Ppo2 protein having tolerance to ppo inhibitor herbicides and use thereof

Abstract

The present application relates to the field of biotechnology, more particularly, the present application relates to PPO2 protein with tolerance to PPO inhibitor herbicide and application. The application of the present application 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

Technical Field

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

[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 a key target for herbicides. Inhibition of PPO in plants leads to the accumulation of protoporphyrinogen, the substrate catalyzing this reaction, within cells. The accumulation of protoporphyrinogen in chloroplasts and mitochondria results in its non-enzymatic oxidation by O2. Under light conditions, this non-enzymatic oxidation produces singlet oxygen. Singlet oxygen leads to the oxidation of lipids in intracellular membrane systems, causing their oxidative disintegration and ultimately killing plant cells.

[0005] By studying the evolutionary relationships of PPO enzymes in the biological world through sequence similarity analysis, PPOs 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 migrated to other Proteobacteria classes and cyanobacteria, while HemY is the only type of PPO enzyme distributed in eukaryotes.

[0006] Plants typically possess at least two PPO genes, named PPO1 and PPO2. PPO1 is generally located in the chloroplasts, while PPO2 is typically located in the mitochondria. However, in some Amaranthaceae plants, the PPO2 gene mRNA has different translation initiation sites, resulting in PPO2 polypeptides of varying lengths. For example, the PPO2 gene in spinach (Spinaciaoleracea L.) expresses two PPO2 proteins with 26 polypeptides of different lengths, with molecular weights of approximately 58 kDa and 56 kDa, respectively. The longer one is located in the chloroplast, and the shorter one in the mitochondria.

[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, rapidly leading to plant cell death. Invention Summary

[0008] The present invention provides a PPO2 protein or a bioactive fragment thereof, comprising an amino acid sequence having the following mutation compared to the amino acid sequence of wild-type PPO2 protein: the 417th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from lysine to tyrosine, phenylalanine, serine, methionine, leucine, arginine, tryptophan or valine.

[0009] In one embodiment, the amino acid sequence further has 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; the protein optionally has tolerance to protoporphyrinogen oxidase inhibitor herbicides.

[0010] In one specific embodiment, the amino acid sequence has the mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1:

[0011] The amino acid at position 417 was mutated from lysine to tyrosine and the amino acid at position 419 was mutated from leucine to serine.

[0012] The 417th amino acid was mutated from lysine to tyrosine and the 419th amino acid was mutated from leucine to asparagine.

[0013] The 417th amino acid was mutated from lysine to tyrosine and the 422nd amino acid was mutated from leucine to methionine.

[0014] The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to glutamine, and the amino acid at position 422 was mutated from leucine to isoleucine.

[0015] The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine.

[0016] The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine.

[0017] The amino acid at position 223 was mutated from serine to tryptophan, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0018] The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0019] The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0020] The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0021] The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine.

[0022] The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine.

[0023] The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 from lysine to tyrosine, the amino acid at position 419 from leucine to asparagine, and the amino acid at position 422 from leucine to methionine.

[0024] The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine.

[0025] The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine.

[0026] The amino acid at position 417 was mutated from lysine to phenylalanine and the amino acid at position 419 was mutated from leucine to serine.

[0027] The 417th amino acid was mutated from lysine to phenylalanine and the 419th amino acid was mutated from leucine to asparagine.

[0028] The amino acid at position 417 was mutated from lysine to phenylalanine and the amino acid at position 422 was mutated from leucine to methionine.

[0029] The amino acid at position 417 was mutated from lysine to phenylalanine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine.

[0030] The amino acid at position 417 was mutated from lysine to phenylalanine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine.

[0031] The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0032] The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0033] The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine.

[0034] The amino acid at position 417 was mutated from lysine to serine and the amino acid at position 419 was mutated from leucine to serine.

[0035] The amino acid at position 417 was mutated from lysine to serine and the amino acid at position 419 was mutated from leucine to asparagine.

[0036] The amino acid at position 417 was mutated from lysine to methionine and the amino acid at position 419 was mutated from leucine to cysteine.

[0037] The amino acid at position 417 was mutated from lysine to methionine and the amino acid at position 419 was mutated from leucine to asparagine.

[0038] The amino acid at position 417 was mutated from lysine to leucine, and the amino acid at position 419 was mutated from leucine to cysteine.

[0039] The 417th amino acid is mutated from lysine to leucine, and the 419th amino acid is mutated from leucine to asparagine; or,

[0040] The 417th amino acid was mutated from lysine to arginine, and the 419th amino acid was mutated from leucine to asparagine.

[0041] In another specific embodiment, the PPO2 protein or its bioactive fragment comprises any one of the amino acid sequences shown in SEQ ID NO: 2-39.

[0042] The present invention also provides an isolated polynucleotide comprising a nucleic acid sequence selected from the following:

[0043] (1) A nucleic acid sequence or a partial sequence or a complementary sequence thereof encoding the PPO2 protein or its bioactive fragment thereof;

[0044] (2) A nucleic acid sequence that hybridizes to the sequence shown in (1) under stringent conditions; and / or

[0045] (3) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) due to the degeneracy of the genetic code, or its complementary sequence;

[0046] In one specific embodiment, the polynucleotide is a DNA molecule.

[0047] The present invention also provides a plant genome containing the aforementioned polynucleotides.

[0048] The present invention also provides a vector construct comprising the aforementioned polynucleotide and a homologous or non-homologous promoter operably linked thereto.

[0049] The present invention also provides a host cell containing the aforementioned polynucleotide or the aforementioned vector construct.

[0050] In one specific embodiment, the host cell is a plant cell.

[0051] 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 polynucleotide or the vector construct in plant cells by gene editing, or introducing the polynucleotide or the vector construct into plant cells by transgenic means.

[0052] The present invention also provides a method for producing plants that can generate or improve tolerance to protoporphyrinogen oxidase inhibitor herbicides, and the plants produced by the method, including regenerating plants from the plant cells or plant cells produced by the method.

[0053] The present invention also provides 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 protein or a bioactive fragment thereof.

[0054] 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, comprising expressing the PPO2 protein or its bioactive fragment in the plant cells, plant tissues, plant parts or plants.

[0055] Alternatively, this includes hybridizing a plant expressing the PPO2 protein or a bioactive fragment thereof with another plant, and screening for plants or parts thereof that can produce or enhance tolerance to protoporphyrinogen oxidase herbicides.

[0056] Alternatively, this may include gene editing of the plant cells, plant tissues, plant parts, or a protein of the plant that has protoporphyrinogen oxidase activity to achieve expression of the PPO2 protein or a bioactive fragment thereof therein.

[0057] The present invention also provides the use of the PPO2 protein 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 inhibitor herbicides.

[0058] In one specific embodiment, the host cell is a bacterial cell or a fungal cell.

[0059] The present invention also provides a method for controlling weeds in a plant cultivation site, wherein the plant includes the plant described above or a plant prepared by the method, and the method includes applying a herbicidal amount of a protoporphyrinogen oxidase inhibitor herbicide to the cultivation site.

[0060] In one embodiment, the herbicide is applied in an amount that does not harm the plant containing the recombinant DNA molecule.

[0061] In another specific embodiment, the herbicide is applied to the area at a ratio of about 0.02 g ai / ha to about 2000 g ai / ha, about 1 g ai / ha to about 1200 g ai / ha, about 15 g ai / ha to about 800 g ai / ha, about 30 g ai / ha to about 500 g ai / ha, or about 50 g ai / ha to about 200 g ai / ha.

[0062] In one specific embodiment, the protoporphyrinogen oxidase inhibitor herbicide is applied in combination with one or more other herbicides.

[0063] In one specific embodiment, the herbicide is formulated as a dispersible oil suspension, an aqueous suspension, a suspension emulsion, a wettable powder, an emulsifiable concentrate, a water-dispersible granule, an emulsion, or a microemulsion.

[0064] In one specific embodiment, the herbicide is applied before emergence or after emergence, and the application of the herbicide includes bringing the plant into contact with the herbicide, or the application of the herbicide includes applying the herbicide to the top of the plant or by drip irrigation.

[0065] In one specific embodiment, the application of the herbicide results in an increase in the growth or yield of the plant compared to plants of the same genotype grown in a growth area where the herbicide was not applied.

[0066] In one specific embodiment, the plant is a monocotyledonous plant or a dicotyledonous plant. Invention Details

[0067] Some of the terms used in this specification are defined as follows.

[0068] 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, which have a mode of action including inhibiting the chlorophyll biosynthesis step in plants and belong to Group E of the HRAC classification system (see HRAC, classification of herbicides according to mode of action, http: / / www.plantprotection.org / hrac / MOA.html). Any herbicide satisfying this mode of action is within the scope of protection of this invention. These PPO inhibitor herbicides can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with different chemical structures.

[0069] In one exemplary embodiment, pyrimidine dione herbicides include, but are not limited to, flufenacet (CAS NO: 134605-64-4), bensulfuron-methyl (CAS NO: 372137-35-4), bispyribac-methyl (CAS NO: 158755-95-4), tiafenacil (CAS NO: 1220411-29-9), epyrifenacil (CAS 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-pyrimidine-2,4-dione (CAS NO: 134605-64-4), pyrimidine dione (CAS NO: 158755-95-4), tiafenacil (CAS NO: 1220411-29-9), epoxazin-6-yl)-1H-pyrimidine-2,4-dione ... 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), , , (fluridine) , , Uracil-pyridine disclosed in WO2017 / 202768 and uracil derivatives disclosed in WO2018 / 019842.

[0070] Diphenyl ether herbicides include, but are not limited to, flufenoxuron (CAS NO: 72178-02-0), ethoxyflufen (CAS NO: 42874-03-3), bensulfuron (CAS NO: 74070-46-5), quizalofop-p-ethyl (CAS NO: 77501-63-4), methoxyflufen (CAS NO: 32861-85-1), glufosinate (CAS NO: 1836-77-7), ethoxyflufen (CAS NO: 77501-90-7), trifluralin or its sodium salt (CAS NO: 50594-66-6 or 62476-59-9), methoxyflufen (CAS NO: 42576-02-3), ethoxyfen (CAS NO: 188634-90-4), chlorfluazuron ethyl (CAS NO: 131086-42-5), and fluoronitrofen (CAS NO: 131086-42-5). NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0071] Phenylepiazole herbicides include, but are not limited to, imidacloprid (CAS NO: 129630-19-9), isopyrazosulfuron (CAS NO: 174514-07-9) and ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxo]propionate (CAS: 118237-10-8).

[0072] N-phenylimide herbicides include, but are not limited to, propyzamide (CAS NO: 103361-09-7), indole-3-methyl (CAS NO: 142891-20-1), flumipropyn (CAS NO: 84478-52-4), flumethrin (CAS NO: 87546-18-7), chlorophthalim (CAS: 39985-63-2), and N-(4-chlorophenyl)-3,4,5,6-tetrahydrophenyl-o-dicarboximide (CAS: 7386-21-2).

[0073] 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).

[0074] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0075] Triazoline herbicides include, but are not limited to, oxadiazon (CAS NO: 128621-72-7), oxadiazon / oxadiazon (CAS NO: 128639-02-1), mesotrione (CAS NO: 122836-35-5), oxadiazon (CAS NO: 68049-83-2), and oxadiazon (CAS NO: 173980-17-1).

[0076] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0077] Other herbicides include, but are not limited to, cyclopyranil (CAS NO: 158353-15-2), flupyrazosulfuron (CAS NO: 188489-07-8), cyclopyranil (CAS NO: 1651191-47-7), flupyrazosulfuron (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), phenopylate (CAS: 40575-34-6), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9), and N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 190314-43-3), pyrazole-1-carboxamide (CAS NO: 1258836-72-4), phenopylate (CAS NO: 40575-34-6), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 190314-43-3), ... NO: 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (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: 915396-43-9), N-ethyl ...099-05-7), 3-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7- 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-Diketone (CAS 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: 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 NO: 948893-00-3), ethyl acetate of 3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy] (CAS: 353292-31-6, S-3100), 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4- Dione (CAS: 1258836-72-4), 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: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS: 212754-02-4), The phenylpyridine derivatives disclosed in WO2016 / 120116 and the benzoxazinone derivatives disclosed in EP09163242.2.

[0078] In another exemplary embodiment, the PPO inhibitor herbicide has the typical chemical formula listed in WO2024126560A1.

[0079] 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.

[0080] 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 in the form of agriculturally acceptable derivatives, for example as amides such as mono- and di-C1-C6-alkylamides or arylamides, as esters such as allyl esters, propargyl esters, C1-C10-alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters, and also as thioesters such as C1-C10-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. Examples of straight-chain or branched C1-C10-alkyl thioesters are ethyl thioesters.

[0081] The PPO-inhibiting herbicides described above, which can be used in carrying out the present invention, are generally preferred to be used in combination with one or more other herbicides to achieve control of a variety of unwanted plants. For example, PPO-inhibiting herbicides can also be used in combination with additional herbicides to which the crop is 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.

[0082] Suitable mixture components, for example, are herbicides selected from categories b1) to b15):

[0083] b1) Inhibitors of lipid biosynthesis;

[0084] b2) Acetolactate synthase inhibitors (ALS inhibitors);

[0085] b3) Photosynthesis inhibitors;

[0086] b4) Protoporphyrinogen-IX oxidase inhibitor;

[0087] b5) Bleaching herbicides;

[0088] b6) 5-Enolpyruvylshikimate-3-phosphate synthase inhibitor (EPSP inhibitor);

[0089] b7) Glutamine synthase inhibitor;

[0090] b8) 7,8-Dihydropteranoic acid synthase inhibitor (DHP inhibitor);

[0091] b9) Mitosis inhibitors;

[0092] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0093] b11) Cellulose biosynthesis inhibitor;

[0094] b12) Decoupler herbicides;

[0095] b13) auxinic herbicides;

[0096] b14) Auxin transport inhibitors; and

[0097] b15) is selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, sodium DSMA, dymron, endothal and its salts, etobenzanid, flamprop, and fl. amprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleichydrazide, mefluidide, metam, methiozolin (CAS) NO: 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridiphane, and other herbicides of 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and their salts and esters;

[0098] This includes their agriculturally acceptable salts or derivatives.

[0099] 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.

[0100] In addition, safeners, PPO inhibitory herbicides and / or other herbicide compounds may be applied simultaneously or sequentially.

[0101] PPO inhibitory herbicides and herbicide compounds and safeners of groups b1)-b15) are known herbicides and safeners, see, for example, WO2013 / 189984; The Compendium of Pesticide Common Names (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000, Vol. 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R.R. Schmidt, Herbizide [herbicide], 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.

[0102] The term "expression" and its grammatical variations refer to the biosynthesis or process that produces polynucleotides, including the transcription and / or translation of gene products. For example, nucleic acid molecules disclosed in this invention can be transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the transcribed mRNA can be subsequently translated into a protein. The term "gene product" and its grammatical variations can refer to, for example, transcripts and encoded proteins. The inhibition (or enhancement) of the expression or function of a gene product (i.e., the target gene product) can be in a comparative context between any two plants, for example, the expression or function of a gene product in a genetically modified plant relative to the expression or function of a gene product in a corresponding but susceptible wild-type plant or other susceptible plant. The expression level of the gene product may not be present in wild-type plants. For example, a "wild-type" plant can be a plant, plant cell, or plant part that does not express a foreign resistance gene.

[0103] An "expression cassette" refers to DNA capable of expressing proteins in a host cell. This DNA includes not only promoters that initiate transcription of the protein-encoding gene but also terminators that terminate transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: cauliflower mosaic virus constitutive promoter 35S; a wound-inducible promoter from tomato leucine aminopeptidase (“LAP”) (Chao et al., (1999) Plant Physiol 120:979-992); a chemically induced promoter from tobacco pathogenesis-associated 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both induced by methyl jasmonate); heat shock promoters; tetracycline-inducible promoters; seed-specific promoters, such as the millet seed-specific promoter pF128; seed storage protein-specific promoters (e.g., bean protein, rapeseed protein, oleic acid protein, and soybean β-conglycinin promoters) (Beachy et al., (1985) EMBO J. [Journal of the European Society for Molecular Biology] 4:3047-3053). They can be used alone or in combination with other plant promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al., (1985) Nature, 313:810; Rosenberg et al., (1987) Gene, 56:125; Guerineau et al., (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al., Genes Dev., 5:141; Mogen et al., (1990) Plant Cell, 2:1261; Munroe et al., (1990) Gene, 91:151; Ballad et al., (1989) Nucleic Acids Res., 17:7891; Joshi et al., (1987) Nucleic Acid Res., 15:9627.

[0104] “Vector” means any recombinant DNA molecule that can be used for bacterial or plant transformation purposes, containing any of a variety of nucleic acids containing one or more desired sequences to be delivered to and / or expressed in cells. Vectors are typically made of DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phage particles, viral genomes, synthetic chromosomes, etc. Recombinant DNA molecules, as listed in the sequence listing, can be inserted into vectors as part of a construct having a recombinant DNA molecule operatively linked to a gene expression element that functions in plants to influence the expression of an engineered protein encoded by the recombinant DNA molecule. General methods for manipulating DNA molecules for the preparation and use of recombinant DNA constructs and plant transformation vectors are well known in the art and are described in detail, for example, in manuals and laboratory guides including MR Green and J Sambrook, “Molecular Cloning: A Laboratory Manual” (4th edition), ISBN: 978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012).

[0105] 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.

[0106] The term "transformation" and its grammatical variations are used in this invention to refer, for example, the transfer of a nucleic acid fragment into the genome of a host organism, thereby acquiring genetically stable inheritance. A host organism containing the transformed nucleic acid fragment is called a "transgenic" organism. Methods of vector transformation include introducing recombinant plasmids into plants using methods such as Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption.

[0107] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0108] The term “host cell” and its grammatical variations refer to the cell in which the transformation of recombinant DNA constructs occurs, and may include yeast cells, bacterial cells and / or plant cells.

[0109] 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).

[0110] The term "gene editing" generally refers to a technique that involves the insertion, deletion, modification, or replacement of DNA in the genome. 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.

[0111] 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.

[0112] 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.

[0113] The terms "variant," "mutation," and their grammatical variations refer to substantially similar sequences. For nucleic acid molecules, a variant includes the deletion and / or addition of one or more nucleotides at one or more sites in the native nucleic acid molecule, and / or the substitution of one or more nucleotides at one or more sites in the native nucleic acid molecule. A "mutation" can refer to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to a 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, substitutions, deletions, insertions, or inversions 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, substitutions, deletions, insertions, or inversions may include variations at positions 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

[0114] 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 that possesses this 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, the wild-type rice PPO2 protein can be represented by SEQ ID NO: 1.

[0115] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, particularly monocotyledonous or dicotyledonous plants. Plants of this invention include inbred lines, hybrids, open-pollinated varieties, double-haploid plants, or plants from segregating populations. The term "plant" and its grammatical variations can refer to the whole plant, any part thereof, or cell or tissue cultures derived from a plant. Thus, the term "plant" can refer to any of the following: whole plant, plant part or organ, plant tissue, seed, and / or plant cell. A plant cell is a cell of a plant obtained directly from a seed or plant, or derived from a cell culture obtained from a plant. Progeny, variants, and mutants of regenerated plants are within the scope of this invention, provided that these parts contain the introduced resistance gene.

[0116] The term "plant part" includes, but is not limited to, embryos, pollen, seeds, leaves, flowers (including but not limited to anthers, ovules, etc.), fruits, stems or branches, roots, root tips, cells (including intact cells in plants and / or plant parts), protoplasts, plant cell tissue cultures, plant callus, plant masses, etc. Therefore, plant parts include soybean tissue cultures that can regenerate soybean plants. Furthermore, "plant cell" refers to the structural and physiological unit of a plant, including the cell wall, and may also refer to the protoplast. The plant cells of this invention can be in the form of isolated single cells, or can be cultured cells, or can be part of a higher-level tissue unit (e.g., plant tissue or plant organ). In this invention, "plant cell" should be understood as any cell derived from or found in a plant capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, plants, or seeds.

[0117] The term “introduction” or “insertion” in plants refers to any method of completion, including but not limited to: gene introgression, transgenesis, modification with regularly clustered short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs) (Feng et al. 2013, Joung and Sander 2013), large-scale nucleases, or zinc finger nucleases (ZFNs).

[0118] The terms “increase,” “enhance,” “enhance,” etc., and their grammatical variations, are used to refer to any promotion or gain or increase in the expression, function, or activity of a target gene (e.g., a resistance gene) product compared to a susceptible plant, thereby providing enhanced resistance to one or more pathogens (e.g., *Laminaria*) or diseases (e.g., rust). Furthermore, as used herein, the terms “lead to” or “increase,” and their grammatical variations, can refer to higher expression of a target gene product such that the level is increased by 10% or more, 50% or more, or 100% relative to cells or plants lacking the target gene or protein disclosed herein.

[0119] As used herein, the terms “phenotype,” “phenotypic trait,” or “trait” refer to one or more characteristics and / or manifestations of an organism. A phenotype is a manifestation that can be observed with the naked eye or by any other evaluative means known in the art (e.g., microscopy, biochemical analysis, or electromechanical measurement).

[0120] Alternatively, inhibition (or enhancement) of the expression or function of a target gene product can be achieved in settings where comparisons are made between plant cells, organelles, tissues, or plant parts within the same or different plants, and including comparisons between developmental stages or time periods of the same or different plants. Any method or composition that downregulates the expression of a target gene product or downregulates its functional activity at the transcriptional or translational level can be used to achieve inhibition of the expression or function of a target gene product. Similarly, any method or composition that induces or upregulates the expression of a target gene product, or increases or activates or upregulates its functional activity at the transcriptional or translational level, can be used to achieve increased expression or function of a target gene or protein. Methods for inhibiting or enhancing gene expression are well known in the art.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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, K417S indicates that, relative to the amino acid sequence of SEQ ID NO: 1, lysine at position 417 is replaced by serine. For double or multiple mutations, each mutation is separated by a " / " or "+". For example, K417S + L419S or K417S / L419S indicates that, relative to the amino acid sequence of SEQ ID NO: 1, lysine at position 417 is replaced by serine, and leucine at position 419 is replaced by serine, and both mutations are present in the specific mutant OsPPO2 protein.

[0126] The terms "corresponding to" and "relative to" are used interchangeably. The specific amino acid position (number) within the protein described in this invention is 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 is considered aligned when the alignment score is the highest. 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 databanks. 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 / DNAGAPDIST = 4.

[0127] 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.

[0128] Amino acid sequence identity can be determined using conventional methods, see, for example, Smith and Waterman, 1981.

[0129] The teachings of Adv. Appl. Math. 2:482, Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., can be used to determine the algorithm by computer-running algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group). Alternatively, 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 / ), can be used with default parameters.

[0130] Specific identity / comparison methods

[0131] The following terms are used to describe the sequence relationship between two or more nucleotides or amino acids (peptides, proteins, protein sequences): (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity" and (e) "substantial identity".

[0132] (a) As used herein, a “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0133] (b) As used herein, a “comparison window” refers to a continuous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may include additions or deletions (i.e., vacancies) compared to a reference sequence (which does not include additions or deletions) used for the best alignment of the two sequences. Typically, the length of the comparison window is at least 20 consecutive nucleotides, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art will understand that, due to the presence of gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of matches to avoid high similarity with the reference sequence.

[0134] The alignment methods used for comparing sequences are well known in the art. Therefore, the determination of percentage sequence identity between any two sequences can be performed using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is the work of Myers and Miller (1988) in CABIOS. The algorithms in 4:11-17; the local alignment algorithm in Smith et al. (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482; the global alignment algorithm in Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; the search-based local alignment method in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences] 85:2444-2448; the algorithm in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 872-264; as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:5873-5877.

[0135] Computer implementations of these mathematical algorithms can be used for sequence comparisons to determine sequence identity. These implementations include, but are not limited to: CLUSTAL from the PC / Gene program (available from Intelligenetics, Mountain View, CA); ALIGN program (version 2.0); and GAP, BESTFIT, BLAST, FASTA, and TFASTA from GCG Wisconsin Genetics software package version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, CA, USA). Comparisons using these programs can be performed with default parameters. The following fully describe the CLUSTAL procedure: Higgins et al. (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN procedure is based on the algorithm of Myers and Miller (1988) (ibid.). When comparing amino acid sequences, the ALIGN procedure can use the PAM120 weighted residue table with a nick length penalty of 12 and a nick penalty of 4. The BLAST procedure proposed by Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403 is based on the algorithm in Karlin and Altschul (1990) above. A BLAST nucleotide search can be performed using the BLASTN procedure with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleotide sequence of the protein encoding the example. A BLAST protein search can be performed using the BLASTX procedure with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein or polypeptide of the example. For gapped alignments for comparative purposes, GappedBLAST (in BLAST 2.0) as described by Altschul et al. (1997) Nucleic Acids Res. [Nucleic Acids Research] 25:3389 can be used. Alternatively, PSI-BLAST (in BLAST 2.0) can be used for iterative searches to detect distant relationships between molecules. See Altschul et al. (1997), ibid.When using BLAST, nick BLAST, or PSI-BLAST, the default parameters for each program can be used (e.g., BLASTN for nucleotide sequences, BLASTX for proteins). See the National Center for Biotechnology Information website, ncbi.hlm.nih.gov. Alignment can also be performed manually via inspection.

[0136] (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences means the same residues in two sequences when compared at maximum correspondence on a specified comparison window. When using a percentage of sequence identity for a protein, it is recognized that dissimilar residue positions are generally distinguished by conserved amino acid substitutions, where an amino acid residue is substituted by another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ in terms of conserved substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of that substitution. Sequences differing from these conserved substitutions are referred to as having “sequence similarity” or “similarity.” Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring conserved substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, a score for a conserved substitution is between zero and 1 when the same amino acid scores 1 and a non-conserved substitution scores zero. Scores for conserved substitutions are calculated, for example, as implemented in the program PC / GENE (Edali Genetics, Mountain View, CA).

[0137] (d) As used herein, the sequence “identity” percentage refers to the value determined by comparing two best-aligned sequences within a comparison window, where the polynucleotide sequence portion of the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to produce the sequence identity percentage.

[0138] (e)(i) The term “substantial identity” for a polynucleotide sequence means that when a polynucleotide is compared with a reference sequence using one of the alignment procedures described above with standard parameters, it includes sequences having at least 70%, 80%, 90%, or 95% or higher sequence identity. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. For these purposes, substantial identity of the amino acid sequence generally means sequence identity with at least 60%, 70%, 80%, 90%, or 95% or higher sequence identity.

[0139] Another indicator of substantially identical nucleotide sequences is whether the two molecules hybridize with each other under stringent conditions. Typically, stringent conditions are chosen to be about 5°C lower than the Tm of a particular sequence at defined ionic strengths and pH. However, stringent conditions encompass temperatures ranging from about 1°C to about 20°C lower than Tm, depending on the desired level of stringency as limited elsewhere in this document. If nucleic acids that do not hybridize with each other under stringent conditions encode substantially identical polypeptides, then these nucleic acids remain substantially identical. This can occur, for example, when a single copy of a nucleic acid is produced using the maximum codon degeneracy allowed by genetic coding. Another indicator of substantially identical nucleic acid sequences is that the polypeptide encoded by the first nucleic acid is immunely cross-reactive with the polypeptide encoded by the second nucleic acid.

[0140] (e)(ii) In the context of peptides, the term “substantially similar” means that a peptide contains a sequence within a specified comparison window that has at least 70%, 80%, 85%, 90%, 95%, or higher sequence identity with a reference sequence. Optimal alignment for these purposes can be performed using the global alignment algorithm described above by Needleman and Wunsch (1970). One indication that two peptide sequences are substantially identical is that one peptide is immunoreactive with an antibody against the second peptide. Thus, for example, when one peptide is not identical to a second peptide only because of conserved substitutions, the two peptides are substantially identical. Aside from the possibility that the positions of not entirely identical residues may differ due to conserved amino acid changes, “substantially similar” peptides share the sequence as described above.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] The obtained PPO protein can be transferred to plants to enhance their herbicide resistance.

[0147] 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.

[0148] 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 adenine transphosphoribosylase (APRT) promoter, octopine synthase promoter, and BCB (copper-binding protein) promoter.

[0149] 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.

[0150] 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.

[0151] 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.

[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. Attached Figure Description

[0155] Figure 1 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants transformed with wild-type OsPPO2 gene (WT) or various OsPPO2 mutant genes when treated with flurbimethrin at concentrations of 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM, respectively.

[0156] Figure 2 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants converted from the OsPPO2 wild-type gene (WT) or various OsPPO2 mutant genes when treated with propyzamide at concentrations of 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM.

[0157] Figure 3 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants converted from the OsPPO2 wild-type gene (WT) or various OsPPO2 mutant genes when treated with flusulfanilamide at concentrations of 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM.

[0158] Figure 4 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants transformed with wild-type OsPPO2 gene (WT) or various OsPPO2 mutant genes when treated with 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM respectively.

[0159] Figure 5 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants converted from the OsPPO2 wild-type gene (WT) or various OsPPO2 mutant genes when treated with ethoxyflufenicol at concentrations of 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM.

[0160] Figure 6 Cell growth levels of PPO-deficient Escherichia coli (ΔhemG) transformants transformed with wild-type OsPPO2 gene (WT) or various OsPPO2 mutant genes when treated with 0 μM, 5 μM, 20 μM, 50 μM, 100 μM and 200 μM respectively.

[0161] Figure 7Enzyme activity assay of OsPPO2 mutants. Enzyme kinetic curves of rice OsPPO2 wild type (WT), 417Y+419N+422M (3M), and 223K+279Q+417Y+419N+422M (5M).

[0162] Figure 8 The growth of rice seedlings was investigated by treating them with fluroxypyr containing 45 g ai / ha and 360 g ai / ha, respectively.

[0163] Figure 9 The growth of rice seedlings treated with fluroxypyr at concentrations of 45 g ai / ha and 360 g ai / ha were investigated. 1: Wild-type rice, untreated; 2: Wild-type; 3: OsPPO2-WT overexpressed; 4 / 5: OsPPO2 223K+279Q+417Y+419N+422M overexpressed.

[0164] Figure 10 The growth of rice seedlings treated with fluroxypyr containing 45 g ai / ha. From left to right: wild-type recipient untreated blank control, wild-type recipient rice treated with fluroxypyr (WT), and gene-edited rice varieties K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, and K417Y+L419N+L422M treated with fluroxypyr.

[0165] Figure 11 The growth of rice seedlings treated with fluroxypyr containing 45 g ai / ha. From left to right, the wild-type recipient rice (untreated blank control), wild-type recipient rice treated with fluroxypyr (WT), and gene-edited 223K+279Q+417Y+419N+422M rice treated with fluroxypyr (3 plants on the right). Sequence Description

[0166]

[0167] Detailed Implementation

[0168] 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.

[0169] Example 1: Cloning of the rice protoporphyrinogen oxidase PPO2 gene

[0170] 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 (SEQ ID NO: 1) and the sequence of the vector pET15b (Novagen), primers were designed and synthesized, and a wild-type rice OsPPO2 coding region DNA expression vector was successfully constructed, named pET15b-OsPPO2 WT.

[0171] Example 2: Screening for OsPPO2 mutation sites in rice using PPO-deficient Escherichia coli (ΔhemG)

[0172] To screen for sites in the rice OsPPO2 gene that exhibit tolerance to fluroxypyr, amino acid saturation mutations were performed on the rice OsPPO2 gene site (Kille S, Acevedo-Rocha CG, Parra LP, Zhang ZG, OppermanDJ, ReetzMT, AcevedoJP (2013) Reducing codon redundancy and screening effortof combinatorial protein libraries created by saturation mutagenesis. ACSSynth Biol 2(2):83-92;Directed EvolutionLibrary Creation: methods and protocols 2nd ed. Edited by Elizabeth MJ Gillam, Janine N. Copp and David F. Ackerley New York, NY United States: Springer, 2014. doi:10.1007 / 978-1-4939-1053-3), constructing mutant libraries at different sites. PPO-deficient Escherichia coli (ΔhemG) was used to screen for sites in rice OsPPO2 that exhibit resistance to flurbimethrin. The ΔhemG strain is an Escherichia coli strain lacking the hemG type PPO gene and exhibiting kanamycin resistance (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.).

[0173] Plasmids from preconstructed saturated libraries at different sites were transformed into ΔhemG competent cells. The herbicide tolerance at different sites of the rice PPO2 gene was tested using an E. coli screening system. Normally growing resistant clones were selected from plates containing fluroxypyr and their genotypes were identified. The screened resistant mutants grew normally on LB medium containing fluroxypyr compared to the wild type. Specific growth data for the mutants are shown in Table 1; "-" indicates no normal growth at a 5 μM fluroxypyr concentration, and "+" indicates normal growth at a 5 μM fluroxypyr concentration. These results indicate that mutations at different sites in the rice OsPPO2 gene confer tolerance to the herbicide fluroxypyr.

[0174] Table 1. Tolerance of different OsPPO2 mutants to flurbimethrin in rice

[0175]

[0176] Example 3: Herbicide tolerance of rice OsPPO2 resistance site combinations

[0177] To further enhance the tolerance of rice OsPPO2 to PPO inhibitor herbicides, relevant sites were combined, and herbicide tolerance was screened using PPO-deficient *E. coli* (ΔhemG). The herbicides were cultured on plates containing different concentrations of fluroxypyr (0 μM, 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM), and growth inhibition was observed. The specific screening results are shown in Table 2. Additionally, the tolerance of rice OsPPO2 mutants K417F+L419N, K417Y+419N, K417F+L419N+L422M, and K417Y+L419N+L422M is shown in Table 2. Figure 1 The symbols indicate the growth rate. "-" indicates that the plant cannot grow normally at a drug concentration of 5 μM; "+" indicates that the plant can grow normally at a drug concentration of 5 μM; "++" indicates that the plant can grow normally at a drug concentration of 20 μM; "+++" indicates that the plant can grow normally at a drug concentration of 50 μM; "++++" indicates that the plant can grow normally at a drug concentration of 100 μM; and "+++++" indicates that the plant can grow normally at a drug concentration of 200 μM.

[0178] Table 2. Tolerance of OsPPO2 mutant combinations to flurbimethrin

[0179]

[0180] Example 4: Verifying the tolerance of different rice OsPPO2 mutants to different PPO herbicides.

[0181] To verify whether the mutants in Example 3 were also tolerant to other PPO herbicides, tolerance was verified using five different types of PPO herbicides. The vector plasmids of the constructed mutants were transformed into ΔhemG competent cells and plated onto LB agar plates containing 0 μM, 5 μM, 20 μM, 50 μM, 100 μM, and 200 μM of five PPO herbicides (propyzoxystrobin, flumetsulam, oxyflufenoxam, ethoxysulfuron, and pyrimethanil), respectively. After incubation at 37°C overnight, growth inhibition was observed. Figures 2 to 6 The table shows the tolerance of rice OsPPO2 mutants K417F+L419N, K417Y+419N, K417F+L419N+L422M, and K417Y+L419N+L422M to other PPO herbicides. The test results for other representative OsPPO2 mutant resistance sites are shown in Table 3. "-" indicates no normal growth at a 5 μM herbicide concentration; "+" indicates normal growth at a 5 μM herbicide concentration; "++" indicates normal growth at a 20 μM herbicide concentration; "++++" indicates normal growth at a 50 μM herbicide concentration; "++++" indicates normal growth at a 100 μM herbicide concentration; "++++" indicates normal growth at a 200 μM herbicide concentration; and "NT" indicates not tested.

[0182] The results showed that, compared with wild-type PPO2, each mutant OsPPO2 was also tolerant to other PPO types of herbicides.

[0183] Table 3. Tolerance of different OsPPO2 mutants of rice to different types of PPO herbicides

[0184]

[0185] Example 5: In vitro enzyme activity assay of rice OsPPO2 resistance site combinatorial protein (peptide)

[0186] To further verify the in vitro enzyme activity of rice OsPPO2 resistance combinatorial protein (peptide), the activity of different rice OsPPO2 mutants was tested (Rangani G, Salas-Perez RA, Aponte RA, et al. A Novel Single-Site Mutation in the Catalytic Domain of ProtoporphyrinogenOxidase IX (PPO) Confers Resistance to PPO-Inhibiting Herbicides[J].other,2019.DOI:10.3389 / fpls.2019.00568.).

[0187] Protein expression vectors were constructed from wild-type rice OsPPO2, OsPPO2 417Y+419N+422M, and S223K+R279Q+K417Y+L419N+L422M mutants. After isolating and purifying the OsPPO2 enzyme, reaction solutions containing different concentrations of the substrate protoporphyrinogen were prepared at concentrations of 0.125, 0.5, 2, 4, 8, and 16 μM. The OsPPO2 enzyme was diluted to 10 μM, and 5 μL was pipetted into a 96-well black ELISA plate. The final working concentration of the enzyme was 500 nM. The plates were immediately mixed, and the reaction was detected using a fluorescence microplate reader. Excitation was performed at 410 nm, and detection was performed at 630 nm. Reaction curves were plotted. Results are shown below. Figure 7 As shown, compared with the wild type of rice OsPPO2, the in vitro enzyme activities of the rice OsPPO2 gene mutants K417Y+L419N+L422M and S223K+R279Q+K417Y+L419N+L422M were significantly higher than those of the wild type of rice PPO2.

[0188] Example 6: Validation of tolerance to PPO herbicides by overexpressing different mutants of rice OsPPO2 in Arabidopsis plants

[0189] To rapidly validate the tolerance of rice OsPPO2 triple mutants K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, K417Y+L419N+L422M and pentamutation S223K+R279Q+K417Y+L419N+L422M to PPO inhibitor herbicides in plants, vectors overexpressing wild-type and mutant PPO2 genes in rice were constructed using conventional methods and overexpressed in Arabidopsis thaliana.

[0190] The obtained rice OsPPO2 mutant and wild-type Arabidopsis seeds were subjected to resistance tests on MS medium (petries) containing different concentrations of PPO inhibitor herbicides. As shown in Table 4, compared with wild-type Arabidopsis, Arabidopsis plants overexpressing rice OsPPO2 tri-mutant, penta-mutant, and WT all exhibited certain tolerance / resistance to flufenoxuron. Their resistance levels were similar at a treatment concentration of 50 nM. However, at a higher application concentration of 2 μM, mutant Arabidopsis plants overexpressing rice OsPPO2 tri-mutant K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, and penta-mutant S223K+R279Q+K417Y+L419N+L422M still showed resistance. Meanwhile, those overexpressing OsPPO2... All WT plants and wild-type Arabidopsis thaliana controls died, indicating that overexpression of various OsPPO2 mutants in Arabidopsis thaliana resulted in higher tolerance to PPO inhibitor herbicides.

[0191] Table 4. Tolerance of transgenic Arabidopsis seedlings to flurbimethrin

[0192]

[0193] Note: 0 - No difference, 1 - Slight burn, 2 - Significant burn, 3 - Severe burn, 4 - Slight drying, 5 - Significant drying, 6 - Severe drying, 7 - Few deaths, 8 - Most deaths, 9 - All deaths.

[0194] Example 7: Obtaining herbicide resistance by overexpressing different types of OsPPO2 mutants in rice

[0195] To further test the herbicide tolerance of the obtained mutants in plants, the rice mutants OsPPO2K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M and S223K+R279Q+K417Y+L419N+L422M were overexpressed in rice, while rice overexpressing OsPPO2 WT was used as a control.

[0196] Resistance tests were conducted by spraying different concentrations of fluroxypyr on T0 generation rice seedlings overexpressing OsPPO2 K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, S223K+R279Q+K417Y+L419N+L422M and OsPPO2 WT. Compared to the wild type, all OsPPO2 overexpression mutants and OsPPO2 overexpression WT rice showed some tolerance / resistance to fluroxypyr. Resistance levels were similar at an application concentration of 45 g ai / ha, but at a higher concentration of 360 g ai / ha, the OsPPO2 overexpression mutants still showed resistance. The OsPPO2 overexpression WT plants exhibited leaf necrosis, which was not different from the wild-type control. This indicates that the OsPPO2 overexpression mutants showed higher tolerance to fluroxypyr. Representative data can be found in [link to relevant data]. Figure 8 and Figure 9 .

[0197] Example 8: Gene-edited rice with PPO-resistant herbicides

[0198] To obtain herbicide-resistant non-transgenic rice, homologous recombination was performed on the OsPPPO2 K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M and S223K+R279Q+K417Y+L419N+L422M mutants using gene editing. After identification, homozygous rice lines of the above-mentioned homologous recombination gene-edited mutants were successfully obtained. To verify the tolerance of homozygous gene-edited rice seedlings to fluroxypyr, rice seedlings were treated with fluroxypyr at a dose of 45 g ai / ha. Compared with wild-type lines, lines with homologous recombination at the K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, and S223K+R279Q+K417Y+L419N+L422M mutation sites could still survive at the 45 g ai / ha dose, while wild-type lines withered and died. Figure 10 and 11 As shown.

[0199] Example 9: Testing of resistance to other PPO-inhibiting herbicides in overexpressed and gene-edited rice

[0200] To test the tolerance of overexpressing rice and gene-edited rice in Examples 7 and 8 to other PPO inhibitor herbicides, wild-type and OsPPO2 overexpressing WT rice were used as controls.

[0201] Pre-emergence sealing for direct-seeded rice: Sow rice after germination, spray the soil before seedling emergence, and keep the soil moist thereafter.

[0202] Post-seedling spraying of rice seedlings: After the rice seedlings emerge, spray the stems and leaves of weeds when they have 2-4 leaves (apply to the top).

[0203] After treatment, the test materials were routinely cultured, and an investigation was conducted 14 days later.

[0204] 45g ai / ha propyzamide, 75g ai / ha mesotrione, 75g ai / ha flusulfanilamide, 30g ai.i. / ha cyclopyridazine, 30g ai / ha bensulfuron-methyl, 30g ai / ha 75g ai / ha epyrifenacil, 7.5g ai / ha methyl methacrylate, 30g ai / ha trifludimoxazin, 90g ai / ha bispyribac-sodium, 30g ai / ha tiafenacil, 300g ai / ha pendioxaflutole, 60g ai / ha flufenoxuron, 450g ai / ha trifluralin, 1000g ai / ha methyl methoxyfenozide, 10g ai / ha pyrazosulfuron, 75g ai / ha ethoxyflufenozide, 108g ai / ha quizalofop-p-ethyl, 360g ai / ha oxadiazon, 75g ai / ha propyzoxystrobin, 15g ai / ha As a field baseline, herbicide tolerance levels of various rice varieties were tested using 2x and 4x doses of the aforementioned herbicides (herbicide damage level: numbers 0-5 represent resistance levels from no herbicide damage to death). The results showed that both the overexpressing and gene-edited rice varieties exhibited excellent safety against PPO inhibitor herbicides and effectively controlled common weeds in paddy fields, demonstrating good selectivity. Representative data are as follows: In the pre-emergence sealing test of direct-seeded rice, the K417Y+L419N+L422M mutant overexpressing herbicide emerged normally when 4x dose of propyzamide was applied, while wild-type and OsPPO2 overexpressing WT rice varieties died completely; in the post-emergence spray test, the K417Y+L419N+L422M mutant overexpressing herbicide was at level 1 when 2x dose of propyzamide was applied, while the wild-type and OsPPO2 overexpressing WT rice varieties were at levels 5 and 4, respectively.

[0205] Example 10: Resistance test of OsPPO2 overexpression mutant maize and cotton to different PPO inhibitor herbicides

[0206] The OsPPO2 mutants K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, and S223K+R279Q+K417Y+L419N+L422M were overexpressed in maize or cotton, with maize and cotton overexpressing OsPPO2 WT serving as controls.

[0207] Different methods were applied to corn or cotton overexpressing OsPPO2 K417F+L419N+L422I, K417Y+L419N+L422I, K417F+L419N+L422M, K417Y+L419N+L422M, S223K+R279Q+K417Y+L419N+L422M and OsPPO2 WT before and after emergence.

[0208] Referring to Example 9, 45g ai / ha fluroxypyr, 45g ai / ha propyzoxystrobin, 75g ai / ha mesotrione, 75g ai / ha flusulfanilamide, 30g ai / ha pyrazosulfuron, 30g ai / ha pyrazosulfuron, 30g ai / ha pyrazosulfuron, 30g ai / ha 75g ai / ha epyrifenacil, 7.5g ai / ha methyl methacrylate, 30ga.i. / ha trifludimoxazin, 90g ai / ha bispyribac-sodium, 30g ai / ha tiafenacil, 300g ai / ha pendioxaflutole, 60g ai / ha flufenoxuron, 450g ai / ha trifluralin, 1000g ai / ha methyl methoxyfenozide, 10g ai / ha pyrazosulfuron, 75g ai / ha ethoxyflufenozide, 108g ai / ha quizalofop-p-ethyl, 360g ai / ha oxadiazon, 75g ai / ha propyzoxystrobin, 15g ai / ha As a field baseline, herbicide tolerance levels in corn / cotton were tested using 2x and 4x doses of the aforementioned herbicides (herbicide damage level: numbers 0-5 represent resistance levels from no herbicide damage to death). The tests revealed that corn / cotton overexpressing various OsPPO2 mutants also exhibited high tolerance to PPO inhibitor herbicides, demonstrating excellent safety and selectivity.

[0209] Furthermore, numerous tests revealed that introducing the mutant described in this invention into model plants such as Arabidopsis thaliana and Brachypodium distichum resulted in increased resistance to PPO-inhibiting herbicides at corresponding levels. Therefore, it is evident that transgenic or gene-edited versions of this mutant can also produce corresponding resistance traits in other aforementioned plants, demonstrating significant industrial value.

[0210] 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.

[0211] 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 protein or a bioactive fragment thereof, comprising an amino acid sequence having the following mutation compared to the amino acid sequence of wild-type PPO2 protein: the 417th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from lysine to tyrosine, phenylalanine, serine, methionine, leucine, arginine, tryptophan or valine.

2. The PPO2 protein or its bioactive fragment according to claim 1, wherein the amino acid sequence further has 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; and the protein optionally has tolerance to protoporphyrinogen oxidase inhibitor herbicides.

3. The PPO2 protein or its bioactive fragment according to claim 1 or 2, wherein the amino acid sequence has the mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: The amino acid at position 417 was mutated from lysine to tyrosine and the amino acid at position 419 was mutated from leucine to serine. The amino acid at position 417 was mutated from lysine to tyrosine and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 417 was mutated from lysine to tyrosine and the amino acid at position 422 was mutated from leucine to methionine. The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to glutamine, and the amino acid at position 422 was mutated from leucine to isoleucine. The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine. The amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine. The amino acid at position 223 was mutated from serine to tryptophan, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine. The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine. The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 from lysine to tyrosine, the amino acid at position 419 from leucine to asparagine, and the amino acid at position 422 from leucine to methionine. The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine. The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to tyrosine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine. The 417th amino acid was mutated from lysine to phenylalanine and the 419th amino acid was mutated from leucine to serine. The 417th amino acid was mutated from lysine to phenylalanine and the 419th amino acid was mutated from leucine to asparagine. The amino acid at position 417 was mutated from lysine to phenylalanine and the amino acid at position 422 was mutated from leucine to methionine. The amino acid at position 417 was mutated from lysine to phenylalanine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to isoleucine. The amino acid at position 417 was mutated from lysine to phenylalanine, the amino acid at position 419 was mutated from leucine to asparagine, and the amino acid at position 422 was mutated from leucine to methionine. The amino acid at position 223 was mutated from serine to lysine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 223 was mutated from serine to arginine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 279 was mutated from arginine to glutamine, the amino acid at position 417 was mutated from lysine to phenylalanine, and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 417 was mutated from lysine to serine and the amino acid at position 419 was mutated from leucine to serine. The amino acid at position 417 was mutated from lysine to serine and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 417 was mutated from lysine to methionine and the amino acid at position 419 was mutated from leucine to cysteine. The amino acid at position 417 was mutated from lysine to methionine and the amino acid at position 419 was mutated from leucine to asparagine. The amino acid at position 417 was mutated from lysine to leucine, and the amino acid at position 419 was mutated from leucine to cysteine. The 417th amino acid is mutated from lysine to leucine, and the 419th amino acid is mutated from leucine to asparagine; or, The 417th amino acid was mutated from lysine to arginine, and the 419th amino acid was mutated from leucine to asparagine.

4. The PPO2 protein or its bioactive fragment according to any one of claims 1-3, comprising any one of the amino acid sequences shown in SEQ ID NO: 2-39.

5. An isolated polynucleotide comprising a nucleic acid sequence selected from the following: (1) A nucleic acid sequence or a partial sequence or a complementary sequence thereof encoding the PPO2 protein or a biologically active fragment thereof as described in any one of claims 1-4; (2) A nucleic acid sequence that hybridizes to the sequence shown in (1) under stringent conditions; and / or (3) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) due to the degeneracy of the genetic code, or its complementary sequence; Preferably, the polynucleotide is a DNA molecule.

6. A plant genome comprising the polynucleotide as described in claim 5.

7. A vector construct comprising the polynucleotide of claim 5 and a homologous or non-homologous promoter operatively linked thereto.

8. A host cell comprising the polynucleotide of claim 5 or the vector construct of claim 7, preferably, the host cell being a plant cell.

9. A method for producing plant cells capable of generating or improving tolerance to protoporphyrinogen oxidase inhibitor herbicides, comprising generating the polynucleotide as described in claim 5 or the vector construct as described in claim 7 in plant cells by gene editing, or introducing the polynucleotide as described in claim 5 or the vector construct as described in claim 7 into plant cells by transgenic means.

10. A method for producing or enhancing the tolerance of plants to protoporphyrinogen oxidase inhibitor herbicides, and the plants produced by the method, comprising regenerating plants from plant cells of claim 8 or plant cells produced by the method of claim 9.

11. 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 protein or a bioactive fragment thereof as described in any one of claims 1-4.

12. A method for producing or enhancing the tolerance of plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase inhibitor herbicides, comprising expressing the PPO2 protein or a bioactive fragment thereof as described in any one of claims 1-4 in the plant cells, plant tissues, plant parts or plants; Alternatively, this includes hybridizing a plant expressing the PPO2 protein or a bioactive fragment thereof as described in any one of claims 1-4 with another plant, and screening for plants or parts thereof that can produce or improve tolerance to protoporphyrinogen oxidase inhibitor 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 express therein the PPO2 protein or a bioactive fragment thereof as described in any one of claims 1-4.

13. The use of the PPO2 protein or its bioactive fragment as described in any one of claims 1-4 or the polynucleotide as described in claim 5 for producing or enhancing the tolerance of host cells, plant cells, plant tissues, plant parts or plants to protoporphyrinogen oxidase inhibitor herbicides, preferably, wherein the host cell is a bacterial cell or a fungal cell.

14. A method for controlling weeds in a plant cultivation site, wherein the plant comprises the plant of claim 10 or the plant prepared by the method of any one of claims 9-12, the method comprising applying a herbicidally effective amount of a protoporphyrinogen oxidase inhibitor herbicide to the cultivation site.

15. The method according to claim 9, 10, 11, 12 or 14, or the use according to claim 13, 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, triazolinones, oxazolidinones and others; preferably, (1) Pyrimidine diones include: Flupyrifenacil, pyrimethanil, sulfadiazine, flupyrifenacil, Epyrifenacil, 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, ; (2) Diphenyl ethers include: flusulfanilamide, ethoxyflufenican, bensulfuron-methyl, quizalofop-P-ethyl, methoxyflufenican, ethoxyflufenican, trifluralin or sodium salt, methoxyflufenican, ethoxyfen, chlorofluorofen, fluoronitrofen, furyloxyfen, nitrofluorfen, halosafen. (3) Phenylated pyrazoles include: imidacloprid, isopyrazosulfuron, and ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxo]propionate; (4) N-phenylimides include: propyzamide, indole-3-methylpropylate, flumipropyn, flumethrin, chlorophthalim, and N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide. (5) Thiadiazoles include: methyl methazine, methazine, and thiamethoxam; (6) Oxadiazoles include: propyzil and oxadiazine; (7) Triazoline ketones include: oxadiazon, oxadiazon / oxadiazon, mesotrione, oxadiazon, and oxadiazon; (8) Oxazolidinones include: cyclopentoxazolone; (9) Others include: bispyribac-methyl, flupyridaben, cyclopyranil, flupyribac-methyl, trifluralin, phenopylate, 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-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazine-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, 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, 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(propyl-2-) -alkynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione, 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-alkynyl-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.

16. The method or use according to claim 15, wherein the herbicide is applied in an amount that does not harm the plant containing the recombinant DNA molecule, preferably, the herbicide is applied to the area at a ratio of about 0.02 g ai / ha to about 2000 g ai / ha, about 1 g ai / ha to about 1200 g ai / ha, about 15 g ai / ha to about 800 g ai / ha, about 30 g ai / ha to about 500 g ai / ha, or about 50 g ai / ha to about 200 g ai / ha.

17. The method according to claim 9, 10, 11, 12 or 14, or the use according to claim 13, wherein the protoporphyrinogen oxidase inhibitor herbicide is applied in combination with one or more other herbicides.

18. The method according to claim 9, 10, 11, 12 or 14, or the use according to claim 13, wherein the herbicide is specifically formulated as a dispersible oil suspension, an aqueous suspension, a suspension emulsion, a wettable powder, an emulsifiable concentrate, a water-dispersible granule, an emulsion, or a microemulsion.

19. The method according to claim 9, 10, 11, 12 or 14, or the use according to claim 13, wherein the herbicide is applied before emergence, or after emergence, and the application of the herbicide comprises contacting the plant with the herbicide, or the application of the herbicide comprises application of the herbicide to the top of the plant or by drip irrigation.

20. The method or use according to claim 19, wherein the application of the herbicide results in an increase in the growth or yield of the plant relative to plants of the same genotype cultivated in a growth area where the herbicide has not been applied.

21. The plant genome according to claim 6, the host cell according to claim 8, the plant according to claim 10, the method according to any one of claims 9-12, or the use according to claim 13, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

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