Method for controlling or preventing weed growth within plant growth area resistant to ppo inhibitor herbicide

Abstract

The present invention relates to the fields of agriculture, plant biotechnology, and molecular biology. Specifically, the present invention relates to a method for controlling or preventing weed growth within a plant growth area resistant to a protoporphyrinogen oxidase inhibitor herbicide.

Description

Methods for controlling or preventing weed growth in areas where plants are resistant to PPO inhibitor herbicides Technical Field

[0001] This invention relates to the fields of agriculture, plant biotechnology, and molecular biology. Specifically, this invention relates to a method for controlling or preventing weed growth in plant growing areas resistant to protoporphyrinogen oxidase (PPO) inhibitors. Background Technology

[0002] In production processes, chemical herbicides are commonly used to control the growth and spread of unwanted weeds or other plants in specific environments. These chemicals are active at one or more target sites within the plant, disrupting its normal functions. Herbicides vary in their mechanisms of action, their effects on weeds and crops, and their application methods. While herbicides are highly effective in controlling the growth of unwanted vegetation, their use can cause collateral damage to desired plants (such as crops) located in or near the same environment. To minimize crop damage, extensive research has been conducted on developing herbicide-tolerant plants, particularly through transgenic traits to induce herbicide tolerance in crops, including glyphosate tolerance, glufosinate tolerance, and dicamba tolerance.

[0003] With the increasing number of weed species resistant to commonly used herbicides (especially glyphosate), growers have turned to herbicides with different modes of action, thus creating a need for new herbicide tolerance traits in field production. Herbicides of particular interest include those that inhibit protoporphyrinogen oxidase (PPO, EC1.3.3.4), known as PPO inhibitor herbicides. PPO inhibitor herbicides provide control over a range of herbicide-resistant weeds, making herbicide tolerance traits particularly useful in planting systems combined with one or more other herbicide tolerance traits.

[0004] Invention Summary

[0005] To solve the above problems, the technical solution provided by the present invention is as follows:

[0006] This invention provides a method for controlling or preventing the growth of weeds in a plant growth area, comprising the following steps:

[0007] (a) Providing a plant or seed containing a recombinant DNA molecule in a plant growth area, the DNA molecule containing a nucleotide sequence encoding a PPO protein, wherein the plant is tolerant to at least one PPO inhibitor herbicide.

[0008] The PPO protein has at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1;

[0009] (b) Apply an effective amount of the compound to the area to control or prevent weed growth in the area.

[0010] In one specific embodiment, the PPO protein has protoporphyrinogen oxidase activity that is insensitive to herbicides.

[0011] In another specific embodiment, the amino acid sequence of the PPO protein is shown in SEQ ID NO:2.

[0012] In one embodiment, the nucleotide sequence encoding the PPO protein has at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0013] In another specific embodiment, the nucleotide sequence encoding the PPO protein is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0014] In one embodiment, the recombinant DNA molecule further comprises a heterologous promoter operatively linked to a nucleotide sequence encoding the PPO protein.

[0015] In one specific embodiment, the PPO inhibitor herbicide is selected from one or more of the following types of compounds: pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, etc.

[0016] In one specific embodiment, the compound is applied in an amount that does not harm the plant containing the recombinant DNA molecule. Preferably, the compound is applied to the area at a rate 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.

[0017] In one embodiment, the application of an effective amount of at least one other type of herbicide compound to the area is also included.

[0018] In one specific embodiment, the compound 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.

[0019] In one specific embodiment, the application of the compound is performed before emergence, or after emergence, and the application of the compound includes contacting the plant with the compound, or the application of the compound includes application of the compound to the top of the plant or application via drip irrigation.

[0020] In one specific embodiment, the application of the compound 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 compound was not applied.

[0021] In one specific embodiment, the plant is a monocotyledonous or dicotyledonous plant.

[0022] In another specific embodiment, the plant comprises a transgenic event having a single copy of the recombinant DNA.

[0023] In another specific embodiment, the event is selected from genetically modified maize events. Or the genetically modified soybean incident It is preserved in the form of seeds at the China Center for Type Culture Collection, with accession numbers CCTCC NO: P202403 and CCTCC NO: P202427, respectively.

[0024] Invention Details

[0025] Some of the terms used in this specification are defined as follows.

[0026] In this invention, "herbicide" refers to an active ingredient capable of killing, controlling, or adversely altering plant growth. "Herbicide tolerance" or "herbicide resistance" in this invention refers to the continued growth of a plant even after the use of a herbicide that kills common or wild plants, inhibits plant growth, or weakens or stops the plant's growth compared to wild plants. The aforementioned herbicides include protoporphyrinogen oxidase (PPO) inhibitors. These PPO inhibitors can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with different chemical structures.

[0027] 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.

[0028] PPO inhibitor 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.

[0029] In one exemplary embodiment, pyrimidinid herbicides include, but are not limited to, flufenacet (CAS NO: 134605-64-4), fensulfuron-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-pyrimidin-2,4-dione (CAS NO: 1220411-29-9), Epyrifenacil (CAS NO: 353292-31-6), and 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 134605-64-4), pyrimidinidyl-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1220411-29-9). 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), Uracil-pyridine disclosed in WO2017 / 202768 and uracil derivatives disclosed in WO2018 / 019842.

[0030] 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).

[0031] 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).

[0032] 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).

[0033] 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).

[0034] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0035] 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).

[0036] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0037] Other herbicides include, but are not limited to, bispyribac-methyl (CAS NO: 158353-15-2), flupyrazosulfuron-methyl (CAS NO: 188489-07-8), cyclopyranil (CAS NO: 1651191-47-7), flupyrazosulfuron-methyl (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: 452098-92-9). 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 Benzoxazinone derivatives disclosed in WO2016 / 120116 and EP09163242.2.

[0038] 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.

[0039] Suitable mixture components, for example, are herbicides selected from categories b1) to b15):

[0040] b1) Inhibitors of lipid biosynthesis;

[0041] b2) Acetolactate synthase inhibitors (ALS inhibitors);

[0042] b3) Photosynthesis inhibitors;

[0043] b4) Protoporphyrinogen-IX oxidase inhibitor

[0044] b5) Bleaching herbicides;

[0045] b6) Enolpyruvylshikimate 3-phosphate synthase inhibitor (EPSP inhibitor);

[0046] b7) Glutamine synthase inhibitor;

[0047] b8) 7,8-Dihydropteranoic acid synthase inhibitor (DHP inhibitor);

[0048] b9) Mitosis inhibitors;

[0049] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0050] b11) Cellulose biosynthesis inhibitor;

[0051] b12) Decoupler herbicides;

[0052] b13) auxinic herbicides;

[0053] b14) Auxin transport inhibitors; and

[0054] b15) is selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, and etobenzyl sulfadiazine. zanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic Hydrazine, 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 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and other herbicides and their salts and esters;

[0055] This includes their agriculturally acceptable salts or derivatives.

[0056] 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.

[0057] In addition, safeners, PPO inhibitory herbicides and / or other herbicide compounds may be applied simultaneously or sequentially.

[0058] PPO inhibitory herbicides and herbicide compounds and safeners in groups b1)-b15) are known herbicides and safeners, see, for example, WO2013 / 189984; The Compendium of Pesticide Common Names (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000, Vol. 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R.R. Schmidt, Herbizide, Georg Thieme Verlag, Stuttgart, 1995; W.H. Ahrens, Herbicide Handbook, 7th edition, Weed Science Society of America, 1994; and K.K. Hatzios, Herbicide Handbook, 7th edition supplement, Weed Science Society of America, 1998.

[0059] The term "weed control" will be understood as killing weeds and / or delaying or inhibiting their normal growth. In the broadest sense, weeds are understood as all plants known to grow in unwanted locations, such as (crop) plant cultivation sites. The weeds included in this invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds from the following genera: *Sinapis*, *Lepidium*, *Galium*, *Stellaria*, *Matricaria*, *Anthemis*, *Galinsoga*, *Chenopodium*, *Urtica*, *Senecio*, *Amaranthus*, *Portulaca*, *Xanthium*, *Convolvulus*, *Ipomoea*, *Polygonum*, *Sesbania*, *Aragula*. The genera *mbrosia*, *Cirsium*, *Carduus*, *Sonchus*, *Solanum*, *Rorippa*, *Rotala*, *Lindernia*, *Lamium*, *Veronica*, *Abutilon*, *Emex*, *Datura*, *Viola*, *Galeopsis*, *Papaver*, *Centaurea*, *Trifolium*, *Ranunculus*, and *Taraxacum*.Monocotyledonous weeds include, but are not limited to, the following genera: *Echinochloa*, *Setaria*, *Panicum*, *Digitaria*, *Phleum*, *Poa*, *Festuca*, *Eleusine*, *Brachiaria*, *Lolium*, *Bromus*, *Avena*, *Cyperus*, *Sorghum*, and *Agropyron*. The genera *Cynodon*, *Monochoria*, *Fimbristyslis*, *Sagittaria*, *Eleocharis*, *Scirpus*, *Paspalum*, *Ischaemum*, *Sphenoclea*, *Dactyloctenium*, *Agrostis*, *Alopecurus*, and *Apera*.

[0060] The term "plant" is used in its broadest sense because it refers to organic matter and is intended to encompass eukaryotes belonging to the plant kingdom, including but not limited to vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones, suckers, and plant parts used for asexual reproduction (e.g., cuttings, tubes, seedlings, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses the whole plant, the ancestor and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, pedicels, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of a plant, and tissues and organs therein that each contains the target gene / nucleic acid. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing contains the target gene / nucleic acid.

[0061] Plants particularly useful in the method of this invention include all plants belonging to the superfamily of the Kingdom Viridiplantae, especially monocots and dicots, including legumes used for fodder or feed, ornamental plants, food crops, trees or shrubs, wherein said plants are selected from a list containing the following species: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., and Artocarpus spp. spp.), Asparagus officinalis, Avenas spp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Star fruit (Averrhoacarambola), Bambusa sp., Benincasa hispida, Brazil chestnut (Bertholletia excelsea), Beetroot (Beta vulgaris), Brassica spp. (e.g., Brassicanapus, Brassica rapa ssp.), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, papaya (Caricapapaya), large-fruited false tiger thorn (Carissa macrocarpa), species of the genus Carya (Carya spp.), red flower (Carthamus tinctorius), species of the genus Castanea (Castanea spp.).), American kapok (Ceiba pentandra), chicory (Cichorium endivia), Cinnamomum species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos spp.), coffee species (Coffea spp.), taro (Colocasia esculenta), African sycamore species (Cola spp.), jute (Corchorus sp.), coriander (Coriandrum sativum), hazel species (Corylus spp.), hawthorn species (Crataegus spp.), saffron (Crocus sativus), squash species (Cucurbita spp.), cantaloupe species (Cucumis spp.), artichoke species (Cynara spp.), carrot (Daucus carota), grasshopper species (Desmodium spp.), longan (Dimocarpus) species of the genera *Dioscorea*, *Diospyros*, *Echinochloa*, *Elaeis* (e.g., *Elaeis guineensis*, *Elaeis oleifera*), *Eleusine coracana*, *Eragrostis tef*, *Erianthus* sp., *Eriobotrya japonica*, *Eucalyptus* sp., *Eugenia uniflora*, *Fagopyrum* sp., *Fagus* sp., *Festuca arundinacea*, *Ficus carica*, *Fortunella* sp., *Fragaria* sp., and *Ginkgo*. genus *Bibona*, genus *Glycine* (e.g., *Glycine max*, *Soja hispida*, or *Soja max*), upland cotton (*Gossypiumhirstum*), and species of the genus *Helianthus* (*Helianthus* spp.).(e.g., sunflower (Helianthus annuus)), daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), barley (Hordeum spp.) (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut species (Juglans spp.), lettuce (Lactuca sativa), pea species (Lathyrus spp.), lentil (Lens culinari), flax (Linumusitatissimum), litchi (Litchichinensis), lotus species (Lotus spp.), loofah (Luffaacutangula), lupinus species (Lupinus spp.), Luzula sylvatica, tomato species (Lycopersicon spp.) (e.g., tomato (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), species of the genera *Macrotyloma*, *Malus*, *Malpighia emarginata*, avocado (*Mammea americana*), mango (*Mangifera indica*), cassava (*Manihotspp.*), sapodilla (*Manilkara zapota*), alfalfa (*Medicago sativa*), species of the genera *Melilotus*, *Mentha*, mango (*Miscanthus sinensis*), bitter melon (*Momordica*), black mulberry (*Morus nigra*), banana (*Musa*), tobacco (*Nicotiana*), olea (*Olea*), cactus (*Opuntia*), bird's foot bean (*Ornithopus*), rice (*Oryza*) (e.g., rice (Oryza sativa), broadleaf rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), passion fruit (Passiflora edulis), parsnip (Pastinaca sativa), species of the genus Pennisetum (Pennisetum sp.), and species of the genus Persea (Persea spp.).Parsley (Petroselinum crispum), *Phalaris arundinacea*, *Phaseolus* spp., *Phleum pratense*, *Phoenix* spp., *Phragmites australis*, *Physalis* spp., *Pinus* spp., *Pistacia vera*, *Pisum* spp., *Poa* spp., *Populus* spp., *Prosopis* spp., *Prunus* spp., *Psidium* spp., *Punica granatum*, *Pyrus communis*, *Quercus* spp., *Raphanus* *Rheum sativus*, *Rheum rhabarbarum*, *Ribes* spp., *Ricinus communis*, *Rubus* spp., *Saccharum* spp., *Salix* sp., *Sambucus* spp., rye (Secale cereale), *Sesamum* spp., *Sinapis* sp., *Solanum* spp. (e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium), or tomato), *Sorghum bicolor*, *Spinacia* spp., *Syzygium* spp., *Tagetes* spp., tamarind (Tamarindus indica), *Theobroma* Species of the genera *Trifolium* (cacao), *Trifolium* spp., *Tripsacum dactyloides*, *Triticosecale rimpaui*, and *Triticum* spp.(For example, common wheat (Triticum aestivum), durum wheat (Triticum durum), cylindrical wheat (Triticum turgidum), Triticum hybernum, macha wheat (Triticum macha), common wheat (Triticum sativum), emmer wheat (Triticum monococcum), or common wheat (Triticum vulgare)), tarope (Tropaeolumminus), tarope (Tropaeolum majus), species of the genus *Vaccinium* (Vaccinium spp.), species of the genus *Vicias* (Vicias spp.), species of the genus *Vigna* (Vigna spp.), violet (Viola odorata), species of the genus *Vitis* (Vitis spp.), maize (Zea mays), *Zizania palustris*, species of the genus *Ziziphus* This includes, but is not limited to, spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower, celery, kale, flax, kale, lentils, rapeseed, okra, onions, potatoes, rice, soybeans, strawberries, sugar beets, sugarcane, sunflowers, tomatoes, squash, tea, and algae, along with others. According to a preferred embodiment of the invention, the plants are crop plants. Examples of crop plants particularly include soybeans, corn, rice, and cotton.

[0062] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0063] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0064] In this invention, "host organism" should be understood as any single-celled or multi-celled organism into which mutant protein-encoding nucleic acids can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0065] 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.

[0066] The specific amino acid positions (numbers) within the protein described in this invention are determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:1, for example, using the Smith-Waterman algorithm or the CLUSTALW2 algorithm to align two sequences. The sequence 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 data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0067] 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.

[0068] Amino acid sequence identity can be determined using conventional methods with the BLAST algorithm (Altschul et al., 1990, Mol.Biol. 215:403-10) available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) using default parameters.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the regular sequence, wild-type sequence, or reference sequence. In some embodiments, a mutation refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the nucleotide or amino acid sequence of a non-herbicide-resistant PPO protein. In some embodiments, a mutation refers to one or more mutations at amino acid positions relative to the reference PPO amino acid sequence as shown in SEQ ID NO:1 or at homologous positions in its different species homologs. In some embodiments, a mutation may include substitution, deletion, inversion, or insertion. In some embodiments, substitution, deletion, insertion, or inversion may include variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, substitution, deletion, insertion, or inversion may include variations at amino acid positions 1, 2, 3, 4, 5, 6, 7, or 8.

[0073] The terms "wildtype" and "mutation" are relative and refer to the phenotype that is most frequent in a particular population, or the system, organism, or gene that possesses that phenotype. In some cases, the wild-type allele refers to the standard allele at the locus, or the allele that is most frequent in a particular population, which can be represented by a specific amino acid or nucleic acid sequence.

[0074] 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.

[0075] 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.

[0076] 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).

[0077] 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.

[0078] 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.

[0079] 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.

[0080] The obtained PPO protein can be transferred to plants to enhance their herbicide resistance.

[0081] 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.

[0082] Using any appropriate promoter, including vectors, is a common practice in the industry for plant transgenic or gene editing. For example, commonly used promoters in plant transgenic or gene editing include, but are not limited to, the SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, alpha-linolenic acid synthase (NOS) promoter, Scrophularia mosaic virus 35S promoter, sugarcane stalk virus promoter, bamboo mottle virus promoter, light-induced ribulose-1,5-ketocarboxylase (ssRUBISCO small subunit) promoter, rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis thaliana adenine transphosphoribosylase (APRT) promoter, octopine synthase promoter, and BCB (copper-binding protein) promoter.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants.

[0087] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0088] 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 nucleotide sequence encoding a PPO variant polypeptide resistant to PPO inhibitor herbicides provided in this invention, offspring obtained through sexual and asexual reproduction containing the aforementioned nucleotide sequence encoding a PPO variant polypeptide resistant 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.

[0089] 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.

[0090] 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.

[0091] The terms "first," "second," and "third" used in this article are to describe a variety of different factors and components, and are not limited by terminology. These terms are used to distinguish one factor or component from another.

[0092] 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 terms “a,” “an,” and “the” as used in the foregoing include 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.

[0093] 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. Attached Figure Description

[0094] Figure 1. Corn V7 period Six days after field dose treatment, maize recipient control materials and transgenic maize events The growth status of the material.

[0095] Figure 2 Soybean V5 stage usage Events involving 4× field dose treatment of soybean recipient control and 8× field dose treatment of transgenic soybean The growth of the material after 6 days.

[0096] sequence list Detailed Implementation

[0097] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention, and these embodiments are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent or imply that the experiments described below are all or only the experiments performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention shown in specific aspects without departing from the spirit or scope broadly described herein. Therefore, this document is to be considered illustrative rather than restrictive in various respects.

[0098] Example 1: Obtaining the OsPPO protoporphyrinogen oxidase mutant

[0099] The amino acids of wild-type OsPPO2 (SEQ ID NO: 1) were mutated to screen for a mutant OsPPO2-k1 (amino acid sequence shown in SEQ ID NO: 2) that showed significantly improved tolerance to PPO inhibitor herbicides. DNA sequences encoding OsPPO and its variants were synthesized to optimize expression in monocotyledonous or dicotyledonous plants. The DNA sequences optimized for OsPPO2-k1 in maize and soybean were SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0100] Example 2: Evaluation of crop herbicide tolerance to OsPPO2-k1 expression

[0101] 1. Obtaining genetically modified crops

[0102] Plant expression vectors expressing OsPPO2-k1 (SEQ ID NO: 2) were constructed for soybean and maize, and introduced into soybean and maize explants using standard Agrobacterium-mediated transformation. The regenerated T0 generation plants were then analyzed to screen and identify transgenic maize events. And the genetically modified soybean incident The T1 generation was cultured and seeds were obtained. This included the transgenic maize incident. And the genetically modified soybean incident The samples are deposited as seeds at the China Center for Type Culture Collection, with accession numbers CCTCC NO: P202403 and CCTCC NO: P202427, respectively.

[0103] 2. Indoor herbicide tolerance evaluation of transgenic crops using different PPO inhibitor herbicides.

[0104] (1) Foliar spraying

[0105] The active ingredients required for the test were purchased from reagent companies or synthesized using conventional methods. All technical grade reagents were diluted with acetone as a solvent and diluted with a 0.1% Tween-80 emulsifier aqueous solution, and diluted immediately before use. Water containing the same solvent and emulsifier but without the reagent served as a blank control.

[0106] The seeds of the aforementioned transgenic plants and their wild-type recipients were cultured in greenhouse pots using crop seeds with good germination rates (corn and soybean). Two plants were retained per pot. When the corn reached the 3-leaf stage and the soybean reached the 1-2 compound leaf stage, foliar spraying (top application) was performed with a water concentration of 450 L / ha. Each treatment was replicated four times, with three pots per treatment. After 14 days of treatment, a survey was conducted using the following methods and the rating criteria described in Table 1. Representative statistical results are shown in Table 2.

[0107] The absolute number survey method was used. Surviving weeds or crop seedlings were cut along the soil surface with a blade, and their fresh weight was measured using an analytical balance. Dead weeds or crops were counted as having a fresh weight of zero.

[0108] Table 1 Data Rating Criteria

[0109] Table 2. Statistics on Crop Efficacy Grades

[0110] Note: N represents no data.

[0111] The results showed that transgenic maize expressing OsPPO2-k1 and genetically modified soybeans It exhibits significantly improved tolerance to PPO inhibitor herbicides.

[0112] In addition, the efficacy data of the PPO inhibitor herbicides against different weeds at corresponding dosages were tested. The herbicides were applied as foliar sprays (top application) at a concentration of 450 L / ha. Based on the rating criteria in Table 1, some representative data are shown in Table 3.

[0113] Table 3. Representative statistical results of the control efficacy of PPO inhibitor herbicides against various weeds.

[0114] Note: N represents no data.

[0115] (2) Drip irrigation application of pesticides

[0116] Select crop seeds (corn) with good germination rate and plant them in plastic boxes, 8 seeds per box. When the corn has 3 leaves, apply pesticides by drip irrigation.

[0117] Simulated drip irrigation application method: The drip irrigation water volume is 75,000 L / ha. The amount of pesticide and water is calculated according to the treatment area. The prepared pesticide solution is drip-irrigated from the soil surface. The crop efficacy grade or weed efficacy grade is evaluated using the absolute number survey method in (1) and the data rating standard in Table 1. Some representative data are shown in Tables 4 and 5.

[0118] Table 4. Representative statistical results of crop phytotoxicity levels caused by PPO inhibitor herbicides.

[0119] Table 5. Representative statistical results of the control efficacy of PPO inhibitor herbicides against various weeds.

[0120] Note: N represents no data.

[0121] 3. Evaluation of herbicide tolerance in field crops of genetically modified crops

[0122] To test genetically modified corn events And the genetically modified soybean incident To investigate herbicide resistance in field crops, homotypic non-GMO maize and soybean receptors were used as controls, with a water control group included. Tolerance to PPO inhibitor herbicides was tested in level, uniform experimental fields. Foliar treatment (top application) was performed at the 2-4 compound leaf stage of soybeans, the 3-5 leaf stage of maize, and the 3-5 leaf stage of weeds, using 450 L / ha of water. Test plots were approximately 10-20 square meters in size, arranged sequentially, and replicated three times. Representative data are shown in Table 6.

[0123] Table 6. Effects of transgenic crops and some weeds on... Tolerance test results

[0124] Additionally, at 22.5 ai / ha Using 45 ai / ha of propyzamide, 15 ai / ha of oxychloride, 30 ai / ha of bensulfuron-methyl, and 30 ai / ha of ethoxysulfuron as the field baseline, transgenic maize was treated with 2, 4, and 8 times the recommended doses of these herbicides. And the genetically modified soybean incident Herbicide tolerance level tests were conducted (herbicide damage level: numbers 0-5 represent resistance levels from no herbicide damage to death). Results showed that at the V2 stage with a field dose of 2×, both recipient maize control materials exhibited some herbicide damage, while transgenic maize events... No obvious phytotoxicity was observed in any of the materials; during the V5 and V7 periods, under the above five herbicides at an 8× dose, the recipient maize control materials showed varying degrees of phytotoxicity and even death, while the transgenic maize incident... No significant phytotoxicity was observed in any of the materials. Representative results are shown in Figure 1, regarding the herbicide application during the V7 stage of maize. Six days after treatment with an 8× field dose, the recipient maize control material and weeds showed significant phytotoxicity, even death; while in the transgenic maize incident... No significant phytotoxicity was observed in any of the materials. However, during the VC stage, under a 2× field dose treatment, the recipient soybean control materials all exhibited significant phytotoxicity, with a phytotoxicity grade of 4-5. (This is related to the transgenic soybean event.) No obvious phytotoxicity was observed in the materials; however, during the V3 and V5 stages, the recipient soybean control materials all showed significant phytotoxicity under treatment with the above five herbicides at 4× field doses. (This is related to the transgenic soybean event.) No significant phytotoxicity was observed in the materials. Representative results are shown in Figure 2, regarding herbicides used during the soybean V5 stage. Six days after treatment with a 4× field dose, the recipient soybean control and weeds showed significant phytotoxicity, even death; while during the same period, under treatment with an 8× field herbicide dose, the transgenic soybean event... The material is still growing normally, with no obvious pesticide damage observed. This indicates the genetically modified corn incident. And soybean incident It exhibits significantly improved resistance levels to both PPO inhibitor herbicides, demonstrating excellent safety and selectivity.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for controlling or preventing the growth of weeds in a plant growth area, comprising the following steps: (a) Providing a plant or seed containing a recombinant DNA molecule in a plant growth area, the DNA molecule containing a nucleotide sequence encoding a PPO protein, wherein the plant is tolerant to at least one PPO inhibitor herbicide. The PPO protein has at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1; (b) Apply an effective amount of the compound to the area to control or prevent weed growth in the area.

2. The method according to claim 1, wherein the PPO protein has protoporphyrinogen oxidase activity that is insensitive to herbicides; preferably, the amino acid sequence of the PPO protein is shown in SEQ ID NO:

2.

3. The method according to claim 1 or 2, wherein the nucleotide sequence encoding the PPO protein has at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence shown in SEQ ID NO:3 or SEQ ID NO:4, preferably, the nucleotide sequence encoding the PPO protein is as shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. The method according to any one of claims 1-3, wherein the recombinant DNA molecule further comprises a heterologous promoter operatively linked to a nucleotide sequence encoding the PPO protein.

5. The method according to any one of claims 1-4, wherein the PPO 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: Flupropacil, pyrimisulfuron, bispyribac-methyl, flupropazine, 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, flupropacil (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: propyne oxadiazon, oxadiazon; (7) Triazoline ketones include: oxadiazon, oxadiazon / oxadiazon, mesotrione, oxadiazon, and oxadiazon; (8) Oxazolidinones include: cyclopentoxane; (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.

6. The method according to any one of claims 1-5, wherein the compound is applied in an amount that does not harm the plant containing the recombinant DNA molecule, preferably, the compound 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.

7. The method according to any one of claims 1-6, further comprising applying an effective amount of at least one other type of herbicide compound to the area.

8. The method according to any one of claims 1-7, wherein the specific formulation of the compound is 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.

9. The method according to any one of claims 1-8, wherein the application of the compound is performed before emergence, or after emergence, and the application of the compound comprises contacting the plant with the compound, or the application of the compound comprises applying the compound to the top of the plant or by drip irrigation.

10. The method according to any one of claims 1-9, wherein the application of the compound 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 compound has not been applied.

11. The method according to claims 1-10, wherein the plant is a monocotyledonous or dicotyledonous plant; preferably, the plant contains a transgenic event having a single copy of the recombinant DNA; more preferably, the event is selected from transgenic maize events. 15-1 or the genetically modified soybean incident 16-7 are deposited as seeds at the China Center for Type Culture Collection, with accession numbers CCTCC NO: P202403 and CCTCC NO: P202427, respectively.

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