Ppo2 polypeptide with tolerance to ppo inhibitor herbicides and application

Abstract

The present application relates to the field of biotechnology, more particularly, the present application relates to PPO2 polypeptide with tolerance to PPO inhibitor herbicide and application. The application of the polypeptide to plants can greatly improve the resistance of plants to PPO inhibitor herbicide, and can be used on plants including economic crops, according to the herbicide resistance characteristics and the selection of herbicide, so as to achieve the purpose of economically controlling the growth of weeds.

Description

Invention Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a PPO2 polypeptide resistant to PPO inhibitor herbicides and its applications. Background of the Invention

[0002] Weeds are one of the core factors affecting crop yield in agricultural production. Herbicides are the main technical means of weed control. The American Weed Science Society (weedscience.org) classifies herbicides into 28 categories based on their different target sites within plants and their mechanisms of action. Group 14 (HRAC GROUP E) consists of inhibitors of protoporphyrinogen IX oxidase (http: / / www.weedscience.org / ).

[0003] Protoporphyrinogen oxidase (PPOX, PPX, or PPO; EC1.3.3.4) is the last common enzyme in the chlorophyll and heme synthesis pathway. Under aerobic conditions, protoporphyrinogen oxidase (PPO) catalyzes the conversion of protoporphyrinogen (Protoporphyrinogen IX) to protoporphyrin (Protoporphyrin IX).

[0004] In plants, protoporphyrinogen oxidase (PPO) is an important target for herbicides. Inhibition of PPO in plants leads to the accumulation of protoporphyrinogen, the substrate that catalyzes this reaction, within cells. The accumulation of protoporphyrinogen in chloroplasts and mitochondria results in the non-enzymatic oxidation of protoporphyrinogen by O2. Under light conditions, the non-enzymatic oxidation of protoporphyrinogen produces singlet oxygen. Singlet oxygen leads to the oxidation of lipids in the intracellular membrane system and causes the oxidative disintegration of these membrane systems, thereby killing plant cells (Future Med Chem. 2014 Apr; 6(6):597–599. doi:10.4155 / fmc.14.29).

[0005] By studying the evolutionary relationships of PPO enzymes in the biological world through sequence similarity, PPO can be divided into three categories: HemG, HemJ, and HemY. In most cases, a single species possesses only one of these categories. Among them, HemG is generally distributed in the γ-Proteobacteria class, HemJ is distributed in the α-Proteobacteria class and has been transferred to other Proteobacteria classes and cyanobacteria, while HemY is the only type of PPO enzyme distributed in eukaryotes (Genome Biol Evol. 2014 Aug; 6(8):2141-55. doi:10.1093 / gbe / evu170).

[0006] Plants generally have at least two PPO genes, named PPO1 and PPO2. PPO1 is usually located in the chloroplasts of plants, while PPO2 is usually located in the mitochondria of plant cells. However, the mRNA of the PPO2 gene in some Amaranthaceae plants has different translation initiation sites, which can produce PPO2 polypeptides of different lengths. For example, the PPO2 gene in spinach (Spinaciaoleracea L.) can express two PPO2 proteins with 26 polypeptides of different lengths, with molecular weights of approximately 58 KD and 56 KD, respectively. The longer one is located in the chloroplast, and the shorter one is located in the mitochondria (J Biol Chem. 2001 Jun 8; 276(23):20474-81. doi:10.1074 / jbc.M101140200.Epub 2001 Mar 23).

[0007] When PPO activity is inhibited by a certain compound, chlorophyll and heme production will also be inhibited. Matrix protoporphyrin IX will then deviate from the normal porphyrin biosynthesis pathway, rapidly detaching from the chloroplast and entering the cytoplasm, where it is oxidized to protoporphyrin IX and accumulates on the cell membrane. The accumulated protoporphyrin IX, under the influence of light and oxygen molecules, produces highly reactive singlet oxygen (…). 1 O2 damages cell membranes and rapidly leads to plant cell death. Due to the use of PPO herbicides, there have been cases worldwide of weeds developing resistance to certain types of PPO herbicides (Pest Manag Sci. 2014 Sep; 70(9):1358-66. doi:10.1002 / ps.3728.Epub2014 Feb 24).

[0008] For example, the deletion of glycine at position 210 (ΔG210) in the PPO2L gene of Amaranthus tuberculatus produces resistance to the herbicide haloxyfop-R-methyl (Proc Natl Acad Sci U SA. 2006 Aug 15; 103(33): 12329-34. doi: 10.1073 / pnas.0603137103. Epub 2006 Aug 7).

[0009] A mutation of arginine at position 98 of the PPO2 gene in Amaranthus palmeri to glycine or methionine (R98G, R98M) produced resistance to the herbicide flumetsulam (Pest Manag Sci. 2017 Aug; 73(8):1559-1563. doi:10.1002 / ps.4581.Epub 2017May 16).

[0010] A mutation of glycine at position 399 of the PPO2 gene in Amaranthus palmeri to alanine (G399A) produced resistance to the herbicide fomesafen (Front Plant Sci. 2019 May 15; 10:568. doi:10.3389 / fpls.2019.00568.eCollection 2019).

[0011] A mutation of arginine at position 98 of the PPO2 gene in ragweed (Ambrosia artemisiifolia) to leucine (R98L) produced resistance to the herbicide flumioxazin (Weed Science, 60(3):335-344(2012)). Invention Overview

[0012] This invention relates to a PPO2 polypeptide or its bioactive fragment that is resistant to PPO inhibitor herbicides.

[0013] The present invention also relates to an isolated polynucleotide, and the corresponding plant genome, vector construct or host cell.

[0014] On the other hand, the present invention also provides a method for producing plant cells or plants that can generate or improve tolerance to protoporphyrinogen oxidase herbicides, and plants produced by said method.

[0015] In another aspect, the present invention provides a method for enabling plants to produce or enhance their tolerance to protoporphyrinogen oxidase-type herbicides.

[0016] The present invention also provides a method for generating or improving the tolerance of plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase herbicides.

[0017] The present invention further provides the use of the protein or its bioactive fragment or the polynucleotide for producing or enhancing the tolerance of host cells, plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase herbicides.

[0018] The present invention also relates to a method for controlling weeds in plant cultivation sites. Attached Figure Description

[0019] Figure 1The values ​​represent the cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants transformed with the wild-type OsPPO2 gene (denoted as OsPPO2 WT) or various OsPPO2 mutant genes when treated with compound A at concentrations of 0 μM, 5 μM, 50 μM, 100 μM, and 200 μM.

[0020] Figure 2 This indicates the cell growth levels of PPO-deficient *E. coli* (ΔhemG) transformants converted from various OsPPO2 F442 mutant genes when treated with compound A at concentrations of 0 μM, 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM. The resistance to OsPPO2 F442M varies with other factors such as culture time. Figure 1 They showed differences.

[0021] Figure 3 The values ​​represent the cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants transformed with the wild-type OsPPO2 gene (denoted as OsPPO2 WT) or various OsPPO2 L422 mutant genes when treated with compound A at concentrations of 0 μM and 5 μM, respectively.

[0022] Figure 4-6 The numbers represent treatments of different concentrations of compound A, propyzamide, and pyrimethanil overexpressing rice PPO2 WT and L422M / F442M Arabidopsis seeds. Compared to wild-type Arabidopsis, both rice PPO2 WT and L422M / F442M overexpressing in Arabidopsis showed some tolerance to the compounds, but L422M / F442M overexpressing showed stronger tolerance than wild-type overexpressing. Here, "wild-type" refers to wild-type Arabidopsis; "pHSE-OsPPO2 WT" represents rice PPO2 overexpression; and "pHSE-OsPPO2 L422M / F442M" represents rice PPO2 L422M / F442M overexpression.

[0023] Figure 7 The results indicate that spraying rice seedlings overexpressing PPO2 WT and L422M / F442M with different concentrations of compound A showed that, compared with wild-type lines, both PPO2 WT and L422M / F442M seedlings exhibited a certain degree of tolerance to compound A. Here, WT represents the wild-type Jingeng 818.

[0024] Figure 8 This indicates the results of screening wild-type and mutant PPO2 genes in maize and soybean using compound A.

[0025] Figure 9 A schematic diagram showing a transgenic recombinant vector for maize.

[0026] Figure 10 This indicates the resistance test of compound A in maize overexpressing rice OsPPO2 L422M / F442M.

[0027] Figure 11 A schematic diagram showing a soybean transgenic recombinant vector.

[0028] Figure 12 This indicates the resistance test of compound A in soybeans overexpressing rice OsPPO2 L422M / F442M.

[0029] Figure 13 This is a peak diagram representing the gene editing sequencing peaks at the PPO2 L422M / F442M locus in rice.

[0030] Figure 14 The resistance test of rice materials with OsPPO2 L422M / F442M site gene editing to compound A.

[0031] Serial Number name SEQ ID NO: 1 The amino acid sequence of wild-type rice PPO2 (OsPPO2 WT) SEQ ID NO: 2 Amino acid sequence of rice PPO2 mutant (OsPPO2 L422M) SEQ ID NO: 3 Amino acid sequence of rice PPO2 mutant (OsPPO2 F442M) SEQ ID NO: 4 Amino acid sequence of rice PPO2 mutant (OsPPO2 L422M / F442M) SEQ ID NO: 5 The amino acid sequence of wild-type PPO2 in maize (ZmPPO2 WT) SEQ ID NO: 6 Amino acid sequence of maize PPO2 mutant (ZmPPO2 L411M / F431M) SEQ ID NO: 7 Amino acid sequence of soybean wild-type PPO2 (GmPPO2 WT) SEQ ID NO: 8 Amino acid sequence of soybean PPO2 mutant (GmPPO2 L370M / F390M) SEQ ID NO: 9 Rice Act1 promoter nucleotide sequence SEQ ID NO: 10 CTP-MDH nucleotide sequence SEQ ID NO: 11 OsPPO2-422-442 optimizes the nucleotide sequence for maize codons. SEQ ID NO: 12 T-NoS nucleotide sequence SEQ ID NO: 13 P-E35S nucleotide sequence SEQ ID NO: 14 Pat nucleotide sequence SEQ ID NO: 15 CaMV poly(A)signal nucleotide sequence SEQ ID NO: 16 P-CsVMV nucleotide sequence SEQ ID NO: 17 Pat nucleotide sequence SEQ ID NO: 18 T-E9 nucleotide sequence SEQ ID NO: 19 P-AtNt1 nucleotide sequence SEQ ID NO: 20 OsPPO2-422-442-Gm1 optimizes the nucleotide sequence for soybean codons. SEQ ID NO: 21 T-Nos nucleotide sequence SEQ ID NO: 22 Maize transgenic vector full sequence SEQ ID NO: 23 Soybean transgenic vector full sequence Invention Details

[0033] Some of the terms used in this specification are defined as follows.

[0034] In this invention, "herbicide" refers to an active ingredient capable of killing, controlling, or adversely altering plant growth. "Herbicide tolerance" or "herbicide resistance" in this invention refers to the continued growth of a plant even after the use of a herbicide that kills common or wild plants, inhibits plant growth, or weakens or stops the plant's growth compared to wild plants. The aforementioned herbicides include protoporphyrinogen oxidase (PPO) inhibitors. These PPO inhibitors can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with different chemical structures.

[0035] Generally, if the PPO-inhibiting herbicides and / or other herbicides, as described herein and usable in the context of this invention, are capable of forming geometric isomers, such as E / Z isomers, then both, pure isomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have one or more chiral centers and are thus present as enantiomers or diastereomers, then both, pure enantiomers, diastereomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have ionizable functional groups, then they may also be used in the form of their agriculturally acceptable salts. Typically, salts of those cations and acid addition salts of those acids are suitable, whose cations and anions do not have adverse effects on the activity of the active compound, respectively. The preferred cations are alkali metal ions, preferably lithium, sodium, and potassium ions; alkaline earth metal ions, preferably calcium and magnesium ions; and transition metal ions, preferably manganese, copper, zinc, and iron ions, further preferably ammonium and substituted ammonium ions, wherein one to four hydrogen atoms are substituted by C1-C4-alkyl, hydroxy-C1-C4-alkyl, C1-C4-alkoxy-C1-C4-alkyl, hydroxy-C1-C4-alkoxy-C1-C4-alkyl, phenyl, or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2 - Hydroxyethylammonium (olamine salt), 2-(2-hydroxyethyl-1-oxy)ethyl-1-ylammonium (diethylene glycolamine salt), di(2-hydroxyethyl-1-yl)ammonium (diethylene glycolamine salt), tri(2-hydroxyethyl)ammonium (trinitroethanolamine salt), tri(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), in addition to phosphonium ions, sulfonium ions, preferably tri(C1-C4-alkyl)sulfonium such as trimethylsulfonium, and sulfonium oxide ions, preferably tri(C1-C4-alkyl)sulfonium oxide ions, and finally, salts of polyamines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. The main anions that can be used for acid addition salts are chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and anions of C1-C4-alkanoic acids, with formate, acetate, propionate, and butyrate being preferred.

[0036] PPO-inhibiting herbicides and / or other herbicidal compounds having carboxyl groups, as described herein, can be used in the form of acids, agriculturally suitable salts as mentioned above, or otherwise agriculturally acceptable derivatives, for example as amides such as mono- and di-C1-C6-alkylamides or arylamides, and as esters such as allyl esters, propargyl esters, C1-C6-alkyl esters, etc.10 -Alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters, and also as thioesters, for example as C1-C 10 -Alkyl thioesters. Preferred mono- and di-C1-C6-alkylamides are methyl and dimethylamides. Preferred arylamides are, for example, N-anilide and 2-chloroanilide. Preferred alkyl esters are, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl), meptyl (1-methylheptyl), heptyl, octyl, or isooctyl (2-ethylhexyl) esters. Preferred C1-C4-alkoxy-C1-C4-alkyl esters are straight-chain or branched C1-C4-alkoxyethyl esters, such as 2-methoxyethyl ester, 2-ethoxyethyl ester, 2-butoxyethyl ester, 2-butoxypropyl ester, or 3-butoxypropyl ester. Straight-chain or branched C1-C 10 An example of an alkyl thioester is an ethyl thioester.

[0037] In one exemplary embodiment, pyrimidinid herbicides include, but are not limited to, flufenoxuron (CAS NO: 134605-64-4), fenproxuron (CAS NO: 372137-35-4), bispyribac-methyl (CAS NO: 158755-95-4), tiafenacil (CAS NO: 1220411-29-9), and ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]ethyl acetate (CAS NO: 1220411-29-9). NO: 353292-31-6), 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS NO: 212754-02-4), flupropacil (CAS NO: 120890-70-2), and isoxazoline-containing uracil derivatives (such as compounds) disclosed in CN105753853A. Uracil-pyridine disclosed in WO2017 / 202768 and uracil-like compounds disclosed in WO2018 / 019842.

[0038] Diphenyl ether herbicides include, but are not limited to, flufenoxuron (CAS NO: 72178-02-0), ethoxyflufenoxuron (CAS NO: 42874-03-3), bensulfuron-methyl (CAS NO: 74070-46-5), quizalofop-p-ethyl (CAS NO: 77501-63-4), methoxyflufenoxuron (CAS NO: 32861-85-1), glufosinate (CAS NO: 1836-77-7), ethoxyflufenoxuron (CAS NO: 77501-90-7), trifluralin or its sodium salt (CAS NO: 50594-66-6 or 62476-59-9), methoxyflufenoxuron (CAS NO: 42576-02-3), chlorfluazuron (CAS NO: 188634-90-4), and chlorfluazuron ethyl (CAS NO: 188634-90-4). NO: 131086-42-5), fluoronitrofen (CAS NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0039] Phenylepiazole herbicides include, but are not limited to, imidacloprid (CAS NO: 129630-19-9) and isopyrazosulfuron (CAS NO: 174514-07-9).

[0040] N-phenylimide herbicides include, but are not limited to, propyzoxystrobin (CAS NO: 103361-09-7), indole-3-propyzate (CAS NO: 142891-20-1), Flumipropyn (CAS NO: 84478-52-4), and flufenoxuron (CAS NO: 87546-18-7).

[0041] Thiadiazole herbicides include, but are not limited to, methyl methacrylate (CAS NO: 117337-19-6), methoxyfenozide (CAS NO: 149253-65-6), and thiamethoxam (CAS NO: 123249-43-4).

[0042] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0043] Triazoline herbicides include, but are not limited to, acetamiprid (CAS NO: 128621-72-7), acetamiprid ethyl ester (CAS NO: 128639-02-1), mesotrione (CAS NO: 122836-35-5), acetamiprid (CAS NO: 68049-83-2), and acetamiprid (CAS NO: 173980-17-1).

[0044] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0045] Other herbicides include, but are not limited to, bispyribac-sodium (CAS NO: 158353-15-2), flupyridaben (CAS NO: 188489-07-8), flupyrazosulfuron (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), and N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 1258836-72-4). NO: 452098-92-9), N-Tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CASNO: 915396-43-9), N-Ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CASNO: 452099-05-7), N-Tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CASNO: 452098-92-9), N-Tetrahydrofurfuryl-3-(2-chloro-6-fluoro ...8-92-9), CAS NO: 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazin-2,4-dione (CAS NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3 ...2100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl)-4,5,6, NO: 1300118-96-0), (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester (CAS NO: 948893-00-3), phenylpyridines disclosed in WO2016 / 120116, benzoxazinone derivatives disclosed in EP09163242.2, and compounds represented by general formula I. (See patent CN202011462769.7);

[0046] In another exemplary embodiment, Q represents

[0047] Y represents halogen, halogenated C1-C6 alkyl, or cyano;

[0048] Z represents halogen;

[0049] M represents CH or N;

[0050] X represents -CX1X2-(C1-C6 alkyl). n -、-(C1-C6 alkyl)-CX1X2-(C1-C6 alkyl) n -or-(CH2) r -, n represents 0 or 1, r represents an integer greater than 2;

[0051] X1 and X2 independently represent hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxy-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, phenyl, or benzyl.

[0052] X3 and X4 represent O or S independently, respectively;

[0053] W represents hydroxyl, C1-C6 alkoxy, C2-C6 alkenoxy, C2-C6 alkynoxy, halo-C1-C6 alkoxy, halo-C2-C6 alkenoxy, halo-C2-C6 alkynoxy, C3-C6 cycloalkyloxy, phenoxy, mercapto, C1-C6 alkylthio, C2-C6 alkenthio, C2-C6 alkynthio, halo-C1-C6 alkylthio, halo-C2-C6 alkenthio, halo-C2-C6 alkynthio, C3-C6 cycloalkylthio, phenylthio, amino, or C1-C6 alkylamino.

[0054] In another exemplary embodiment, the compound represented by general formula I is selected from compound A: Q represents Y represents chlorine; Z represents fluorine; M represents CH; X represents -C*X1X2-(C1-C6 alkyl). n -(C* is the chiral center, R configuration), n represents 0; X1 represents hydrogen; X2 represents methyl; X3 and X4 each independently represent O; W represents methoxy.

[0055] The PPO-inhibiting herbicides described above, which can be used in carrying out the present invention, are generally preferred to be applied in combination with one or more other herbicides to achieve control of a variety of undesirable plants. For example, PPO-inhibiting herbicides can also be applied in combination with additional herbicides to which the crop plants are naturally resistant or resistant via the expression of one or more additional transgenes as described above. When used in combination with other targeted herbicides, the compounds claimed in this invention can be formulated with one or more other herbicides, mixed with one or more other herbicides, or applied sequentially with one or more other herbicides.

[0056] Suitable mixture components, for example, are herbicides selected from categories b1) to b15):

[0057] b1) Inhibitors of lipid biosynthesis;

[0058] b2) Acetolactate synthase inhibitors (ALS inhibitors);

[0059] b3) Photosynthesis inhibitors;

[0060] b4) Protoporphyrinogen-IX oxidase inhibitor

[0061] b5) Bleaching herbicides;

[0062] b6) Enolpyruvylshikimate 3-phosphate synthase inhibitor (EPSP inhibitor);

[0063] b7) Glutamine synthase inhibitor;

[0064] b8) 7,8-Dihydropteranoic acid synthase inhibitor (DHP inhibitor);

[0065] b9) Mitosis inhibitors;

[0066] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0067] b11) Cellulose biosynthesis inhibitor;

[0068] b12) Decoupler herbicides;

[0069] b13) auxinic herbicides;

[0070] b14) Auxin transport inhibitors; and

[0071] b15) is selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, and benzoylmethrin.

[0072] (difenzoquat), difenzoquat-metilsulfate, dimethipin, sodium DSMA, dymron, endothal and its salts, etobenzanid, flamprop, flamprop

[0073] (flamprop-isopropyl), flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozolin (CAS NO: 403640-27-7), methyl azide, methylbromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid Other herbicides including acid, pyributicarb, quinoclamine, triaziflam, tridiphane, and 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and their salts and esters;

[0074] This includes their agriculturally acceptable salts or derivatives.

[0075] Furthermore, when used in combination with other herbicide compounds as described above, it may be useful to apply PPO inhibitory herbicides in combination with safeners. Safeners are compounds that prevent or reduce damage to beneficial plants but do not significantly affect the herbicidal effect of the herbicide on unwanted plants. They can be applied before sowing (e.g., at seed treatment, on branches or seedlings) or before or after germination of the beneficial plants.

[0076] In addition, safeners, PPO inhibitory herbicides and / or other herbicide compounds may be applied simultaneously or sequentially.

[0077] PPO inhibitory herbicides and herbicide compounds and safeners in groups b1)-b15) are known herbicides and safeners, see, for example, WO2013 / 189984; The Compendium of Pesticide Common Names

[0078] (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000, Volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R.R. Schmidt, Herbizide, Georg Thieme Verlag, Stuttgart, 1995; W.H. Ahrens, Herbicide Handbook, 7th Edition, Weed Science Society of America, 1994; and K.K. Hatzios, Herbicide Handbook, 7th Edition Supplement, Weed Science Society of America, 1998.

[0079] The term "weed control" will be understood as killing weeds and / or delaying or inhibiting their normal growth. In the broadest sense, weeds are understood as all plants known to grow in unwanted locations, such as (crop) plant cultivation sites. The weeds included in this invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds from the following genera: *Sinapis*, *Lepidium*, *Galium*, *Stellaria*, *Matricaria*, *Anthemis*, *Galinsoga*, *Chenopodium*, *Urtica*, *Senecio*, *Amaranthus*, *Portulaca*, *Xanthium*, *Convolvulus*, *Ipomoea*, *Polygonum*, *Sesbania*, *Aragula*. The genera *mbrosia*, *Cirsium*, *Carduus*, *Sonchus*, *Solanum*, *Rorippa*, *Rotala*, *Lindernia*, *Lamium*, *Veronica*, *Abutilon*, *Emex*, *Datura*, *Viola*, *Galeopsis*, *Papaver*, *Centaurea*, *Trifolium*, *Ranunculus*, and *Taraxacum*.Monocotyledonous weeds include, but are not limited to, the following genera: *Echinochloa*, *Setaria*, *Panicum*, *Digitaria*, *Phleum*, *Poa*, *Festuca*, *Eleusine*, *Brachiaria*, *Lolium*, *Bromus*, *Avena*, *Cyperus*, *Sorghum*, and *Agropyron*. The genera include *Cynodon*, *Monochoria*, *Fimbristyslis*, *Sagittaria*, *Eleocharis*, *Scirpus*, *Paspalum*, *Ischaemum*, *Sphenoclea*, *Dactyloctenium*, *Agrostis*, *Alopecurus*, and *Apera*. Additionally, the weeds in this invention can include, for example, crop plants growing in unwanted locations. For instance, if corn plants are not desired in a soybean field, free-growing corn plants present in a field primarily containing soybeans can be considered a weed.

[0080] The term "plant" is used in its broadest sense because it refers to organic matter and is intended to encompass eukaryotes belonging to the plant kingdom, including but not limited to vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones, suckers, and plant parts used for asexual reproduction (e.g., cuttings, tubes, seedlings, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses the whole plant, the ancestor and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, pedicels, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of a plant, and tissues and organs therein that each contains the target gene / nucleic acid. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing contains the target gene / nucleic acid.

[0081] Plants particularly useful in the method of this invention include all plants belonging to the superfamily of the Kingdom Viridiplantae, especially monocots and dicots, including legumes used for fodder or feed, ornamental plants, food crops, trees or shrubs, wherein said plants are selected from a list containing the following species: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., and Artocarpus spp. spp.), Asparagus officinalis, species of the genus Avenaspp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), star fruit

[0082] (Averrhoacarambola), Bambusa sp., Benincasa hispida, Brazil chestnut

[0083] Bertholletia excelsea, beet (Beta vulgaris), Brassica species (e.g., Brassicanapus, Brassica rapa ssp.), Cadaba farinosa, tea (Camellia sinensis), canna (Canna indica), hemp (Cannabis sativa), Capsicum species (Capsicum spp.), Carex elata, papaya (Caricapapaya), large-fruited false tiger thorn (Carissa macrocarpa), pecan species (Carya spp.), safflower (Carthamus tinctorius), chestnut species (Castanea spp.), kapok (Ceiba pentandra), endive (Cichorium endivia), camphor species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos) spp.), Coffea spp., Taro (Colocasia esculenta), Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbitas spp., Cucumis spp., Cynara spp., Carrot (Daucus carota), Desmodium spp., Longan (Dimocarpus longan), Dioscoreas spp., Diospyros spp., Echinochloa spp., Oil Palm (Elaeis) (e.g., oil palm (Elaeis) guineensis), American oil palm (Elaeis oleifera), millet (Eleusine coracana), Ethiopian thrush (Eragrostis tef), species of the genus Erianthus (Erianthus sp.), loquat (Eriobotryajaponica), species of the genus Eucalyptus (Eucalyptus sp.).), red berries (Eugenia uniflora), buckwheat species (Fagopyrum spp.), beech species (Fagus spp.), reed fescue (Festuca arundinacea), fig (Ficus carica), kumquat species (Fortunella spp.), strawberry species (Fragaria spp.), ginkgo (Ginkgo biloba), soybean species (Glycine spp.) (e.g., soybean (Glycine max), soybean (Soja hispida) or soybean (Soja max)), upland cotton (Gossypium hirstum), sunflower species (Helianthus spp.) (e.g., sunflower (Helianthus annuus)), daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), barley species (Hordeum spp.) (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut species (Juglans) spp.), lettuce (Lactuca sativa), species of the genera *Lathyrus*, lentils (Lens culinari), flax (Linumusitatissimum), litchi (Litchi chinensis), species of the genera *Lotus*, loofah (Luffaacutangula), species of the genera *Lupinus*, *Luzulasylvatica*, species of the genera *Lycopersicon* (e.g., tomato (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme)), species of the genera *Macrotyloma*, species of the genera *Malus*, *Malpighia emarginata*, avocado (Mammea americana), mango (Mangifera indica), species of the genera *Manihot*. spp.), sapodilla (Manilkarazapota), alfalfa (Medicago sativa), species of the genus *Melilotus* (Spp.), species of the genus *Mentha* (Spp.), mango (Miscanthus sinensis), and species of the genus *Momordica* (Spp.).), Black Mulberry (Morus nigra), Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Millet (Panicum miliaceum), Switchgrass (Panicum virgatum), Passiflora edulis, Pastinaca sativa, Pennisetum sp., Avocado spp., Parsley (Petroselinum crispum), Phalaris arundinacea, Bean spp., Phleum pratense, Phoenix spp. spp.), Southern reed (Phragmites australis), Physalis spp., Pinus spp., Pistachio (Pistacia vera), Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Pomegranate (Punica granatum), Pyrus communis, Quercus spp., Radish (Raphanus sativus), Rheum rhabarbarum, Ribes spp., Castor bean (Ricinus communis), Rubus spp., Saccharum spp.), Salix sp., Sambucus sp., Secale cereale, Sesamum sp., Sinapis sp., Solanum sp.(e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium), or tomato), bicolor sorghum (Sorghum bicolor), spinach species (Spinaciaspp.), syzygium species (Syzygium spp.), marigold species (Tagetes spp.), tamarind (Tamarindus indica), cacao (Theobroma cacao), trifolium species (Trifolium spp.), orchardgrass (Tripsacum dactyloides), Triticosecale rimpaui, wheat (Triticum spp.) (e.g., common wheat (Triticum aestivum), durum wheat (Triticum durum), cylindrical wheat (Triticum turgidum), Triticum hybernum, maca wheat (Triticum macha), common wheat (Triticum sativum), emmer wheat (Triticum monococcum), or common wheat (Triticum *Vulgare*, *Tropaeolumminus*, *Tropaeolum majus*, *Vaccinium* spp., *Vicia* spp., *Vigna* spp., *Viola odorata*, *Vitis* spp., *Zea mays*, *Zizania palustris*, *Ziziphus* spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, kale, flax, kale, lentils, rapeseed, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugarcane, sunflower, tomato, squash, tea, and algae, along with others. According to a preferred embodiment of the invention, the plants are crop plants. Examples of crop plants include, in particular, soybeans, sunflowers, canola, alfalfa, rapeseed, cotton, tomatoes, potatoes, or tobacco. More preferably, the plant is a monocotyledonous plant, such as sugarcane. More preferably, the plant is a cereal, such as rice, corn, wheat, barley, millet, rye, sorghum, or oats.

[0084] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0085] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0086] In this invention, "host organism" should be understood as any single-celled or multi-celled organism into which mutant protein-encoding nucleic acids can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0087] In one aspect, the present invention provides a PPO2 polypeptide or a bioactive fragment thereof that is resistant to PPO inhibitor herbicides, comprising an amino acid sequence having / only having the following mutations compared to the amino acid sequence shown in SEQ ID NO:1: the amino acid at position 422 in the amino acid sequence corresponding to SEQ ID NO:1 is mutated from leucine to methionine and / or the amino acid at position 442 is mutated from phenylalanine to methionine.

[0088] The amino acid sequence comprising, compared to the amino acid sequence shown in SEQ ID NO:5, having / only having the following mutations: amino acid position 411 in the amino acid sequence corresponding to SEQ ID NO:5 is mutated from leucine to methionine and / or amino acid position 431 is mutated from phenylalanine to methionine; or,

[0089] The amino acid sequence having / only having the following mutations compared to the amino acid sequence shown in SEQ ID NO:7: the 370th amino acid in the amino acid sequence corresponding to SEQ ID NO:7 is mutated from leucine to methionine and / or the 390th amino acid is mutated from phenylalanine to methionine.

[0090] In one embodiment, the amino acid sequence further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1, 5, or 7.

[0091] In another embodiment, the PPO2 polypeptide or its bioactive fragment comprises an amino acid sequence having at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 6, or 8. Preferably, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 2, 3, 4, 6, and 8.

[0092] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0093] For the terminology related to amino acid substitutions used in the specification, the first letter represents a naturally occurring amino acid at a specific position in a particular sequence, the following number represents the position relative to SEQ ID NO:1, and the second letter represents the different amino acid that replaces that natural amino acid. For example, L422M indicates that, relative to the amino acid sequence of SEQ ID NO:1, leucine at position 422 is replaced by methionine. For double or multiple mutations, the mutations are separated by " / ". For example, L422M / F442M indicates that, relative to the amino acid sequence of SEQ ID NO:1, leucine at position 422 is replaced by methionine, and phenylalanine at position 442 is replaced by methionine, and both mutations are present in the specific mutant OsPPO2 protein.

[0094] The specific amino acid positions (numbers) within the protein described in this invention are determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:1, for example, using the Smith-Waterman algorithm or the CLUSTALW2 algorithm to align two sequences. The sequence with the highest alignment score is considered aligned. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0095] The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10; gap extension penalty: 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID NO:1.

[0096] Amino acid sequence identity can be determined using conventional methods with the BLAST algorithm (Altschul et al., 1990, Mol.Biol. 215:403-10) available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) using default parameters.

[0097] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitutions in which an amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided that the substitution does not impair the biological activity of the protein.

[0098] Therefore, in addition to the mutations described above, the mutant proteins of the present invention may also contain one or more other mutations, such as conserved substitutions, in their amino acid sequences. Furthermore, the present invention also covers mutant proteins containing one or more other non-conserved substitutions, provided that such non-conserved substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0099] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of mutant proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a bioactive fragment of a mutant protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0100] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the regular sequence, wild-type sequence, or reference sequence. In some embodiments, a mutation refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the nucleotide or amino acid sequence of a non-herbicide-resistant PPO protein. In some embodiments, a mutation refers to one or more mutations at amino acid positions relative to the reference PPO2 amino acid sequence as shown in SEQ ID NO:1 or at homologous positions in its different species homologs. In some embodiments, a mutation may include substitution, deletion, inversion, or insertion. In some embodiments, substitution, deletion, insertion, or inversion may include variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, substitution, deletion, insertion, or inversion may include variations at amino acid positions 1, 2, 3, 4, 5, 6, 7, or 8.

[0101] The terms "wildtype" and "mutation" are relative and refer to the phenotype with the highest frequency in a particular population, or the system, organism, or gene possessing that phenotype. In some cases, a wild-type allele refers to the standard allele at a locus, or the allele with the highest frequency in a particular population, which can be represented by a specific amino acid or nucleic acid sequence. For example, wild-type rice PPO2 protein can be represented by SEQ ID NO:1, wild-type maize PPO2 protein by SEQ ID NO:5, and wild-type soybean PPO2 protein by SEQ ID NO:7.

[0102] In another aspect, the present invention also provides an isolated polynucleotide comprising a nucleic acid sequence selected from the following:

[0103] (1) The nucleic acid sequence or a portion thereof or its complementary sequence encoding the PPO2 polypeptide or its bioactive fragment thereof;

[0104] (2) The nucleic acid sequence shown in SEQ ID NO:11 or 20 or its complementary sequence;

[0105] (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or

[0106] (4) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence;

[0107] In one embodiment, the polynucleotide is a DNA molecule.

[0108] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” or “nucleic acid sequence” are used interchangeably to refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The single-letter codes for nucleotides are as described in Table 1 of Section 2422 of the U.S. Patent Examination Procedure Manual. At this point, the nucleotide name “R” indicates a purine, such as guanine or adenine; “Y” indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); “M” indicates adenine or cytosine; “K” indicates guanine or thymine; and “W” indicates adenine or thymine. The term "isolated," when referring to nucleic acids, means a nucleic acid that is separate from the substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany it. For example, any nucleic acid that has been synthesized (e.g., by sequential base condensation) is considered isolated. Similarly, recombinantly expressed nucleic acids, cloned nucleic acids, nucleic acids produced by primer extension reactions (e.g., PCR), or other nucleic acids excised from the genome are also considered isolated.

[0109] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0110] The present invention also provides a plant genome containing the aforementioned polynucleotides.

[0111] In one embodiment, the plant genome is modified with at least one mutation. In another embodiment, the plant genome is modified with at least two mutations.

[0112] The present invention also provides a vector construct comprising the aforementioned polynucleotide and a homologous or non-homologous promoter operably linked thereto.

[0113] The present invention also provides a carrier construct comprising:

[0114] (1) Genes with nucleotide sequences as shown in SEQ ID NO:11 and genes with nucleotide sequences as shown in SEQ ID NO:14;

[0115] (2) Genes with nucleotide sequences as shown in SEQ ID NO:17 and genes with nucleotide sequences as shown in SEQ ID NO:20;

[0116] (3) Two tandem expression frames, one of which contains the promoter Rice Act1 promoter (nucleotide sequence as shown in SEQ ID NO:9), the chloroplast localization peptide CTP-MDH (nucleotide sequence as shown in SEQ ID NO:10), the gene (nucleotide sequence as shown in SEQ ID NO:11), and the terminator T-NOS (nucleotide sequence as shown in SEQ ID NO:12); the other expression frame contains the promoter P-E35S (nucleotide sequence as shown in SEQ ID NO:13), the gene (nucleotide sequence as shown in SEQ ID NO:14), and the terminator CaMV poly(A)signal (nucleotide sequence as shown in SEQ ID NO:15); or,

[0117] (4) Two tandem expression frames, one of which contains the promoter P-CsVMV with the nucleotide sequence shown in SEQ ID NO:16, the gene with the nucleotide sequence shown in SEQ ID NO:17, and the terminator T-E9 with the nucleotide sequence shown in SEQ ID NO:18; the other expression frame contains the promoter P-AtNt1 with the nucleotide sequence shown in SEQ ID NO:19, the gene with the nucleotide sequence shown in SEQ ID NO:20, and the terminator T-Nos with the nucleotide sequence shown in SEQ ID NO:21.

[0118] In one specific embodiment, the nucleotide sequence of the vector construct is shown in SEQ ID NO:22 or SEQ ID NO:23.

[0119] The present invention also provides a host cell comprising the aforementioned polynucleotide or vector construct.

[0120] In one implementation, the host cell is a plant cell.

[0121] The present invention also provides a method for producing plant cells that can generate or improve tolerance to protoporphyrinogen oxidase inhibitor herbicides, including generating the above-mentioned polynucleotides in plant cells by gene editing, or introducing the above-mentioned polynucleotides or the above-mentioned vector constructs into plant cells by transgenic methods.

[0122] The present invention also provides a method for producing plants that can generate or improve tolerance to protoporphyrinogen oxidase inhibitor herbicides, comprising regenerating plants from the above-described plant cells or plant cells produced by the above-described method.

[0123] The present invention also provides plants produced by the above-described method.

[0124] In one implementation, the plant or plant cells described above are non-GMO.

[0125] In another embodiment, the plant or plant cells described above are genetically modified.

[0126] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" as used herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., autofertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0127] The terms “gene-edited plant,” “gene-edited plant part,” or “gene-edited plant cell” refer to a plant, part, or cell containing one or more endogenous genes edited by a gene-editing system. A “gene-editing system” refers to a protein, nucleic acid, or combination thereof that, when introduced into a cell, is capable of modifying a target locus of an endogenous DNA sequence. Many gene-editing systems suitable for use in the methods of this invention are known in the art, including, but not limited to, zinc finger nuclease (ZFN) systems, transcription activation-like effector nuclease (TALEN) systems, and CRISPR / Cas systems. As used herein, the term “gene editing” generally refers to a technique for inserting, deleting, modifying, or replacing DNA in the genome. For example, said gene editing may include knock-in. The knock-in method may be a technique commonly used by those skilled in the art; for example, see “Gene Targeting: A Practical Approach,” edited by Joyner, Oxford University Press Ltd, 2000.

[0128] The present invention also provides a method for enabling plants to produce or enhance their tolerance to protoporphyrinogen oxidase herbicides, comprising introducing a modification into a gene encoding a protein having protoporphyrinogen oxidase activity to produce the PPO2 polypeptide or a bioactive fragment thereof.

[0129] The present invention also provides a method for generating or improving the tolerance of plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase herbicides, including expressing the PPO2 polypeptide or its bioactive fragment in the plant cells, plant tissues, plant parts or plants.

[0130] Alternatively, this includes hybridizing a plant expressing the PPO2 polypeptide or a bioactive fragment thereof with another plant, and screening for plants or parts thereof that can produce or improve tolerance to protoporphyrinogen oxidase herbicides.

[0131] Alternatively, this may include gene editing of the plant cells, plant tissues, plant parts, or proteins of the plant that have protoporphyrinogen oxidase activity to achieve expression of the PPO2 polypeptide or its bioactive fragments therein.

[0132] The present invention also provides the use of the PPO2 polypeptide or its bioactive fragment or the polynucleotide for producing or improving the tolerance of host cells, plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase herbicides.

[0133] In one embodiment, the host cell is a bacterial cell or a fungal cell.

[0134] The aforementioned herbicide-resistant PPO protein is obtained through the most common natural extraction and refining methods in the industry. It can also be obtained through chemical synthesis or recombinant protein technology. When using chemical synthesis, the protein is obtained using industry-standard peptide synthesis methods. When using recombinant protein technology, the nucleic acid encoding the herbicide-resistant PPO protein is inserted using a suitable expression vector, which is then transformed into host cells. After culturing these host cells to express the target protein, the herbicide-resistant PPO protein can be found and obtained within the host cells. After the protein is expressed in the selected host cells, it is separated using common biochemical methods. These include protein precipitation (salting out), centrifugation, ultrasonic ablation, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other similar treatments for isolation and purification. To obtain highly pure separated proteins, several of these methods may be combined.

[0135] Herbicide-resistant PPO nucleic acid molecules can be isolated and prepared using standard molecular biology methods, such as chemical synthesis or recombinant techniques. One of these methods can be chosen for commercial applications.

[0136] The obtained PPO protein can be transferred to plants to enhance their herbicide resistance.

[0137] The aforementioned herbicide resistance PPO gene can be introduced into plants using methods commonly used in the industry, and can be transgenic or gene-edited using appropriate plant transformation expression vectors.

[0138] Using any appropriate promoter, including vectors, is a common practice in the industry for plant transgenic or gene editing. For example, commonly used promoters in plant transgenic or gene editing include, but are not limited to, the SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, alpha-linolenic acid synthase (NOS) promoter, Scrophularia mosaic virus 35S promoter, sugarcane stalk virus promoter, bamboo mottle virus promoter, light-induced ribulose-1,5-ketocarboxylase (ssRUBISCO small subunit) promoter, rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis thaliana adenine transphosphoribosylase (APRT) promoter, octopine synthase promoter, and BCB (copper-binding protein) promoter.

[0139] Plant transgenic or gene-editing vectors include polyadenylate signal sequences that can induce 3'-terminal polyadenylation. Examples include, but are not limited to, the NOS 3'-terminal derivative of the Agrobacterium tumefaciens synthase gene, the octopine synthase 3'-terminal derivative of the Agrobacterium tumefaciens synthase gene, the 3'-terminus of the tomato or potato protease resistance I or II gene, the CaMVPoly A signal sequence, the 3'-terminus of the rice α-amylase gene, and the 3'-terminus of the betaine gene.

[0140] The aforementioned transgenic vector expresses the herbicide resistance PPO gene in chloroplasts, and the transport peptide labeled on the chloroplasts can be linked to the 5' end of the PPO gene.

[0141] Vectors also include gene encodings that can be selectively labeled as reporter molecules. Examples of selective labeling include, but are not limited to, antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistant (glyphosate, glufosinate, glufosinate, etc.) genes.

[0142] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants.

[0143] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0144] The scope of transgenic or gene-edited plants includes not only contemporary plants from which genes have been introduced, but also their clones and offspring (T1, T2, or subsequent generations). For example, transgenic or gene-edited plants containing the PPO2 polypeptide-encoding nucleotide sequence for resistance to PPO inhibitor herbicides provided in this invention, offspring obtained through sexual and asexual reproduction containing the aforementioned PPO2 polypeptide-encoding nucleotide sequence for resistance to PPO inhibitor herbicides, and plants possessing genetic herbicide resistance are also included. The scope of this invention also includes all mutants and variants of the aforementioned transgenic or gene-edited plants that exhibit characteristics of the primary transgenic or gene-edited plant after hybridization and fusion. The scope of this invention also includes parts of a plant, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and rhizomes, derived from plants that have been previously transgenic or gene-edited using the methods mentioned in this invention, or their offspring, which must consist at least of a portion of transgenic or gene-edited cells.

[0145] The present invention also provides a method for controlling weeds in a plant cultivation site, wherein the plant includes the aforementioned plant or a plant prepared by the aforementioned method, and the method includes applying a herbicide-effective amount of a protoporphyrinogen oxidase inhibitor herbicide to the cultivation site.

[0146] In one implementation, a protoporphyrinogen oxidase inhibitor herbicide is used to control weeds.

[0147] In another implementation, two or more protoporphyrinogen oxidase inhibitor herbicides are used sequentially or simultaneously to control weeds.

[0148] In yet another embodiment, the protoporphyrinogen oxidase inhibitor herbicide is applied in combination with one or more other herbicides.

[0149] In this invention, the term "site" includes the location where the plants of this invention are cultivated, such as soil, and also includes, for example, plant seeds, seedlings, and mature plants. The term "effective herbicide amount" refers to an amount of herbicide sufficient to affect the growth or development of a target weed, such as preventing or inhibiting the growth or development of the target weed, or killing the weed. Advantageously, the effective herbicide amount does not significantly affect the growth and / or development of the plant seeds, seedlings, or plants of this invention. Such effective herbicide amounts can be determined by those skilled in the art through conventional experiments.

[0150] This invention can be implemented in many different forms, and the methods of implementation are not limited to those described herein. The embodiments provided herein are provided to achieve thorough and complete effects, and those skilled in the art will fully understand the scope of the invention. The same reference numerals refer to the same elements throughout this invention.

[0151] The terms "first," "second," and "third" used in this article are to describe a variety of different factors and components, and are not limited by terminology. These terms are used to distinguish one factor or component from another.

[0152] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Unless otherwise expressly stated herein, the use of “a,” “an,” and “the” in the foregoing includes their plural forms. The terms “comprises” and / or “comprising,” or “includes” and / or “including” as used herein specifically refer to the presence of the features, factors, and / or ingredients described herein, without excluding the presence and addition of one or more other features, factors, and ingredients. The term “and / or” as used above includes all or one of the items in the list of combinations.

[0153] This invention has been described in detail through a series of embodiments, but the invention is not limited to the disclosed embodiments. Any variations, substitutions, or replacements that fall within the scope of this invention, not described herein, may be modified as needed.

[0154] The beneficial effects of this invention are as follows: the mutant forms can reduce the inhibitory effect of protoporphyrinogen oxidase inhibitor herbicides on protoporphyrinogen oxidase containing mutant forms, but at the same time, these mutations do not reduce the catalytic activity of protoporphyrinogen oxidase itself. By modifying the endogenous protoporphyrinogen oxidase (PPO2) in plants into these mutant forms through gene editing or by introducing genes carrying these mutant forms of protoporphyrinogen oxidase into plants through transgenic means, the resistance of plants to protoporphyrinogen oxidase inhibitor herbicides can be greatly improved. It can be used on plants including economic crops, and can be used according to the herbicide resistance characteristics and herbicide selection, thereby achieving the purpose of economically controlling weed growth. Detailed Implementation

[0155] The present invention will be further described below with reference to embodiments. All methods and operations described in these embodiments are provided by way of example and should not be construed as limiting.

[0156] Example 1: Cloning of the rice protoporphyrinogen oxidase PPO2 gene

[0157] The rice (Oryza sativa Japonica Group) mitochondrial protoporphyrinogen oxidase (PPO2) gene is located on chromosome 4, with NCBI gene number LOC4336237. Based on its cDNA sequence and the sequence of the vector pET15b (Novagen), primers were designed and synthesized, and a DNA expression vector for the coding region of wild-type rice OsPPO2 (SEQ ID NO: 1) was successfully constructed. The vector was named pET15b-OsPPO2 WT.

[0158] Example 2: Testing the tolerance of different mutants of rice OsPPO2 to compound A using PPO-deficient Escherichia coli (ΔhemG).

[0159] The herbicide tolerance of rice OsPPO2 was tested using PPO-deficient Escherichia coli (ΔhemG). The ΔhemG strain is an Escherichia coli strain lacking the hemG-type PPO gene and exhibiting kanamycin tolerance (Watanabe N, Che FS, Iwano M, et al. Dual Targeting of Spinach Protoporphyrinogen Oxidase II to Mitochondria and Chloroplasts by Alternative Use of Two In-frame Initiation Codons[J]. Journal of Biological Chemistry, 2001, 276(23):20474-20481.). The prepared rice OsPPO2 cloning plasmid was added to ΔhemG competent cells, and the cells were transformed by electroporation to restore PPO activity, enabling growth on standard LB agar medium supplemented with ampicillin and kanamycin.

[0160] To test the tolerance of different mutants of rice OsPPO2 to PPO inhibitor herbicides, the tolerance of L422M (SEQ ID NO: 2), F442M (SEQ ID NO: 3), and L422M / F442M (SEQ ID NO: 4) to compound A was tested using an E. coli screening system. The screening results are as follows: Figure 1-3 As shown, compared with wild type and other published mutants, rice PPO2-L422M, F442M and L422M / F442M mutants all have a certain tolerance to compound A. Among them, the mutant combination L422M / F442M can still grow normally under 200 μM compound A treatment, showing high tolerance.

[0161] Example 3: Tolerance to different compounds by overexpression of rice OsPPO2 L422M / F442M mutant in Arabidopsis thaliana

[0162] To rapidly verify the tolerance of the rice OsPPO2 L422M / F442M mutant to different compounds in plants, vectors overexpressing the wild-type and mutant PPO2 genes were constructed using conventional methods and overexpressed in Arabidopsis thaliana.

[0163] The obtained rice OsPPO2-overexpressing mutant and wild-type Arabidopsis seeds were subjected to resistance tests on MS medium (petries) containing different concentrations of PPO inhibitor herbicides. Figure 4-6As shown, compared with wild-type Arabidopsis, both rice overexpression of OsPPO2 L422M / F442M and OsPPO2 overexpression of WT exhibited certain tolerance / resistance to compounds A, propyzamide, and pyrimethanil. The resistance levels were similar at treatment concentrations (50 nM, 10 nM, and 40 nM, respectively). However, at higher concentrations (2 μM, 1 μM, and 1 μM, respectively), the rice OsPPO2 L422M / F442M mutant still showed resistance, while OsPPO2 overexpression of WT showed no difference from the wild-type Arabidopsis control. This indicates that overexpression of the OsPPO2 L422M / F442M mutant in Arabidopsis resulted in higher tolerance to PPO inhibitor herbicides.

[0164] Example 4: Obtaining herbicide resistance by overexpressing the rice OsPPO2 L422M / F442M mutant

[0165] To further test the herbicide tolerance of the obtained mutants in plants, the rice OsPPO2L422M / F442M mutant was overexpressed in rice.

[0166] Resistance tests were conducted by spraying different concentrations of compound A onto T0 generation rice seedlings overexpressing OsPPO2 L422M / F442M and OsPPO2 WT. For example... Figure 7 As shown, compared with the wild-type Jingeng 818, both rice overexpressing OsPPO2L422M / F442M and OsPPO2 WT showed some tolerance / resistance to compound A. At an application concentration of 2 g / mu (1 mu = 1 / 15 hectare), the resistance levels of both were similar. However, at a higher application concentration of 8 g / mu, OsPPO2L422M / F442M still showed resistance, while OsPPO2 WT showed leaf necrosis, which was not different from the wild-type control. This indicates that rice overexpressing OsPPO2L422M / F442M had a higher tolerance level to compound A.

[0167] Example 5: Rice gene-edited herbicide resistant to PPO inhibition

[0168] To obtain herbicide-resistant non-GMO rice, CRISPR / Cas9-mediated homologous substitution was performed on the above-mentioned L422M / F442M mutation combination. After identification, homologous substitution-adapted OsPPO2 L422M / F442M homozygous rice lines were successfully obtained. Sequencing results are shown below. Figure 13To further verify the tolerance of the above-mentioned gene-edited homozygous rice seedlings to compound A, rice seedlings (4-leaf stage) were treated with 1 g, 2 g, and 4 g / mu of compound A. Compared with wild-type lines, the lines with homologous substitution at the L422M / F442M site could still survive at a dose of 4 g / mu, while the wild-type lines suffered severe phytotoxicity and eventually died at a dose of 1 g / mu (see...). Figure 14 The resistance rates are shown in Table 1. Furthermore, major weeds in paddy fields, such as barnyard grass, Echinochloa crus-galli, Monochoria esculenta, Sagittaria sagittifolia, and Alisma plantago-aquatica, all died at a dosage of 1 gram per acre. In addition, numerous tests revealed that when other PPO-inhibiting herbicides such as pyrimethanil, propyzoxystrobin, ethoxysulfuron, cyprodinil, acetamiprid, epyrifenacil, metolachlor, flupyrimisulfuron, flusulfanilamide, and trifluralin were applied... At the same time, it also has excellent crop safety and can establish better selectivity on crops.

[0169] Table 1. Statistical results of resistance ratios in gene-edited materials with the L422M / F442M mutation.

[0170]

[0171] Example 6: Verification of tolerance to compound A at the L422M / F442M site corresponding to the OsPPO2 mutation in rice in maize and soybean PPO2 genes.

[0172] To verify whether mutations at the corresponding rice PPO2 L422M / F442M locus in other plants could also induce herbicide resistance, the monocotyledonous maize PPO2 gene (ZmPPO2 L411M / F431M) and the dicotyledonous soybean PPO2 gene (GmPPO2 L370M / F390M) corresponding to the rice OsPPO2 L422M / F442M locus were tested in an E. coli screening system using LB medium containing herbicide compound A, and growth inhibition was observed. Figure 8 As shown, compared with wild-type ZmPPO2-WT (SEQ ID NO: 5) and GmPPO2-WT (SEQ ID NO: 7), the mutant maize and soybean PPO2 genes showed certain tolerance / resistance to compound A. They grew normally on plates containing 500 nM of compound A without any inhibition. Furthermore, the mutants showed strong tolerance with increasing concentration of compound A, indicating that the mutations corresponding to the L422M / F442M sites of rice OsPPO2 have a consistent effect on herbicide tolerance in different plants.

[0173] Example 7: Obtaining herbicide resistance by overexpressing the rice OsPPO2 L422M / F442M mutant in maize

[0174] To further test the herbicide tolerance of the obtained mutants in other plants, the rice OsPPO2L422M / F442M mutant was overexpressed in maize.

[0175] like Figure 9 As shown, a maize transgenic recombinant vector (SEQ ID NO: 22) was constructed, and maize was transformed using Agrobacterium-mediated embryo transformation. The transformants were screened with glufosinate to obtain transgenic maize seedlings that overexpressed rice PPO2 422M / 442M in maize. The resistance of T0 generation maize seedlings overexpressing rice OsPPO2 L422M / F442M was tested by spraying compound A.

[0176] The results are as follows Figure 10 As shown, compared with wild-type maize, maize overexpressing rice OsPPO2 L422M / F442M exhibits a certain degree of tolerance / resistance to compound A. Under the condition of application concentration of 8 g / mu, maize plants overexpressing OsPPO2L422M / F442M still showed resistance, while wild-type plants showed leaf necrosis, indicating that the tolerance level of maize to compound A was improved after overexpressing OsPPO2L422M / F442M.

[0177] Example 8: Obtaining herbicide resistance by overexpressing the rice OsPPO2 L422M / F442M mutant in soybean

[0178] like Figure 11 As shown, a soybean transgenic recombinant vector (SEQ ID NO: 23) was constructed, and soybeans were transformed using Agrobacterium-mediated embryo transformation. The transformants were screened with glufosinate to obtain transgenic soybean seedlings that overexpressed rice PPO2 422M / 442M. The resistance of T0 generation soybean seedlings overexpressing rice OsPPO2 L422M / F442M was tested by spraying compound A.

[0179] The results are as follows Figure 12 As shown, compared with wild-type soybean, soybean overexpression of rice OsPPO2 L422M / F442M exhibits a certain degree of tolerance / resistance to compound A. Under the condition of application concentration of 12 g / mu, soybean plants overexpressing OsPPO2L422M / F442M still showed resistance, while wild-type plants showed leaf necrosis, indicating that the tolerance level of soybean to compound A was improved after overexpression of OsPPO2L422M / F442M.

[0180] Furthermore, numerous tests have shown that introducing the gene described in this invention into model plants such as Arabidopsis thaliana and Brachypodium distichum resulted in increased resistance to corresponding levels of PPO-inhibiting herbicides. In addition, editing the aforementioned mutation sites and combinations using the CRISPR / Cpf1 system is also in use. Therefore, it is evident that transgenic or gene-edited versions of this gene can also produce corresponding resistance traits in other aforementioned plants, demonstrating significant industrial value.

[0181] All publications and patent applications mentioned in the specification are incorporated herein by reference as if each publication or patent application were individually and specifically incorporated herein by reference.

[0182] Although the invention has been described in considerable detail by way of example and embodiments for clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims, and all such changes and modifications are within the scope of the invention.

Claims

1. A PPO2 polypeptide resistant to PPO inhibitor herbicides, comprising an amino acid sequence having only the following mutations compared to the amino acid sequence shown in SEQ ID NO: 1: amino acid position 422 of the amino acid sequence shown in SEQ ID NO: 1 is mutated from leucine to methionine; or, amino acid position 422 of the amino acid sequence shown in SEQ ID NO: 1 is mutated from leucine to methionine and amino acid position 442 is mutated from phenylalanine to methionine; The amino acid sequence comprising, compared to the amino acid sequence shown in SEQ ID NO: 5, only having the following mutations: amino acid position 413 of the amino acid sequence shown in SEQ ID NO: 5 is mutated from leucine to methionine; or, amino acid position 413 of the amino acid sequence shown in SEQ ID NO: 5 is mutated from leucine to methionine and amino acid position 433 is mutated from phenylalanine to methionine; or, The amino acid sequence comprising, compared to the amino acid sequence shown in SEQ ID NO: 7, only having the following mutations: the amino acid at position 370 of the amino acid sequence shown in SEQ ID NO: 7 is mutated from leucine to methionine; or, the amino acid at position 370 of the amino acid sequence shown in SEQ ID NO: 7 is mutated from leucine to methionine and the amino acid at position 390 is mutated from phenylalanine to methionine.

2. The PPO2 polypeptide according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8.

3. The PPO2 polypeptide according to claim 1 or 2, wherein the amino acid sequence is as shown in SEQ ID NO: 2, 4, 6 or 8.

4. An isolated polynucleotide comprising the following nucleic acid sequence: a nucleic acid sequence encoding the PPO2 polypeptide of any one of claims 1-3 or its complementary sequence.

5. The polynucleotide according to claim 4, comprising a nucleic acid sequence selected from the following: the nucleic acid sequence shown in SEQ ID NO: 11 or 20 or its complementary sequence.

6. The polynucleotide according to claim 4, wherein the nucleotide sequence is shown in SEQ ID NO: 11 or 20.

7. The polynucleotide according to any one of claims 4-6, wherein the polynucleotide is a DNA molecule.

8. A plant genome comprising the polynucleotides as described in any one of claims 4-7.

9. A vector construct comprising the polynucleotide as described in any one of claims 4-7 and a homologous or non-homologous promoter operatively linked thereto.

10. A vector construct comprising: a gene with nucleotide sequences as shown in SEQ ID NO: 11 and a gene with nucleotide sequences as shown in SEQ ID NO:

14.

11. A vector construct comprising: a gene with nucleotide sequences as shown in SEQ ID NO: 17 and a gene with nucleotide sequences as shown in SEQ ID NO:

20.

12. The carrier construct according to claim 10, comprising: Two tandem expression frames, one containing the promoter Rice Act1 promoter (nucleotide sequence as shown in SEQ ID NO: 9), the chloroplast localization peptide CTP-MDH (nucleotide sequence as shown in SEQ ID NO: 10), the gene (nucleotide sequence as shown in SEQ ID NO: 11), and the terminator T-NOS (nucleotide sequence as shown in SEQ ID NO: 12); the other containing the promoter P-E35S (nucleotide sequence as shown in SEQ ID NO: 13), the gene (nucleotide sequence as shown in SEQ ID NO: 14), and the terminator CaMV poly (A)signal (nucleotide sequence as shown in SEQ ID NO: 15).

13. The carrier construct according to claim 11, comprising: Two tandem expression frames, one containing the promoter P-CsVMV (nucleotide sequence as shown in SEQ ID NO: 16), the gene (nucleotide sequence as shown in SEQ ID NO: 17), and the terminator T-E9 (nucleotide sequence as shown in SEQ ID NO: 18); the other containing the promoter P-AtNt1 (nucleotide sequence as shown in SEQ ID NO: 19), the gene (nucleotide sequence as shown in SEQ ID NO: 20), and the terminator T-Nos (nucleotide sequence as shown in SEQ ID NO: 21).

14. The vector construct according to claim 12, wherein the nucleotide sequence is shown in SEQ ID NO:

22.

15. The vector construct according to claim 13, wherein the nucleotide sequence is shown in SEQ ID NO:

23.

16. A host cell comprising a polynucleotide as described in any one of claims 4-7 or a vector construct as described in any one of claims 9-15, wherein the host cell is a fungal cell or a bacterial cell.

17. A method for producing plant cells capable of generating or improving tolerance to protoporphyrinogen oxidase inhibitor herbicides, comprising generating a polynucleotide as described in any one of claims 4-7 or a vector construct as described in any one of claims 9-15 in plant cells by gene editing, or introducing a polynucleotide as described in any one of claims 4-7 or a vector construct as described in any one of claims 9-15 into plant cells by transgenic means.

18. A method for producing or enhancing the tolerance of plants to protoporphyrinogen oxidase inhibitor herbicides, comprising producing a plant cell regeneration plant using the method of claim 17.

19. A method for enabling plants to produce or enhance tolerance to protoporphyrinogen oxidase inhibitor herbicides, comprising introducing a modification into a gene encoding a protein having protoporphyrinogen oxidase activity to produce the PPO2 polypeptide as described in any one of claims 1-3.

20. A method for producing or enhancing the tolerance of plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase herbicides, comprising expressing the PPO2 polypeptide of any one of claims 1-3 in the plant cells, plant tissues, plant parts or plants; Alternatively, this includes hybridizing a plant expressing the PPO2 polypeptide of any one of claims 1-3 with another plant, and screening for plants or parts thereof that can produce or improve tolerance to protoporphyrinogen oxidase herbicides. Alternatively, it may include gene editing of the plant cells, plant tissues, plant parts or a protein of the plant having protoporphyrinogen oxidase activity to achieve expression of the PPO2 polypeptide of any one of claims 1-3 therein.

21. Use of the PPO2 polypeptide of any one of claims 1-3 or the polynucleotide of any one of claims 4-7 for producing or enhancing the tolerance of a host cell, plant cell, plant tissue, plant part or plant to protoporphyrinogen oxidase herbicides, wherein the host cell is a bacterial cell or a fungal cell.

22. A method for controlling weeds in a plant cultivation site, wherein the plant is a plant prepared by the method according to any one of claims 17-20, the method comprising applying a herbicidally effective amount of a protoporphyrinogen oxidase inhibitor herbicide to the cultivation site.

23. The method of claim 22, wherein the protoporphyrinogen oxidase inhibitor herbicide is applied in combination with one or more other herbicides.

24. The plant genome of claim 8, the host cell of claim 16, the method of claim 17, 18, 19, 20, 22 or 23, or the use of claim 21, wherein the plant is a monocotyledonous or dicotyledonous plant.

25. The method according to claim 17, 18, 19, 20, 22 or 23, or the use according to claim 21, wherein the protoporphyrinogen oxidase inhibitor herbicide is selected from one or more of the following types of compounds: pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylimides, thiadiazoles, oxadiazoles, triazolones, oxazolidinones and others, wherein the others include: Bismuth subtilis, flupyridaben, flupyrazosulfuron, trifluralin, N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, 3-[7-fluoro-3-oxo-4-(prop-2-yne)] (E)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazin-2,4-dione, 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester, phenylpyridines, benzoxazinones, and compounds represented by general formula I. ,in, Q represents , , , , , , , , , , , , , , , or ; Y represents halogen, halogenated C1-C6 alkyl, or cyano; Z represents halogen; M represents CH or N; X represents -CX1X2-(C1-C6 alkyl). n -、-(C1-C6 alkyl)-CX1X2-(C1-C6 alkyl) n -or-(CH2) r -, n represents 0 or 1, r represents an integer greater than 2; X1 and X2 independently represent hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxy-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, phenyl, or benzyl. X3 and X4 represent O or S independently, respectively; W represents hydroxyl, C1-C6 alkoxy, C2-C6 alkenoxy, C2-C6 alkynoxy, halo-C1-C6 alkoxy, halo-C2-C6 alkenoxy, halo-C2-C6 alkynoxy, C3-C6 cycloalkyloxy, phenoxy, mercapto, C1-C6 alkylthio, C2-C6 alkenthio, C2-C6 alkynthio, halo-C1-C6 alkylthio, halo-C2-C6 alkenthio, halo-C2-C6 alkynthio, C3-C6 cycloalkylthio, phenylthio, amino, or C1-C6 alkylamino.

26. The method according to claim 17, 18, 19, 20, 22 or 23, or the use according to claim 21, wherein the protoporphyrinogen oxidase inhibitor herbicide is selected from one or more of the following types of compounds: (1) Pyrimidinediones include: Flupropacil, pyrimisulfuron, bispyribac-methyl, flupyrifenacil, 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione, 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione, flupropacil ; (2) Diphenyl ethers include: flusulfanilamide, oxyfluorfen, bensulfuron-methyl, quizalofop-P-ethyl, methoxyfen, glufosinate, ethoxyfluorfen, trifluralin or its sodium salt, methoxyfen, chlorfluazuron, chlorfluazuron ethyl ester, fluoronitrofen, furyloxyfen, nitrofluorfen. (3) Phenylepiazoles include: pyrazosulfuron and isopyrazosulfuron; (4) N-phenylimides include: propyzamide, indole-3-methyl, and fluoxetine; (5) Thiadiazoles include: methyl methacrylate, methacin, and thiamethoxam; (6) Oxadiazoles include: propyzoxystrobin and oxadiazon; (7) Triazoline ketones include: acetochlor, acetochlor ethyl ester, mesotrione, acetochlor, and acetochlor; (8) Oxazolidinones include: cyclopentoxazolone; (9) Others include: bispyribac-methyl, flupyridazine, flupyrazosulfuron, trifluralin, N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, 3-[7-fluoro-3-oxo-4-(propanediol)] (-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazinane-2,4-dione, 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester, phenylpyridines, benzoxazinones, and compounds of general formula I. ,in, Q represents , , , , , , , , , , , , , , , or ; Y represents halogen, halogenated C1-C6 alkyl, or cyano; Z represents halogen; M represents CH or N; X represents -CX1X2-(C1-C6 alkyl). n -、-(C1-C6 alkyl)-CX1X2-(C1-C6 alkyl) n -or-(CH2) r -, n represents 0 or 1, r represents an integer greater than 2; X1 and X2 independently represent hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxy-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, phenyl, or benzyl. X3 and X4 represent O or S independently, respectively; W represents hydroxyl, C1-C6 alkoxy, C2-C6 alkenoxy, C2-C6 alkynoxy, halo-C1-C6 alkoxy, halo-C2-C6 alkenoxy, halo-C2-C6 alkynoxy, C3-C6 cycloalkyloxy, phenoxy, mercapto, C1-C6 alkylthio, C2-C6 alkenthio, C2-C6 alkynthio, halo-C1-C6 alkylthio, halo-C2-C6 alkenthio, halo-C2-C6 alkynthio, C3-C6 cycloalkylthio, phenylthio, amino, or C1-C6 alkylamino.

27. The method according to claim 17, 18, 19, 20, 22 or 23, or the use according to claim 21, wherein the protoporphyrinogen oxidase inhibitor herbicide is selected from: compound A, propyzoxystrobin, sulfadiazine, ethoxysulfuron, cyprodinil, acetamiprid, epyrifenacil, mesotrione, flupyrazosulfuron, flusulfanilamide, trifluralin and... The compound A is as shown in general formula I. As shown, where Q represents Y represents chlorine; Z represents fluorine; M represents CH; X represents -C*X1X2-(C1-C6 alkyl). n -, n represents 0; X1 represents hydrogen; X2 represents methyl; X3 and X4 each independently represent O; W represents methoxy; and C* is a chiral center, so the compound has the R configuration.

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