIG. 8. In the leaves of the lines 6-S-1, S-6-1, P-6-1, 6-P-1, 3-6-1 and 6-3-1 of the recombinant tobacco, the protein of 44 kDa was detected.
Example 11
Crossing the Recombinant Tobacco Line in which the Variant Soybean PPO (sPPOav) Gene has been Introduced with the Recombinant Tobacco Line to which the Cytochrome P450 Gene Encoding the Amino Acid Sequence of SEQ ID NO: 2 has been Introduced, and Spray Test of the Present Herbicidal Compound to the Hybrid Line
It was carried out the crossing the homozygote line 1584soy#16 which was selected in Example 4 from among the recombinant tobacco lines to which DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 has been introduced, with the line P023 to which plasmid pBI-NdG6-sPPOav has been introduced or the line 35S-2 to which plasmid pBI-35S-sPPOav has been introduced which are homozygote lines selected in Example 6 from among the recombinant tobacco lines to which the variant soybean PPO (sPPOav) gene has been introduced, according to the operations described in Example 8. Name of the produced crossed lines, the male lines and female lines are shown in Table 3.











TABLE 3







crossed





line
female line
male line









5-S-1
SR-1
1584soy#16



S-5-1
1584soy#16
SR-1



P-5-1
1584soy#16
P023



5-P-1
P023
1584soy#16



3-5-1
1584soy#16
35S-2



5-3-1
35S-2
1584soy#16










Next, as to the lines SR-1, 5-S-1, S-P-1 and 5-P-1, according to the method described in Example 9 (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying), their sensitivities to the compound (II) were investigated. As the results, in the hybrid line 5-P-1, remarkable improvement of the resistance was recognized as compared to the line 5-S-1 or S-P-1.
Example 12
Spray Test of the Present Herbicidal Compound to the Hybrid Line Between the Recombinant Tobacco Line to which the Variant Soybean PPO (sPPOav) Gene has been Introduced and the Recombinant Tobacco Line to which the Gene of the Cytochrome P450 Having the Amino Acid Sequence of SEQ ID NO: 1 has been Introduced (ver. 2)
The tobacco individuals were obtained from the seeds of the wild-type tobacco line SR-1 and the recombinant tobacco lines 6-S-1, S-P-1 and 6-P-1 produced by crossing in Example 8, and applied to the spray test of butafenacil, flufenpyr-ethyl, the compound (III) and calfentolazonethyl, according to the operations described in Example 9. As to each herbicidal compound,
(1) in the case of flufenpyr-ethyl and the compound (III), the spray liquid was prepared by dissolving each herbicidal compound separately to be contained 0.89 mg or 1.78 mg in the Solvesso cocktail 0.5 mL respectively;
(2) in the case of butafenacil, the spray liquid was prepared by dissolving the herbicidal compound to be contained 8.9 mg or 17.8 mg in the Solvesso cocktail 0.5 mL;
(3) in the case of carfentrazone-ethyl, the spray liquid was prepared by dissolving dry flowable preparation of the herbicidal compound (AIMTM HERBICIDE manufactured by FMC, containing 40% (w/w) of carfentrazone-ethyl) in Agri dex (manufactured by Valent) 1% aqueous solution to be contained 0.89 mg or 1.78 mg of the active ingredient.
Next, as to the lines SR-1, 6-S-1, S-P-1 and 6-P-1, according to the method described in Example 9 (evaluation by scoring index based on the degree of phytotoxicity as dying of the individual and browning or whitening of the leaves or stems caused by compound spraying), their sensitivities to butafenacil, flufenpyr-ethyl, the compound (III) and carfentrazone-ethyl were investigated. As the results, in the hybrid line 6-P-1, remarkable improvement of the resistance to all the herbicidal compounds was recognized as compared to the line 6-S-1 or S-P-1.
Example 13
Confirmation of Gene Introduction to Chromosome of the Hybrid Recombinant Tobacco Line
It was confirmed DNA having the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, DNA having the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, or DNA having the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 were introduced to chromosome of the crossed recombinant tobacco line according to PCR method described hereinafter.
Seeds of each of the wild-type line SR-1, the recombinant tobacco lines 6-S-1, S-P-1, 6-P-1, 5-S-1 and 5-P-1 were aseptically seeded on MS agar medium that contains no kanamycin. After sampling seedlings after 1 days from seeding, 40 μL PreMan Ultra Reagent (manufactured by Applied Biosystems) was added thereto. The obtained mixture was heated at 100° C. for 10 minutes, and then cooled. By centrifuging the cooled mixture at 15,000 rpm for 1 minute, the supernatant was obtained. The obtained supernatant was used as chromosomal DNA solution to be a template in the following PCR reaction. Using 1 μL of the chromosome DNA solution as a template, and DNA polymerase Ex-Taq-HS (manufactured by TAKARA BIO INC.), total 20 μL of the reaction mixture was prepared according to the attached protocol. The prepared reaction mixture was kept at 94° C. for 2 minutes, and repeated 30 times the cycle which was a set of the steps to keep at 94° C. for 30 seconds, then at 58° C. for 30 seconds, then at 72° C. for 1 minute, and finally kept at 72° C. for 2 minutes. As the primers, 2 kinds of the synthetic oligonucleotide in the combinations of the later (1) to (3) were used. 10 μL of the reaction mixture after the reaction was applied to the electrophoresis using an agarose gel containing 1.5% agarose in TBE buffer, and then stained the agarose gel with ethidium bromide to detect under the irradiation of 254 nm of ultraviolet a DNA amplified in the above PCR reaction. As the results, the introduced gene in chromosomal DNA was detected (FIG. 9). In the chromosome of the recombinant tobacco line 6-S-1, it was confirmed a DNA amplified in the above PCR from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. In the chromosome of the recombinant tobacco line S-P-1, it was confirmed a DNA amplified in the above PCR from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. In the chromosome of the recombinant tobacco line 6-P-1, it was confirmed DNAs amplified respectively in the above PCR from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. In the chromosome of the recombinant tobacco line 5-S-1, it was confirmed a DNA amplified in the above PCR from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. In the chromosome of the recombinant tobacco line 5-P-1, it was confirmed DNAs amplified respectively in the above PCR from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 and from a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.
(1) In the Case of SEQ ID NO: 1:
It was used as primers the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 7, and the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 8. By detecting a 503 bp DNA amplified in the PCR, it was confirmed a DNA containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 was introduced.
(2) In the Case of SEQ ID NO: 2
It was used as primers the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 9, and the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 10. By detecting a 656 bp DNA amplified in the PCR, it was confirmed a DNA containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 was introduced.
(3) In the Case of SEQ ID NO: 3
It was used as primers the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 11, and the synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 12. By detecting a 686 bp DNA amplified in the PCR, it was confirmed a DNA containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 was introduced.
Free Text in Sequence Listing
SEQ ID No: 4


    Part of a plasmid containing a Protoporphyrinogen IX oxydase gene derived from Glycine max 

SEQ ID No: 5

    Part of an expression plasmid containing a Protoporphyrinogen IX oxydase gene derived from Glycine max 

SEQ ID No: 6

    Part of an expression plasmid containing a Protoporphyrinogen IX oxydase gene derived from Glycine max 

SEQ ID No: 7

    Designed oligonucleotide primer for PCR

SEQ ID No: 8

    Designed oligonucleotide primer for PCR

SEQ ID No: 9

    Designed oligonucleotide primer for PCR

SEQ ID No: 10

    Designed oligonucleotide primer for PCR

SEQ ID No: 11

    Designed oligonucleotide primer for PCR

SEQ ID No: 12

    Designed oligonucleotide primer for PCR

SEQ ID No: 13

    Designed oligonucleotide to produce an adaptor DNA

SEQ ID No: 14

    Designed oligonucleotide to produce an adaptor DNA

## Claims

1. A plant on which resistance to a protoporphyrinogen IX oxidase inhibitory-type herbicidal compounds is conferred, wherein to said plant both of the following DNAs have been introduced and expressed:
(1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein that shows protoporphyrinogen IX oxidase activity, and
(2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 selected from the group consisting of:
(a) cytochrome P450 having activity of metabolizing a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound and comprising an amino acid sequence that has 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or 2,
(b) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 1, and
(c) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 2.

2. The plant of claim 1, wherein said protein showing protoporphyrinogen IX oxidase activity and said cytochrome P450 showing activity of metabolizing said herbicidal compound in combination in said plant results in synergistic resistance to said herbicidal compound.

3. The plant of claim 1, wherein said cytochrome P450 is cytochrome P450 derived from actinomyces.

4. The plant of claim 1, wherein said protein showing protoporphyrinogen IX oxidase activity is a protein derived from a plant and showing protoporphyrinogen IX oxidase activity.

5. The plant of claim 1, wherein said protein showing protoporphyrinogen IX oxidase activity is
(1a) a protein derived from a plant and showing protoporphyrinogen IX oxidase activity that is inhibited by said herbicidal compound, or
(1b) a protein derived from a plant and showing protoporphyrinogen IX oxidase activity that is not inhibited by said herbicidal compound.

6. A method for producing a herbicidal compound resistant plant comprising propagating the plant of claim 1.

7. A method for controlling weeds comprising applying to a cultivation area of the plant of claim 1, a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound to which said plant exhibit resistance.

8. A method for selecting a herbicidal compound resistant plant, said method comprising:
(1) applying or adding to a cultivation area of the plant of claim 1, a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound to which said plant exhibit resistance, and
(2) selecting a plant which has survived the weed control effect of said applied or added compound.

9. A method for conferring resistance to a protoporphyrinogen IX oxidase inhibitory-type herbicidal compounds on a plant, said method comprising introducing to and expressing in said plant both of the following DNAs:
(1) a DNA comprising a nucleotide sequence encoding an amino acid sequence of a protein showing protoporphyrinogen IX oxidase activity, and
(2) a DNA comprising a nucleotide sequence encoding an amino acid sequence of cytochrome P450 selected from the group consisting of:
(a) cytochrome P450 having activity of metabolizing a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound and comprising an amino acid sequence that has 90% or more sequence homology to the amino acid sequence of SEQ ID NO: 1 or 2,
(b) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 1 and
(c) cytochrome P450 comprising the amino acid sequence of SEQ ID NO: 2.