Application of HemG PPO enzyme to production or improvement of tolerance to PPO inhibitory herbicides

Abstract

The invention relates to the fields of agriculture, plant biotechnology and molecular biology. Specifically, the invention relates to an application of a HemG PPO enzyme in production or improvement of tolerance to PPO inhibitory herbicides. The amino acid sequence of the enzyme has at least 98%, at least 99% or 100% sequence identity with at least one sequence of SEQ ID NO: 1-13.

Description

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 enhancing tolerance to PPO-inhibiting herbicides. Background Art

[0002] Crop production often utilizes transgenic traits formed using biotechnological methods. Heterologous genes can be introduced into plants (also known as transgenes) to produce transgenic traits. The expression of heterologous genes in plants confers traits such as herbicide tolerance to the plants. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. As the number of weed species resistant to commonly used herbicides increases, there is a need in the art for new herbicide tolerance traits. Herbicides of particular interest include herbicides that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), called PPO herbicides. PPO herbicides provide control of a range of herbicide-resistant weeds, making traits that confer tolerance to these herbicides particularly useful in cropping systems incorporating one or more other herbicide tolerance traits. Summary of the Invention

[0003] The present invention provides the use of a HemG PPO enzyme (HemG protein, homologous to the PPO enzyme in plants) in producing or enhancing tolerance to PPO-inhibiting herbicides, and its amino acid sequence has at least 98%, at least 99%, or 100% sequence identity with at least one of the sequences of SEQ ID NO: 1-13.

[0004] In a specific embodiment, the nucleotide encoding the amino acid sequence has 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 the sequences of SEQ ID NO: 14-26.

[0005] The present invention also provides a method for conferring tolerance to PPO-inhibiting herbicides on plants, seeds, cells, or plant parts, including: heterologously expressing the HemG PPO enzyme in the plants, seeds, cells, or plant parts.

[0006] The present invention also provides a method for producing a PPO-inhibiting herbicide-tolerant plant and the transgenic plant obtained thereby, the method comprising the following steps:

[0007] a) transforming a plant cell with a recombinant DNA molecule encoding a HemG PPO enzyme; and

[0008] b) regenerating a plant from the plant cell containing the recombinant DNA molecule.

[0009] In one specific embodiment, the method further comprises the step of selecting the plant or its progeny for PPO-inhibiting herbicide tolerance.

[0010] In another specific embodiment, the method further comprises the step of crossing the regenerated plant with itself or with a second plant to produce progeny.

[0011] The present invention also provides a method for controlling or preventing weed growth in a plant growth area, comprising applying an effective amount of at least one PPO-inhibiting herbicide to a plant growth area containing the transgenic plant or seed, wherein the transgenic plant or seed is tolerant to the PPO-inhibiting herbicide.

[0012] The present invention also provides a method for identifying a nucleotide sequence encoding a protein having PPO-inhibiting herbicide tolerance protoporphyrinogen oxidase activity, comprising:

[0013] a) transforming an Escherichia coli strain lacking herbicide-tolerant PPO enzyme activity with a bacterial expression vector of a recombinant DNA molecule encoding a HemG PPO enzyme; and

[0014] b) growing the transformed Escherichia coli to identify a protein having herbicide-tolerant protoporphyrinogen oxidase activity.

[0015] The present invention also provides a method for screening for genes tolerant to PPO-inhibiting herbicides, comprising:

[0016] a) expressing a recombinant DNA molecule encoding a HemG PPO enzyme in a plant cell; and

[0017] b) identifying a plant cell that exhibits tolerance to a PPO-inhibiting herbicide.

[0018] The present invention also provides a method for producing a plant tolerant to a PPO-inhibiting herbicide and at least one other herbicide, comprising:

[0019] a) obtaining a transgenic plant by the method;

[0020] b) crossing the plant with a second plant containing tolerance to the at least one other herbicide, and

[0021] c) selecting progeny plants resulting from the crossing that contain tolerance to the PPO-inhibiting herbicide and the at least one other herbicide.

[0022] The present invention also provides a method for reducing the development of herbicide-tolerant weeds, comprising:

[0023] a) growing the transgenic plant obtained by the method in a crop growth environment; and

[0024] b) applying a PPO-inhibiting herbicide and at least one other herbicide to the crop growth environment, wherein the crop plant is tolerant to the PPO-inhibiting herbicide and the at least one other herbicide.

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

[0026] As used herein, "herbicide" refers to an active ingredient capable of killing, controlling, or adversely affecting plant growth. "Herbicide tolerance" or "herbicide resistance" in the context of the present invention means that the plant continues to grow even when a herbicide that can kill or control or adversely affect the growth of normal or wild plants is used. The above herbicides include protoporphyrinogen oxidase (PPO) inhibitor herbicides. Such PPO inhibitor herbicides can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and herbicides with other different chemical structures.

[0027] Generally, if a PPO-inhibiting herbicide and / or other herbicidal compounds as described herein that can form geometric isomers such as E / Z isomers, both the pure isomers and their mixtures may be used in the compositions according to the invention. If a PPO-inhibiting herbicide and / or other herbicidal compounds as described herein have one or more chiral centers and thus exist as enantiomers or diastereomers, both the pure enantiomers and diastereomers and their mixtures may be used in the compositions according to the invention. If a PPO-inhibiting herbicide and / or other herbicidal compounds as described herein have ionizable functional groups, they may also be used in the form of their agriculturally acceptable salts. Generally, the salts of those cations and the acid addition salts of those acids are suitable, and their cations and anions have no side effects on the activity of the active compound. Preferred cations are the ions of alkali metals, preferably lithium, sodium, and potassium ions, the ions of alkaline earth metals, preferably calcium and magnesium ions, and the ions of transition metals, preferably manganese, copper, zinc, and iron ions, further ammonium and substituted ammonium, wherein 1 to 4 hydrogen atoms are replaced by C 1 -C 4 -alkyl, hydroxy-C 1 -C4 -alkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, hydroxy-C 1 -C 4 -alkoxy-C 1 -C 4 -substituted by 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 (diethanolamine salt), bis(2-hydroxyeth-1-yl)ammonium (diolamine salt), tris(2-hydroxyethyl)ammonium (trisethanolamine salt), tris(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), furthermore phosphonium ions, sulfonium ions, preferably tris(C 1 -C 4 -alkyl)sulfonium such as trimethylsulfonium, and sulfoxide ions, preferably tris(C 1 -C 4 -alkyl)sulfoxide ions, and finally salts of polyamines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. The anions of the acid addition salts are mainly chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, hydrogen carbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate and also the anions of C 1 -C 4 -alkanoic acids, preferably formate, acetate, propionate and butyrate.

[0028] The PPO-inhibiting herbicides and / or other herbicide compounds having a carboxyl group as described herein can be used in the form of the acid, in the form of the agriculturally suitable salts mentioned above or alternatively in the form of agriculturally acceptable derivatives, for example as amides such as mono- and di-C 1 -C 6 -alkylamides or arylamides, as esters such as allyl esters, propargyl esters, C 1 -C 10 -alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters and also as thioesters such as C 1 -C 10 -alkyl thioesters. Preferred mono- and di-C 1 -C 6-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 C 1 -C 4 -alkoxy - C 1 -C 4 -alkyl esters are straight - chain or branched - chain C 1 -C 4 -alkoxyethyl esters, such as 2 - methoxyethyl, 2 - ethoxyethyl, 2 - butoxyethyl (butotyl) ester, 2 - butoxypropyl or 3 - butoxypropyl esters. Straight - chain or branched - chain C 1 -C 10 -alkyl thioesters examples are ethyl thioester.

[0029] In one exemplary embodiment, the pyrimidine - dione herbicides include, but are not limited to, flupropacil (CAS NO: 134605 - 64 - 4), benzobicyclon (CAS NO: 372137 - 35 - 4), bispyribac - sodium (CAS NO: 158755 - 95 - 4), tiafenacil (CAS NO: 1220411 - 29 - 9), 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 (Epyrifenacil, CAS NO: 353292 - 31 - 6), 1 - methyl - 6 - trifluoromethyl - 3-(2,2,7 - trifluoro - 3 - oxo - 4 - prop - 2 - yn - 1 - yl - 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), isoxazoline - containing uracils disclosed in CN105753853A (such as compound ), uracil pyridines disclosed in WO2017 / 202768 and uracils disclosed in WO2018 / 019842.

[0030] Diphenyl ether herbicides include but are not limited to fomesafen (CAS NO: 72178-02-0), oxyfluorfen (CAS NO: 42874-03-3), acifluorfen (CAS NO: 74070-46-5), lactofen (CAS NO: 77501-63-4), methoxyfen (CAS NO: 32861-85-1), nitrofen (CAS NO: 1836-77-7), fluoroglycofen-ethyl (CAS NO: 77501-90-7), acifluorfen or its sodium salt (CAS NO: 50594-66-6 or 62476-59-9), bentazone (CAS NO: 42576-02-3), flurochloridone (CAS NO: 188634-90-4), flurochloridone ethyl ester (CAS NO: 131086-42-5), fluoronitrofen (CAS NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0031] Phenylpyrazole herbicides include but are not limited to pyraflufen-ethyl (CAS NO: 129630-19-9) and isopropyl pyraflufen (CAS NO: 174514-07-9).

[0032] N-phenylimide herbicides include but are not limited to flumioxazin (CAS NO: 103361-09-7), cinidon-ethyl (CAS NO: 142891-20-1), Flumipropyn (CAS NO: 84478-52-4) and flumiclorac-pentyl (CAS NO: 87546-18-7).

[0033] Thiadiazole herbicides include but are not limited to fluthiacet-methyl (CAS NO: 117337-19-6), fluthiacet (CAS NO: 149253-65-6) and thidiazimin (CAS NO: 123249-43-4).

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

[0035] Triazolinone herbicides include, but are not limited to, carfentrazone-ethyl (CAS NO: 128621-72-7), carfentrazone-ethyl ester (CAS NO: 128639-02-1), sulfentrazone (CAS NO: 122836-35-5), picolinafen (CAS NO: 68049-83-2), and benzofenap (CAS NO: 173980-17-1).

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

[0037] Other herbicides include, but are not limited to, bipyrazonitrile (CAS NO: 158353-15-2), flufenpyr-ethyl (CAS NO: 188489-07-8), fluzoletermine (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), N-ethyl-3-(2,6-dichloro-4-(trifluoromethyl)phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-(trifluoromethyl)phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-(trifluoromethyl)phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-(trifluoromethyl)phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione (CAS NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS NO: 1300118-96-0), methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-pyrazol-3-yl]-4-fluorophenoxy]-3-methoxy-but-2-enoate (CAS NO: 948893-00-3), the pyridinylbenzenes disclosed in WO2016 / 120116, the benzoxazinone derivatives disclosed in EP09163242.2, and the compounds represented by general formula I (see Patent CN202011462769.7);

[0038] In another exemplary embodiment, Q represents

[0039] Y represents halogen, halo-C1-C6 alkyl or cyano;

[0040] Z represents halogen;

[0041] M represents CH or N;

[0042] X represents -CX 1 X 2 -(C1-C6 alkyl) n -, -(C1-C6 alkyl)-CX 1 X 2 -(C1-C6 alkyl) n - or -(CH 2 ) r -, n represents 0 or 1, r represents an integer greater than 2;

[0043] X 1 、X 2 each independently represents hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxy-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, phenyl or benzyl;

[0044] X 3 、X 4 each independently represents O or S;

[0045] W represents hydroxy, C1-C6 alkoxy, C2-C6 alkenyloxy, C2-C6 alkynyloxy, halo-C1-C6 alkoxy, halo-C2-C6 alkenyloxy, halo-C2-C6 alkynyloxy, C3-C6 cycloalkyloxy, phenoxy, mercapto, C1-C6 alkylthio, C2-C6 alkenylthio, C2-C6 alkynylthio, halo-C1-C6 alkylthio, halo-C2-C6 alkenylthio, halo-C2-C6 alkynylthio, C3-C6 cycloalkylthio, phenylthio, amino or C1-C6 alkylamino.

[0046] In another exemplary embodiment, the compound represented by the general formula I is selected from compound A: Q represents Y represents chlorine; Z represents fluorine; M represents CH; X represents -C*X 1 X 2 -(C1-C6 alkyl) n -(C* is a chiral center, R configuration), n represents 0; X 1represents hydrogen; X 2 represents methyl; X 3 、X 4 each independently represents O; W represents methoxy.

[0047] The PPO-inhibiting herbicides useful in carrying out the present invention as described above are generally preferably applied in combination with one or more other herbicides to obtain control of a variety of undesired plants. For example, the PPO-inhibiting herbicides can also be used in combination with additional herbicides to which the crop plants are naturally tolerant or to which they are resistant via expression of one or more additional transgenes as described above. When used in combination with other targeted herbicides, the compounds claimed in the present invention can be formulated together with one or more other herbicides, tank-mixed with one or more other herbicides, or applied sequentially with one or more other herbicides.

[0048] Suitable mixture components are, for example, herbicides selected from categories b1) to b15):

[0049] b1) Lipid biosynthesis inhibitors;

[0050] b2) Acetolactate synthase inhibitors (ALS inhibitors);

[0051] b3) Photosynthesis inhibitors;

[0052] b4) Protoporphyrinogen-IX oxidase inhibitors,

[0053] b5) Bleaching herbicides;

[0054] b6) Enolpyruvylshikimate 3-phosphate synthase inhibitors (EPSP inhibitors);

[0055] b7) Glutamine synthetase inhibitors;

[0056] b8) 7,8-Dihydropteroate synthase inhibitors (DHP inhibitors);

[0057] b9) Mitosis inhibitors;

[0058] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0059] b11) Cellulose biosynthesis inhibitors;

[0060] b12) Decoupler herbicides;

[0061] b13) Auxinic herbicides;

[0062] b14) Auxin transport inhibitors; and

[0063] b15) selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleichydrazide, mefluidide, metam, methiozolin (CAS NO: 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridiphane, and other herbicides of 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and its salts and esters;

[0064] including their agriculturally acceptable salts or derivatives.

[0065] In addition, when used in combination with other herbicide compounds as described above, it may be useful to apply the PPO-inhibiting herbicide in combination with a safener. A safener is a compound that prevents or reduces damage to useful plants but has no significant effect on the herbicidal action of the herbicide on unwanted plants. They can be applied before sowing (e.g., during seed treatment, on branches or seedlings) or before or after the germination of useful plants.

[0066] In addition, the safener, the PPO-inhibiting herbicide, and / or other herbicide compounds can be applied simultaneously or sequentially.

[0067] The PPO-inhibiting herbicide, the herbicide compounds of groups b1)-b15), and the safener are known herbicides and safeners. See, for example, WO2013 / 189984; The Compendium of Pesticide Common Names

[0068] (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000, Volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R. R. Schmidt, Herbizide [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 "controlling weeds" will be understood to mean killing weeds and / or retarding or inhibiting the normal growth of weeds. In the broadest sense, weeds are understood to mean all plants that are known to grow in locations where they are not wanted, such as in the location of (crop) plant cultivation. The weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the following genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, 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, weeds of the following genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. Additionally, the weeds of the present invention may include, for example, crop plants growing in an unwanted location. For example, if corn plants are not desired in a soybean plant field, volunteer corn plants present in a field mainly containing soybean plants can be regarded as a kind of weed.

[0070] The term "plant" is used in its broadest sense as it relates to organic matter and is intended to cover eukaryotes belonging to the plant kingdom, examples of which include, but are not limited to, vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, etc., as well as clones, offsets, and plant parts used for asexual reproduction (such as cuttings, tubes, seedlings, rhizomes, stolons, tufts, crowns, bulbs, corms, tubers, rhizomorphs, plants / tissues produced in tissue culture, etc.). The term "plant" also covers whole plants, ancestors and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, peduncles, 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 plants, and tissues and organs each containing the gene / nucleic acid of interest therein. The term "plant" also covers plant cells, suspension cultures, callus, embryos, meristematic zones, gametophytes, sporophytes, pollen, and microspores, again, each of the foregoing containing the gene / nucleic acid of interest.

[0071] Plants that are particularly useful in the method of the present invention include all plants belonging to the superfamily Viridiplantae, especially monocotyledonous and dicotyledonous plants, including forage or feed legumes, ornamental plants, food crops, trees or shrubs, wherein said plants are 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., Asparagus officinalis, Avena spp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsa, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp.), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp.) Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus spp.

[0072] (Eucalyptus sp.), Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (such as Glycine max, Soja hispida or Soja max), Gossypium hirstum, Helianthus spp. (such as Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (such as Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (such as Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot 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, Pastinaca sativa.

[0073] (Pastinaca sativa), Pennisetum sp., Persea spp., 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 sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (such as 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., common wheat (Triticum aestivum), durum wheat (Triticum durum), emmer wheat (Triticum turgidum), Triticum hybernum, macha wheat (Triticum macha), common wheat (Triticum sativum), einkorn wheat (Triticum monococcum) or common wheat (Triticum vulgare)), nasturtium (Tropaeolum minus), garden nasturtium (Tropaeolum majus), Vaccinium spp., Vicia spp., Vigna spp., sweet violet (Viola odorata), Vitis spp., maize (Zea mays), wild rice (Zizania palustris), Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, kale, flax, collard greens, lentils, rapeseed, 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 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 monocotyledon, such as sugar cane. Further preferably, the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

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

[0075] As used herein, "ΔhemG strain" and "hemG knockout strain" refer to an organism (such as Escherichia coli) or a somatic cell thereof that lacks HemG activity and is thus unable to grow on a growth medium without heme, or whose growth is detectably impaired in the absence of heme relative to an otherwise isogenic strain containing functional HemG. For example, a hemG knockout strain of Escherichia coli can be prepared according to knowledge in the art, such as according to the Escherichia 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 "recombinant" refers to a DNA, protein, cell, seed or organism that is non-naturally occurring as a result of genetic engineering and formed by human intervention. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that is non-naturally occurring and thus the result of human intervention, such as a DNA molecule containing at least two DNA molecules that are heterologous to each other. An example of a recombinant DNA molecule is the DNA molecule provided herein that encodes a herbicide-tolerant protoporphyrinogen oxidase and is operably linked to a heterologous promoter. A recombinant cell, seed or organism is a cell, seed or organism that contains a transgene or heterologous DNA or protein, such as a transgenic plant cell, seed or plant that contains a DNA construct or engineered protein of the present invention.

[0077] In the present invention, "control" refers to an experimental control designed for comparison purposes and is divided into a positive control and a negative control. For example, a control plant in transgenic plant analysis is a plant of the same type as the experimental plant (i.e., the plant to be tested). Herein, the negative control is a non-transgenic wild-type Col control, and the positive control is an isogenic plant control 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 cultures, plant calli, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0079] In the present invention, "plant cell" should be understood to mean any cell derived from or found in a plant that is capable of forming, for example: undifferentiated tissues such as calli, differentiated tissues such as embryos, components of a plant, a plant or a seed.

[0080] In the present invention, "host organism" is to be understood as any unicellular or multicellular organism into which a mutant protein-coding nucleic acid 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 in the present invention and refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogues of natural amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0082] The identity of amino acid sequences can be determined by conventional methods using the BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215: 403-10), which is available from the National Center for Biotechnology Information www.ncbi.nlm.nih.gov / (National Center for Biotechnology Information), using default parameters.

[0083] The term "wild type" refers to the phenotype with the highest frequency in a particular population, or a system, organism and gene having such a phenotype.

[0084] The terms "polynucleotide", "nucleic acid", "nucleic acid molecule", "DNA molecule" or "nucleic acid sequence" are used interchangeably and refer to oligonucleotides, nucleotides or polynucleotides and their fragments or portions, which can be single-stranded or double-stranded and represent sense or antisense strands. Nucleic acids include DNA, RNA or their hybrids and can have natural or synthetic origins. For example, nucleic acids can include mRNA or cDNA. Nucleic acids can 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 Manual of Patent Examining Procedure of the United States Patent and Trademark Office. In this regard, the nucleotide name "R" means purine such as guanine or adenine; "Y" means pyrimidine such as cytosine or thymine (uracil if RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0085] Those skilled in the art are well aware that due to the degeneracy of the genetic code, there are multiple different nucleic acid sequences that can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences that encode the same protein is within the capabilities of those of ordinary skill in the art, and thus the present invention encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic codons. For example, in order to achieve high expression of a heterologous gene in a target host organism such as a plant, the codons preferred by the host organism can be used to optimize the gene so that it can be better expressed.

[0086] The term "transgenic" plant refers to a plant that contains a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can 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 heterologous nucleic acid, including those initially altered transgenic organisms or cells, as well as those produced by hybridization or asexual propagation from the initial transgenic organism or cell. The term "transgenic" as used herein is not intended to include alterations to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by natural events such as, self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0087] The herbicide-resistant PPO protein is obtained by the most common natural extraction and refining methods in the industry. It can also be obtained by chemical synthesis methods to obtain synthetic proteins, or by recombinant protein methods through genetic recombination technology. When using chemical synthesis methods, the protein is obtained by the general polypeptide synthesis methods in the industry. When using genetic recombination technology, the nucleic acid encoding of the herbicide-resistant PPO protein will be inserted with the aid of an appropriate expression vector, and the above vector will be transformed into a host cell. After culturing the host cell to express the target protein, the herbicide-resistant PPO protein can be found and obtained in the host cell. After the protein is expressed in the selected host cell, general biochemical separation is used. For example, protein precipitants (salting out), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other types of treatments are used for isolation and purification. Usually, in order to obtain a highly purified separated protein, several of the above methods can be combined.

[0088] The herbicide-resistant PPO nucleic acid molecule can be isolated and prepared by standard molecular biological methods. For example: chemical synthesis or recombinant technology. One of the commercialized methods can be selected for use.

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

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

[0091] Selecting any appropriate promoter, including the vector, 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, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, figwort mosaic virus 35S promoter, sugarcane bacilliform virus promoter, commelina yellow mottle virus promoter, light-induced promoter ribulose-1,5-bisphosphate carboxylase (ssRUBISCO small subunit), rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, octopine synthase promoter, and BCB (blue copper-binding protein) promoter.

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

[0093] To express the herbicide-resistant PPO gene in chloroplasts, a transit peptide targeted to chloroplasts can be linked to the 5'-end of the PPO gene.

[0094] The vector also includes genes encoding selectable markers as reporter molecules. Examples of selectable markers include, but are not limited to, antibiotics (such as: neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide-resistant (glyphosate, glufosinate, bialaphos, etc.) genes.

[0095] Methods for vector transformation include using Agrobacterium-mediated transformation, electroporation, particle bombardment, polyethylene glycol-mediated absorption, and other methods to introduce the recombinant plasmid into plants.

[0096] Plant transformation receptors in the present invention include plant cells (including suspension-cultured cells), protoplasts, callus, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0097] The scope of transgenic plants includes not only the plants obtained in the current generation into which genes are introduced, but also their clones and progeny (T1 generation, T2 generation or subsequent generations). For example: transgenic plants containing the HemG PPO enzyme-encoding nucleotide sequence that is tolerant to PPO inhibitor herbicides provided in the present invention, and progeny 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 that show the characteristics of the primary transgenic plants after hybridization and fusion of the above-mentioned transgenic plants. The scope of the present invention also includes parts of plants, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and corms, which are from plants that have been previously genetically modified by the methods mentioned in the present invention, or their progeny, and at least consist of a part of the genetically modified cells.

[0098] In the present invention, the term "site" includes the site for cultivating the plants of the present invention, such as soil, and also includes, for example, plant seeds, plant seedlings, and grown plants. The term "herbicidally effective amount" refers to an amount of herbicide sufficient to affect the growth or development of target weeds, such as preventing or inhibiting the growth or development of target weeds, or killing the weeds. Advantageously, the herbicidally effective amount does not significantly affect the growth and / or development of the plant seeds, plant seedlings or plants of the present invention. Those skilled in the art can determine such a herbicidally effective amount through routine experiments.

[0099] The present invention can be implemented in many different forms, and the implementation methods are not limited by the methods described herein. The examples provided herein are for achieving thorough and complete effects, and those in the industry can fully understand the scope of the present invention. The same reference numerals in the present invention refer to the same elements.

[0100] The "first", "second", "third" used herein are for describing various different elements and components, and these elements and components are not limited by the terms. These terms are used to distinguish one element and component from another.

[0101] The terms used herein are for describing specific embodiments and are not intended to set limitations. Unless specifically stated otherwise in the text, the plural forms of "a," "an," and "the" used in the above content in both Chinese and English versions are also included. The terms "comprises - comprises" and / or "comprising - comprising," or "includes - includes" and / or "including - including" used herein specifically refer to the presence of the characteristics, elements, and / or components described herein, and do not exclude the presence and addition of one or more other characteristics, elements, 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 elaborated in detail through a series of embodiments, but the present invention is not limited to the disclosed embodiments. Any number of variations, substitutions, permutations, etc. within the scope of the present invention that are not elaborated herein or can be modified as needed. Description of the Drawings

[0103] Results of the resistance test of 30 candidate HemG PPO genes to compound A in the E. coli system.

[0104] Figure 2a 、 2b Growth status of transgenic seedlings after 10 days of screening on the compound A medium.

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

[0106] Figure 4 Growth status of transgenic materials 14 days after foliar treatment with carfentrazone-ethyl.

[0107] Figure 5 Growth status of transgenic materials 14 days after foliar treatment with flumioxazin.

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

[0109] Figure 7 Growth status of transgenic materials 14 days after foliar treatment with sulfentrazone.

[0110] Figure 8 Growth status of transgenic materials 14 days after foliar treatment with fomesafen.

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

[0112] Figure 10 Growth status of transgenic foxtail millet 5 days after spraying with different concentrations of compound A. Under the same treatment dose, the leftmost one in the first row is the untreated with compound A, the second one from the left is the wild-type control foxtail millet, the third one from the left is the transgenic line A1 S9M8, and the fourth and fifth ones from the left are the transgenic line A1S1J2.

[0113] Figure 11 Growth status of transgenic sorghum 5 days after spraying with different concentrations of compound A. Under the same treatment dose, the leftmost one in the first row is the untreated with compound A, the second one from the left is the wild-type control sorghum, the third one from the left is the transgenic line A1 S9M8, and the fourth and fifth ones from the left are the transgenic line A1S1J2.

[0114] Figure 12Growth status of genetically modified soybeans 7 days after spraying compound A. The left figure shows the test results of A1 S9M8 gene - transformed soybeans, and the right figure shows the test results of A1 S1J2 gene - transformed soybeans. In the same group of experiments, the first one on the left is the wild - type control soybean, and the second one on the left is the genetically modified soybean.

[0115]

[0116] Detailed implementation mode

[0117] Example 1: Using PPO - defective Escherichia coli (ΔhemG) to test the tolerance of different microbial HemG PPO genes to compound A

[0118] The PPO enzyme family is usually called the HemY family in microorganisms. However, in some microorganisms, especially Proteobacteria, they do not have HemY but have HemG PPO enzymes (Larue C T, Ream J E, 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, an Escherichia coli screening system was used to test the tolerance of these genes to compound A. The ΔhemG strain is an Escherichia coli strain lacking the hemG - type PPO gene and having kanamycin tolerance. The ΔhemG - defective Escherichia coli strain hardly grows on ordinary LB bacterial medium, but can resume normal growth when the medium is supplemented with free heme or when a functional protoporphyrinogen oxidase is expressed in the cells.

[0119] Thirty expression vectors containing different microbial HemG PPO genes (the amino acid sequences encoded by them are shown in SEQ ID NO: 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. Then the transformants were spread on LB agar medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl) containing ampicillin and kanamycin for growth. At the same time, 100 μL of the transformants were respectively spread on LB agar medium (petri dishes) containing 0 μM, 50 μM, 100 μM and 200 μM of compound A. After culturing overnight at 37 °C, the growth situation was observed, as shown in Figure 1 .

[0120] Through testing, it was found that 13 genes (nucleotide sequences such as SEQ ID NO: 14-26) could grow on LB agar medium containing ampicillin and kanamycin, indicating that these genes could complement the activity of the defective strain. At the same time, they could also grow normally on the medium containing different concentrations (50 μM, 100 μM, 200 μM) of compound A, indicating that these genes also had a certain resistance or tolerance to compound A.

[0121] Example 2: Construction of Arabidopsis overexpression vectors and screening of transgenic lines

[0122] Thirteen genes screened in Example 1 were selected, and overexpression vectors were respectively constructed and transformed into the model crop Arabidopsis thaliana for resistance verification.

[0123] 1. Construction of overexpression vectors

[0124] The gene fragments recovered after digestion with XbaI and SacI and the plasmid pHSE401V were used to construct overexpression vectors with the HB-in fusionTM seamless cloning kit of Hanheng Biotechnology (Shanghai) Co., Ltd. After transformation into competent E. coli DH5α, positive clones were obtained. After verification by sequencing and restriction endonuclease digestion, they were transformed into Agrobacterium tumefaciens GV3101 for standby.

[0125] At the same time, the wild-type rice OsPPO2-WT gene was overexpressed in Arabidopsis thaliana as a positive control, and the tolerance to PPO inhibitor herbicides was tested together subsequently.

[0126] The vector numbers and gene information are shown in Table 1:

[0127] Table 1 Information of overexpression vectors of HemG PPO genes

[0128] Gene information Name of transgenic material pHSEA1S9M8 1752 pHSEA6TGM8 1753 pHSEA1SRF1 1758 pHSEA1S1J2 1759 pHSEA4H4U0 1760 pHSEB8F801 1761 pHSEC4L776 1762 pHSEA0KEL6 1763 pHSED4ZDA5 1764 pHSEQ086Z7 1765 pHSEQ08A35 1766 pHSEQ12TB0 1767 pHSEQ6LW03 1770 pHSE-OsPPO2WT 870

[0129] 2. Transformation of Arabidopsis thaliana and screening of transgenic lines

[0130] The floral dip method was used for the transformation of Arabidopsis thaliana. After the seeds matured, they could be harvested. After harvesting, they were placed in an oven at 30 °C for about a week to be dried to obtain T1 seeds. The harvested T1 generation seeds were sown on MS screening solid medium (containing 50 mg / L Hyg) after disinfection to screen for positive plants. 10 - 15 independent transgenic lines were selected for each vector for planting. The screened positive seedlings were transplanted into flower pots filled with nutrient soil and placed in an artificial climate chamber for cultivation. T2 generation seeds were harvested after 2 months.

[0131] Example 3. Tolerance test of overexpressing the complementation gene to compound A in Arabidopsis thaliana

[0132] 1. Medium resistance test

[0133] The obtained Arabidopsis thaliana seeds overexpressing different genes were subjected to resistance tests on MS medium containing different concentrations of the PPO inhibitor herbicide compound A.

[0134] After testing, the critical lethal concentration of wild - type Arabidopsis thaliana seedlings was about 10 nM. MS media with 4 concentration gradients of 0 nM, 20 nM, 200 nM, and 1 μM of the original drug of compound A were prepared. The T2 generation seeds of 13 overexpressing transgenic materials of the HemG PPO gene in Example 2 were disinfected and then spread on the corresponding compound - concentration MS media respectively. Using wild - type Col as a control, they were vernalized in a 4 °C refrigerator for 2 days, and then placed in a light incubator for vertical cultivation. After 10 days, the growth of the seedlings was observed ( Figure 2a 、 2b ).

[0135] For each vector of the tested lines, 4 plants were selected for replication, and Col (wild - type) was used as a negative control. Through testing, it was found that wild - type Col seedlings had completely died on MS medium containing 20 nM compound A, while all transgenic lines grew normally on MS medium containing 1 μM compound A. Compared with wild - type Col, the transgenic materials overexpressing 13 HemG PPO genes and OsPPO2WT(870) of microorganisms had a significantly improved resistance to compound A, indicating that these HemG PPO genes have a certain resistance or tolerance to compound A.

[0136] 2. Shoot - leaf treatment test

[0137] After 20 days of treatment with 0.25 g a.i. / mu of Compound A, most of the wild-type Arabidopsis plants had died, and individual plants showed signs of regreening. Therefore, it was speculated that the critical lethal concentration of foliar treatment of wild-type seedlings with Compound A was approximately 0.3 g a.i. / mu.

[0138] Prepare OD preparations of Compound A with effective concentrations of 0 g a.i / mu, 0.5 g a.i / mu, 12 g a.i / mu, 24 g a.i / mu, 48 g a.i / mu, 72 g a.i / mu, 96 g a.i / mu, 144 g a.i / mu, 168 g a.i / mu, 192 g a.i / mu, 216 g a.i / mu, 240 g a.i / mu, and 288 g a.i / mu, a total of thirteen concentration gradients. Foliar spray tests were carried out on seedlings of wild-type Col, 13 HemG PPO genes, and OsPPO2WT(870) overexpression materials that had grown for 15 days after sowing. The observation results on the 12th day after treatment are shown in Figure 3 .

[0139] Through foliar treatment tests, it was found that after 12 days of foliar treatment, wild-type seedlings had completely died under the treatment of 0.5 g a.i / mu of Compound A, and the positive control OsPPO2 WT(870) had also completely died under the treatment of low-concentration Compound A, while the above 13 transgenic materials were still able to survive under the treatment of 288 g a.i / mu of Compound A. Compared with 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 had certain resistance to Compound A. Example 3. Tolerance test of overexpressing complementary genes in Arabidopsis to other PPO inhibitor herbicides

[0140] To further prove that these HemG PPO genes are resistant to PPO inhibitor herbicides, foliar treatment tests of PPO inhibitor herbicides of different types were carried out on Arabidopsis seedlings overexpressing different genes obtained.

[0141] Foliar treatment tests of the PPO herbicides carfentrazone-ethyl, flumioxazin, oxyfluorfen, sulfentrazone, and fomesafen were carried out on the T2 or T3 generation seedlings of overexpressing transgenic materials 1752, 1753, 1758-1767, 1770 and the plant-source overexpressing material 870 (OsPPO2 WT) to determine whether they had broad-spectrum resistance to PPO herbicides.

[0142] The test concentrations of the compounds were as follows: carfentrazone-ethyl: 0, 4 (critical), 20, 40, 80, 160, 320 g a.i. / mu; flumioxazin: 0, 20 (critical), 100, 200, 400, 800, 1600 g a.i. / mu; oxyfluorfen: 0, 50 (critical), 250, 500, 1000, 2000, 4000 g a.i. / mu; sulfentrazone: 0, 0.4 (critical), 2, 4, 8, 16, 32 g a.i. / mu; and fomesafen: 0, 1 (critical), 5, 10, 20, 40, 80 g a.i. / mu. The test results were observed 14 days after the test, see Figures 4 - 8 .

[0143] The results showed that in the foliar treatment test of carfentrazone-ethyl, the critical lethal concentration was 4 g a.i. / mu. The HemG PPO transgenic Arabidopsis thaliana materials had a certain degree of increased resistance compared with the wild type Col and the positive control OsPPO2 WT(870), and could still survive under the highest treatment concentration of 320 g a.i. / mu.

[0144] In the foliar treatment test of flumioxazin, the critical lethal concentration was 20 g a.i. / mu. The HemG PPO transgenic Arabidopsis thaliana materials had strong resistance compared with the wild type Col and the positive control OsPPO2 WT(870), and most of them could still survive well under the highest treatment concentration of 1600 g a.i. / mu.

[0145] In the foliar treatment test of oxyfluorfen, the critical lethal concentration was 50 g a.i. / mu. The HemG PPO transgenic Arabidopsis thaliana materials had strong resistance compared with the wild type Col and the positive control OsPPO2 WT(870), and most of them could still survive well under the highest treatment concentration of 4000 g a.i. / mu.

[0146] In the foliar treatment test of sulfentrazone, the critical lethal concentration was 0.4 g a.i. / mu. The HemG PPO transgenic materials still had resistance and could still survive under the treatment of the highest concentration of 32 g a.i. / mu compared with the wild type Col and the positive control OsPPO2 WT(870).

[0147] In the foliar treatment test of fomesafen, the critical lethal concentration was 1 g a.i. / mu. The HemG PPO transgenic Arabidopsis thaliana materials still had resistance and could still survive under the treatment of the highest concentration of 80 g a.i. / mu compared with the wild type Col and the positive control OsPPO2 WT(870).

[0148] In summary, the overexpressed microbial HemG PPO transgenic materials have strong resistance to carfentrazone-ethyl, flumioxazin, oxyfluorfen, sulfentrazone, and fomesafen, which is sufficient to demonstrate their broad-spectrum resistance to PPO herbicides.

[0149] Example 4. Tolerance Test of Transgenic Maize to Compound A

[0150] The gene sequence screened in Example 1 was synthesized and ligated to an overexpression vector, which was then transferred into competent Escherichia coli cells. Positive clones were selected and subjected to sequencing detection. The positive clones with correct sequencing were used for plasmid extraction. The extracted plasmid was transferred into competent Agrobacterium tumefaciens EHA105 cells for standby. Through the Agrobacterium-mediated immature embryo transformation method, the vector was transferred into maize receptors to obtain transgenic maize plants. The representative vector numbers and gene information are shown in Table 2.

[0151] Table 2 Representative Vector Numbers and Gene Information

[0152] Vector number Vector name pQY7193 pOsAct1-CTP-MDH-A1S9M8+PAT pQY7194 pOsAct1-CTP-MDH-A6TGM8+PAT pQY7195 pOsAct1-CTP-MDH-A1SRF1+PAT pQY7196 pOsAct1-CTP-MDH-A1S1J2+PAT pQY7197 pOsAct1-CTP-MDH-A4H4U0+PAT pQY7198 pOsAct1-CTP-MDH-B8F801+PAT pQY7199 pOsAct1-CTP-MDH-C4L776+PAT pQY7200 pOsAct1-CTP-MDH-A0KEL6+PAT pQY7201 pOsAct1-CTP-MDH-D4ZDA5+PAT pQY7202 pOsAct1-CTP-MDH-Q086Z7+PAT pQY7203 pOsAct1-CTP-MDH-Q08A35+PAT pQY7204 pOsAct1-CTP-MDH-Q12TB0+PAT pQY7205 pOsAct1-CTP-MDH-Q6LW03+PAT

[0153] The transgenic maize seedlings obtained by tissue culture were acclimatized in the greenhouse for 10 days and then subjected to the resistance test of compound A. The critical lethal concentration of compound A for wild-type maize (control) was 1.5 g a.i / mu. Herbicidal working solutions with three concentrations of 3 g a.i / mu, 6 g a.i / mu, and 9 g a.i / mu were prepared from compound A formulations, and the wild-type maize plants and the T0 generation transgenic maize plants of the above 13 vectors were sprayed on the stems and leaves with a spray tower, respectively, and then transferred to the greenhouse for resistance observation. The growth status of transgenic maize plants and wild-type plants on the 15th day after gradient application of compound A is shown in Figure 9 .

[0154] The results showed that the wild-type plants had completely died under the treatment of 3 g a.i / mu of compound A. However, the transgenic plants containing the above 13 representative vectors could still survive under the treatment of 9 g a.i / mu of compound A and could all grow normally, and their resistance to compound A was 6 times that of the wild-type. It can be seen that the overexpression of the microbial HemG PPO gene described in this application can greatly improve the resistance of maize to compound A.

[0155] Example 5. Tolerance Test of Transgenic Foxtail Millet, Sorghum, and Soybean to Compound A

[0156] The gene sequences A1S9M8 and A1S1J2 screened in Example 1 were randomly selected, synthesized and ligated to an overexpression vector, and were respectively transformed into foxtail millet, sorghum, and soybean by Agrobacterium-mediated genetic transformation. After obtaining transgenic plants, their tolerance to compound A was tested.

[0157] The growth states of transgenic foxtail millet and sorghum on the 5th day after gradient application of Compound A are shown respectively as Figure 10 and 11 . The wild-type foxtail millet and sorghum had completely died under the treatment of 1.5 g a.i / mu of Compound A, while the transgenic plants could still survive under the treatment of 6 g a.i / mu of Compound A and both could grow normally, and the resistance to Compound A was at least 4 times that of the wild type. The growth state of transgenic soybean on the 7th day after application of Compound A is shown as Figure 12 . The wild-type soybean had completely died under the treatment of 1.5 g a.i / mu of Compound A, while the transgenic soybean could still survive and grow normally under the treatment of 1.5 g a.i / mu of Compound A. Thus, it further illustrates that the overexpression of the microbial HemG PPO gene described in this application can greatly improve the resistance of plants to Compound A.

[0158] Although the present invention is satisfied by many different forms of embodiments, as detailed in connection with the preferred embodiments of the present invention, it should be understood that the present 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 described and illustrated 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 determined by the appended claims and their equivalents. The abstract and the title should not be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities 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 each independently represent O; W represents methoxy; wherein C* is a chiral center, and the compound is of R configuration.

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