Gene combination for improving herbicide resistance and use thereof

Abstract

Provided are a gene combination for improving herbicide resistance and use thereof. The gene combination comprises a promoter and a gene coding region, wherein the promoter is a UBI2 gene promoter, and the gene coding region is a PPO2 gene mutant coding region. The herbicide resistance of a plant comprising the gene combination is improved.

Description

A gene combination to enhance herbicide resistance and its application Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically, to a gene combination that enhances herbicide resistance and its application. Background Technology

[0002] When weeds develop resistance to specific herbicides, those herbicides become unusable for weed control. Therefore, creating crop materials with higher herbicide resistance has always been a key focus of agricultural research, as improving crop herbicide resistance greatly benefits crop production. Obtaining and incorporating genes or combinations of genes that enhance herbicide resistance into the rice genome while maintaining beneficial traits to preserve or improve health is challenging, unpredictable, time-consuming, and expensive, but essential for meeting the world's growing food needs.

[0003] Invention Summary

[0004] To address the aforementioned problems in the prior art, this invention provides a gene combination for improving herbicide resistance and its application.

[0005] The technical solution adopted in this invention is as follows:

[0006] A gene combination comprising a promoter and a gene coding region, wherein the promoter is a UBI2 gene promoter and the gene coding region is a PPO2 gene mutant coding region.

[0007] In one specific embodiment, the UBI2 gene promoter sequence is shown in SEQ ID NO:4, 5, 6, 7, 29 or 30.

[0008] In one specific embodiment, the coding region of the PPO2 gene mutant contains an amino acid sequence as shown in SEQ ID NO:22.

[0009] In another specific embodiment, the nucleotide sequence of the coding region of the PPO2 gene mutant comprises the sequence shown in SEQ ID NO:23.

[0010] In one embodiment, the gene combination further comprises the sequence shown in SEQ ID NO:19, 25 or 26.

[0011] In another specific embodiment, its nucleotide sequence comprises the sequence shown in SEQ ID NO:24, 27, 28 or 31.

[0012] In one specific implementation, the gene combination is obtained through a gene editing system or transgenic technology.

[0013] In another specific embodiment, the gene editing system is a meganuclease, zinc finger nuclease, TALEN, or CRISPR / Cas system.

[0014] In another specific embodiment, the gene editing system is a CRISPR / Cas9 or CRISPR / Cas12 system.

[0015] The present invention also provides a recombinant genome comprising the aforementioned gene combination.

[0016] The present invention also provides a host cell comprising the aforementioned gene combination or comprising the aforementioned recombinant genome.

[0017] The present invention also provides the application of the aforementioned gene combination, the aforementioned recombinant genome, or the aforementioned host cell in improving plant resistance to PPO-inhibiting herbicides.

[0018] The present invention also provides a primer pair for detecting the aforementioned gene combination, the sequences of which are shown in SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:18.

[0019] The present invention also provides a method for cultivating plants with or enhanced tolerance to PPO-inhibiting herbicides, characterized in that the method comprises the following steps:

[0020] (1) DNA breaks were simultaneously generated at specific locations upstream of the start codon of the PPO2 gene and downstream of the promoter of the UBI2 gene in plant cells. The DNA breaks were connected to each other through intracellular repair pathways. Gene combinations of the UBI2 gene promoter and the PPO2 gene coding region were screened by designing primer pairs. Then, the L422+F442 site of the PPO2 gene coding region in the gene combination was mutated to L422M+F442M.

[0021] First, the L422+F442 site in the coding region of the PPO2 gene in plant cells is mutated to L422M+F442M. Then, DNA breaks are simultaneously generated at specific locations upstream of the start codon of the PPO2 gene and downstream of the promoter of the UBI2 gene in the plant cells. These DNA breaks are interconnected through intracellular repair pathways. Primer pairs are designed to screen for gene combinations between the UBI2 gene promoter and the coding region of the PPO2 gene mutant. Alternatively...

[0022] A recombinant expression vector containing the UBI2 gene promoter and the expression cassette of the L422M+F442M mutant coding region of the PPO2 gene was constructed, and the vector was transformed into plant callus tissue. Gene combinations of the UBI2 gene promoter and the coding region of the PPO2 gene mutant were screened by screening markers or designed primer pairs.

[0023] (2) Regenerate plants from the obtained plant cells or tissues.

[0024] The present invention also provides a plant containing the gene combination described herein obtained by the method.

[0025] The present invention also provides a method for controlling weeds in a plant cultivation site, wherein the plant comprises a plant prepared by the method, the method comprising applying a herbicidally effective amount of a PPO inhibitory herbicide to the plant cultivation site.

[0026] In one specific embodiment, the PPO inhibitory herbicide is applied in combination with one or more other herbicides.

[0027] In one specific embodiment, the PPO inhibitory herbicide is selected from one or more of the following types of compounds: pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinones, and others.

[0028] In one embodiment, the PPO inhibitory herbicide is applied in an amount that does not harm the plant.

[0029] In another specific embodiment, the PPO inhibitory herbicide compound is applied to the plant cultivation site at a ratio of about 0.02 g ai / ha to about 2000 g ai / ha, about 1 g ai / ha to about 1200 g ai / ha, about 15 g ai / ha to about 800 g ai / ha, about 30 g ai / ha to about 500 g ai / ha, or about 50 g ai / ha to about 200 g ai / ha.

[0030] In one specific embodiment, the PPO inhibitory herbicide compound is formulated as a dispersible oil suspension, an aqueous suspension, a suspension emulsion, a wettable powder, an emulsifiable concentrate, a water-dispersible granule, an emulsion, or a microemulsion.

[0031] In one specific embodiment, the application of the PPO suppressant herbicide is carried out before emergence or after emergence. The application of the PPO suppressant herbicide includes contacting the plant with the PPO suppressant herbicide, or the application of the PPO suppressant herbicide includes soil sealing, foliar spraying, or water application.

[0032] In one specific embodiment, the plant is cultivated by direct seeding in water, direct seeding in dry land, or transplanting.

[0033] In another specific embodiment, the plant is rice.

[0034] This invention obtained herbicide-resistant gene combinations through knock-up and point mutation methods, resulting in rice with significantly enhanced herbicide resistance, exhibiting unexpected resistance effects.

[0035] Invention Details

[0036] Some of the terms used in this specification are defined as follows.

[0037] In this invention, "herbicide" refers to an active ingredient capable of killing, controlling, or adversely altering plant growth. "Herbicide tolerance" or "herbicide resistance" in this invention refers to the continued growth of a plant even after the use of a herbicide that kills common or wild plants, inhibits plant growth, or weakens or stops the plant's growth compared to wild plants. The aforementioned herbicides include protoporphyrinogen oxidase (PPO) inhibitors, which have a mode of action including inhibiting the chlorophyll biosynthesis step in plants and belong to Group E of the HRAC classification system (see HRAC, classification of herbicides according to mode of action, http: / / www.plantprotection.org / hrac / MOA.html). Any herbicide satisfying this mode of action is within the scope of protection of this invention. These PPO inhibitor herbicides can be classified into pyrimidinediones, diphenyl-ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, and other herbicides with different chemical structures.

[0038] In one exemplary embodiment, pyrimidinid herbicides include, but are not limited to, flufenacet (CAS NO: 134605-64-4), fensulfuron-methyl (CAS NO: 372137-35-4), bispyribac-methyl (CAS NO: 158755-95-4), tiafenacil (CAS NO: 1220411-29-9), epyrifenacil (CAS NO: 353292-31-6), 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1220411-29-9), Epyrifenacil (CAS NO: 353292-31-6), and 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 134605-64-4), pyrimidinidyl-6-yl)-1H-pyrimidin-2,4-dione (CAS NO: 1220411-29-9). NO: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS NO: 212754-02-4), flupropacil (CAS NO: 120890-70-2), Uracil-pyridine disclosed in WO2017 / 202768 and uracil derivatives disclosed in WO2018 / 019842.

[0039] Diphenyl ether herbicides include, but are not limited to, flufenoxuron (CAS NO: 72178-02-0), ethoxyflufen (CAS NO: 42874-03-3), bensulfuron (CAS NO: 74070-46-5), quizalofop-p-ethyl (CAS NO: 77501-63-4), methoxyflufen (CAS NO: 32861-85-1), glufosinate (CAS NO: 1836-77-7), ethoxyflufen (CAS NO: 77501-90-7), trifluralin or its sodium salt (CAS NO: 50594-66-6 or 62476-59-9), methoxyflufen (CAS NO: 42576-02-3), ethoxyfen (CAS NO: 188634-90-4), chlorfluazuron ethyl (CAS NO: 131086-42-5), and fluoronitrofen (CAS NO: 131086-42-5). NO: 13738-63-1), furyloxyfen (CAS NO: 80020-41-3), nitrofluorfen (CAS NO: 42874-01-1) and halosafen (CAS NO: 77227-69-1).

[0040] Phenylepiazole herbicides include, but are not limited to, imidacloprid (CAS NO: 129630-19-9), isopyrazosulfuron (CAS NO: 174514-07-9) and ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxo]propionate (CAS: 118237-10-8).

[0041] N-phenylimide herbicides include, but are not limited to, propyzamide (CAS NO: 103361-09-7), indole-3-methyl (CAS NO: 142891-20-1), flumipropyn (CAS NO: 84478-52-4), flumethrin (CAS NO: 87546-18-7), chlorophthalim (CAS: 39985-63-2), and N-(4-chlorophenyl)-3,4,5,6-tetrahydrophenyl-o-dicarboximide (CAS: 7386-21-2).

[0042] Thiadiazole herbicides include, but are not limited to, methyl methacrylate (CAS NO: 117337-19-6), methoxyfenozide (CAS NO: 149253-65-6), and thiamethoxam (CAS NO: 123249-43-4).

[0043] Oxadiazole herbicides include, but are not limited to, propyzinoxadiazon (CAS NO: 39807-15-3) and oxadiazon (CAS NO: 19666-30-9).

[0044] Triazoline herbicides include, but are not limited to, oxadiazon (CAS NO: 128621-72-7), oxadiazon / oxadiazon (CAS NO: 128639-02-1), mesotrione (CAS NO: 122836-35-5), oxadiazon (CAS NO: 68049-83-2), and oxadiazon (CAS NO: 173980-17-1).

[0045] Oxazolidinone herbicides include, but are not limited to, cyclooxadiazon (CAS NO: 110956-75-7).

[0046] Other herbicides include, but are not limited to, bispyribac-methyl (CAS NO: 158353-15-2), flupyrazosulfuron-methyl (CAS NO: 188489-07-8), cyclopyranil (CAS NO: 1651191-47-7), flupyrazosulfuron-methyl (CAS NO: 190314-43-3), trifludimoxazin (CAS NO: 1258836-72-4), phenopylate (CAS: 40575-34-6), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9), and N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452098-92-9). NO: 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazin-2,4-dione (CAS NO: 915396-43-9), N-ethyl ...099-05-7), 3-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS NO: 452100-03-7), 3-[7- NO: 451484-50-7), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-Diketone (CAS NO: 1300118-96-0), (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester (CAS NO: 1300118-96-0), (E)-4 ...[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester (CAS NO NO: 948893-00-3), ethyl acetate of 3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy] (CAS: 353292-31-6, S-3100), 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4- Dione (CAS: 1258836-72-4), 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione (CAS: 1304113-05-0), 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione (CAS: 212754-02-4), The phenylpyridine derivatives disclosed in WO2016 / 120116 and the benzoxazinone derivatives disclosed in EP09163242.2.

[0047] In another exemplary embodiment, the PPO inhibitor herbicide has the typical chemical formula listed in WO2024126560A1.

[0048] Generally, if the PPO-inhibiting herbicides and / or other herbicides, as described herein and usable in the context of this invention, are capable of forming geometric isomers, such as E / Z isomers, then both, pure isomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have one or more chiral centers and are thus present as enantiomers or diastereomers, then both, pure enantiomers, diastereomers, and mixtures thereof may be used in compositions according to the invention. If the PPO-inhibiting herbicides and / or other herbicides, as described herein, have ionizable functional groups, then they may also be used in the form of their agriculturally acceptable salts. Typically, salts of those cations and acid addition salts of those acids are suitable, whose cations and anions do not have adverse effects on the activity of the active compound, respectively. The preferred cations are alkali metal ions, preferably lithium, sodium, and potassium ions; alkaline earth metal ions, preferably calcium and magnesium ions; and transition metal ions, preferably manganese, copper, zinc, and iron ions, further preferably ammonium and substituted ammonium ions, wherein one to four hydrogen atoms are substituted by C1-C4-alkyl, hydroxy-C1-C4-alkyl, C1-C4-alkoxy-C1-C4-alkyl, hydroxy-C1-C4-alkoxy-C1-C4-alkyl, phenyl, or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2 - Hydroxyethylammonium (olamine salt), 2-(2-hydroxyethyl-1-oxy)ethyl-1-ylammonium (diethylene glycolamine salt), di(2-hydroxyethyl-1-yl)ammonium (diethylene glycolamine salt), tri(2-hydroxyethyl)ammonium (trinitroethanolamine salt), tri(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), in addition to phosphonium ions, sulfonium ions, preferably tri(C1-C4-alkyl)sulfonium such as trimethylsulfonium, and sulfonium oxide ions, preferably tri(C1-C4-alkyl)sulfonium oxide ions, and finally, salts of polyamines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. The main anions that can be used for acid addition salts are chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and anions of C1-C4-alkanoic acids, with formate, acetate, propionate, and butyrate being preferred.

[0049] PPO-inhibiting herbicides and / or other herbicidal compounds having carboxyl groups as described herein can be used in the form of acids, agriculturally suitable salts as mentioned above, or otherwise in the form of agriculturally acceptable derivatives, for example as amides such as mono- and di-C1-C6-alkylamides or arylamides, as esters such as allyl esters, propargyl esters, C1-C10-alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters, and also as thioesters such as C1-C10-alkyl thioesters. Preferred mono- and di-C1-C6-alkylamides are methyl and dimethylamides. Preferred arylamides are, for example, N-anilide and 2-chloroanilide. Preferred alkyl esters are, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl), meptyl (1-methylheptyl), heptyl, octyl, or isooctyl (2-ethylhexyl) esters. Preferred C1-C4-alkoxy-C1-C4-alkyl esters are straight-chain or branched C1-C4-alkoxyethyl esters, such as 2-methoxyethyl ester, 2-ethoxyethyl ester, 2-butoxyethyl ester, 2-butoxypropyl ester, or 3-butoxypropyl ester. Examples of straight-chain or branched C1-C10-alkyl thioesters are ethyl thioesters.

[0050] The PPO-inhibiting herbicides described above, which can be used in carrying out the present invention, are generally preferred to be used in combination with one or more other herbicides to achieve control of a variety of unwanted plants. For example, PPO-inhibiting herbicides can also be used in combination with additional herbicides to which the crop is naturally resistant or resistant via the expression of one or more additional transgenes as described above. When used in combination with other targeted herbicides, the compounds claimed in this invention can be formulated with one or more other herbicides, mixed with one or more other herbicides, or applied sequentially with one or more other herbicides.

[0051] Suitable mixture components, for example, are herbicides selected from categories b1) to b15):

[0052] b1) Inhibitors of lipid biosynthesis;

[0053] b2) Acetolactate synthase inhibitors (ALS inhibitors);

[0054] b3) Photosynthesis inhibitors;

[0055] b4) Protoporphyrinogen-IX oxidase inhibitor;

[0056] b5) Bleaching herbicides;

[0057] b6) 5-Enolpyruvylshikimate-3-phosphate synthase inhibitor (EPSP inhibitor);

[0058] b7) Glutamine synthase inhibitor;

[0059] b8) 7,8-Dihydropteranoic acid synthase inhibitor (DHP inhibitor);

[0060] b9) Mitosis inhibitors;

[0061] b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

[0062] b11) Cellulose biosynthesis inhibitor;

[0063] b12) Decoupler herbicides;

[0064] b13) auxinic herbicides;

[0065] b14) Auxin transport inhibitors; and

[0066] b15) is selected from bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, and etobenzyl sulfadiazine. zanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic Hydrazine, mefluidide, metam, methiozolin (CAS NO: 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridiphane, and 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS NO: 499223-49-3) and other herbicides and their salts and esters;

[0067] This includes their agriculturally acceptable salts or derivatives.

[0068] Furthermore, when used in combination with other herbicide compounds as described above, it may be useful to apply PPO inhibitory herbicides in combination with safeners. Safeners are compounds that prevent or reduce damage to beneficial plants but do not significantly affect the herbicidal effect of the herbicide on unwanted plants. They can be applied before sowing (e.g., at seed treatment, on branches or seedlings) or before or after germination of the beneficial plants.

[0069] In addition, safeners, PPO inhibitory herbicides and / or other herbicide compounds may be applied simultaneously or sequentially.

[0070] PPO inhibitory herbicides and herbicide compounds and safeners of groups b1)-b15) are known herbicides and safeners, see, for example, WO2013 / 189984; The Compendium of Pesticide Common Names (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000, Vol. 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R.R. Schmidt, Herbizide, 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.

[0071] As used in this article, “genome” refers to the complete complement of genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism, and / or the complete set of chromosomes inherited as a unit (haploid) from a parent.

[0072] The term "gene editing" refers to strategies and techniques for targeted and specific modifications to any genetic information or genome of a living organism. Therefore, the term includes editing of gene-coding regions, but also editing of regions other than the gene-coding regions of the genome. It also includes editing or modifying the nucleus (if present) and other genetic information within the cell.

[0073] The term “CRISPR / Cas” can refer to a CRISPR-based nuclease or a nucleic acid sequence encoding it, including but not limited to: 1) Cas9, including SpCas9, ScCas9, SaCas9, xCas9, VRER-Cas9, EQR-Cas9, SpG-Cas9, SpRY-Cas9, SpCas9-NG, NG-Cas9, NGA-Cas9 (VQR), etc.; 2) Cas12, including LbCpf1, FnCpf1, AsCpf1, MAD7, KingCas12 (SEQ ID NO:12), etc.; or any variant or derivative of the aforementioned CRISPR-based nucleases. Preferably, the at least one of the CRISPR-based nucleases contains a mutation compared to the corresponding wild-type sequence, such that the obtained CRISPR-based nuclease recognizes a different PAM sequence.

[0074] The term "CRISPR" refers to a sequence-specific genetic manipulation technique that relies on clustered, regularly spaced short palindromic repeats, unlike RNA interference which regulates gene expression at the transcriptional level.

[0075] In this invention, guide RNA is used to guide the Cas protein to a specific DNA sequence.

[0076] In some embodiments, guide RNA(one or more) and Cas9 or KingCas12 protein can be delivered to cells as a ribonucleoprotein (RNP) complex. The RNP consists of purified Cas9 or KingCas12 protein complexed with the guide RNA, and it is well known in the art that RNPs can be efficiently delivered to a variety of cell types, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA; Mirus Bio LLC, Madison, WI).

[0077] In one specific implementation, the guide RNA is crRNA.

[0078] In one specific embodiment, the guide RNA consists of two parts: crRNA (CRISPR RNA) containing a sequence complementary to the target DNA; and transducer RNA (tracrRNA) that helps the crRNA bind to the Cas protein. The combination of these two components is referred to in this invention as guide RNA (gRNA).

[0079] In one specific implementation, crRNA and tracrRNA are designed into a single chimeric RNA, called sgRNA (single guide RNA).

[0080] The term "crRNA" or "CRISPR RNA" refers to a guide RNA suitable for the CRISPR system, which includes a recognition sequence (or spacer sequence) and a backbone region. The backbone region interacts with a CRISPR protein (or Cas protein), thereby forming a complex between the Cas protein and the crRNA, and guiding the complex to bind to the target sequence. As described in this invention, crRNA is a short RNA that guides the KingCas12 protein / enzyme (SEQ ID NO:12) to bind to a target genomic DNA sequence, and its recognition sequence is complementary to the target sequence.

[0081] The terms “target genomic DNA sequence,” “target sequence,” or “genomic target locus” refer to any locus in the nucleic acids (e.g., the genome) of a cell or population of cells, in vitro or in vivo, where it is desired to alter at least one nucleotide using a nucleic acid-guided nuclease editing system. A target sequence can be a genomic locus or an extrachromosomal locus.

[0082] The terms “complementary,” “complementarity,” “reverse complementarity,” and “reverse complementarity” are used interchangeably to refer to a nucleotide sequence that is complementary to a given nucleotide sequence, but in reverse order. For example, the sequence “5'-CAGT-3'” is complementary to the sequence “3'-GTCA-5'.” The term “complementary sequence” refers to a nucleic acid sequence that can form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick or other non-traditional types.

[0083] As used in this article, the term "biological" includes animals, plants, fungi, bacteria, etc.

[0084] As used in this article, the term "host cell" includes plant cells, animal cells, fungal cells, bacterial cells, etc.

[0085] The term "plant" is used in its broadest sense because it refers to organic matter and is intended to encompass eukaryotes belonging to the plant kingdom, including but not limited to vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones, suckers, and plant parts used for asexual reproduction (e.g., cuttings, tubes, seedlings, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses the whole plant, the ancestor and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, pedicels, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of a plant, and tissues and organs therein each containing the said gene combination. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing contains the said gene combination.

[0086] Plants particularly useful in the method of this invention include all plants belonging to the superfamily of the Kingdom Viridiplantae, especially monocots and dicots, including legumes used for fodder or feed, ornamental plants, food crops, trees or shrubs, wherein said plants are selected from a list containing the following species: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., and Artocarpus spp. spp.), Asparagus officinalis, Avena spp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Star fruit (Averrhoacarambola), Bambusa sp., Benincasa hispida, Brazil chestnut (Bertholletia excelsea), Beetroot (Beta vulgaris), Brassica spp. (e.g., Brassicanapus, Brassica rapa ssp.), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, papaya (Caricapapaya), large-fruited false tiger thorn (Carissa macrocarpa), species of the genus Carya (Carya spp.), red flower (Carthamus tinctorius), species of the genus Castanea (Castanea spp.).), American kapok (Ceiba pentandra), chicory (Cichorium endivia), Cinnamomum species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos spp.), coffee species (Coffea spp.), taro (Colocasia esculenta), African sycamore species (Cola spp.), jute (Corchorus sp.), coriander (Coriandrum sativum), hazel species (Corylus spp.), hawthorn species (Crataegus spp.), saffron (Crocus sativus), squash species (Cucurbita spp.), cantaloupe species (Cucumis spp.), artichoke species (Cynara spp.), carrot (Daucus carota), grasshopper species (Desmodium spp.), longan (Dimocarpus) species of the genera *Dioscorea*, *Diospyros*, *Echinochloa*, *Elaeis* (e.g., *Elaeis guineensis*, *Elaeis oleifera*), *Eleusine coracana*, *Eragrostis tef*, *Erianthus* sp., *Eriobotrya japonica*, *Eucalyptus* sp., *Eugenia uniflora*, *Fagopyrum* sp., *Fagus* sp., *Festuca arundinacea*, *Ficus carica*, *Fortunella* sp., *Fragaria* sp., and *Ginkgo*. genus *Bibona*, genus *Glycine* (e.g., *Glycine max*, *Soja hispida*, or *Soja max*), upland cotton (*Gossypiumhirstum*), and species of the genus *Helianthus* (*Helianthus* spp.).(e.g., sunflower (Helianthus annuus)), daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), barley (Hordeum spp.) (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut species (Juglans spp.), lettuce (Lactuca sativa), pea species (Lathyrus spp.), lentil (Lens culinari), flax (Linumus itatisimum), litchi (Litchi chinensis), lotus species (Lotus spp.), loofah (Luffaacutangula), lupinus species (Lupinus spp.), Luzula sylvatica, tomato species (Lycopersicon spp.) (e.g., tomato (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), species of the genera *Macrotyloma*, *Malus*, *Malpighia emarginata*, *Mammea americana*, *Mangifera indica*, species of the genera *Manihot*, *Manilkara zapota*, *Medicago sativa*, species of the genera *Melilotus*, *Mentha*, *Miscanthus sinensis*, species of the genera *Momordica*, *Morus nigra*, species of the genera *Musa*, species of the genera *Nicotiana*, species of the genera *Olea*, species of the genera *Opuntia*, species of the genera *Ornithopus*, and species of the genera *Oryza*. (e.g., rice (Oryza sativa), broadleaf rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), passion fruit (Passiflora edulis), European parsnip (Pastinaca sativa), species of the genus Pennisetum (Pennisetum sp.)), avocado species (Persea spp.), parsley (Petroselinum crispum), purslane (Phalaris arundinacea), common bean species (Phaseolus spp.), cat's tail grass (Phleum pratense), prickly ash species (Phoenix spp.), southern reed (Phragmites australis), groundcherry species (Physalis spp.), pine species (Pinus spp.), pistachio species (Pistacia vera), pea species (Pisum spp.), Kentucky bluegrass species (Poa spp.), poplar species (Populus spp.), prosopis species (Prosopis spp.), plum species (Prunus spp.), guava species (Psidium spp.), pomegranate (Punica granatum), European pear (Pyrus communis), oak species (Quercus spp.) Radish (Raphanus sativus), Rheum rhabarbarum, Ribes spp., Castor bean (Ricinus communis), Rubus spp., Saccharum spp., Salix sp., Sambucus spp., rye (Secale cereale), Sesamum spp., Sinapis sp., Solanum spp. (e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium), or tomato), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarind (Tamarindus) (Indica), cacao (Theobroma cacao), Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp.(For example, common wheat (Triticum aestivum), durum wheat (Triticum durum), cylindrical wheat (Triticum turgidum), Triticum hybernum, macha wheat (Triticum macha), common wheat (Triticum sativum), emmer wheat (Triticum monococcum), or common wheat (Triticum vulgare)), tarope (Tropaeolumminus), tarope (Tropaeolum majus), species of the genus *Vaccinium* (Vaccinium spp.), species of the genus *Vicias* (Vicias spp.), species of the genus *Vigna* (Vigna spp.), violet (Viola odorata), species of the genus *Vitis* (Vitis spp.), maize (Zea mays), *Zizania palustris*, species of the genus *Ziziphus* This includes, but is not limited to, spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower, celery, kale, flax, kale, lentils, rapeseed, okra, onions, potatoes, rice, soybeans, strawberries, sugar beets, sugarcane, sunflowers, tomatoes, squash, tea, and algae, along with others. According to a preferred embodiment of the invention, the plants are crop plants. Examples of crop plants particularly include soybeans, corn, rice, and cotton.

[0087] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0088] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0089] In this invention, the term "site" includes the location where the plants of this invention are cultivated, such as soil, and also includes, for example, plant seeds, seedlings, and mature plants. The term "effective amount for weed control" refers to an amount of herbicide sufficient to affect the growth or development of a target weed, such as preventing or inhibiting the growth or development of the target weed, or killing the weed. Advantageously, the effective amount for weed control does not significantly affect the growth and / or development of the seeds, seedlings, or plants of this invention. Such effective amounts for weed control can be determined by those skilled in the art through routine experiments.

[0090] The term "gene" includes a segment of nucleic acid that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence).

[0091] The DNA sequence that “encodes” a specific RNA is the DNA nucleic acid sequence that is transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into proteins, or DNA polynucleotides can encode RNA that is not translated into proteins (such as tRNA, rRNA, or RNA that targets DNA; also known as “non-coding RNA” or “ncRNA”).

[0092] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0093] For the terminology related to CDS positioning used in the specification, numbers indicate a specific position relative to the nucleotide sequence, ".." indicates all consecutive nucleotides including two numbers and the position, and "," indicates that nucleotides at all positions are connected end-to-end; for example, SEQ ID NO:1, 69..262,386..521,1101..1166,1261..1311,1400..1456,1743..1828,2233..2269,2386..2451,2745..2785,4051..4183,4858..5046,5146..5220,6229..6355,6964..7072,7156..7230,7386..7492,7749..7855bp represents SEQ ID NO:1. NO: 1 Nucleotides 69 to 262 are linked to nucleotides 386 to 521, nucleotides 1101 to 1166, nucleotides 1261 to 1311, nucleotides 1400 to 1456, nucleotides 1743 to 1828, nucleotides 2233 to 2269, nucleotides 2386 to 2451, and nucleotides 2745 to 2... Nucleotide 785, linked to nucleotides 4051 to 4183, linked to nucleotides 4858 to 5046, linked to nucleotides 5146 to 5220, linked to nucleotides 6229 to 6355, linked to nucleotides 6964 to 7072, linked to nucleotides 7156 to 7230, linked to nucleotides 7386 to 7492, linked to nucleotides 7749 to 7855.

[0094] The specific amino acid positions (numbers) within the protein described in this invention are determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:2, etc. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, with the sequence considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0095] For the terminology related to amino acid substitutions used in the specification, the first letter represents a naturally occurring amino acid at a specific position in a particular sequence, the following number represents the position relative to SEQ ID NO:2, and the second letter represents the different amino acid that replaces that natural amino acid. For example, L422M indicates that, relative to the amino acid sequence of SEQ ID NO:2, leucine at position 422 is replaced by methionine. For double or multiple mutations, the mutations are separated by " / " or "+". For example, L422M+F442M indicates that, relative to the amino acid sequence of SEQ ID NO:2, leucine at position 422 is replaced by methionine, and phenylalanine at position 442 is replaced by methionine, and both mutations are present in the specific mutant PPO2 protein.

[0096] The terms "corresponding to" and "relative to" are used interchangeably. In this invention, the specific amino acid position (number) within the protein is determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:2, etc. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, where the sequence is considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0097] The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10; gap extension penalty: 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID NO:2.

[0098] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitutions in which an amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided that the substitution does not impair the biological activity of the protein.

[0099] Therefore, in addition to the mutations described above, the mutant proteins of the present invention may also contain one or more other mutations, such as conserved substitutions, in their amino acid sequences. Furthermore, the present invention also covers mutant proteins containing one or more other non-conserved substitutions, provided that such non-conserved substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0100] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of mutant proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a bioactive fragment of a mutant protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0101] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the normal sequence, wild-type sequence, or reference sequence. In some embodiments, a mutation refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the nucleotide or amino acid sequence of a non-herbicide-resistant PPO2 protein. In some embodiments, a mutation refers to one or more mutations at amino acid positions relative to the reference PPO2 amino acid sequence as shown in SEQ ID NO:2 or at homologous positions in its different species homologs. In some embodiments, a mutation may include substitution, deletion, inversion, or insertion. In some embodiments, substitution, deletion, insertion, or inversion may include variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, substitution, deletion, insertion, or inversion may include variations at amino acid positions 1, 2, 3, 4, 5, 6, 7, or 8.

[0102] The terms "wildtype" and "mutation" are relative and refer to the phenotype that is most frequent in a particular population, or the system, organism, or gene that possesses that phenotype. In some cases, the wild-type allele refers to the standard allele at the locus, or the allele that is most frequent in a particular population, which can be represented by a specific amino acid or nucleic acid sequence.

[0103] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” or “nucleic acid sequence” are used interchangeably to refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The single-letter codes for nucleotides are as described in Table 1 of Section 2422 of the U.S. Patent Examination Procedure Manual. At this point, the nucleotide name “R” indicates a purine, such as guanine or adenine; “Y” indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); “M” indicates adenine or cytosine; “K” indicates guanine or thymine; and “W” indicates adenine or thymine. The term "isolated," when referring to nucleic acids, means a nucleic acid that is separate from the substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany it. For example, any nucleic acid that has been synthesized (e.g., by sequential base condensation) is considered isolated. Similarly, recombinantly expressed nucleic acids, cloned nucleic acids, nucleic acids produced by primer extension reactions (e.g., PCR), or other nucleic acids excised from the genome are also considered isolated.

[0104] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0105] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" as used herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., autofertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0106] As used in this invention, "expression cassette," "expression vector," and "expression construct" refer to vectors, such as recombinant vectors, suitable for expressing nucleotide sequences of interest in plants. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into precursor or mature proteins.

[0107] The "expression construct" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0108] The "expression construct" of the present invention may contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those normally found in nature.

[0109] The "highly expressed gene" in this invention refers to a gene whose expression level is higher than that of a normal gene in a specific tissue.

[0110] The terms “recombinant expression vector” or “DNA construct” are used interchangeably herein and refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically created for the purpose of expressing and / or propagating the insert or for constructing other recombinant nucleotide sequences. The insert may be operatively or inoperably linked to a promoter sequence and may be operatively or inoperably linked to a DNA regulatory sequence.

[0111] The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to nucleotide sequences located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that influence the transcription, RNA processing, stability, or translation of the relevant coding sequence. Plant expression regulatory elements are nucleotide sequences that can control the transcription, RNA processing, stability, or translation of the nucleotide sequence of interest in plants. Depending on their proximity to the sequence or gene they control, regulatory elements are also called cis- or trans-regulatory elements.

[0112] Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0113] A “promoter” refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from a plant cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter. It should also be recognized that because the exact range of the regulatory sequence cannot be fully defined in most cases, DNA fragments of different lengths can have the same promoter activity. For example, it should be understood that “UBI2 gene promoter” can be used to refer to a fragment derived from the promoter region of the UBI2 gene.

[0114] The term "strong promoter" is well-known and widely used in the art, and many strong promoters are known in the art or can be identified by routine experiments. The promoter activity is higher than that of a promoter effectively linked to the nucleic acid molecule to be overexpressed in a wild-type organism, for example, a promoter with higher activity than the promoter of an endogenous gene. Preferably, the strong promoter activity is about 2%, 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than 1000% higher than that of a promoter effectively linked to the nucleic acid molecule to be overexpressed in a wild-type organism. Those skilled in the art know how to determine promoter activity and compare the activities of different promoters.

[0115] "Constraint promoters" refer to promoters that generally cause gene expression in most cell types and under most conditions. "Tissue-specific promoters" and "tissue-preferred promoters" are used interchangeably and refer to promoters that are primarily, but not necessarily, expressed specifically in one tissue or organ, and may also be expressed in a specific cell type. "Developmental regulatory promoters" are promoters whose activity is determined by developmental events. "Inducible promoters" selectively express manipulated DNA sequences in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).

[0116] The term "enhancer" refers to a DNA sequence that can stimulate promoter activity and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the promoter level or tissue specificity.

[0117] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0118] "Introducing" nucleic acid molecules (such as plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into plants refers to transforming plant cells with the nucleic acids or proteins so that the nucleic acids or proteins can function in the plant cells. The term "transformation" as used in this invention includes both stable transformation and transient transformation.

[0119] "Stable transformation" refers to the introduction of a foreign nucleotide sequence into a plant genome, resulting in the stable inheritance of the foreign gene. Once stable transformation occurs, the foreign nucleic acid sequence is stably integrated into the genome of the plant and its subsequent generations.

[0120] "Transient transformation" refers to the introduction of nucleic acid molecules or proteins into plant cells to perform their functions without the foreign gene being stably inherited. In transient transformation, the foreign nucleic acid sequence does not integrate into the plant genome.

[0121] Altering the expression of endogenous genes in organisms involves both intensity and spatiotemporal characteristics. Intensity includes upregulation (knock-up), downregulation (knock-down), and zeroing (knockout); spatiotemporal specificity includes time (reproductive stage) specificity and space (tissue) specificity, as well as inducibility. Furthermore, altering protein targeting can be achieved, for example, changing a protein located in the cytoplasm to one located in chloroplasts or the nucleus.

[0122] The terms "knock-up" and "upregulation of gene expression level" in this invention refer to an increase in the expression level of a new gene relative to the corresponding endogenous wild-type gene in the organism, preferably an increase of at least 0.5-fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold. "Highly expressed gene" refers to a gene whose expression level is higher than that of a normal gene in a specific tissue.

[0123] In this invention, "herbicide" refers to an active ingredient capable of killing, controlling, or adversely altering plant growth. "Herbicide tolerance" or "herbicide resistance" in this invention refers to the continued growth of a plant even after the use of a herbicide that kills common or wild plants, inhibits plant growth, or weakens or stops the plant's growth compared to wild plants.

[0124] The term "weed control" will be understood as killing weeds and / or delaying or inhibiting their normal growth. In the broadest sense, weeds are understood as all plants known to grow in unwanted locations, such as (crop) plant cultivation sites. The weeds included in this invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds from the following genera: *Sinapis*, *Lepidium*, *Galium*, *Stellaria*, *Matricaria*, *Anthemis*, *Galinsoga*, *Chenopodium*, *Urtica*, *Senecio*, *Amaranthus*, *Portulaca*, *Xanthium*, *Convolvulus*, *Ipomoea*, *Polygonum*, *Sesbania*, *Aragula*. The genera *mbrosia*, *Cirsium*, *Carduus*, *Sonchus*, *Solanum*, *Rorippa*, *Rotala*, *Lindernia*, *Lamium*, *Veronica*, *Abutilon*, *Emex*, *Datura*, *Viola*, *Galeopsis*, *Papaver*, *Centaurea*, *Trifolium*, *Ranunculus*, and *Taraxacum*.Monocotyledonous weeds include, but are not limited to, the following genera: *Echinochloa*, *Setaria*, *Panicum*, *Digitaria*, *Phleum*, *Poa*, *Festuca*, *Eleusine*, *Brachiaria*, *Lolium*, *Bromus*, *Avena*, *Cyperus*, *Sorghum*, and *Agropyron*. The genera include *Cynodon*, *Monochoria*, *Fimbristylis*, *Sagittaria*, *Eleocharis*, *Scirpus*, *Paspalum*, *Ischaemum*, *Sphenoclea*, *Dactyloctenium*, *Agrostis*, *Alopecurus*, and *Apera*. Additionally, the weeds in this invention can include, for example, crop plants growing in unwanted locations. For instance, if corn plants are not desired in a soybean field, free-growing corn plants present in a field primarily containing soybeans can be considered a weed.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter.

[0126] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the publications referenced. Any reference to a publication is made prior to the filing date and should not be construed as an admission that the invention does not predate such publication. Furthermore, the publication date provided may differ from the actual publication date, which may require independent verification.

[0127] Unless specifically stated or implied, as used herein, the terms “a,” “an,” and “described” mean “at least one.” All patents, patent applications, and publications mentioned or cited herein are incorporated herein by reference in their entirety as if they were individually cited separately. Attached Figure Description

[0128] Figure 1. Schematic diagram of OsPPO2 gene knock-up editing driven by the OsUbi2 gene promoter;

[0129] Figure 2. T1 generation P1 rice. Herbicide resistance test results 14 days after treatment; 1,5: wild type; 2,3,4: P1;

[0130] Figure 3. T1 generation P2 rice. Results of herbicide resistance test 14 days after treatment;

[0131] Figure 4. Schematic diagram of L422M / F442M double-point mutation editing of P1;

[0132] Figure 5. T1 generation P2@P1 rice Herbicide resistance test results 14 days after treatment; 1: wild type; 2: P2@P1;

[0133] Figure 6. Transgenic rice P3 used Herbicide resistance test results 14 days after treatment; 1-5: 0, 22.5, 45, 90, 180 g ai / ha treatments.

[0134] Sequence Description Detailed Implementation

[0135] The present invention will be further illustrated below with reference to examples. The following description is by way of example, but the scope of protection of the present invention should not be limited thereto.

[0136] Example 1: Gene function analysis and promoter activity test

[0137] The OsPPO2 gene (Gene ID: LOC4336237, SEQ ID NO:1) is located on chromosome 4 of rice at positions 24977590-24985688. The gene sequence, from start to stop codon, is 7787 bp in length, containing 17 exons and 16 introns, encoding the protein OsPPO2 (XP_025880545.1, SEQ ID NO:2), which contains 551 amino acids. The rice Ubiquitin2 (OsUbi2) gene sequence is shown in SEQ ID NO:3, and its promoter is a constitutively strong promoter. Analysis of rice gene expression profile data provided by the International Rice Genome Sequencing Project shows that the expression intensity of the OsUbi2 gene in rice leaves is hundreds of times higher than that of the OsPPO2 gene. Therefore, this invention uses the promoter of the OsUbi2 gene to drive the expression of the OsPPO2 gene, thereby increasing the expression level of OsPPO2.

[0138] The transcriptional activity of OsUbi2 promoters of different truncated lengths was detected in rice protoplasts using a dual-fluorescence reporter system. The promoter sequences of different truncated lengths are shown in SEQ ID NO:4-7. The test results showed that OsUbi2 promoters of different lengths all had transcriptional activity.

[0139] Example 2: Obtaining rice with high expression of endogenous PPO2 gene via gene gun-mediated transformation

[0140] As shown in Figure 1, OsPPO2 gene knock-up expression was achieved by selecting a chromosome translocation method, resulting in OsPPO2 gene knock-up rice with OsUbi2 gene promoter driving OsPPO2 gene expression, which was then identified and tested for herbicide resistance.

[0141] 1. RNP design and preparation

[0142] Based on the results of OsUbi2 promoter transcriptional activity assays, suitable crRNA targets were designed near the OsUbi2 gene promoter and upstream of the OsPPO2 gene start codon using the CRISPOR online tool (http: / / crispor.tefor.net / crispor.py). The downstream target of the OsUbi2 gene promoter was named OsUbi2crRNA1:5'-TTTGtgaagacattgaccggcaagact-3' (SEQ ID NO:8), and the upstream target of the OsPPO2 gene start codon was named OsPPO2-crRNA1:5'-TTTCccgtatgaccggccgtccccacc-3' (SEQ ID NO:9). After synthesis, these sequences were assembled with purified KingCas12 protein (SEQ ID NO:12) to form RNP complexes.

[0143] 2. Gene gun-mediated genetic transformation of rice

[0144] Wild-type rice callus was transformed using a gene gun-mediated transformation method. After callus recovery culture, it was transferred to a selection medium for three rounds of selection. Primers were designed to sample and detect whether the expected editing occurred. The OsPPO2 gene knockout high expression OsUbi2p:OsPPO2 chromosomal structural variation interface was detected using P-OsUbi2-F: 5'-gttggtgcaaacacaggctt-3' (SEQ ID NO:13) and OsPPO2-R: 5'-cagaactgaacccacggagag-3' (SEQ ID NO:14), and the OsPPO2p:OsUbi2 chromosomal structural variation interface was detected using P-OsPPO2-F: 5'-ccactcccagtccacttctc-3' (SEQ ID NO:15) and OsUbi2-R: 5'-gggatgccctccttatcttggatc-3' (SEQ ID NO:16). Positive bands were subjected to Sanger first-generation sequencing, and the sequencing results are shown in SEQ ID NO:19-20.

[0145] Positive, well-developed, yellowish-white callus tissue was selected and transferred to differentiation medium for differentiation. After 3-4 weeks, seedlings approximately 1 cm in length were obtained. These differentiated seedlings were then transferred to rooting medium for rooting culture. After successful rooting, the seedlings underwent hardening treatment and were then transplanted into pots filled with soil and placed in a greenhouse for further cultivation. Molecular identification of the T0 generation seedlings successfully yielded rice with high expression of the OsPPO2 gene driven by the OsUbi2 gene promoter, which was named P1.

[0146] 3. T1 generation homozygous rice herbicide resistance test

[0147] T0 generation positive seedlings were transferred to a greenhouse for cultivation, and individual plants were harvested to obtain T1 generation seeds. These seeds were then sown in Qingdao during the peak season, and individual plants were tested for genotype, yielding several homozygous edited individual plants. The identified homozygous individual plants were transplanted into the field according to plot size, with wild-type plants planted next to them as a control. Herbicide testing was conducted on these plants at the 4-leaf stage.

[0148] Apply medicine The results after 14 days are shown in Figure 2. The wild-type control showed obvious phytotoxicity at a dose of 22.5 g ai / ha and was basically dead at a dose of 45 g ai / ha. P1 showed almost no or slight phytotoxicity (small patches of pesticide on leaves) at doses of 22.5 and 45 g ai / ha. Growth slowed down at a dose of 90 g ai / ha and the area of ​​pesticide patches on leaves increased. Severe phytotoxicity occurred at a dose of 180 g ai / ha, gradually drying out and even dying.

[0149] Example 3: Obtaining PPO2 point mutant rice via Agrobacterium-mediated transformation

[0150] By introducing L422M / F442M amino acid substitutions at the OsPPO2 site through homologous recombination-mediated gene knock-in editing, OsPPO2 double-point mutant rice was obtained, and its identification and herbicide resistance testing were carried out.

[0151] 1. Construction of gene editing vectors and genetic transformation in rice:

[0152] First, a suitable target site was selected using the CRISPOR online tool (http: / / crispor.tefor.net / crispor.py). The target site near the L422M / F442M site of the OsPPO2 gene was named OsPPO2-crRNA2: 5'-TTTCattggggggagccataatagaga-3' (SEQ ID NO:10). The 1000bp upstream of L422, along with the 1000bp downstream of F442, was selected as the repair template OsPPO2-donor (SEQ ID NO:11). The repair template introduced amino acid substitutions of L422M / F442M.

[0153] Vectors were constructed using conventional methods. Primers were designed to amplify the target fragments, and crRNA expression cassettes were constructed. KingCas12 was used as the Cas enzyme, and its amino acid sequence is shown in SEQ ID NO:12. The OsPPO2-donor repair template was ligated into the intermediate vector containing the target fragments to obtain the final vector.

[0154] 2. Agrobacterium-mediated genetic transformation of rice

[0155] Rice callus was transformed using Agrobacterium-mediated genetic transformation. Specific primers were designed to amplify the transformed material, and Sanger sequencing was performed. OsPPO2 double point mutations were amplified using OsPPO2-5780F: 5'-ccactatctctcatggtaacagc-3' (SEQ ID NO:17) and OsPPO2-8097R: 5'-cacccaagagctttcttagg-3' (SEQ ID NO:18). Callus with point mutation substitution editing was retained. Sequencing results are shown in SEQ ID NO:21.

[0156] Positive callus samples were transferred to a new screening medium for a third round of screening and expansion culture. Once the callus diameter exceeded 5 mm, a second round of molecular identification was performed using primers from the first round of identification. Yellowish-white callus tissue showing good growth and positive results from the second round of identification was selected and transferred to differentiation medium for differentiation. After 3-4 weeks, seedlings approximately 1 cm in diameter were obtained. The differentiated seedlings were then transferred to rooting medium for rooting culture. After successful rooting, the seedlings underwent hardening treatment and were then transferred to pots filled with soil and placed in a greenhouse for further cultivation. Molecular identification of the T0 generation seedlings successfully yielded rice with a double-point mutation substitution in OsPPO2 (amino acid sequence as shown in SEQ ID NO:22, nucleotide sequence as shown in SEQ ID NO:23), named P2.

[0157] 3. T1 generation homozygous rice herbicide resistance test

[0158] T0 generation positive seedlings were transferred to a greenhouse for cultivation, and individual plants were harvested to obtain T1 generation seeds. These seeds were then sown in Qingdao during the peak season, and individual plants were tested for genotype, yielding several homozygous edited individual plants. The identified homozygous individual plants were transplanted into the field according to plot size, with wild-type plants planted next to them as a control. Herbicide testing was conducted on these plants at the 4-leaf stage.

[0159] Apply medicine The results after 14 days are shown in Figure 3. The wild-type control showed obvious phytotoxicity at a dose of 22.5 g ai / ha and was basically dead at a dose of 45 g ai / ha. P2 showed almost no or slight phytotoxicity (small patches on the leaves) at a dose of 22.5 g ai / ha. At a dose of 45 g ai / ha, growth was inhibited, leaves turned yellow, and the proportion of patches increased. At a dose of 90 g ai / ha, severe phytotoxicity occurred, and the leaves gradually dried out and even died.

[0160] Example 4: Obtaining Knockout and Point Mutant Polymer-Edited Rice via Agrobacterium-Mediated Transformation

[0161] As shown in Figure 4, P1 was edited with amino acid substitutions of L422M / F442M in the OsPPO2 gene to obtain OsPPO2 knock-up and double-point mutation polymerized rice, which were then identified and tested for herbicide resistance.

[0162] 1. Agrobacterium-mediated genetic transformation of rice

[0163] After inducing callus with homozygous edited seeds of generation T2 of P1, the P2 vector was transformed into callus using Agrobacterium-mediated transformation. Differentiated T0 seedlings were transferred to pots filled with soil and cultured in a greenhouse. Samples were taken for identification, and polymer-edited rice with OsUbi2 gene promoter-driven OsPPO2 gene knockout and OsPPO2 gene L422M / F442M double point mutations was successfully obtained and named P2@P1. The sequencing results of the OsUbi2p:OsPPO2 interface are the same as SEQ ID NO:19. The OsUbi2 promoter sequence is shown in SEQ ID NO:6, and the full length of the OsUbi2 promoter-driven OsPPO2 double mutant gene is shown in SEQ ID NO:24.

[0164] In addition, using the same method, polymer-edited rice with other genotypes of OsUbi2 gene promoter-driven OsPPO2 gene knock-up and OsPPO2 gene L422M / F442M double-point mutations were obtained and named P4 and P5, respectively. The sequencing results of the OsUbi2p:OsPPO2 interface of P4 and P5 are shown in SEQ ID NO:25-26, the OsUbi2 promoter sequence is shown in SEQ ID NO:29-30, and the full length of the OsUbi2 promoter-driven OsPPO2 double-mutant gene is shown in SEQ ID NO:27-28.

[0165] 2. T1 generation homozygous rice herbicide resistance test

[0166] T0 generation positive seedlings were harvested individually to obtain T1 generation seeds. These seeds were then sown in Qingdao during the peak season, and individual plant genotypes were tested to obtain several homozygous edited plants. The identified homozygous plants were transplanted into the field in plots, with wild-type plants planted alongside them as a control. Herbicide testing was conducted on these plants at the 4-leaf stage.

[0167] Apply medicine The results after 14 days are shown in Figure 5. The wild-type control showed significant herbicide damage at a dose of 22.5 g ai / ha and essentially died at a dose of 45 g ai / ha. P2@P1 showed no significant herbicide damage at doses of 22.5, 45, 90, and 180 g ai / ha; however, at a dose of 360 g ai / ha, growth was slow, showing only mild herbicide damage. The test results for P4 and P5 were the same as those for P2@P1. These results indicate that rice with OsPPO2 gene knockout and L422M / F442M point mutation aggregation editing exhibits significantly improved herbicide resistance compared to wild-type WT, the knockout-only P1, and the point mutation-only P2.

[0168] Example 5: Obtaining Knockout and Point Mutation-Induced Polymer-Edited Rice via Transgenic Transplantation

[0169] The OsUbi2 gene start codon is used as a promoter 2000 bp upstream (SEQ ID NO:4) to drive the OsPPO2 genome sequence, in which an L422M / F442M amino acid substitution is introduced at the OsPPO2 site. A binary vector containing the above elements and genes, and a HYG selection marker, is constructed for genetic transformation of rice callus. The obtained T0 generation seedlings are treated with 22.5 g ai / ha. After screening and transplanting of resistant seedlings, a number of T1 generation seeds were obtained through self-pollination and seed collection.

[0170] T1 generation seeds were sown, and molecular analysis yielded homozygous single-copy plants P3. The full-length OsPPO2 double mutant gene, driven by the OsUbi2 promoter, is shown in SEQ ID NO:31. Different concentrations of [unclear text - likely a continuation of the previous sentence] were used. Tests. The results 14 days after application are shown in Figure 6. The wild type (OS11) was essentially dead at a dose of 22.5 g ai / ha, while no phytotoxicity was observed in P3 at a dose of 180 g ai / ha.

[0171] Example 6: Evaluation of herbicide tolerance in rice by polymer editing of knock-up and point mutations.

[0172] 1. Indoor herbicide tolerance evaluation using different PPO inhibitor herbicides.

[0173] The active ingredients required for the test were purchased from reagent companies or synthesized using conventional methods. All technical grade reagents were diluted with acetone as a solvent and diluted with a 0.1% Tween-80 emulsifier aqueous solution, and diluted immediately before use. Water containing the same solvent and emulsifier but without the reagent served as a blank control.

[0174] The greenhouse pot cultivation method was adopted. Seeds of P2@P1 and their wild-type varieties with good germination rates were selected and sown. When the rice had 3.5 leaves, a foliar spray treatment was applied, diluted with 450 L / ha of water. Each treatment was repeated four times, with three pots per treatment. After 14 days of treatment, a survey was conducted using the following methods and the rating criteria described in Table 1. Representative statistical results are shown in Table 2.

[0175] The absolute number survey method was used. Surviving weeds or crop seedlings were cut along the soil surface with a blade, and their fresh weight was measured using an analytical balance. Dead weeds or crops were counted as having a fresh weight of zero.

[0176] Table 1 Data Rating Criteria

[0177] Table 2 Statistics of P2@P1 Crop Efficacy Grades

[0178] The results showed that the knock-up and point-mutation polymerized rice P2@P1 exhibited excellent tolerance to PPO inhibitor herbicides.

[0179] 2. Evaluation of field herbicide tolerance using different PPO inhibitor herbicides.

[0180] To test the field herbicide resistance of P2@P1, different varieties of wild-type rice were used as controls. The tolerance test for PPO inhibitor herbicides was conducted in a level, uniform experimental field using direct seeding. Foliar spraying with 450 L / ha of water was applied at the 3.5-leaf stage of the rice. Test plots were approximately 10-20 square meters in size, arranged sequentially, and repeated three times. Representative data are shown in Table 3.

[0181] Table 3. Evaluation results of field herbicide tolerance of PPO inhibitor herbicides to rice varieties.

[0182] In addition, the efficacy data of the PPO inhibitor herbicides against different weeds at corresponding dosages were tested. The herbicides were applied as foliar sprays and diluted with 450 L / ha of water. Some representative data are shown in Table 4.

[0183] Table 4. Representative statistical results of the control efficacy of PPO inhibitor herbicides against various weeds.

[0184] The results showed that, at doses that effectively control weeds, the knock-up and point-mutated polymer-edited rice P2@P1 also exhibited significantly improved tolerance to PPO inhibitor herbicides in field tests.

[0185] Meanwhile, other cultivation methods (such as transplanted fields and direct-seeded rice) were adopted, and the tolerance of rice to various types of PPO inhibitor herbicides was evaluated at different stages. The results showed that the knock-up and point-mutation polymer-edited rice P2@P1 exhibited excellent herbicide resistance.

[0186] All publications and patent applications mentioned in the specification are incorporated herein by reference as if each publication or patent application were individually and specifically incorporated herein by reference.

[0187] Although the invention has been described in considerable detail by way of example and embodiments for clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims, and all such changes and modifications are within the scope of the invention.

Claims

1. A gene combination comprising a promoter and a gene coding region, wherein the promoter is a UBI2 gene promoter and the gene coding region is a PPO2 gene mutant coding region.

2. The gene combination according to claim 1, wherein the UBI2 gene promoter sequence is as shown in SEQ ID NO:4, 5, 6, 7, 29 or 30, and / or the PPO2 gene mutant coding region contains the amino acid sequence shown in SEQ ID NO:22; preferably, the nucleotide sequence of the PPO2 gene mutant coding region contains the sequence shown in SEQ ID NO:

23.

3. The gene combination according to claim 1 or 2, further comprising the sequence shown in SEQ ID NO: 19, 25 or 26.

4. The gene combination according to any one of claims 1-3, wherein the nucleotide sequence comprises the sequence shown in SEQ ID NO:24, 27, 28 or 31.

5. The gene combination according to any one of claims 1-4, wherein it is obtained by a gene editing system or transgenic technology; preferably, the gene editing system is a meganuclease, zinc finger nuclease, TALEN or CRISPR / Cas system; more preferably, the gene editing system is a CRISPR / Cas9 or CRISPR / Cas12 system.

6. A recombinant genome comprising the gene combination as described in any one of claims 1-5.

7. A host cell comprising the gene combination as described in any one of claims 1-5 or comprising the recombinant genome as described in claim 6.

8. The application of a gene combination as described in any one of claims 1-5, a recombinant genome as described in claim 6, or a host cell as described in claim 7 in improving plant resistance to PPO-inhibiting herbicides.

9. A primer pair for detecting the gene combination as described in any one of claims 1-5, the sequences of which are shown in SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:

18.

10. A method for breeding a plant having or improved ability to tolerate a PPO-inhibiting herbicide, characterized in that, The method includes the following steps: (1) DNA breaks were simultaneously generated at specific locations upstream of the start codon of the PPO2 gene and downstream of the promoter of the UBI2 gene in plant cells. The DNA breaks were connected to each other through intracellular repair pathways. Gene combinations of the UBI2 gene promoter and the PPO2 gene coding region were screened by designing primer pairs. Then, the L422+F442 site of the PPO2 gene coding region in the gene combination was mutated to L422M+F442M. First, the L422+F442 site in the coding region of the PPO2 gene in plant cells was mutated to L422M+F442M. Then, DNA breaks were simultaneously generated at specific locations upstream of the start codon of the PPO2 gene and downstream of the promoter of the UBI2 gene in the plant cells. The DNA breaks were connected to each other through intracellular repair pathways. Gene combinations of the coding regions of the UBI2 gene promoter and the PPO2 gene mutant were screened by designing primer pairs. or, A recombinant expression vector containing the UBI2 gene promoter and the expression cassette of the L422M+F442M mutant coding region of the PPO2 gene was constructed, and the vector was transformed into plant callus tissue. Gene combinations of the UBI2 gene promoter and the coding region of the PPO2 gene mutant were screened by screening markers or designed primer pairs. (2) Regenerate plants from the obtained plant cells or tissues; Preferably, a plant containing the gene combination as described in any one of claims 1-5 is obtained.

11. A method for controlling weeds in a plant cultivation site, wherein the plant comprises a plant prepared by the method of claim 10, the method comprising applying a herbicidally effective amount of a PPO inhibitory herbicide to the plant cultivation site; preferably, the PPO inhibitory herbicide is applied in combination with one or more other herbicides.

12. The use of claim 8 or the method of claim 10 or 11, wherein, The PPO inhibitory herbicide is selected from one or more of the following types of compounds: pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinones, and others; preferably, (1) Pyrimidine diones include: flupropachlor, pyrimisulfuron, bispyribac-methyl, flupropachlor, Epyrifenacil, 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-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: flusulfanilamide, ethoxyflufenican, bensulfuron-methyl, quizalofop-P-ethyl, methoxyflufenican, ethoxyflufenican, trifluralin or sodium salt, methoxyflufenican, ethoxyfen, chlorofluorofen, fluoronitrofen, furyloxyfen, nitrofluorfen, halosafen. (3) Phenylated pyrazoles include: imidacloprid, isopyrazosulfuron, and ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxo]propionate; (4) N-phenylimides include: propyzamide, indole-3-methylpropylate, flumipropyn, flumethrin, chlorophthalim, and N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide. (5) Thiadiazoles include: methyl methazine, methazine, and thiamethoxam; (6) Oxadiazoles include: propyzil and oxadiazine; (7) Triazoline ketones include: oxadiazon, oxadiazon / oxadiazon, mesotrione, oxadiazon, and oxadiazon; (8) Oxazolidinones include: cyclopentoxane; (9) Others include: bispyribac-methyl, flupyridaben, cyclopyranil, flupyribac-methyl, trifluralin, phenopylate, N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide N-Tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide, 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thio-[1,3,5]triazine-2,4-dione, 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7- Tetrahydro-isoindole-1,3-dione, (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoic acid methyl ester, 3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]ethyl acetate, 1,5-dimethyl-6-thio-3-(2,2,7-trifluoro-3-oxo-4-(propyl-2-) (-alkynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione, 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-alkynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidin-2,4-dione, 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidin-2,4-dione, 13. The method according to claim 11 or 12, wherein the PPO inhibitory herbicide is applied in an amount that does not harm the plant; preferably, the PPO inhibitory herbicide compound is applied to the plant cultivation site at a ratio of about 0.02 g ai / ha to about 2000 g ai / ha, about 1 g ai / ha to about 1200 g ai / ha, about 15 g ai / ha to about 800 g ai / ha, about 30 g ai / ha to about 500 g ai / ha, or about 50 g ai / ha to about 200 g ai / ha.

14. The method according to any one of claims 11-13, wherein the specific formulation of the PPO inhibitory herbicide compound is a dispersible oil suspension, an aqueous suspension, a suspension emulsion, a wettable powder, an emulsifiable concentrate, a water-dispersible granule, an emulsion, or a microemulsion.

15. The method according to any one of claims 11-14, wherein the application of the PPO suppressant herbicide is carried out before emergence, or after emergence, and the application of the PPO suppressant herbicide comprises contacting the plant with the PPO suppressant herbicide, or the application of the PPO suppressant herbicide comprises soil sealing, foliar spraying, or hydroponic application.

16. The application according to claim 8 or the method according to any one of claims 10-15, wherein the plant is cultivated by direct seeding in water, direct seeding in dry land, or transplanting; preferably, the plant is rice.

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