Use of hemg PPO enzyme in generating or improving tolerance to PPO-inhibiting herbicides

Abstract

The invention relates to the fields of agriculture, plant biotechnology and molecular biology. In particular, the invention relates to a use of HemG PPO enzyme in generating or improving the tolerance to PPO-inhibiting herbicides. The amino acid sequence of the enzyme has at least 98%, at least 99% or 100% sequence identity to at least one sequence of SEQ ID NO: 1-13.

Description

Application of HemG PPO enzyme in producing or improving tolerance to PPO-inhibiting herbicides Technical Field

[0001] The present invention relates to the fields of agriculture, plant biotechnology and molecular biology. Specifically, the present invention relates to the use of a HemG PPO enzyme in producing or improving tolerance to PPO-inhibiting herbicides. Background Art

[0002] Crop production typically utilizes transgenic traits formed using biotechnology methods. Heterologous genes can be introduced into plants (also referred to as transgenics) to produce transgenic traits. Heterologous genes are expressed in plants to confer traits such as herbicide tolerance on plants. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. As the types of weeds resistant to conventional herbicides increase, new herbicide tolerance traits are needed in this area. Particularly concerning herbicides include herbicides that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), known as PPO herbicides. PPO herbicides provide control of a range of herbicide-resistant weeds, thereby making the traits imparting tolerance to these herbicides particularly useful in farming systems combined with one or more other herbicide tolerance traits.

[0003] Summary of the Invention

[0004] The present invention provides a HemG PPO enzyme (HemG protein, homologous to the PPO enzyme in plants) for use in producing or improving tolerance to PPO-inhibiting herbicides, wherein the amino acid sequence thereof has at least 98%, at least 99% or 100% sequence identity with at least one of SEQ ID NOs: 1-13.

[0005] In one embodiment, the nucleotides encoding the amino acid sequence have a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 14-26.

[0006] The present invention also provides a method for conferring tolerance to a PPO-inhibiting herbicide on a plant, seed, cell or plant part, comprising heterologously expressing the HemG PPO enzyme in the plant, seed, cell or plant part.

[0007] The present invention also provides a method for producing a PPO-inhibiting herbicide-tolerant plant and the transgenic plant obtained therefrom, the method comprising the following steps:

[0008] a) transforming a plant cell with a recombinant DNA molecule encoding a HemG PPO enzyme; and

[0009] b) regenerating a plant from said plant cell comprising said recombinant DNA molecule.

[0010] In one embodiment, the method further comprises the step of selecting the plant or its progeny for tolerance to a PPO-inhibiting herbicide.

[0011] In another embodiment, the method further comprises the step of crossing the regenerated plant with itself or with a second plant to produce progeny.

[0012] The present invention also provides a method for controlling or preventing the growth of weeds in a plant-growing area, comprising applying an effective amount of at least one PPO-inhibiting herbicide to a plant-growing area comprising the transgenic plant or seed, wherein the transgenic plant or seed is tolerant to the PPO-inhibiting herbicide.

[0013] The present invention also provides a method for identifying a nucleotide sequence encoding a protein having PPO-inhibiting herbicide-tolerant protoporphyrinogen oxidase activity, comprising:

[0014] a) transforming an E. coli strain lacking herbicide-tolerant PPO enzyme activity with a bacterial expression vector encoding a recombinant DNA molecule of the HemG PPO enzyme; and

[0015] b) growing the transformed E. coli to identify a protein having herbicide-tolerant protoporphyrinogen oxidase activity.

[0016] The present invention also provides a method for screening a PPO-inhibiting herbicide tolerance gene, comprising:

[0017] a) expressing a recombinant DNA molecule encoding a HemG PPO enzyme in a plant cell; and

[0018] b) Identification of plant cells exhibiting tolerance to PPO-inhibiting herbicides.

[0019] The present invention also provides a method for producing a plant tolerant to a PPO-inhibiting herbicide and at least one other herbicide, comprising:

[0020] a) obtaining transgenic plants by the method;

[0021] b) crossing said plant with a second plant comprising tolerance to said at least one other herbicide, and

[0022] c) selecting progeny plants resulting from said cross that comprise tolerance to the PPO-inhibiting herbicide and said at least one other herbicide.

[0023] The present invention also provides a method for reducing the development of herbicide-tolerant weeds, comprising:

[0024] a) cultivating the transgenic plant obtained by the method in a crop growth environment; and

[0025] b) applying a PPO-inhibiting herbicide and at least one other herbicide to the crop growing environment, wherein the crop plants are tolerant to the PPO-inhibiting herbicide and the at least one other herbicide.

[0026] Some terms used in this specification are defined below.

[0027] "Herbicide" as used herein refers to an active ingredient that kills, controls, or adversely alters plant growth. "Herbicide tolerance" or "herbicide resistance" as used herein refers to the continued growth of a plant despite exposure to a herbicide that kills common or wild plants, resists plant growth, or weakens or halts growth compared to wild plants. These herbicides include protoporphyrinogen oxidase (PPO) inhibitors. These PPO inhibitors can be categorized as pyrimidinediones, diphenylethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with varying chemical structures.

[0028] In general, if the PPO-inhibiting herbicides and / or other herbicidal compounds described herein that can be used in the context of the present invention are capable of forming geometric isomers, such as E / Z isomers, it is possible to use both, pure isomers, and mixtures thereof, in the compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicidal compounds described herein have one or more chiral centers and thus exist as enantiomers or diastereomers, it is possible to use both, pure enantiomers and diastereomers, and mixtures thereof, in the compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicidal compounds described herein have ionizable functional groups, they can also be used in the form of their agriculturally acceptable salts. Generally, salts of those cations and acid addition salts of those acids are suitable, whose cations and anions, respectively, have no adverse effect on the activity of the active compound. Preferred cations are ions of alkali metals, preferably lithium, sodium and potassium, ions of alkaline earth metals, preferably calcium and magnesium, and ions of transition metals, preferably manganese, copper, zinc and iron, further ammonium and substituted ammonium in which 1 to 4 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-hydroxyeth-1-oxy)eth-1-ylammonium (diglycolamine salt), di(2-hydroxyeth-1-yl)ammonium (diolamine salt), tri(2-hydroxyethyl)ammonium (trinitolamine salt), tri(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), furthermore phosphonium ions, sulfonium ions, preferably tri(C1-C4-alkyl)sulfonium such as trimethylsulfonium, and sulfoxonium ions, preferably tri(C1-C4-alkyl)sulfoxonium ions, and finally salts of polyamines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. Anions of usable acid addition salts are primarily chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, hydrogen carbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate and also anions of C1-C4-alkanoic acids, preferably formate, acetate, propionate and butyrate.

[0029] The PPO-inhibiting herbicides and / or other herbicidal compounds having a carboxyl group as described herein can be used in the form of an acid, in the form of the agriculturally suitable salts mentioned above or else 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-C6-10 -alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl) methyl) esters and also as thioesters, for example as C1-C 10 -alkylthioesters. Preferred mono- and di-C1-C6-alkylamides are methyl and dimethylamides. Preferred arylamide is, 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, for example, 2-methoxyethyl, 2-ethoxyethyl, 2-butoxyethyl (butotyl) ester, 2-butoxypropyl or 3-butoxypropyl ester. Straight-chain or branched C1-C 10 An example of an alkylthioester is ethylthioester.

[0030] In an exemplary embodiment, the pyrimidinedione herbicide includes but is not limited to fluazifop-butyl (CAS NO: 134605-64-4), benzylpyrimidine (CAS NO: 372137-35-4), benzylpyrimidine (CAS NO: 158755-95-4), fluazifop-butyl (CAS NO: 1220411-29-9), [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 ... [3-[2-chloro- 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: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS NO: 212754-02-4), flupropacil (CAS NO: 120890-70-2), uracils containing isoxazoline disclosed in CN105753853A (such as compounds ), uracil pyridine disclosed in WO2017 / 202768 and uracils disclosed in WO2018 / 019842.

[0031] Diphenyl ether herbicides include, but are not limited to, fomesafen (CAS NO: 72178-02-0), oxyfluorfen (CAS NO: 42874-03-3), aclonifen (CAS NO: 74070-46-5), lactofen (CAS NO: 77501-63-4), oxyfluorfen (CAS NO: 32861-85-1), fenvalerate (CAS NO: 1836-77-7), fluazifop-butyl (CAS NO: 77501-90-7), acifluorfen or sodium salt (CAS NO: 50594-66-6 or 62476-59-9), oxyfluorfen (CAS NO: 42576-02-3), chlorfenapyr (CAS NO: 188634-90-4), chlorfenapyr ethyl (CAS NO: 131086-42-5), fluoronitrofen (CAS NO: 131086-42-5), fluoronitrofen (CAS NO: 131086-42-5), fluoronitrofen (CAS NO: 131086-42-5), fluorofluor ... NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0032] Phenylpyrazole herbicides include, but are not limited to, pyraclostrobin (CAS NO: 129630-19-9) and isopyraclostrobin (CAS NO: 174514-07-9).

[0033] N-phenylimide herbicides include, but are not limited to, flumipyralid (CAS NO: 103361-09-7), fenoxaline (CAS NO: 142891-20-1), Flumipropyn (CAS NO: 84478-52-4), and flufenoxal (CAS NO: 87546-18-7).

[0034] Thiadiazole herbicides include, but are not limited to, fluthiacet-methyl (CAS NO: 117337-19-6), fluthiacet-methyl (CAS NO: 149253-65-6), and thiamethoxam (CAS NO: 123249-43-4).

[0035] Oxadiazole herbicides include, but are not limited to, oxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0036] Triazolinone herbicides include, but are not limited to, foramsulfuron (CAS NO: 128621-72-7), foramsulfuron ethyl (CAS NO: 128639-02-1), sulfentrazone (CAS NO: 122836-35-5), pyraclostrobin (CAS NO: 68049-83-2) and acetaminophen (CAS NO: 173980-17-1).

[0037] Oxazolidinedione herbicides include, but are not limited to, oxadiazon (CAS NO: 110956-75-7).

[0038] Other herbicides include, but are not limited to, bispyribac (CAS NO: 158353-15-2), fluazifop-butyl (CAS NO: 188489-07-8), flufenacet (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 1258836-72-4), 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-thioxo-[1,3,5]triazinane-2,4-dione (CAS NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (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: 948893-00-3), phenylpyridines disclosed in WO2016 / 120116, benzoxazinone derivatives disclosed in EP09163242.2, and compounds represented by general formula I (See patent CN202011462769.7);

[0039] In another exemplary embodiment, Q represents

[0040] Y represents halogen, halogenated C1-C6 alkyl or cyano;

[0041] Z represents halogen;

[0042] M represents CH or N;

[0043] X represents -CX1X2-(C1-C6 alkyl) n -、-(C1-C6 alkyl)-CX1X2-(C1-C6 alkyl) n -or-(CH2) r -, n represents 0 or 1, and r represents an integer greater than 2;

[0044] X1 and X2 each independently represent hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxyC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, phenyl or benzyl;

[0045] X3 and X4 independently represent O or S;

[0046] W represents hydroxy, C1-C6 alkoxy, C2-C6 alkenyloxy, C2-C6 alkynyloxy, halogenated C1-C6 alkoxy, halogenated C2-C6 alkenyloxy, halogenated C2-C6 alkynyloxy, C3-C6 cycloalkyloxy, phenoxy, thiol, C1-C6 alkylthio, C2-C6 alkenylthio, C2-C6 alkynylthio, halogenated C1-C6 alkylthio, halogenated C2-C6 alkenylthio, halogenated C2-C6 alkynylthio, C3-C6 cycloalkylthio, phenylthio, amino or C1-C6 alkylamino.

[0047] In another exemplary embodiment, the compound represented by general formula I is selected from compound A: Q represents Y represents chlorine; Z represents fluorine; M represents CH; X represents -C*X1X2-(C1-C6 alkyl) n -(C* is a chiral center, R configuration), n represents O; X1 represents hydrogen; X2 represents a methyl group; X3 and X4 each independently represent O; and W represents a methoxy group.

[0048] The PPO-inhibiting herbicides described above for use in practicing the present invention are generally best used in combination with one or more other herbicides to achieve control of a variety of undesirable vegetation. For example, PPO-inhibiting herbicides can also be used in combination with additional herbicides to which crop plants are naturally tolerant or have been rendered resistant through the expression of one or more additional transgenes as described above. When used in combination with other targeted herbicides, the presently claimed compounds can be formulated with the other herbicide(s), tank-mixed with the other herbicide(s), or applied sequentially with the other herbicide(s).

[0049] Suitable mixture components are, for example, herbicides from classes b1) to b15):

[0050] b1) lipid biosynthesis inhibitors;

[0051] b2) acetolactate synthase inhibitors (ALS inhibitors);

[0052] b3) photosynthesis inhibitors;

[0053] b4) protoporphyrinogen-IX oxidase inhibitors,

[0054] b5) bleaching herbicides;

[0055] b6) Enolpyruvylshikimate 3-phosphate synthase inhibitors (EPSP inhibitors);

[0056] b7) glutamine synthetase inhibitors;

[0057] b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors);

[0058] b9) mitotic inhibitors;

[0059] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0060] b11) cellulose biosynthesis inhibitors;

[0061] b12) Decoupler herbicides;

[0062] b13) auxinic herbicides;

[0063] b14) auxin transport inhibitors; and

[0064] b15) is selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, sodium doxymethyl (DSMA), dymron, endothal and its salts, etobenzyl oxalate, zanid), flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic other herbicides including 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and their salts and esters;

[0065] These include agriculturally acceptable salts or derivatives thereof.

[0066] Furthermore, when used in combination with other herbicidal compounds as described above, it may be useful to apply the PPO-inhibiting herbicides in combination with safeners. Safeners are compounds that prevent or reduce damage to useful plants but do not significantly affect the herbicidal action of the herbicide on undesirable plants. They can be applied before sowing (e.g., during seed treatment, on branches or seedlings) or before or after germination of the useful plants.

[0067] Furthermore, the safener, PPO-inhibiting herbicide and / or other herbicidal compounds may be applied simultaneously or sequentially.

[0068] PPO-inhibiting herbicides and herbicide compounds of groups b1) to b15) and safeners are known herbicides and safeners, see, for example, WO 2013 / 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, RR Schmidt, Herbizides [Herbicides], 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.

[0069] The term "control weeds" will be understood as killing weeds and / or retarding or inhibiting the normal growth of weeds. In the broadest sense, weeds are understood to be all plants known to grow in locations where they are not desired, such as (crop) plant cultivation sites. The weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, These include sedge, lycoris, cycad, dandelion, iris, iris, sedge, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family,Monocotyledonous weeds include, but are not limited to, weeds of the following genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, and The weeds of the present invention include, for example, crop plants that are growing in unwanted locations. For example, if corn plants are undesirable in a field of soybean plants, the presence of volunteer corn plants in a field primarily containing soybean plants can be considered a weed.

[0070] The term "plant" is used in its broadest sense as it relates to organic matter and is intended to encompass eukaryotic organisms belonging to the kingdom Plantae, examples of which include, but are not limited to, vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones, offsets and plant parts used for asexual propagation (e.g., cuttings, tubes, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses complete plants, ancestors and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, stalks, 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 plants, and tissues and organs each of which comprises the gene / nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing comprises the gene / nucleic acid of interest.

[0071] Plants that are particularly useful in the methods of the invention include all plants belonging to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including forage or fodder legumes, ornamental plants, food crops, trees or shrubs, wherein the plant is selected from the list comprising 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., Artocarpus spp. spp.), Asparagus officinalis, Avena species (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa species, Benincasa hispida, Brazil nut (Bertholletia excelsea), Beta vulgaris, Brassica species (e.g., Brassica napus, Brassica rapa ssp.), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum species, Carex elata, Caricapapaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp.), American cotton (Ceiba pentandra), chicory (Cichorium endivia), 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.), cilantro (Coriandrum sativum), hazel species (Corylus spp.), hawthorn species (Crataegus spp.), saffron (Crocus sativus), pumpkin species (Cucurbita spp.), cantaloupe species (Cucumis spp.), cardoon species (Cynara spp.), carrot (Daucus carota), mountain locust species (Desmodium spp.), longan (Dimocarpus longan), Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, and Eucalyptus spp.

[0072] Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida, or Soja max), Gossypium hirstum, Helianthus spp. (e.g., Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Sweet potato (Ipomoea batatas), walnut species (Juglans spp.), lettuce (Lactuca sativa), Lathyrus spp., lentil (Lens culinari), flax (Linumusitatissimum), litchi (Litchi chinensis), Lotus spp., Luffa acutangula, lupinus species (Lupinus spp.), Luzula sylvatica, tomato species (Lycopersicon spp.) (e.g., Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), macrotyloma species, apple species (Malus spp.), Malpighia emarginata, avocado (Mammea americana), mango (Mangifera indica), cassava species (Manihot spp.), spp.), Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), black mulberry (Morus nigra), Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, and Parsnip.

[0073] Pastinaca sativa), Pennisetum sp., Persea spp., Parsley (Petroselinum crispum), Phalaris arundinacea, Phaseolus spp., Cattail (Phleum pratense), Phoenix spp., Southern bulrush (Phragmites australis), Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, granatum), Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium, or Tomato), Sorghum bicolor, Spinacia spp., Syzygium spp.), Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp.) (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum, or Triticum vulgare), Tropaeolumminus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp.), amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, kale, flax, collard greens, lentils, rapeseed oilseed, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, pumpkin, tea and algae, among others. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include, in particular, soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant is a monocotyledonous plant, such as sugar cane. Further preferably, the plant is a cereal, such as rice, corn, wheat, barley, millet, rye, sorghum or oats.

[0074] In the present invention, "herbicide-tolerant protox" means the ability of a protox enzyme to maintain at least some of its protox activity in the presence of one or more PPO herbicides. The term "protox activity" means the ability to catalyze the six-electron oxidation (electron removal) of protoporphyrinogen IX to form protoporphyrin IX, i.e., to catalyze the dehydrogenation of protox to form protoporphyrin. The enzymatic activity of protox can be measured by any means known in the art, such as by an enzyme assay in which the production of a product of protox or the consumption of a substrate of protox in the presence of one or more PPO herbicides is measured by fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS). An example of an assay for measuring protox enzyme activity is a bacterial assay, such as the assay described herein, whereby recombinant protox is expressed in bacterial cells that otherwise lack PPO activity, and the ability of the recombinant protox to complement this knockout phenotype is measured.

[0075] As used herein, "ΔhemG strain" or "hemG knockout strain" refers to an organism (such as E. coli) or an organism cell that lacks HemG activity such that it cannot grow on a growth medium without heme, or that has detectable impairment of growth in the absence of heme relative to an otherwise isogenic strain containing functional HemG. For example, a hemG knockout strain of E. coli can be prepared according to knowledge in the art, for example, according to the E. coli HemG PPO sequence (Ecogene accession number EG11485; Sasarman et al., "Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of E. coli K12," Can J Microbiol 39: 1155-1161, 1993).

[0076] In the present invention, the term "recombination" refers to a non-naturally occurring DNA, protein, cell, seed or organism as a result of genetic engineering and formed by human intervention. A "recombinant DNA molecule" is a DNA molecule comprising a DNA sequence that is non-naturally occurring and therefore a result of human intervention, such as a DNA molecule comprising at least two DNA molecules that are heterologous to each other. The example of a recombinant DNA molecule is a DNA molecule provided herein that is operably linked to a coding herbicide tolerance protoporphyrinogen oxidase of a heterologous promoter. Recombinant cells, seeds or organisms are cells, seeds or organisms that comprise transgenic or heterologous DNA or protein, for example, transgenic plant cells, seeds or plants that comprise a DNA construct of the present invention or engineered protein.

[0077] As used herein, "control" refers to an experimental control designed for comparative purposes, and is categorized as either a positive control or a blank control. For example, a control plant in a transgenic plant assay is a plant of the same type as the experimental plant (i.e., the plant being tested). Here, a blank control is a non-transgenic wild-type Col control, while a positive control is a control plant of the same type containing the OsPPO2-WT transgenic insertion, recombinant DNA molecule, or DNA construct.

[0078] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue culture, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0079] In the present invention, "plant cell" is understood to be any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0080] In the present invention, "host organism" should be understood as any unicellular or multicellular 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, etc.

[0081] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues is a chemical analog of a naturally occurring amino acid residue. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0082] Amino acid sequence identity can be determined conventionally using 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.

[0083] The term "wild type" refers to the phenotype that occurs at the highest frequency in a particular population, or to the system, organism, or gene that possesses this phenotype.

[0084] The terms "polynucleotide," "nucleic acid," "nucleic acid molecule," "DNA 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 represent sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may be of natural or synthetic origin. For example, a nucleic acid may include mRNA or cDNA. A nucleic acid may include a nucleic acid that has been amplified (e.g., using the polymerase chain reaction). The single-letter codes for nucleotides are as described in Table 1 of Section 2422 of the U.S. Patent Office Manual of Patent Examining Procedures. In this regard, the nucleotide designation "R" means a purine such as guanine or adenine; "Y" means a pyrimidine such as cytosine or thymine (uracil in the case of RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0085] It is well known to those skilled in the art 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 alternative nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art, and thus, the present invention encompasses 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 achieve better expression.

[0086] The term "transgenic" plant refers to a plant comprising a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated in the genome so that the polynucleotide is passed to successive generations. The heterologous polynucleotide can be integrated into the genome separately or integrated as a part of a recombinant expression cassette." transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, whose genotype is changed due to the presence of heterologous nucleic acids, including those initially changed transgenic organisms or cells, and those produced from initial transgenic organisms or cell hybridization or asexual reproduction. As used herein, the term "transgenic" is not intended to include changing genomes (chromosomes or chromosomes) by conventional plant breeding methods (for example, hybridization) or by naturally occurring events (such as, self-fertilization, random cross fertilization, non-recombinant virus infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation).

[0087] The herbicide-resistant PPO protein is obtained through the most common natural extraction and refining methods in the industry. Synthetic proteins can also be obtained through chemical synthesis methods, or recombinant proteins can be obtained through genetic recombination technology. When chemical synthesis is used, the protein is obtained through the peptide synthesis methods commonly used in the industry. When genetic recombination technology is used, the nucleic acid encoding the herbicide-resistant PPO protein will be inserted with the help of an appropriate expression vector, and the above-mentioned vector will be transformed into a host cell. After culturing the host cells to express the target protein, the herbicide-resistant PPO protein can be found and obtained in the host cells. After the protein is expressed in the selected host cells, it is separated by common biochemical methods. For example, protein precipitation agents (salting out), centrifugation, ultrasonic ablation, ultrafiltration, dialysis, molecular sieve chromatography analysis (gel filtration), adsorption chromatography analysis, ion exchange chromatography analysis, affinity chromatography analysis and other types of treatments are used for isolation and purification. In order to obtain a high-purity isolated protein, several of the above methods can be combined.

[0088] Herbicide-resistant PPO nucleic acid molecules can be isolated and prepared by standard molecular biology methods, such as chemical synthesis or recombinant techniques. Commercially available methods can be used.

[0089] The obtained PPO protein can be transferred to plants to enhance the plants' herbicide resistance.

[0090] The herbicide-resistant PPO gene can be introduced into plants according to common methods in the industry, and can be used for transgenic manipulation through appropriate plant transformation expression vectors.

[0091] The use of any appropriate promoter, including vectors, is a common method in the plant transgenic industry. For example, commonly used promoters in plant transgenics include, but are not limited to, the SP6 promoter, the T7 promoter, the T3 promoter, the PM promoter, the maize ubiquitin promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase (nos) promoter, the figwort mosaic virus 35S promoter, the sugarcane stalk-shaped virus promoter, the bamboo yellow mottle virus promoter, the light-inducible promoter ribulose-1,5-ketose carboxylase (ssRUBISCO small subunit), the rice cytoplasmic triosephosphate isomerase (TPI) promoter, the Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, the octopine synthase promoter, and the BCB (blue copper binding protein) promoter.

[0092] Plant transgenic vectors include a polyadenylation signal sequence that can cause 3'-end polyadenylation, including, but not limited to, the NOS 3'-terminal derivative of the nopaline synthase gene of Agrobacterium tumefaciens, the octopine synthase 3'-terminal derivative of the octopine synthase gene of Agrobacterium tumefaciens, the 3'-terminal end of the tomato or potato protease inhibitor I or II gene, the CaMVPolyA signal sequence, the 3'-terminal end of the rice α-amylase gene, and the 3'-terminal end of the phaseolin gene.

[0093] The transgenic vector is to express the herbicide-resistant PPO gene in the chloroplast, and the transit peptide targeted to the chloroplast can be connected to the 5'-end of the PPO gene.

[0094] The vector also includes a gene encoding a selectable marker as a reporter molecule. Examples of selectable markers include, but are not limited to, antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistance (glyphosate, glufosinate, phosphinothricin, etc.) genes.

[0095] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, polyethylene glycol-medium absorption, and the like to introduce recombinant plasmids into plants.

[0096] The plant transformation recipients of the present invention include plant cells (including suspension culture cells), protoplasts, callus tissues, hypocotyls, seeds, cotyledons, buds and mature plants.

[0097] The scope of transgenic plants includes not only the plant bodies obtained at the same time as the gene is introduced, but also its clones and offspring (T1 generation, T2 generation or subsequent generations). For example: a transgenic plant comprising the HemG PPO enzyme encoding nucleotide sequence that is tolerant to PPO inhibitor herbicides provided in the present invention, and offspring containing the above-mentioned HemG PPO enzyme encoding nucleotide sequence that is tolerant to PPO inhibitor herbicides obtained through sexual and asexual reproduction, and plants with genetic herbicide resistance characteristics are also included. The scope of the present invention also includes all mutants and variants of the above-mentioned transgenic plants that show the characteristics of the first-generation transgenic plants after hybridization and fusion. The scope of the present invention also includes parts of plants, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and tuberous stems. These parts come from plants that have been genetically modified in advance by the method mentioned in the present invention, or its offspring, and must consist of at least a part of the transgenic modified cells.

[0098] As used herein, the term "site" includes the site, such as soil, where the plants of the present invention are cultivated, and also includes, for example, plant seeds, seedlings, and grown plants. The term "herbicidally effective amount" refers to an amount of herbicide sufficient to affect the growth or development of the target weeds, such as to prevent or inhibit the growth or development of the target weeds, or to kill the weeds. Advantageously, the herbicidally effective amount does not significantly affect the growth and / or development of the plant seeds, seedlings, or plants of the present invention. Such a herbicidally effective amount can be determined by those skilled in the art through routine experimentation.

[0099] The present invention can be implemented in a variety of different forms, and the implementation methods are not limited to the methods described herein. The examples provided herein are provided for thorough and complete effectiveness, and those skilled in the art will fully understand the scope of the present invention. The same reference numbers refer to the same elements throughout the present invention.

[0100] The terms "first", "second" and "third" used herein are intended to describe a variety of factors and components, and these factors and components are not limited by the terms. These terms are used to distinguish one factor or component from another.

[0101] The terms used herein are intended to describe specific embodiments and are not intended to set limitations. Unless otherwise expressly stated herein, the terms "a," "an," and "the" used in the English version of the above content also include their plural forms. The terms "comprises" and / or "comprising," or "includes" and / or "including" used herein specifically refer to the presence of the features, factors, and / or components described herein, and do not exclude the presence or addition of one or more other features, factors, and components. The term "and / or" used in the above content includes all items in one or more combination lists.

[0102] The present invention has been described in detail through a series of embodiments, but the present invention is not limited to the disclosed embodiments. Any quantitative changes, substitutions, replacements, etc. within the scope of the present invention are not described herein and may be modified as needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Fig. 1 Resistance test results of 30 candidate HemG PPO genes to compound A in Escherichia coli system.

[0104] Figures 2a and 2b show the growth status of transgenic seedlings after 10 days of screening on Compound A medium.

[0105] Figure 3 Growth status of transgenic materials 12 days after stem and leaf treatment with compound A.

[0106] Figure 4 Growth status of transgenic materials after stems and leaves were treated with foramsulfuron for 14 days.

[0107] Figure 5 Growth status of transgenic materials 14 days after stem and leaf treatment with fluazifop-butyl.

[0108] Figure 6 Growth status of transgenic materials 14 days after stem and leaf treatment with oxyfluorfen.

[0109] Figure 7 Growth status of transgenic materials 14 days after stem and leaf treatment with saflufenacil.

[0110] Figure 8 Growth status of transgenic materials 14 days after stems and leaves were treated with fomesafen.

[0111] Figure 9 Growth status of transgenic corn 15 days after spraying with different concentrations of Compound A.

[0112] Figure 10 shows the growth of transgenic millet 5 days after spraying with different concentrations of Compound A. At the same treatment dose, the first left is untreated with Compound A, the second left is the wild-type control millet, the third left is A1S9M8 transgenic millet, and the fourth and fifth left are A1S1J2 transgenic millet.

[0113] Figure 11 shows the growth of transgenic sorghum 5 days after spraying with different concentrations of Compound A. At the same treatment dose, the first left image shows the sorghum plant not sprayed with Compound A, the second left image shows the wild-type control, the third left image shows the transgenic A1S9M8 plant, and the fourth and fifth left images show the transgenic A1S1J2 plant.

[0114] Figure 12 shows the growth of transgenic soybeans seven days after spraying with Compound A. The left image shows the results for transgenic soybeans with the A1S9M8 gene, and the right image shows the results for transgenic soybeans with the A1S1J2 gene. In the same experiment, the first image on the left shows the wild-type control soybean, and the second image on the left shows the transgenic soybean. DETAILED DESCRIPTION

[0115] Example 1: Testing the Tolerance of Different Microorganisms' HemG PPO Genes to Compound A Using PPO-Deficient Escherichia coli (ΔhemG)

[0116] The PPO enzyme family is commonly referred to as the HemY family in microorganisms. However, in some microorganisms, especially Proteobacteria, HemY is not present, but HemG PPO enzymes are present (Larue CT, Ream JE, Zhou X, et al. Microbial HemG-type protoporphyrinogen IX oxidase enzymes for biotechnology applications in plant herbicide tolerance traits[J]. Pest Management Science, 2020, 76(3).). In order to test the tolerance of different microbial HemG PPO genes to PPO inhibitor herbicides, the tolerance of these genes to compound A was tested using the Escherichia coli screening system. ΔhemG strains are Escherichia coli strains that lack hemG-type PPO genes and have kanamycin tolerance. ΔhemG-deficient Escherichia coli strains do not grow substantially on ordinary LB bacterial culture medium, but can resume normal growth when the culture medium is supplemented with free heme or when functional protoporphyrinogen oxidase is expressed in the cell.

[0117] Thirty expression vectors containing HemG PPO genes from different microorganisms (their encoded amino acid sequences are shown in SEQ ID NOs: 1-13, UNIPROT: C4K718, Q8FQR3, Q8NRQ4, D1AQS0, A5I205, Q8YA31, A5N0E4, B0TBR6, A5I0L4, D1C958, A9B6N3, D1CI80, C5C6Q2, B5Y9M9, Q28U43, D3ST86, and A0K106) were transformed into the prepared ΔhemG competent cells by electroporation, and the transformants were then plated on ordinary LB agar medium (5 g / L yeast powder, 10 g / L tryptone, 10 g / L At the same time, 100 μL of the transformant was spread on LB agar medium (petri dish) containing 0 μM, 50 μM, 100 μM and 200 μM compound A, and cultured at 37°C overnight. The growth was observed, as shown in Figure 1.

[0118] Through testing, it was found that 13 genes (nucleotide sequences such as SEQ ID NOs: 14-26) were able to grow on ordinary LB agar medium supplemented with ampicillin and kanamycin, indicating that these genes can complement the activity of the defective strain. At the same time, they can also grow normally on culture medium containing different concentrations (50uM, 100uM, 200uM) of Compound A, indicating that these genes also have a certain degree of resistance or tolerance to Compound A.

[0119] Example 2: Construction of Arabidopsis overexpression vector and screening of transgenic lines

[0120] The 13 genes screened in Example 1 were selected, overexpression vectors were constructed, and the genes were transformed into the model crop Arabidopsis thaliana for resistance verification.

[0121] 1. Construction of overexpression vector

[0122] The gene fragment recovered after digestion with XbaI and SacI and the plasmid pHSE401V were used to construct an overexpression vector using the HB-in fusion™ seamless cloning kit from Hanbio Biotech (Shanghai) Co., Ltd. Positive clones were obtained after transformation into competent E. coli DH5α and verified by sequencing and restriction enzyme digestion before transformation into Agrobacterium tumefaciens GV3101 for use.

[0123] At the same time, the wild-type rice OsPPO2-WT gene was overexpressed in Arabidopsis as a positive control, and tolerance to PPO inhibitor herbicides was subsequently tested.

[0124] The vector number and gene information are shown in Table 1:

[0125] Table 1 HemG PPO gene overexpression vector information

[0126] 2. Arabidopsis transformation and screening of transgenic lines

[0127] Arabidopsis thaliana is transformed using the floral dip method. Seeds are harvested upon maturity and then dried in a 30°C oven for approximately one week to obtain T1 seeds. The T1 seeds are sterilized and sown on MS screening solid medium (containing 50 mg / L Hydration) to screen for positive plants. Ten to 15 independent transformed lines are selected for each vector and planted. Positive seedlings are potted in nutrient soil and cultured in an artificial climate chamber. T2 seeds are harvested after two months.

[0128] Example 3: Testing the Tolerance of Overexpressed Complementary Genes to Compound A in Arabidopsis

[0129] 1. Culture medium resistance test

[0130] The obtained Arabidopsis seeds overexpressing different genes were tested for resistance on MS medium containing different concentrations of PPO inhibitor herbicide compound A.

[0131] Testing showed that the critical lethal concentration of wild-type Arabidopsis seedlings was approximately 10 nM. Four concentration gradients of Compound A (0 nM, 20 nM, 200 nM, and 1 μM) were prepared in MS medium. The T2 generation seeds of the 13 HemG PPO gene-overexpressing transgenic materials described in Example 2 were sterilized and plated on MS medium with the corresponding compound concentrations. Wild-type Col was used as a control. The seeds were vernalized in a 4°C refrigerator for 2 days and then cultured vertically in a light incubator for 10 days. The growth of the seedlings was observed (Figures 2a and 2b).

[0132] Four strains were selected for each vector for the test, with Col (wild type) as a negative control. The test found that wild type Col seedlings were completely dead on MS medium containing 20nM compound A, while all transgenic strains grew normally on MS medium containing 1μM compound A. Compared with wild type Col, transgenic materials that overexpressed 13 HemG PPO genes and OsPPO2WT (870) of the microorganism had significantly improved resistance to compound A, indicating that these HemG PPO genes have a certain resistance or tolerance to compound A.

[0133] 2. Stem and leaf treatment test

[0134] After 20 days of treatment with 0.25 g ai / mu of compound A, most wild-type Arabidopsis plants had died, and some plants showed signs of turning green again. Therefore, it is speculated that the critical lethal concentration of compound A for stem and leaf treatment of wild-type seedlings is about 0.3 g ai / mu.

[0135] Compound A OD preparations were prepared with effective concentrations of 0 g ai / mu, 0.5 g ai / mu, 12 g ai / mu, 24 g ai / mu, 48 g ai / mu, 72 g ai / mu, 96 g ai / mu, 144 g ai / mu, 168 g ai / mu, 192 g ai / mu, 216 g ai / mu, 240 g ai / mu, and 288 g ai / mu. A stem and leaf spray test was performed on seedlings grown 15 days after sowing of wild-type Col, 13 HemG PPO genes, and OsPPO2 WT (870) overexpressing materials. The observation results on day 12 after treatment are shown in Figure 3.

[0136] The stem and leaf treatment test found that after 12 days of stem and leaf treatment, the wild-type seedlings were completely dead under the treatment of 0.5g ai / mu compound A. The positive control OsPPO2 WT (870) also completely died under the treatment of low concentration of compound A, while the above 13 transgenic materials were still able to survive under the treatment of 288g ai / mu compound A. Compared with the wild-type seedlings and the positive control, the above 13 transgenic materials overexpressing the microbial HemG PPO gene had extremely strong resistance to compound A, further indicating that these HemG PPO genes have a certain resistance to compound A.

[0137] Example 3: Testing the Tolerance of Overexpressed Complementary Genes to Other PPO Inhibitor Herbicides in Arabidopsis

[0138] To further demonstrate that these HemG PPO genes are resistant to PPO inhibitor herbicides, Arabidopsis seedlings overexpressing different genes were subjected to stem and leaf treatment tests with different types of PPO inhibitor herbicides.

[0139] The T2 or T3 seedlings of the overexpressing transgenic materials 1752, 1753, 1758-1767, and 1770, as well as the plant-derived overexpressing material 870 (OsPPO2 WT), were treated with the PPO herbicides foramsulfuron, fluazifop-butyl, oxyfluorfen-butyl, saflufenacil, and fomesafen to determine whether they had broad-spectrum resistance to PPO herbicides.

[0140] The test concentrations of the compounds were: foramsulfuron: 0, 4 (critical), 20, 40, 80, 160, and 320 g ai / mu; fluazifop: 0, 20 (critical), 100, 200, 400, 800, and 1600 g ai / mu; oxyfluorfen: 0, 50 (critical), 250, 500, 1000, 2000, and 4000 g ai / mu; saflufenacil: 0, 0.4 (critical), 2, 4, 8, 16, and 32 g ai / mu; and fomesafen: 0, 1 (critical), 5, 10, 20, 40, and 80 g ai / mu. The test results were observed after 14 days, as shown in Figures 4-8.

[0141] The results showed that in the stem and leaf treatment test of foramsulfuron, the critical lethal concentration was 4g ai / mu. Compared with the wild type Col and the positive control OsPPO2 WT (870), the resistance of HemG PPO transgenic Arabidopsis materials was improved to a certain extent, and they could still survive under the highest treatment concentration of 320g ai / mu.

[0142] In the stem and leaf treatment test of fluazifop-butyl, the critical lethal concentration was 20 g ai / mu. Compared with the wild type Col and the positive control OsPPO2 WT (870), the HemG PPO transgenic Arabidopsis materials had stronger resistance, and most of them could still survive well under the highest treatment concentration of 1600 g ai / mu.

[0143] In the stem and leaf treatment test of oxyfluorfen, the critical lethal concentration was 50 g ai / mu. Compared with the wild type Col and the positive control OsPPO2 WT (870), the HemG PPO transgenic Arabidopsis materials had stronger resistance, and most of them could still survive well under the highest treatment concentration of 4000 g ai / mu.

[0144] In the stem and leaf treatment test of saflufenacil, the critical lethal concentration was 0.4 g ai / mu. Compared with the wild type Col and the positive control OsPPO2 WT (870), the HemG PPO transgenic material was still resistant and survived under the highest treatment concentration of 32 g ai / mu.

[0145] In the stem and leaf treatment test of fomesafen, the critical lethal concentration was 1 g ai / mu. Compared with the wild type Col and the positive control OsPPO2 WT (870), the HemG PPO transgenic Arabidopsis material was still resistant and survived under the highest treatment concentration of 80 g ai / mu.

[0146] In summary, the transgenic materials overexpressing microorganism HemG PPO have strong resistance to mesotrione, fluazifop-butyl, oxyfluorfen, saflufenacil, and fomesafen, which is sufficient to show that they have broad-spectrum resistance to PPO herbicides.

[0147] Example 4: Test of tolerance of transgenic corn to compound A

[0148] The gene sequence screened in Example 1 was synthesized and ligated to an overexpression vector, which was then transformed into competent E. coli cells. Positive clones were selected and sequenced. Positive clones with correct sequencing were subjected to plasmid extraction. The extracted plasmids were then transferred into competent Agrobacterium tumefaciens EHA105 cells for later use. Using Agrobacterium-mediated immature embryo transformation, the vectors were then transferred into maize recipient cells to obtain transgenic maize plants. Representative vector numbers and gene information are shown in Table 2.

[0149] Table 2 Representative vector numbers and gene information

[0150] Transgenic corn seedlings obtained through tissue culture were hardened in a greenhouse for 10 days before being tested for resistance to Compound A. The critical lethal concentration of Compound A for wild-type corn (control) is 1.5 g ai / mu. Compound A formulations were prepared into herbicide working solutions at three concentrations: 3 g ai / mu, 6 g ai / mu, and 9 g ai / mu. Wild-type corn plants and T0-generation transgenic corn plants bearing the 13 aforementioned vectors were sprayed on their stems and leaves using a spray tower. The plants were then transferred to a greenhouse for resistance observation. Figure 9 shows the growth status of transgenic corn plants and wild-type plants 15 days after gradient application of Compound A.

[0151] The results showed that wild-type plants completely died after treatment with 3 g ai / mu of Compound A. However, transgenic plants containing the 13 representative vectors described above survived and grew normally after treatment with 9 g ai / mu of Compound A, demonstrating six times the resistance to Compound A compared to the wild-type plants. This demonstrates that overexpression of the HemG PPO gene, a microorganism described herein, can significantly enhance corn's resistance to Compound A.

[0152] Example 5: Tolerance test of transgenic millet, sorghum and soybean to compound A

[0153] The gene sequences A1S9M8 and A1S1J2 screened in Example 1 were randomly selected, synthesized, and ligated into overexpression vectors. Millet, sorghum, and soybean were transformed using Agrobacterium-mediated genetic transformation, and the transgenic plants were obtained and tested for their tolerance to compound A.

[0154] The growth status of transgenic millet and sorghum on the 5th day after the gradient application of compound A is shown in Figures 10 and 11 respectively. The wild-type millet and sorghum have completely died under the treatment of 1.5g ai / mu of compound A, while the transgenic plants can still survive under the treatment of 6g ai / mu of compound A and can grow normally. The resistance to compound A is at least 4 times that of the wild type. The growth status of transgenic soybean on the 7th day after the application of compound A is shown in Figure 12. The wild-type soybean has completely died under the treatment of 1.5g ai / mu of compound A, while the transgenic soybean can still survive under the treatment of 1.5g ai / mu of compound A and can grow normally. This further illustrates that the overexpression of the microbial HemG PPO gene described in the present application can greatly improve the plant's resistance to compound A.

[0155] Although the present invention is satisfied by many different forms of embodiments, as described in detail in conjunction with the preferred embodiments of the present invention, it should be understood that this disclosure should be considered as an example of the principles of the present invention and is not intended to limit the present invention to the specific embodiments illustrated and described herein. Those skilled in the art can make many changes without departing from the spirit of the present invention. The scope of the present invention will be judged by the appended claims and their equivalents. The abstract and title should not be interpreted as limiting the scope of the present invention, as their purpose is to enable appropriate institutions and the general public to quickly determine the general nature of the present invention.

Claims

1. A use of a HemG PPO enzyme for producing or improving tolerance to a PPO-inhibiting herbicide, characterized in that: The amino acid sequence of the HemG PPO enzyme has at least 98%, at least 99% or 100% sequence identity with at least one of SEQ ID NOs: 1-13.

2. The use according to claim 1, wherein the nucleotide encoding the amino acid sequence has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with at least one of SEQ ID NOs: 14-26.

3. A method of conferring tolerance to a PPO-inhibiting herbicide on a plant, seed, cell or plant part, the method comprising: The HemG PPO enzyme of claim 1 or 2 is heterologously expressed in the plant, seed, cell or plant part.

4. A method for producing a PPO-inhibiting herbicide-tolerant plant and a transgenic plant obtained therefrom, the method comprising the following steps: a) transforming plant cells with a recombinant DNA molecule encoding the HemG PPO enzyme as claimed in claim 1 or 2; and b) regenerating a plant from said plant cell comprising said recombinant DNA molecule.

5. The method of claim 4, further comprising the step of selecting the plant or its progeny for tolerance to a PPO-inhibiting herbicide.

6. The method of claim 4 or 5, further comprising the step of crossing the regenerated plant with itself or with a second plant to produce progeny.

7. A method of controlling or preventing the growth of weeds in a plant growing area, the method comprising applying an effective amount of at least one PPO-inhibiting herbicide to a plant growing area comprising a transgenic plant or seed according to any one of claims 4 to 6, wherein the transgenic plant or seed is tolerant to the PPO-inhibiting herbicide.

8. A method for identifying a nucleotide sequence encoding a protein having PPO-inhibiting herbicide-tolerant protoporphyrinogen oxidase activity, the method comprising: a) transforming an E. coli strain lacking herbicide-tolerant PPO enzyme activity with a bacterial expression vector encoding a recombinant DNA molecule of the HemG PPO enzyme as claimed in claim 1 or 2; and b) growing the transformed E. coli to identify a protein having herbicide-tolerant protoporphyrinogen oxidase activity.

9. A method for screening a PPO-inhibiting herbicide tolerance gene, the method comprising: a) expressing the recombinant DNA molecule encoding the HemG PPO enzyme according to claim 1 or 2 in a plant cell; as well as b) Identification of plant cells exhibiting tolerance to PPO-inhibiting herbicides.

10. A method of producing a plant tolerant to a PPO-inhibiting herbicide and at least one other herbicide, the method comprising: a) obtaining a transgenic plant by the method according to any one of claims 4 to 6; b) crossing said plant with a second plant comprising tolerance to said at least one other herbicide, and c) selecting progeny plants resulting from said cross that comprise tolerance to the PPO-inhibiting herbicide and said at least one other herbicide.

11. A method of reducing the development of herbicide-tolerant weeds, the method comprising: a) cultivating the transgenic plant obtained by the method according to any one of claims 4 to 6 in a crop growth environment; as well as b) applying a PPO-inhibiting herbicide and at least one other herbicide to the crop growing environment, wherein the crop plants are tolerant to the PPO-inhibiting herbicide and the at least one other herbicide.

12. The use according to claim 1 or 2, or the method according to any one of claims 3 to 11, wherein the PPO-inhibiting herbicide is selected from one or more of the following types of compounds: pyrimidinediones, diphenyl ethers, phenylpyrazoles, N-phenylimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones and others; preferably, (1) Pyrimidinediones include: Flupyrimidine-butyl, flupyrimidine-butyl, flupyrimidine-butyl, flupyrimidine-butyl, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate, 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, 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione, flupropacil, (2) Diphenyl ethers include: fomesafen, oxyfluorfen, benifloxybutyl, lactofen, methoxyfenoxam, chlorfenapyr, fluazifop-butyl, acifluorfen or sodium salt, chlorfenapyr, chlorfenapyr, ethyl chlorfenapyr, fluoronitrofen, furyloxyfen, nitrofluorfen, and halosafen; (3) Phenylpyrazoles include: pyraclostrobin and isopyraclostrobin; (4) N-phenylimides include fluazifop-butyl, indolizole, Flumipropyn, and flufenoxal; (5) Thiadiazoles include: methomyl, methomyl, and thiamethoxam; (6) Oxadiazoles include: oxadiazole, oxadiazole; (7) Triazolinones include: mesotrione, mesotrione ethyl, sulfentrazone, oxazolidinone, and acetaminophen; (8) Oxazolidinedione includes: cyclopentadione; (9) Others include: bispyribac, fluazifop-butyl, fluazifop-butyl, trifluoxetine, 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-(propanoic acid)]- 2-(2-alkynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinane-2,4-dione, 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester, phenylpyridines, benzoxazinone derivatives and compounds represented by the general formula I in, Q stands for Y represents halogen, halogenated C1-C6 alkyl or cyano; Z represents halogen; M represents CH or N; X represents -CX1X2-(C1-C6 alkyl) n -、-(C1-C6 alkyl)-CX1X2-(C1-C6 alkyl) n -or-(CH2) r -, n represents 0 or 1, and r represents an integer greater than 2; X1 and X2 each independently represent hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxyC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, phenyl or benzyl; X3 and X4 independently represent O or S; W represents hydroxy, C1-C6 alkoxy, C2-C6 alkenyloxy, C2-C6 alkynyloxy, halogenated C1-C6 alkoxy, halogenated C2-C6 alkenyloxy, halogenated C2-C6 alkynyloxy, C3-C6 cycloalkyloxy, phenoxy, mercapto, C1-C6 alkylthio, C2-C6 alkenylthio, C2-C6 alkynylthio, halogenated C1-C6 alkylthio, halogenated C2-C6 alkenylthio, halogenated C2-C6 alkynylthio, C3-C6 cycloalkylthio, phenylthio, amino or C1-C6 alkylamino; More preferably, Q represents Y represents chlorine; Z represents fluorine; M represents CH; X represents -C*X1X2-(C1-C6 alkyl) n -, n represents 0; X1 represents hydrogen; X2 represents methyl; X3 and X4 independently represent O; W represents methoxy; wherein C* is a chiral center, and the compound is of R configuration.

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