Active polypeptide, method for improving tolerance of crop to arylpicolinate herbicide, and use thereof

Abstract

Disclosed in the present invention are an active polypeptide, a method for improving the tolerance of a crop to an arylpicolinate herbicide, and a use thereof. The amino acid sequence of the active polypeptide is as shown in SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4. In the present invention, by introducing into the crop a heterologous protein having 90% or more amino acid sequence identity to the active polypeptide, a transgenic crop that is tolerant to at least twice the recommended dose of florpyrauxifen-benzyl or halauxifen-methyl is obtained. The transgenic crop of the present invention can also express a gene conferring tolerance to another herbicide, so that the transgenic plant has tolerance to a variety of herbicides. Thus, an arylpicolinate herbicide can be used alone for field weeding, and a compounded herbicide of the arylpicolinate herbicide and other herbicides (such as glyphosate and nicosulfuron) can also be used for field weeding, so as to obtain a better weeding effect and a better protection time. The method of the present invention can effectively reduce the probability of the occurrence of resistant weeds.

Description

Active polypeptide and method for improving crop tolerance to arylpicolinic acid ester herbicide and application thereof (I)TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an active polypeptide, a method for obtaining a transgenic arylpicolinic acid ester herbicide-tolerant crop and application thereof in weed control. (II)BACKGROUND

[0002] Weeds interfere with the growth and development of crops, and breed various diseases and pests, affecting the yield and quality of crops. In the 1940s, with the discovery of auxin herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (dimethyl tetrachloride), the mode of weed management has undergone a great change; the use of chemical herbicides has rapidly improved the efficiency of weed control in the process of agricultural production and effectively reduced the cost. At the same time, crops can be improved by genetic engineering to obtain resistance to herbicides, for example, crops can obtain resistance to glyphosate by transgenic expression of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, long-term continuous high-dose use of single variety or single mode of action of chemical herbicides is prone to cause problems such as weed tolerance and resistance evolution. In order to control the occurrence of herbicide resistance of weeds, improve the resistance level of transgenic crops and increase the diversity of resistance genes, it is very useful in production to develop diversified herbicide-resistant transgenic crops.

[0003] Cytochrome P450 is considered as an important protein involved in the degradation metabolism of pesticides, including herbicides. Cytochrome P450 family contains a very large number of members, at least 1000 P450 sequences are known to be disclosed, but the function of most P450s is still unknown. In the past 20 years, 30 cytochrome P450 proteins from different plant sources have been proved to have the function of metabolizing herbicides (Dimaano, N. G. and Iwakami, S. Cytochrome P450-mediated herbicide metabolism in plants: current understanding and prospects. Pest Manag Sci, 2021, 77: 22-32.). The current research found that P450 protein metabolizes herbicides with the following characteristics: (1) P450s of different families can metabolize similar herbicides; (2) a P450 gene can be involved in the metabolism of multiple herbicides from similar or different chemical mechanism families; (3) cytochrome P450 monooxygenases (P450s) involved in biosynthetic pathways can use herbicides as their substrates. In this regard, we can believe that even if a plant cytochrome P450 has a certain degradation metabolism ability for a pesticide, its degradation metabolism activity cannot necessarily reach the level that can be heterologously expressed in other plants to make the plants obtain sufficient tolerance to this herbicide.

[0004] In 1994, the first P450s (SU1 and SU2) genes involved in herbicide metabolism were reported to be not derived from plants, but isolated from bacteria and mammals, and expression of the SU1 (CYP105A1) gene in tobacco could be involved in the metabolism of sulfonylurea herbicides (O’Keefe, D. P. et al. Plant expression of a bacterial cytochrome P450 that catalyses activation of a sulfonylurea pro-herbicide. Plant Physiol. 1994, 105, 473-482). The first class of reported plant-derived P450s were CYP76B1 and CYP71A10, derived from Jerusalem artichoke (Helianthus tuberosus) and soybean, respectively, and had metabolic effects on phenylurea herbicides. A cytochrome P450 protein Cyp81A6 from rice was found to have detoxification function on bensulfuron and sulfonylurea herbicides (US8049063B2; Pan, G., Zhang, X., Liu, K. et al. Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides. Plant Mol Biol. 2006, 61, 933-943.). A cytochrome P450 protein NSF1 from maize was found to have metabolic detoxification ability on herbicides such as bensulfuron and nicosulfuron (CN200610155661; Pataky, J. K., Williams, M., Riechers, D. E. et al. A common genetic basis for cross-sensitivity to mesotrione and nicosulfuron in sweet corn hybrid cultivars and inbreds grown throughout North America. J. Am. Soc. Hortic. Sci. 2009, 134, 252-260.).CdP450 cloned from Bermuda Grass (Cynodon dactylon) has detoxification function for multiple herbicides such as nicosulfuron and pyrimisulfan (US9657307B2; Zheng, T., Yu, X., Sun, Y. et al. Expression of a Cytochrome P450 Gene from Bermuda Grass Cynodon dactylon in Soybean Confers Tolerance to Multiple Herbicides. Plants 2022, 11, 949.). There are literatures showing that P450 enzymes in wheat are involved in the metabolism of multiple herbicides such as tembotrione (Forthoffer, N., Helvig, C., Dillon, N. et al. Induction and inactivation of a cytochrome P450 conferring herbicide resistance in wheat seedlings. Eur. J. Drug Metab. Pharmacokinet. 26, 9-16 (2001); S. Sudhakar, S. Nakka, A. Mohammad et al. Metabolism of Tembotrione, a Triketone Herbicide, confers Differential Sensitivity in Winter Wheat (Triticum aestivum). Journal of Agricultural and Food Chemistry 2024 72(13), 6931-6941.).

[0005] The novel aryl pyridine carboxylate herbicide is the latest family of hormone herbicides, and halauxifen-methyl and pyrifluquinazon are the two members of this family. They are new herbicides developed by introducing aryl groups into the pyridine carboxylic acid structure, which can also be regarded as the optimization and derivation of the pyridine carboxylic acid structure, and still retain the characteristics of hormone herbicides. Compared with other hormone herbicides, halauxifen-methyl and pyrifluquinazon have lower dosage, wider weed spectrum and novel mechanism of action, and are friendly to the environment.

[0006] Halauxifen-methyl is the first product in the new chemical type of aryl pyridine acid in synthetic auxin herbicides, which can be absorbed through the stems, leaves and roots of plants, bind to hormone receptors in the plant body, stimulate excessive cell division, block the conduction tissue, and finally lead to the death of the plant due to nutrient depletion.

[0007] Florpyrauxifen-benzyl is a currently used hormone herbicide in rice fields, which has good control effect on gramineous weeds, sedge weeds and broadleaf weeds. Florpyrauxifen-benzyl can provide good control effect at very low dosage, and the effective dosage is as low as 5-50 g a.i. / ha according to different weed species and application methods. Florpyrauxifen-benzyl acts on the two most important sites in the target site of hormone herbicides, TIR1 and AFB5, and has a unique binding mode with the receptors, especially with AFB5. Florpyrauxifen-benzyl is different from the receptors of traditional synthetic hormone herbicides, and thus has no cross-resistance with such herbicides, and also has no cross-resistance with herbicides of other action mechanisms. Florpyrauxifen-benzyl has a novel action mechanism, and can control weeds resistant to glyphosate, propanil, triazine herbicides, acetyl-lactate-synthase (ALS) inhibitors, acetyl-coenzyme A carboxylase (ACCase) inhibitors, hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors and other synthetic hormone herbicides. The dosage of florpyrauxifen-benzyl used in rice has obvious harmful effect on corn, and is highly toxic to soybean; therefore, florpyrauxifen-benzyl cannot be used as a main herbicide for controlling weeds in corn and soybean at present.

[0008] In fact, there is no known protein that can metabolize florpyrauxifen-benzyl and fluorpyrauxifen, and there is no report on a method for improving the tolerance of crops to florpyrauxifen-benzyl and fluorpyrauxifen by transgenic technology and application.

[0009] The present application provides a method for improving the resistance of crops to florpyrauxifen-benzyl and fluorpyrauxifen. The technology introduces a heterologous protein (cytochrome P450) into crops to obtain crops with high tolerance to florpyrauxifen-benzyl and fluorpyrauxifen, and a weed control method for these crops. Further, the present application provides a method for crops tolerant to florpyrauxifen-benzyl and fluorpyrauxifen, and also tolerant to glyphosate, nicosulfuron, pyrimisulfan and other herbicides with different mechanisms, and a weed control method for these crops tolerant to multiple herbicides. (Three) summary of the application

[0010] The application aims to provide an active polypeptide and a method and application for improving the tolerance of crops to arylpicolinic acid ester herbicides. Expression of a polypeptide fragment having more than 90% amino acid sequence identity with the active polypeptide in crops can make the transgenic plants at least tolerate 2 times the recommended dose of picolinafen or flurochloridone, and the polypeptide fragment includes cytochrome P450 of different sources, such as rice Cyp81A6, CdP450, TaP450, HvP450 or recombinant cytochrome P450 containing the DNA molecule of the active polypeptide. Expression of other herbicide-resistant genes in crops can obtain crops resistant to multiple herbicides, such as expression of CdP450 protein in corn can obtain transgenic corn resistant to picolinafen, flurochloridone, nicosulfuron, foramsulfuron, chlorthal-dimethyl, and mesotrione. Expression of rice Cyp81A6 protein in soybean can obtain transgenic soybean with high tolerance to picolinafen. The application provides more options for weed control methods in crops, and also provides a method for controlling weeds resistant to other different types of herbicides in crop fields.

[0011] The technical scheme adopted by the application is:

[0012] In a first aspect, the application provides an active polypeptide for improving the tolerance of crops to arylpicolinic acid ester herbicides, wherein the amino acid sequence of the active polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4; and the arylpicolinic acid ester herbicide includes picolinafen and flurochloridone. Expression of a heterologous protein having more than 90% amino acid sequence identity with the active polypeptide in plants can improve the tolerance of the plants to picolinafen and flurochloridone, and the expression of the heterologous protein is to functionally connect the transcribable DNA sequence of the heterologous protein with a promoter that can initiate the expression of the heterologous protein in plants, and then transfer the promoter into the plant genome.

[0013] The active polypeptide (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) provided by the present application is a characteristic sequence of the heterologous protein of the present application related to the arylpicolinic acid ester herbicide tolerance. Those skilled in the art can search for proteins having more than 90% amino acid sequence identity with the active polypeptide by using database, thereby obtaining the heterologous protein of the present application related to the arylpicolinic acid ester herbicide tolerance. Those skilled in the art can also clone the DNA sequence encoding the heterologous protein of the present application from plants by traditional gene cloning method using the sequence conservation of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. The amino acid identity can be obtained by the existing method, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:3364; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0014] In the second aspect, the present application provides a method for obtaining arylpicolinic acid ester herbicide-tolerant plants using the active polypeptide, which comprises: 1) expressing in plants a heterologous protein comprising a polypeptide fragment having more than 90% amino acid sequence identity with the active polypeptide; 2) selecting transgenic plants with significantly improved tolerance to arylpicolinic acid ester herbicides.

[0015] Further, the amino acid sequence of the heterologous protein in step 1) comprises one of SEQ ID NO: 5-13, preferably one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 13.

[0016] It is known that expression of a protein in a plant is a mature technology. Generally, a promoter functional in a plant and a DNA encoding a protein of interest are functionally linked, and a terminator functional in a plant is linked at the 3' end to form a heterologous gene expression cassette that can be expressed in a plant. The expression cassette can be further linked to a selection gene expression cassette to construct a T-DNA. The T-DNA is introduced into the genome of a recipient plant by an Agrobacterium-mediated method. The plant-expressible promoter can be, but is not limited to, one of the following: a constitutive promoter, a ZmUbi promoter, an AtUbi promoter, an Actin promoter, a CaMV35S promoter, a CsVMV promoter, a FMV promoter, a CMP promoter, and the like. The gene expression cassette of the selection gene is artificially synthesized, and the selection gene can be one of the following: a CP4-EPSPS gene, a G10evo-EPSPS gene, a PPO gene, a Bar gene, a Pat gene, and the like.

[0017] Further, step 2) selects transformants with significantly increased tolerance to aryloxyphenoxypropionate herbicides by spraying the transformants with aryloxyphenoxypropionate herbicides, preferably transgenic plants with a level of tolerance to aryloxyphenoxypropionate herbicides of at least 30 grams of active ingredient per hectare.

[0018] In a third aspect, the present application provides a plant cell containing a gene encoding the active polypeptide, the plant cell expressing a heterologous protein containing a polypeptide fragment having an amino acid sequence identity of more than 90% of the active polypeptide (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) and improving the metabolism of aryloxyphenoxypropionate herbicides.

[0019] The genome of the plant cell is heritable and includes any genetic material, such as nuclear, plastid, and mitochondrial genomes.

[0020] In a fourth aspect, the present application provides a method for obtaining a recombinant cytochrome P450 protein with tolerance to aryloxyphenoxypropionate herbicides using the active polypeptide, the method comprising: 1) replacing a corresponding polypeptide fragment in the cytochrome P450 protein with the active polypeptide (a polypeptide fragment of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) or a polypeptide having an amino acid sequence identity of more than 90% of the active polypeptide; and 2) selecting a recombinant cytochrome P450 protein with activity in metabolizing aryloxyphenoxypropionate herbicides.

[0021] The present application discloses for the first time that the active polypeptide (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) or a polypeptide fragment having more than 90% amino acid sequence identity with the active polypeptide can replace the corresponding amino acid fragment of other cytochrome P450 which originally does not have the activity of resisting aryl pyridine carboxylate herbicides, so as to enable the cytochrome P450 to have the activity of resisting aryl pyridine carboxylate herbicides. It is illustrated that the active polypeptide (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) of the present application is a key region (referred to as an active region) in cytochrome P450 which determines the activity of resisting aryl pyridine carboxylate herbicides. Therefore, the active polypeptide of the present application has important value in discovering and judging whether a cytochrome P450 has resistance to aryl pyridine carboxylate herbicides. A person skilled in the art can obtain the amino acid sequence of cytochrome P450 through database search, literature acquisition and the like, and then compare the amino acid sequences and homology of different cytochrome P450s through a commonly used alignment program (such as commonly used molecular biology software such as Vector NTI, Snapgene, etc.). In theory, any cytochrome P450 can find the corresponding sequence of the active region provided by the present application and replace it through the alignment program. The corresponding sequence is determined by aligning the amino acid sequence of the target protein with the active polypeptide (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) or a polypeptide fragment having more than 90% amino acid sequence identity with the active polypeptide using a standard sequence alignment tool, for example, using the Smith-Waterman operation algorithm or the CLUSTALW2 operation algorithm to align two sequences, wherein the sequences are considered to be aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, W. J. and Lipman, D. J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. In the ClustalW2 (1.82) operation 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.

[0022] In a fifth aspect, the present application provides a method for obtaining plants that are simultaneously tolerant to arylpicolinic acid ester herbicides and glyphosate by using the active polypeptide, the method comprising: 1) simultaneously expressing in plants a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide and a glyphosate-tolerant EPSPS protein; and 2) selecting transgenic plants that have significantly improved tolerance to both arylpicolinic acid ester herbicides and glyphosate.

[0023] Further, the amino acid sequence of the heterologous protein comprises one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, preferably one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13.

[0024] In a sixth aspect, the present application provides a method for obtaining plants that are simultaneously tolerant to multiple herbicides by using the active polypeptide, the method comprising: 1) expressing in plants a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide, and simultaneously expressing another herbicide-tolerant gene-encoding protein; and 2) selecting transgenic plants that have significantly improved tolerance to both arylpicolinic acid ester herbicides and another herbicide.

[0025] Further, the amino acid sequence of the heterologous protein comprises one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, preferably one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13.

[0026] Further, the other herbicide-tolerant gene includes a glyphosate-tolerant EPSPS gene, a PPO inhibitor herbicide-tolerant PPO gene, a glufosinate-tolerant Pat gene, or a glufosinate-tolerant Bar gene. The glyphosate-tolerant EPSPS gene, such as the CP4-EPSPS gene, has been widely used in transgenic corn and soybean plants (Padgette S R, Re D B, Barry G F, et al. New Weed Control Opportunities: Development of Soybeans With A Roundup Ready Gene [M]. 2018.); the PPO inhibitor-tolerant PPO gene has also been reported (US10370677B2); and the glufosinate-tolerant Pat and Bar genes have been widely used. With the corresponding herbicide, transgenic plants with tolerance to multiple herbicides can be selected.

[0027] In a seventh aspect, the present application provides a method for controlling weeds in a plant growth area using the active polypeptide, which comprises spraying an aryl pyridine carboxylate herbicide on a field in which the transgenic plant with significantly improved tolerance to aryl pyridine carboxylate herbicides is planted; the transgenic plant comprises a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide.

[0028] In an eighth aspect, the present application provides a method for controlling multiple weeds in a plant growth area using the active polypeptide, which comprises spraying a complex herbicide on a field in which the transgenic plant is planted; the transgenic plant simultaneously expresses a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide, and a protein with tolerance to other herbicides; the herbicide in the other herbicide-tolerant gene includes one or two of glyphosate, nicosulfuron, pyrimethanil, chlorpyrifos, atrazine, mesotrione, or benzobacotron; the complex herbicide refers to an aryl pyridine carboxylate herbicide and one or more of the following herbicides: glyphosate, nicosulfuron, pyrimethanil, chlorpyrifos, atrazine, mesotrione, or benzobacotron.

[0029] Further, the complex herbicide is a complex herbicide of one of the pyrimethanil or the fluroxypyr and glyphosate; a complex herbicide of one of the pyrimethanil or the fluroxypyr and benzobacotron; a complex herbicide of one of the pyrimethanil or the fluroxypyr and nicosulfuron; a complex herbicide of one of the pyrimethanil or the fluroxypyr and atrazine; and the like.

[0030] In a ninth aspect, the present application provides a method for controlling weeds in a field where transgenic maize is grown, the method comprising spraying an arylpyridine carboxylate herbicide and at least one of the following herbicides: glyphosate, nicosulfuron, azimsulfuron, chlorpyrifos, mesotrione, or carfentrazone-ethyl on a field where transgenic maize heterologously expressing a heterologous protein having a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide and a glyphosate-tolerant EPSPS protein is planted, wherein the amino acid sequence of the heterologous protein having the polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide is shown in SEQ ID NO: 6.

[0031] In a tenth aspect, the present application also provides a method for controlling weeds in a field where transgenic soybean is grown, the method comprising spraying an arylpyridine carboxylate herbicide and at least one of the following herbicides: glyphosate, nicosulfuron, azimsulfuron, or chlorpyrifos on a field where transgenic soybean heterologously expressing a heterologous protein having 90% or more amino acid sequence identity to the active polypeptide and a glyphosate-tolerant EPSPS protein is planted, wherein the amino acid sequence of the heterologous protein having 90% or more amino acid sequence identity to the active polypeptide is shown in SEQ ID NO: 6.

[0032] In an eleventh aspect, the present application provides a method for obtaining a plant that is tolerant to an arylpyridine carboxylate herbicide and at least another herbicide, the method comprising: 1) heterologously expressing in a plant a heterologous protein having a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide to obtain a transgenic plant that is tolerant to an arylpyridine carboxylate herbicide; 2) crossing the transgenic plant of step 1) with a second plant that is tolerant to the at least another herbicide; and 3) selecting a progeny plant that is tolerant to the arylpyridine carboxylate herbicide and the at least another herbicide from the cross.

[0033] Further, the present application provides a method for controlling weeds, the method comprising planting the progeny plant resulting from the cross in a crop growing environment and spraying an arylpyridine carboxylate herbicide and at least another herbicide.

[0034] Further, the at least another herbicide is selected from any one of the following herbicides: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, an HPPD inhibitor, a PPO inhibitor, and a long-chain fatty acid inhibitor.

[0035] Further, the plant includes soybean, maize, sunflower, peanut, oilseed rape, wheat, pasture, and the like.

[0036] Both flurochloridone and chlormidone are developed from pyridine carboxylic acid structure with aryl group introduced. Flurochloridone has a chemical name of 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)pyridine-2-carboxylic acid methyl ester, and chlormidone has a chemical name of 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)-5-fluoropyridine-2-carboxylic acid benzyl ester. When used as herbicides, the active substances actually acting in plants of flurochloridone and chlormidone are flurochloridone acid (4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)pyridine-2-carboxylic acid) and chlormidone acid (4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)-5-fluoropyridine-2-carboxylic acid) respectively (Epp J B, Alexander A L, Balko T W, et al. The Discovery of ArylexTM Active and RinskorTM Active: Two Novel Auxin Herbicides. Bioorganic & Medicinal Chemistry, 2015, 24(3): 362-371.), and other derivative compounds of flurochloridone acid and chlormidone acid are also within the protection scope of the present application.

[0037] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to polymers of amino acid residues, some or all of which can be artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The polypeptides of the present application can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques.

[0038] The term "heterologous" as used herein refers to from another source. In the context of DNA, "heterologous" refers to any foreign "non-self" DNA, including DNA from another plant of the same species.

[0039] The promoter which can initiate its expression in plants as used herein refers to a promoter which ensures the expression of a transcribable DNA sequence connected therewith in plant cells, and the promoter which can be expressed in plants can be a constitutive promoter, a tissue-specific promoter, etc. Examples of the promoter which guides the expression in plants include, but are not limited to, a 35S promoter derived from cauliflower mosaic virus, a plant ubi promoter, an actin promoter, an FMV promoter, a CMP promoter, a CsVMV promoter, etc.

[0040] As used herein, "plant" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue culture from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.

[0041] As used herein, "transgenic methods" means that the transformation can occur by direct transformation methods such as Agrobacterium-mediated transformation of plant tissue, microprojectile bombardment, electroporation, and the like; or by crossing a plant having a heterologous nucleotide sequence with another plant such that the progeny have the nucleotide sequence incorporated into their genome. Such breeding techniques are well known to those of skill in the art.

[0042] As used herein, "herbicide tolerance" or "herbicide-tolerant plant, plant tissue or cell" means the ability of a plant, plant tissue or cell to withstand the effects of a herbicide when applied. For example, a herbicide-tolerant plant can survive or continue to grow in the presence of a herbicide. Herbicide tolerance of a plant, plant tissue or cell can be measured by comparing the plant, plant tissue or cell to a suitable control. For example, herbicide tolerance can be measured or assessed by applying a herbicide to a plant comprising a DNA molecule encoding a protein capable of conferring herbicide tolerance (test plant) and a plant not comprising a DNA molecule encoding a protein capable of conferring herbicide tolerance (control plant), and then comparing the plant damage of the two plants, wherein herbicide tolerance of the test plant is indicated by a reduction in injury as compared to the injury of the control plant. A herbicide-tolerant plant, plant tissue or cell exhibits a reduced response to the toxic effects of a herbicide as compared to a control plant, plant tissue or cell. The "herbicide tolerance trait" refers to a transgenic trait that confers improved herbicide tolerance to a plant as compared to a wild-type plant. Plants having the herbicide tolerance trait of the present application that can be produced include, for example, any plant, including crop plants such as soybean, corn, sunflower, peanut, canola, wheat, and the like.

[0043] The "resistance" in the present invention is heritable and allows the plant to grow and reproduce in the presence of the herbicide under conditions in which the herbicide would normally be effective in killing the given plant. As recognized by those skilled in the art, a plant can be considered "resistant" even if the given plant is damaged to some extent by the herbicide treatment, such as by a small amount of necrosis, lysis, chlorosis, or other damage, but at least not significantly in terms of yield, i.e., the given plant has an increased ability to resist the various levels of damage induced by the herbicide that would normally result in damage to a wild-type plant of the same genotype under the same herbicide dose. The "tolerance" or "resistance" in the present invention is broader than the term "resistance" and includes "resistance".

[0044] The "weeds" in the present invention refer to plants that compete with the cultivated transgenic plants in the environment in which the plants are growing.

[0045] The "control" and / or "prevention" in the present invention refers to the direct application (e.g., by spraying) of an effective amount of fluroxypyr or halosafen to the environment in which the plants are growing to minimize and / or stop the growth of weeds. At the same time, the cultivated transgenic plants should be morphologically normal and can be cultivated under conventional methods for the consumption and / or production of products; preferably, with reduced plant damage and / or with increased plant yield as compared to non-transgenic wild-type plants.

[0046] The method of weed control in the present invention can be performed by using a plurality of herbicides with different mechanisms of action separately, in mixture, or alternately on the herbicide-resistant transgenic plants provided in the present invention.

[0047] The present invention can impart a new herbicide resistance trait to plants and no adverse effects on the phenotype, including yield, have been observed. The plants in the present invention can tolerate at least 2-fold or more of the recommended dose of the subject herbicide. These increased levels of tolerance are within the scope of the present invention.

[0048] Compared to the prior art, the present invention has the following advantages:

[0049] 1. The present invention provides an active polypeptide, by introducing a heterologous protein having 90% or more of the same amino acid sequence of the active polypeptide into crops, a new transgenic crop resistant to aryl pyridine carboxylate herbicides (including fluroxypyr and halosafen) is obtained; the new transgenic crop obtained in the present invention can at least tolerate 2-fold recommended dose of fluroxypyr or halosafen.

[0050] 2、The transgenic crops resistant to the aryloxyphenoxypropionate herbicide according to the application can express resistance to another herbicide gene at the same time, so that the transgenic plants have multiple herbicide resistance, and can be used for field weeding by using only fluroxypyr or fluorodifen, or can be used for field weeding by using a compound herbicide of fluroxypyr and other herbicides (such as glyphosate, nicosulfuron, etc.), so that better weeding effect and protection time limit are obtained.

[0051] 3、The method according to the application can effectively reduce the probability of occurrence of resistant weeds. The transgenic crops obtained by the application are resistant to multiple herbicides of different mechanisms such as fluroxypyr, fluorodifen, glyphosate, nicosulfuron, chlorpyrifos-methyl, mesotrione and benzobicyclon, and have broad-spectrum herbicide resistance; in the process of weed control, the use of fluroxypyr in combination with other herbicide(s) can not only effectively reduce the cost and improve the control efficiency, but also greatly avoid the occurrence of weed resistance. (IV) DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a phenotype photo of transgenic corn and control corn after spraying a medium dose of fluroxypyr in Example 2.

[0053] Figure 2 is a phenotype photo of transgenic corn and control corn after spraying 2 times of medium dose of fluroxypyr and 2 times of medium dose of nicosulfuron in Example 5.

[0054] Figure 3 is an amino acid sequence alignment chart of Example 6.

[0055] Figure 4 is an amino acid sequence alignment chart of Example 8.

[0056] Figure 5 is an amino acid sequence alignment chart of Example 9. (V) DETAILED DESCRIPTION

[0057] The application will be further described below in conjunction with specific examples, but the protection scope of the application is not limited to this:

[0058] The molecular biology and biochemistry methods used in the following examples of the application are known technologies. Detailed descriptions can be found in Current Protocols in Molecular Biology published by John Wiley and Sons, Inc. edited by Ausubel, and Molecular Cloning: A Laboratory Manual, 3rd ED. published by Cold Spring Harbor Laboratory Press (2001) edited by J. Sambrook et al.

[0059] The amino acid sequence of the active polypeptide of the application is:

[0060] SEQ ID NO: 1 (OsP450core)

[0061] SEQ ID NO: 2 (CdP450core)

[0062] SEQ ID NO: 3 (TaP450core)

[0063]

[0064] SEQ ID NO: 4 (HvP450core)

[0065] The heterologous proteins of the present application containing active polypeptides: SEQ ID NO: 5 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 6 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 2; SEQ ID NO: 7 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 8 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 9 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 10 has more than 88% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 11 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 1; SEQ ID NO: 12 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 3; SEQ ID NO: 13 has more than 90% amino acid sequence identity to the active polypeptide described in SEQ ID NO: 4.

[0066] Example 1, obtaining and herbicide tolerance analysis of transgenic corn expressing the active polypeptide described in SEQ ID NO: 1

[0067] 1. Recombinant Agrobacterium

[0068] (1) heterologous gene expression frame: since the active polypeptide shown in SEQ ID NO: 1 is from amino acids 101-170 in SEQ ID NO: 5, the transcribable DNA sequence of rice cytochrome P450 protein Cyp81A6 (amino acid sequence as shown in SEQ ID NO: 5, corresponding transcribable DNA sequence as shown in SEQ ID NO: 17) is selected, and functionally linked with maize ubiquitin promoter pZmUbi (SEQ ID NO: 26) and Tnos terminator (SEQ ID NO: 27). The promoter-transcribable DNA-terminator fragment is artificially synthesized to form a heterologous gene expression frame.

[0069] (2) screening gene expression frame: the 5' end of the screening gene CP4-EPSPS (SEQ ID NO: 25) is linked with maize ubiquitin promoter pZmUbi (SEQ ID NO: 26), and the 3' end is linked with cauliflower mosaic virus 35s terminator TCaMV35S (SEQ ID NO: 29). The pZmUbi-CP4-EPSPS-TCaMV35S fragment is artificially synthesized to form a screening gene expression frame.

[0070] (3) transformation vector: the vector pCambia1300 (NCBI sequence number AF234296) is double digested with Hind III / Xho I to recover a 6783 bp fragment, which is recombined with the two gene expression frames described above by the method of seamless cloning to form a recombinant DNA construct, which is transformed into E. coli TG1 strain, and sequenced to obtain the transformation vector ZA.

[0071] (4) Agrobacterium transformation: the plasmid ZA is electroporated into Agrobacterium EHA105 competent cells, and after correct enzyme digestion identification, it is stored in glycerol bacteria to obtain recombinant Agrobacterium for crop transformation.

[0072] 2. Obtaining of transgenic maize

[0073] Agrobacterium-mediated method is used for maize genetic transformation. Specifically, the method and medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13-22) are used for transformation, and glyphosate is used as a screening reagent. The steps are as follows:

[0074] (1) The recombinant Agrobacterium containing the transformation vector ZA obtained in step 1 is mixed in the infection medium, and the bacterial solution concentration OD660 is adjusted to 0.5-0.6, which is the infection solution containing Agrobacterium.

[0075] (2) Take the common maize ear 8-10 days after pollination, collect the immature embryo with the size of 1.0-1.5mm. Immerse the collected immature embryo in the infection solution containing Agrobacterium of step (1), stand at room temperature for 5min, take out the embryo, suck dry the liquid, place the embryo with the flat surface downward on the co-culture medium, cultivate at 22°C for 3-5 days.

[0076] (3) Transfer the immature embryo cultivated in step (2) to the callus induction medium containing the final concentration of 200mg / L antibiotic of Basta (GlaxoSmithKline, USA), cultivate at 28°C in dark for 10-14 days to kill Agrobacterium.

[0077] (4) Transfer all the callus induced in step (3) to the screening medium containing the final concentration of 2mM glyphosate, cultivate at 28°C in dark for 2-3 weeks. After the induction, transfer all the callus to the fresh screening medium containing 2mM glyphosate, cultivate at 28°C in dark for 2-3 weeks.

[0078] (5) Transfer the viable embryogenic tissue of step (4) to the regeneration medium, cultivate at 28°C in dark for 10-14 days, then transfer to the fresh regeneration medium, cultivate at 26°C in light for 10-14 days.

[0079] (6) Pick the completely developed plant of step (5) to the rooting medium, cultivate at 26°C in light until the root is completely developed, transplant the regenerated plant after rooting to the greenhouse for growth and cultivation. Obtain 93 T0 generation transgenic maize ZA.

[0080] 3. Screening of transgenic maize resistant to flumioxazin

[0081] After transplanting the 93 T0 generation transgenic maize ZA obtained above to the greenhouse, when the plant grows to the stage of 3-5 leaves, spray the transgenic maize with medium dose of flumioxazin (30g a.i. / ha), and preliminarily screen the excellent transformant with resistance. 7 days after the treatment, observe the growth condition of the plant, as shown in Table 1.

[0082] Table 1. Growth condition of transformant 7 days after the treatment

[0083] 4. Herbicide tolerance test of transgenic maize

[0084] Three of the above-mentioned transformants with excellent resistance performance (without any phytotoxicity) were selected (denoted as ZA1, ZA2, and ZA3) for further herbicide tolerance tests. The T1 generation seeds of the transgenic corn ZA1, ZA2, and ZA3 and common corn (as a control) were taken, a randomized block design was adopted, 3 repetitions were performed, each plot had an area of 4 m x 6 m, double-grain sowing was performed, the plant spacing was 25 cm, the row spacing was 50 cm, 1 m interval was provided between plots, herbicides were sprayed at the 3-5 leaf stage, and the treatments were as follows: (1) blank solvent (water); (2) medium dose of flurochloridone (30 g a.i. / ha); (3) 2 times the medium dose of flurochloridone (60 g a.i. / ha); (4) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha); and (5) 2 times the medium dose of flurochloridone (60 g a.i. / ha) + 2 times the medium dose of glyphosate (1800 g a.i. / ha). The injury rates were calculated according to the growth conditions of the plants at 3 days, 7 days, and 14 days after the treatment, respectively. The results are shown in Table 2.

[0085] Compared with the growth conditions of untreated plants, if any one of the following symptoms occurs, such as plant lodging, base deformation, stem yellowing, leaf yellowing, leaf shrinkage, plant deformation, plant swelling, and plant death, the plant is determined to be injured.

[0086] The injury rate = the number of injured plants / the total number of plants * 100%.

[0087] Table 2, herbicide tolerance test results

[0088] The results show that the control corn has a plant injury rate of more than 90% at 3 days after spraying a single herbicide (flurochloridone), and the injury rate reaches 100% at 7 days after spraying; all the plants are injured at 3 days after spraying a composite herbicide (flurochloridone + glyphosate). Compared with the control corn, the transgenic corn does not have plant injury, regardless of whether a single herbicide or a composite herbicide is sprayed, which indicates that the transgenic corn of the present embodiment is at least tolerant to 2 times the medium dose of flurochloridone and glyphosate. The results can indicate that if a polypeptide fragment protein having an amino acid sequence of 90% or more of SEQ ID NO: 1 (preferably SEQ ID NO: 5, including but not limited to) is expressed in corn, the tolerance of the corn to flurochloridone can be effectively enhanced; if a glyphosate-tolerant EPSPS protein or other herbicide-tolerant protein is also expressed, the tolerance of the corn to the composite herbicide can be enhanced. It is indicated herein that the herbicide used in combination with flurochloridone includes but is not limited to glyphosate.

[0089] Example 2, obtaining and herbicide tolerance analysis of corn expressing the active polypeptide of SEQ ID NO: 2 Since the active polypeptide of SEQ ID NO: 2 is selected from the amino acids 105-174 in SEQ ID NO: 6, a cytochrome P450 protein CdP450 from Cynodon dactylon (amino acid sequence as SEQ ID NO: 6, corresponding to transcribable DNA sequence preferably SEQ ID NO: 18) is selected; the promoter is changed to a plant-expressible promoter pActin (SEQ ID NO: 30), and recombinant Agrobacterium and corn transformation are constructed according to the method and conditions of Example 1, obtaining 81 T0 generation transgenic corn ZB.

[0090] After the obtained 81 T0 generation transgenic corn ZB is transplanted to the greenhouse, when the plants grow to 3-5 leaf stage, the transgenic corn is sprayed with medium dose of flurochloridone (30 g a.i. / ha), and the transgenic plants with excellent resistance are preliminarily screened. 7 days after the treatment, the plant growth conditions are observed, as shown in Table 3.

[0091] Table 3, growth conditions of the transgenic plants 7 days after the treatment

[0092] Three transgenic plants (denoted as ZB1, ZB2, ZB3) with excellent resistance performance (no any phytotoxicity) are selected for further herbicide tolerance test. The seeds of T1 generation transgenic corn ZB1, ZB2, ZB3 and common corn (as a control) are taken, a randomized block design is adopted, 3 repetitions are performed, each plot area is 4 m x 6 m, double-seed sowing is adopted, the plant spacing is 25 cm, the row spacing is 50 cm, 1 m interval is set between the plots, herbicides are sprayed at 3-5 leaf stage, and the treatments are as follows: (1) blank solvent (water); (2) medium dose of flurochloridone (30 g a.i. / ha); (3) 2 times medium dose of flurochloridone (60 g a.i. / ha); (4) medium dose of flurochloridone (10 g a.i. / ha); (5) 2 times medium dose of flurochloridone (20 g a.i. / ha); (6) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha); (7) 2 times medium dose of flurochloridone (60 g a.i. / ha) + 2 times medium dose of glyphosate (1800 g a.i. / ha); (8) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of carfenazone (35 g a.i. / ha); (9) 2 times medium dose of flurochloridone (60 g a.i. / ha) + 2 times medium dose of carfenazone (70 g a.i. / ha). The injury rates are calculated according to the plant growth conditions at 3 days, 7 days and 14 days after the treatment, respectively. The results are shown in Table 4, and the plant phenotype sprayed with 2 times dose of flurochloridone is shown in FIG. 1.

[0093] If any one of the following symptoms occurs, such as plant lodging, base deformity, stem yellowing, leaf yellowing, leaf shriveling, plant deformity, plant swelling, plant death, etc., compared with the growth status of untreated plants, it is determined that the plant is injured.

[0094] Injury rate = number of injured plants / total number of plants * 100%.

[0095] Table 4, growth status of plants

[0096] The results show that the control corn is injured by about 90% at 3 days after spraying a single dose of herbicide (flurochloridone or picolinafen), and the injury rate reaches 100% at 7 days, and the control corn is obviously injured (as shown in Figure 1); all the plants are injured at 3 days after spraying the composite herbicide (flurochloridone + glyphosate, flurochloridone + carfentrazone-ethyl). Compared with the control corn, the transgenic corn, whether sprayed with a single herbicide or a composite herbicide, has no plant injury, which shows that the transgenic corn of the present embodiment is at least tolerant to 2 times the medium dose of flurochloridone, picolinafen or its composite herbicide. The results can show that the protein expressing a polypeptide fragment having an amino acid sequence of 90% or more of SEQ ID NO: 2 in corn (preferably SEQ ID NO: 6, including but not limited to) can effectively enhance the tolerance of corn to flurochloridone, picolinafen, and also show that the protein expressing a polypeptide fragment having an amino acid sequence of 90% or more of SEQ ID NO: 2 in corn (preferably SEQ ID NO: 6, including but not limited to) can effectively enhance the tolerance of corn to carfentrazone-ethyl; if the glyphosate-tolerant EPSPS protein or other herbicide-tolerant protein is also expressed, the tolerance of corn to composite herbicide can be enhanced. It is explained herein that the herbicide for compounding with flurochloridone of the present application includes but is not limited to glyphosate, carfentrazone-ethyl, etc.

[0097] Example 3, obtaining and herbicide tolerance analysis of transgenic corn expressing the active polypeptide of SEQ ID NO: 3 Since the active polypeptide of SEQ ID NO: 3 is selected from the 104th to 173rd amino acid of SEQ ID NO: 12, the cytochrome P450 protein TaP450 from wheat (amino acid sequence as SEQ ID NO: 12, and the corresponding transcribable DNA sequence is preferably SEQ ID NO: 23) is selected, and the recombinant agrobacterium and corn transformation are constructed according to the method and conditions of Example 1, and 64 T0 generation transgenic corn ZC is obtained.

[0098] The obtained 64 T0 generation transgenic maize ZC were transplanted to the greenhouse, and when the plants grew to 3-5 leaf stage, the transgenic maize was sprayed with medium dose of flurochloridone (10 g a.i. / ha), and the transformants with excellent resistance were preliminarily screened. Seven days after the treatment, the plant growth conditions were observed, as shown in Table 5.

[0099] Table 5, plant growth conditions after treatment of transformants

[0100] Three transformants (denoted as ZC1, ZC2, ZC3) with excellent resistance performance (without any phytotoxicity) were selected for further herbicide tolerance test. The seeds of T1 generation transgenic maize ZC1, ZC2, ZC3 and common maize (as a control) were taken, and a randomized block design was adopted, with 3 repetitions, each plot area of 4m x 6m, double-seed sowing, plant spacing of 25cm, row spacing of 50cm, 1m interval between plots, herbicide spraying at 3-5 leaf stage, and the treatments as follows: (1) blank solvent (water); (2) medium dose of flurochloridone (10 g a.i. / ha); (3) 2 times medium dose of flurochloridone (20 g a.i. / ha); (4) medium dose of flurochloridone (10 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha); (5) 2 times medium dose of flurochloridone (20 g a.i. / ha) + 2 times medium dose of glyphosate (1800 g a.i. / ha). The injury rate was calculated according to the plant growth conditions at 3 days, 7 days, and 14 days after the treatment, respectively. The results are shown in Table 6.

[0101] Compared with the growth conditions of untreated plants, if any of the following symptoms appeared, such as plant lodging, base deformity, stem yellowing, leaf yellowing, leaf shrinkage, plant deformity, plant swelling, plant death, etc., the plant was determined to be injured.

[0102] Injury rate = number of injured plants / total number of plants * 100%.

[0103] Table 6, results of herbicide tolerance test

[0104] The results show that the control corn is harmed by about 90% at 3 days after spraying a single herbicide (flurochloridone), and the harm rate is more than 95% at 7 days after spraying; the transgenic corn is not harmed at 3 days after spraying a compound herbicide (flurochloridone + glyphosate). Compared with the control corn, the transgenic corn is not harmed whether a single herbicide or a compound herbicide is sprayed, which shows that the transgenic corn of the embodiment is at least tolerant to 2 times of the medium dose of flurochloridone and glyphosate. The results can show that if a polypeptide fragment having an amino acid sequence of more than 90% of SEQ ID NO: 3 (preferably SEQ ID NO: 12, including but not limited to) is expressed in the corn, the tolerance of the corn to flurochloridone can be effectively enhanced; if a glyphosate-tolerant EPSPS protein or other herbicide-tolerant protein is also expressed, the tolerance of the corn to the compound herbicide can be enhanced. It is illustrated that the herbicide for compounding with flurochloridone of the embodiment includes but is not limited to glyphosate.

[0105] Example 4, obtaining and herbicide tolerance analysis of transgenic corn expressing the active polypeptide of SEQ ID NO: 4 Since the active polypeptide of SEQ ID NO: 4 is selected from the 104th to 173rd amino acids in SEQ ID NO: 13, the cytochrome P450 protein HvP450 (the amino acid sequence is SEQ ID NO: 13, and the corresponding transcribable DNA sequence is preferably SEQ ID NO: 24) from barley is selected, and the recombinant agrobacterium is constructed according to the method and conditions of Example 1, and the corn transformation is performed to obtain 57 T0 generation transgenic corn ZD.

[0106] After the obtained 57 T0 generation transgenic corn ZD is transplanted to the greenhouse, when the plants grow to the 3-5 leaf stage, the transgenic corn is sprayed with the medium dose of flurochloridone (10 g a.i. / ha), and the excellent transformant is preliminarily screened. At 7 days after spraying, the plant growth condition is observed, as shown in Table 7.

[0107] Table 7, plant growth condition of the transformant at 7 days after spraying

[0108] Three of the above-mentioned transformants with excellent resistance performance (without any phytotoxicity) were selected (denoted as ZD1, ZD2, and ZD3) for further herbicide tolerance tests. The T1 generation seeds of the transgenic corn ZD1, ZD2, and ZD3 and common corn (as a control) were taken, a randomized block design was adopted, 3 repetitions were performed, each plot area was 4 m x 6 m, double-grain sowing was performed, the plant spacing was 25 cm, the row spacing was 50 cm, 1 m interval was provided between plots, herbicides were sprayed at the 3-5 leaf stage, and the treatments were as follows: (1) blank solvent (water); (2) medium dose of flurochloridone (10 g a.i. / ha); (3) 2 times the medium dose of flurochloridone (20 g a.i. / ha); (4) medium dose of flurochloridone (10 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha); and (5) 2 times the medium dose of flurochloridone (20 g a.i. / ha) + 2 times the medium dose of glyphosate (1800 g a.i. / ha). The damage rates were calculated according to the growth conditions of the plants at 3 days, 7 days, and 14 days after the spraying, respectively. The results are shown in Table 8.

[0109] Compared with the growth conditions of the untreated plants, if any of the following symptoms occurs, such as plant lodging, base deformity, stem yellowing, leaf yellowing, leaf shrinkage, plant deformity, plant swelling, and plant death, the plant is determined to be damaged.

[0110] The damage rate = the number of damaged plants / total plant number * 100%.

[0111] Table 8, herbicide tolerance test results

[0112] The results show that the control corn has a damage rate of about 90% at 3 days after the spraying of a single herbicide (flurochloridone), and the damage rate is more than 95% at 7 days after the spraying; all the plants are damaged at 3 days after the spraying of a composite herbicide (flurochloridone + glyphosate). Compared with the control corn, the transgenic corn does not have plant damage, no matter whether a single herbicide or a composite herbicide is sprayed, which indicates that the transgenic corn of the present embodiment is at least tolerant to 2 times the medium dose of flurochloridone and glyphosate. The results can indicate that if a polypeptide fragment protein having a sequence identity of more than 90% with the polypeptide of SEQ ID NO: 4 (preferably SEQ ID NO: 13, including but not limited to) is expressed in corn, the tolerance of the corn to flurochloridone can be effectively enhanced; if a glyphosate-tolerant EPSPS protein or other herbicide-tolerant protein is simultaneously expressed, the tolerance of the corn to the composite herbicide can be enhanced. It is indicated herein that the herbicide used in combination with flurochloridone includes but is not limited to glyphosate.

[0113] Example 5, obtaining of transgenic corn expressing multiple active polypeptides and herbicide tolerance analysis

[0114] The recombinant DNA construct of the present example comprises two or more transcribable DNA sequences encoding cytochrome P450 proteins, and the transcribable DNA sequences are functionally linked to a promoter which can initiate their expression in plants. The cytochrome P450 proteins comprise a protein of a polypeptide fragment having 90% or more amino acid sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, which can be SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, and is exemplified by a combination of one of them, specifically as follows:

[0115] A heterologous gene expression cassette is constructed by functionally linking the transcribable DNA sequence of rice Cyp81A6 (SEQ ID NO: 17) in Example 1 to the CMP promoter (SEQ ID NO: 31) and the Tnos terminator (SEQ ID NO: 27); and a heterologous gene expression cassette is constructed by functionally linking the transcribable DNA sequence of CdP450 from Bermudagrass (SEQ ID NO: 18) in Example 2 to the FMV promoter (SEQ ID NO: 32) and the Tnos terminator (SEQ ID NO: 27).

[0116] The two heterologous gene expression cassettes are simultaneously introduced into Agrobacterium with the screening gene expression cassette according to the method of Example 1, and recombinant Agrobacterium is constructed and corn transformation is performed to obtain 102 T0 generation transgenic corn ZAB.

[0117] After the 102 T0 generation transgenic corn ZAB obtained is transplanted to a greenhouse, when the plants grow to the 3-5 leaf stage, the transgenic corn is sprayed with a medium dose of flurochloridone (30 g a.i. / ha), and the transformants with excellent resistance are preliminarily screened. Seven days after the treatment, the plant growth conditions are observed, as shown in Table 9.

[0118] Table 9, growth conditions of transformants 7 days after treatment

[0119] Three transformants (denoted as ZAB1, ZAB2, and ZAB3) with excellent resistance performance (without any phytotoxicity) are selected for further herbicide tolerance tests. Meanwhile, ZA of Example 1 and ZB of Example 2 are crossed to obtain transgenic hybrid corn ZA-B expressing rice Cyp81A6 and CdP450 simultaneously, which is used as a positive control of the present example.

[0120] Seeds of T1 generation transgenic maize ZAB1, ZAB2, ZAB3, common maize (negative control) and ZAB (positive control) were used, and a randomized block design was adopted, with 3 repetitions, each plot area of 4 m x 6 m, double-seed sowing, plant spacing of 25 cm, row spacing of 50 cm, and 1 m interval between plots. At the 3-5 leaf stage, herbicides were sprayed, and the treatments were as follows: (1) blank solvent (water); (2) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of nicosulfuron (24 g a.i. / ha); (3) 2 times medium dose of flurochloridone (60 g a.i. / ha) + 2 times medium dose of nicosulfuron (48 g a.i. / ha); (4) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha) + medium dose of nicosulfuron (24 g a.i. / ha); (5) 2 times medium dose of flurochloridone (60 g a.i. / ha) + 2 times medium dose of glyphosate (1800 g a.i. / ha) + 2 times medium dose of nicosulfuron (48 g a.i. / ha); (6) medium dose of flurochloridone (30 g a.i. / ha) + medium dose of metazachlor (40 g a.i. / ha); (7) 2 times medium dose of flurochloridone (60 g a.i. / ha) + 2 times medium dose of metazachlor (80 g a.i. / ha). The injury rates were calculated according to the growth status of the plants at 3 days, 7 days and 14 days after spraying, respectively. The results are shown in Table 10, and the plant phenotypes of spraying 2 times medium dose of flurochloridone and 2 times medium dose of nicosulfuron are shown in Figure 2.

[0121] Compared with the growth status of untreated plants, if any of the following symptoms occurs, such as plant lodging, base deformity, stem yellowing, leaf yellowing, leaf shrinkage, plant deformity, plant swelling, plant death, etc., the plant is determined to be injured.

[0122] Injury rate = number of injured plants / total number of plants * 100%.

[0123] Table 10, Growth status of plants

[0124] The results show that the negative control corns were all harmed 3 days after spraying the composite herbicide (the harm of the control corns is shown in Figure 2). In comparison with the negative control, the positive control (hybrid transgenic corn) and the transgenic corns of the present example were all not harmed. The results can illustrate that the protein expressing the polypeptide fragment having more than 90% amino acid sequence identity with SEQ ID NO: 1 and SEQ ID NO: 2 (preferably SEQ ID NO: 5 and SEQ ID NO: 6, including but not limited to) or the EPSPS protein resistant to glyphosate in corn can effectively enhance the tolerance of corn to the composite herbicide of one or more of flurochloridone, nicosulfuron and glyphosate or bensulfuron-methyl. It is illustrated herein that the herbicides used in combination with flurochloridone of the present application include but are not limited to glyphosate, nicosulfuron, bensulfuron-methyl, etc.

[0125] Example 6, obtaining and herbicide tolerance analysis of transgenic corn expressing recombinant cytochrome P450 containing active polypeptide of SEQ ID NO: 2

[0126] Using the method of Example 9, the active polypeptide of SEQ ID NO: 2 is recombined into cytochrome P450 genome to construct the protein of the polypeptide fragment having more than 90% amino acid sequence identity with SEQ ID NO: 2, transform crops and detect the tolerance to herbicides.

[0127] The 104th amino acid to the 173rd amino acid of cytochrome P450 gene ZJ3-1 (Chinese patent CN109182369B, amino acid sequence as shown in SEQ ID NO: 15, nucleotide sequence as shown in SEQ ID NO: 21) is replaced by the active polypeptide of SEQ ID NO: 2 (as shown in Figure 3), and the obtained recombinant cytochrome P450 is recorded as NZJ3-1, the amino acid sequence is as shown in SEQ ID NO: 16, and the nucleotide sequence is as shown in SEQ ID NO: 22.

[0128] According to the method of Example 1, recombinant Agrobacterium ZJ3-1 and NZJ3-1 are constructed and corns are transformed to obtain the corresponding T0 generation transgenic corns ZJ3-1 and NZJ3-1.

[0129] After the obtained T0 generation transgenic corns ZJ3-1 and NZJ3-1 are transplanted to the greenhouse, when the plants grow to 3-5 leaf stage, the transgenic corns are sprayed with medium dose of flurochloridone (30 g a.i. / ha), and the transformants with excellent resistance are preliminarily screened. 7 days after the spraying, the growth conditions of the plants are observed, as shown in Table 11.

[0130] Table 11, growth conditions of transformants 7 days after the spraying

[0131] The results show that the T0 generation of the transgenic maize ZJ3-1 has moderate phytotoxicity in a small number of plants and severe phytotoxicity in most plants, which can be considered that the transgenic maize ZJ3-1 has no tolerance to flupicolide; more than 25% of the transgenic maize NZJ3-1 plants have no any phytotoxicity, which indicates that the transgenic maize has high tolerance to flupicolide. Therefore, it can be considered that the replacement of the polypeptide fragment having more than 90% of the amino acid sequence of SEQ ID NO: 2 into the corresponding position of any other P450 gene can make the crops have tolerance to flupicolide, so as to achieve the purpose of making the crops tolerant to flupicolide.

[0132] Example 7, obtaining of transgenic soybean expressing the active polypeptide shown in SEQ ID NO: 1 and analysis of herbicide tolerance

[0133] 1, Recombinant Agrobacterium

[0134] (1) Heterologous gene expression frame: since the active polypeptide shown in SEQ ID NO: 1 is from the 101-170 amino acids in SEQ ID NO: 5, the transcribable DNA sequence of the rice cytochrome P450 protein Cyp81A6 (the amino acid sequence of SEQ ID NO: 5, and the corresponding transcribable DNA sequence is preferably SEQ ID NO: 17) is functionally connected with the CsVMV promoter (SEQ ID NO: 33) and the Tnos terminator (SEQ ID NO: 27), and the promoter-transcribable DNA-terminator fragment is artificially synthesized to form a heterologous gene expression frame.

[0135] (2) Screening gene expression frame: the 5' end of the screening gene CP4-EPSPS (SEQ ID NO: 25) is connected with the Arabidopsis ubiquitin promoter pAtubi (SEQ ID NO: 28), and the 3' end is connected with the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ ID NO: 29), and the pAtubi-CP4-EPSPS-TCaMV35S fragment is artificially synthesized.

[0136] (3) Agrobacterium transformation: the vector pCambia1300 (NCBI sequence number AF234296) is double digested with Hind III / Xho I to recover a 6783 bp fragment, which is recombined with the above two gene expression frames by the method of seamless cloning to construct a recombinant DNA construct, which is transformed into the E. coli TG1 strain, and the sequence is confirmed to obtain the transformation vector SA.

[0137] The plasmid SA is electroporated into the Agrobacterium EHA105 competent cells, and after the correct enzyme digestion is identified, the glycerol bacteria are preserved to obtain the recombinant Agrobacterium, which is used for crop transformation.

[0138] 2, Obtaining of transgenic soybean

[0139] The soybean transformation procedure and medium formula mainly refer to the Agrobacterium-mediated half-seed transformation method of Paz (Paz MM, et al. Plant Cell Reports, 2006, 25(3): 206-213). Glyphosate is used as a screening agent, and the specific process is as follows:

[0140] (1) Soybean seed sterilization: Select high-quality soybean seeds and place them in a desiccator for sterilization with chlorine gas for 16 hours. Chlorine gas is generated by slowly adding 8 mL of concentrated hydrochloric acid to a beaker containing 100 mL of 30% (v / v) sodium hypochlorite aqueous solution in the desiccator.

[0141] (2) Seed germination: Soak the sterilized soybean seeds from step (1) in sterile distilled water at room temperature for 16 hours to obtain germinated soybean seeds.

[0142] (3) Preparation of Agrobacterium liquid: The recombinant Agrobacterium prepared in step 1 is streaked on YEP solid medium containing 50 mg / L Kan (kanamycin) and 25 mg / L rifampicin, and incubated at 28°C for 2 days. The colonies on the YEP plate are scraped and uniformly suspended in the infection medium, and the concentration is adjusted to OD650=0.5-0.6, which is the Agrobacterium liquid. The composition of YEP solid medium: Typtone 10 g, yeast extract 10 g, NaCl 5 g, agar 8 g, add water to 1 L.

[0143] (4) Preparation and infection of explants: Cut the germinated soybean seeds from step (2) along the seed hilum longitudinally into two halves, and remove the seed coat from the half with the hypocotyl and cotyledon, then quickly place it in the Agrobacterium liquid prepared in step (3) for 30 minutes at room temperature, then take it out and place it in a sterile Petri dish lined with filter paper, seal and incubate at 22°C in the dark for 3 days.

[0144] (5) Bud induction culture: Transfer the explants from step (4) after co-culture into bud induction medium containing 2 mM glyphosate, and incubate at 26°C under 16h light / 8h dark conditions for 3 weeks. The composition of bud induction medium: B5 salt (Phytotech, catalog number: G768) 3.21 g, B5 vitamins (Phytotech, catalog number G249-100ML) 1 mL, sucrose 30 g, 4-morpholine ethanesulfonic acid 0.64 g, agar 8 g, add water to 1 L and adjust the pH to 5.6, sterilize and add 200 mg of timentin and 1.67 mg of 6-benzyladenine.

[0145] (6) Bud elongation and screening: The explants after induction in step (5) were cut off the cotyledon, and the part with the cluster buds was transferred to the bud elongation medium with 2 mM glyphosate, and cultured at 26 °C, 16 h light / 8 h dark for 21 days. The bud elongation medium consists of MS salts (Phytotech, Cat# M524) 4.33 g, B5 vitamins (Phytotech, Cat# G249-100ML) 1 mL, sucrose 30 g, 4-morpholineethanesulfonic acid 0.64 g, asparagine 50 mg, glutamine 50 mg, agar 8 g, water to 1 L and pH adjusted to 5.6, after sterilization, 0.5 mg gibberellin, 0.1 mg indole-3-acetic acid, 1 mg zeatin riboside, 200 mg timentin were added.

[0146] (7) Rooting and transplanting: The cluster buds after elongation in step (6) were transferred to the rooting medium, and cultured at 26 °C, 16 h light / 8 h dark. When the roots grew out, the medium adhered to the roots was washed off, and the seedlings were transplanted to the substrate for culture until the T0 generation seeds were harvested. The T0 generation seeds were planted, and the leaf samples were collected for analysis of the gene editing results. The rooting medium consists of MS salts 4.33 g, B5 vitamins 1 mL, sucrose 20 g, 4-morpholineethanesulfonic acid 0.64 g, agar 8 g, water to 1 L and pH adjusted to 5.6, after sterilization, 200 mg timentin was added.

[0147] 108 T0 generation transgenic soybeans SA were obtained.

[0148] 3. Screening of transgenic soybeans

[0149] After the 108 T0 generation transgenic soybeans SA obtained above were transplanted to the greenhouse, when the plants grew to V3 stage, 0.25 times of the medium dose of flurochloridone (7.5 g a.i. / ha) was sprayed on the transgenic soybeans, and the transformants with excellent resistance were preliminarily screened. Seven days after the treatment, the growth conditions of the plants were observed, as shown in Table 12.

[0150] Table 12. Growth conditions of transformants 7 days after treatment

[0151] 4. Herbicide tolerance test of transgenic soybeans

[0152] Three of the above-mentioned transformants with excellent resistance performance (without any phytotoxicity) were selected (denoted as SA1, SA2, and SA3) for further herbicide tolerance tests. The seeds of the T3 generation of the transgenic soybeans SA1, SA2, and SA3 and common soybeans (as a control) were taken, a randomized block design was adopted, 3 repetitions were performed, each plot had an area of 4 m x 6 m, double-seed planting was adopted, the plant spacing was 25 cm, the row spacing was 50 cm, 1 m interval was provided between plots, herbicide spraying was performed at the V3 stage, and the treatments were as follows: (1) blank solvent (water); (2) 0.25 times the medium dose of flurochloridone (7.5 g a.i. / ha); (3) 0.5 times the medium dose of flurochloridone (15 g a.i. / ha); (4) 0.5 times the medium dose of flurochloridone (15 g a.i. / ha) + medium dose of glyphosate (900 g a.i. / ha). The injury rates were calculated according to the growth status of the plants at 3 days, 7 days, and 14 days after the treatment, respectively. The results are shown in Table 13.

[0153] Compared with the growth status of the untreated plants, if any one of the following symptoms occurs, such as leaf yellowing, leaf shrinkage, plant lodging, base deformation, stem yellowing, plant deformation, plant swelling, and plant death, the plant is determined to be injured.

[0154] Injury rate = number of injured plants / total number of plants * 100%.

[0155] Table 13, growth status of plants

[0156] The results show that the control soybeans were all injured 3 days after spraying a single herbicide (flurochloridone) and a composite herbicide (flurochloridone + glyphosate). The transgenic soybeans were not injured whether a single herbicide or a composite herbicide was sprayed, which indicates that the transgenic soybeans of the present embodiment are at least tolerant to 0.5 times the medium dose of flurochloridone. The results can indicate that if a protein expressing a polypeptide fragment having an amino acid sequence of 90% or more of SEQ ID NO: 1 (preferably SEQ ID NO: 5, including but not limited to) in soybeans can effectively enhance the tolerance of soybeans to flurochloridone; if a glyphosate-tolerant EPSPS protein or other herbicide-tolerant protein is simultaneously expressed, the tolerance of soybeans to composite herbicides can be enhanced. It is illustrated herein that the herbicides used in combination with flurochloridone include but are not limited to glyphosate.

[0157] Example 8, obtaining and herbicide tolerance analysis of transgenic soybeans expressing recombinant cytochrome P450 containing the active polypeptide of SEQ ID NO: 1

[0158] The recombinant cytochrome P450 was constructed by replacing the polypeptide fragment having more than 90% amino acid sequence identity with the active polypeptide shown in SEQ ID NO: 1 into the gene of cytochrome P450 according to the method of Example 9, transforming crops and detecting tolerance to flupyradifurone.

[0159] The amino acids 4-68 of the active polypeptide shown in SEQ ID NO: 1 were replaced into the amino acids 111-175 of the NSF gene of cytochrome P450 (NCBI Reference Sequence: NP_001306634.1, the amino acid sequence is shown in SEQ ID NO: 14, and the nucleotide sequence is shown in SEQ ID NO: 20) (as shown in FIG. 4), and the obtained recombinant cytochrome P450 was designated as NZP, the amino acid sequence is shown in SEQ ID NO: 7, and the nucleotide sequence is shown in SEQ ID NO: 19.

[0160] The NSF gene and NZP were respectively constructed into recombinant Agrobacterium ZP and NZP according to the method of Example 6, and were used for soybean transformation; and the corresponding transgenic soybeans SZP and SNZP were obtained according to the method of Example 6.

[0161] After the obtained T0 generation transgenic soybeans SZP and SNZP were transplanted to the greenhouse, when the plants grew to V3 stage, 0.5 times of the medium dose of flupyradifurone (15 g a.i. / ha) was sprayed on the transgenic soybeans, and the transformants with excellent resistance were preliminarily screened. Seven days after the treatment, the growth status of the plants was observed. The growth status of the transformants after the treatment is shown in Table 14.

[0162] Table 14, growth status of transformants after treatment

[0163] The results showed that most of the transgenic soybeans SZP exhibited severe phytotoxicity, and it could be considered that SZP had no tolerance to flupyradifurone; 8 transformants of the transgenic soybeans SNZP had no any phytotoxicity, and had high tolerance to flupyradifurone. It could be considered that replacing the polypeptide fragment having more than 90% amino acid sequence identity with SEQ ID NO: 1 into the corresponding position of any other P450 gene could make crops obtain tolerance to flupyradifurone, and achieve the purpose of making crops tolerant to flupyradifurone.

[0164] Example 9, confirming the active region of cytochrome P450 by amino acid sequence alignment program and replacing

[0165] The skilled person can obtain the amino acid sequences of cytochrome P450s by database search, literature acquisition, etc., and then compare the amino acid sequences of different cytochrome P450s by using common alignment programs (such as common molecular biology software such as Vector NTI, Snapgene, etc.). In theory, any cytochrome P450 can find the corresponding sequence of the active region (i.e., the active polypeptide) provided by the present application by alignment program and replace it. The specific amino acid position (number) in the protein is determined by aligning the amino acid sequence of the target protein with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, etc. using standard sequence alignment tools, for example, using Smith-Waterman algorithm or using CLUSTALW2 algorithm to align two sequences, wherein the sequences are considered to be aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, W. J. and Lipman, D. J. (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. The Align tool (part of Snapgene) is preferably used to determine the position of a specific amino acid in the protein according to the ClustalOmega default parameters suitable for multiple alignment by aligning the amino acid sequence of the protein with SEQ ID NO: 1 or SEQ ID NO: 2.

[0166] For example, by the literature "Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon", the corresponding amino acid sequence (SEQ ID NO: 34) is searched on NCBI, and after alignment by the program, the 97th amino acid-166th amino acid is replaced by SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 provided by the application (as shown in Figure 5).

[0167] Example 10, weed control effect test of different herbicide formulations on transgenic corn

[0168] The weed control effect determination test refers to the method of "Guidelines for Pesticide Field Trials (I) Herbicide Control of Weeds in Corn Crops" (GB / T 17980.42-2000).

[0169] Common corn, transgenic corn ZAB (Example 5), transgenic corn ZA-B (Example 1 transgenic corn ZA and Example 2 transgenic corn ZB hybrid) were used as test materials. A total of 17 treatments were set for each test material: 15 pesticide treatments, 1 artificial weeding treatment, and 1 water blank control treatment. Each treatment had 3 replicates, and the plots were arranged in a randomized block design. Each plot was 24m 2 (Each plot area was 4m x 6m, double-seeded, plant spacing 25cm, row spacing 50cm), and the shape was rectangular. A 1m interval was set between plots, and herbicides were sprayed at the 3-5 leaf stage of corn. The treatments were as follows:

[0170] Table 15, names of herbicides and amounts of active ingredients in different treatments

[0171] Weed control survey method: according to the Z-shaped sampling method, 4 points were investigated in each plot, and 0.25m 2 was surveyed after 14 days and 28 days of application, and the average weed plant control effect of each treatment was calculated. The plant control effect calculation formula: plant control effect = (control area weed plant number - application area weed plant number) / control area weed plant number x 100%.

[0172] The safety of the test agent to corn was investigated 14 days and 28 days after the agent was applied, and the reaction of corn after germination to the agent was observed and recorded. If there was phytotoxicity, the proportion of the damaged plants was investigated. Compared with the growth condition of the untreated plants, if any of the following symptoms occurred, such as plant lodging, base deformation, stem yellowing, leaf yellowing, leaf shrinkage, plant deformation, plant swelling, plant death, and the like, it was determined that the plant was damaged. The damage rate calculation formula was: damage rate = number of damaged plants / total number of plants x 100%.

[0173] The weed control effect results are shown in Table 16.

[0174] Table 16, weed control effect of different herbicides after application

[0175] The damage rate results are shown in Table 17.

[0176] Table 17, effect of different herbicides on corn after application

[0177] The results show that the control corn has a damage rate of more than 95%, and most of them reach 100%, under the action of the composite herbicide; the transgenic corn has no damage phenomenon and is resistant to each composite herbicide. It can also be seen that, compared with the single herbicide (treatments 1-5), the composite herbicide provided by the present application (treatments 6-15) shows better weed control effect, especially the composite treatment containing three herbicides (treatments 11-14) has almost 100% weed control effect. We believe that the transgenic crop provided by the present application effectively resists multiple herbicides, and the weed control method provided by the present application plays a rapid and efficient role in controlling weeds, and can also avoid the occurrence of weed resistance.

[0178] In summary, the polypeptide fragments (preferably rice Cyp81A6, CdP450, TaP450, HvP450 and recombinant cytochrome P450) with more than 90% identity to one or more of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 provided by the present application are introduced into crops, which can effectively improve the tolerance of crops to flumioxazin and acifluorfen, and the transgenic plants can tolerate at least 2 times the recommended dose of flumioxazin (60 g a.i. / ha) or acifluorfen (20 g a.i. / ha). In particular, the expression of CdP450 protein homologous to the active polypeptide shown in SEQ ID NO: 2 in corn can obtain transgenic corn tolerant to flumioxazin, acifluorfen, nicosulfuron, foramsulfuron, chlorthal-dimethyl, mesotrione and other herbicides; the expression of rice Cyp81A6 protein homologous to the active polypeptide shown in SEQ ID NO: 1 in soybean can obtain transgenic soybean with high tolerance to flumioxazin. This method can also be applied to other crops, such as sunflower, peanut, oilseed rape, wheat, pasture, vegetables, etc. The transgenic crops of the present application can be used with flumioxazin, acifluorfen and other herbicides (such as glyphosate, nicosulfuron, etc.) to carry out field weeding, which can efficiently and quickly control weeds, obtain better weeding effect and protection time, avoid weed resistance, and improve the yield and quality of crops, and has a wide application prospect.

[0179] Finally, all materials and methods disclosed and claimed herein can be made and used without undue experimentation in light of the foregoing disclosure. While materials and methods have been described above with regard to preferred embodiments and illustrative examples, those skilled in the art will recognize that changes can be made in form and detail without departing from the spirit, faclure and scope of the invention. All such suitable modifications and equivalents are intended to be covered by the following claims as defined by the appended claims.

Claims

1. An active polypeptide for increasing the tolerance of an arable crop to an arylpyridinecarboxylate herbicide, characterized in that, The amino acid sequence of the active polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO:

4.

2. A method for obtaining a plant tolerant to arylpicolinic acid herbicides using the active polypeptide of claim 1, characterized in that, The method comprises: 1) expressing in a plant a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide; and 2) selecting a transgenic plant having significantly improved tolerance to arylpicolinic acid ester herbicides.

3. The method of claim 2, wherein, The amino acid sequence of the heterologous protein in step 1) comprises one of SEQ ID NO: 5-13.

4. A method for obtaining a recombinant cytochrome P450 protein that is resistant to an arylpicolinic acid herbicide using the active polypeptide of claim 1, characterized in that, The method comprises: 1) replacing a corresponding polypeptide fragment in a cytochrome P450 protein with the active polypeptide or a polypeptide having 90% or more amino acid sequence identity to the active polypeptide; and 2) selecting a recombinant cytochrome P450 protein having metabolic activity for arylpicolinic acid ester herbicides.

5. A plant cell comprising the active polypeptide of claim 1, wherein, The plant cell expresses a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide and has improved metabolism of arylpicolinic acid ester herbicides.

6. A method for obtaining plants that are simultaneously tolerant to an aryl- oxynaphthoate herbicide and glyphosate by using the active polypeptide of claim 1. The method comprises: 1) simultaneously expressing in a plant a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide and a glyphosate-tolerant EPSPS protein; and 2) selecting a transgenic plant having significantly improved tolerance to both arylpicolinic acid ester herbicides and glyphosate.

7. The method of claim 6, wherein, The amino acid sequence of the heterologous protein comprises one of SEQ ID NO: 5-13.

8. A method for obtaining a plant simultaneously tolerant to multiple herbicides using the active polypeptide of claim 1, characterized in that, The method comprises: 1) expressing in a plant a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide, and simultaneously expressing another herbicide-tolerant gene-encoding protein; and 2) selecting a transgenic plant having significantly improved tolerance to both arylpicolinic acid ester herbicides and another herbicide.

9. The method of claim 8, wherein, The amino acid sequence of the heterologous protein is shown in one of SEQ ID NO: 5-13; and the another herbicide-tolerant gene comprises a glyphosate-tolerant EPSPS gene, a PPO gene for PPO inhibitor herbicide tolerance, a Pat gene for glufosinate tolerance, or a Bar gene for glufosinate tolerance.

10. A method of controlling weeds in an area where plants are growing by using the active polypeptide of claim 1. The method is to spray a herbicide comprising arylpicolinic acid ester herbicides on a field planted with a transgenic plant having significantly improved tolerance to arylpicolinic acid ester herbicides; the transgenic plant comprises a heterologous protein comprising a polypeptide fragment having 90% or more amino acid sequence identity to the active polypeptide; and the arylpicolinic acid ester herbicides comprise flurochloridone or flumiclorac-pentyl.

11. A method of controlling a plurality of weeds in an area where plants are growing by using the active polypeptide of claim 1, wherein, The method is spraying a complex herbicide on a field planting a transgenic plant expressing a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide and a protein resistant to another herbicide; the herbicide in the other herbicide-resistant gene includes one or more of glyphosate, nicosulfuron, pyrimisulfan, chlorpyrifos, atrazine, mesotrione or benzobicyclon; the complex herbicide refers to an aryl pyridine carboxylate herbicide and one or more of the following herbicides: glyphosate, nicosulfuron, pyrimisulfan, chlorpyrifos, atrazine, mesotrione or benzobicyclon.

12. A method of controlling weeds in a transgenic corn growing area with the active polypeptide of claim 1, wherein, The method is spraying an aryl pyridine carboxylate herbicide and at least another herbicide on a field planting a transgenic corn expressing a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide and a glyphosate-resistant EPSPS protein; the amino acid sequence of the heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide is shown in SEQ ID NO: 6; the aryl pyridine carboxylate herbicide includes flurochloridone or flurochloridone.

13. A method of controlling weeds in a transgenic soybean growing area with the active polypeptide of claim 1, characterized in that, The method is spraying an aryl pyridine carboxylate herbicide and at least another herbicide on a field planting a transgenic soybean expressing a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide and a glyphosate-resistant EPSPS protein; the amino acid sequence of the heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide is shown in SEQ ID NO:

6.

14. A method of obtaining a plant that is resistant to an aryl oxaloacetate herbicide and at least another herbicide using the active polypeptide of claim 1, wherein, The method comprises: 1) obtaining an aryl pyridine carboxylate herbicide-resistant transgenic plant by planting a heterologous protein expressing a heterologous protein having a polypeptide fragment with more than 90% amino acid sequence identity to the active polypeptide; 2) crossing the transgenic plant of step 1) with a second plant resistant to the at least another herbicide; 3) selecting a progeny plant resistant to the aryl pyridine carboxylate herbicide and the at least another herbicide from the cross.

15. The method of claim 14, wherein, The at least another herbicide is selected from the following: ACCase inhibitor, ALS inhibitor, EPSPS inhibitor, synthetic auxin, photosynthesis inhibitor, glutamine synthesis inhibitor, HPPD inhibitor, PPO inhibitor, long-chain fatty acid inhibitor.

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