Hsl mutants, genes, vectors, cells, compositions and uses thereof

Abstract

This invention relates to the field of genetic engineering, specifically to an HSL mutant, gene, vector, cell, composition, and its applications. The mutant is selected from... Os HSL4 enzyme, Sb HSL1 enzyme, Zm HSL1A enzyme, Hv HSL6D enzyme and Zm A mutant of at least one of the HSL1B enzymes. The HSL mutants, genes, vectors, cells, and compositions provided by this invention can improve plant resistance to HPPD inhibitors.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to an HSL mutant, gene, vector, cell, composition, and its application. Background Technology

[0002] The rice gene HIS1, or 4-hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No. 1, encodes a non-heme ferrous, α-ketoglutarate-dependent oxygenase. This enzyme hydroxylates triketone HPPD inhibitors, thus conferring resistance to HPPD inhibitors in rice. Furthermore, several homologous genes of HIS1 have been found in rice, named HSL (HIS1-Like). HSL genes have also been found in other plants, such as barley, wheat, sorghum, maize, and Arabidopsis thaliana. Current research on HIS1 and HSL shows that proteins encoded by both HIS1 and its homolog HSL can catalyze the hydroxylation of triketone HPPD inhibitors, and the catalytic products exhibit significantly reduced HPPD inhibitory activity.

[0003] Among the more than 20 types of herbicides globally, HPPD inhibitor herbicides have seen rapid growth in recent years, with 13 currently available on the market. The low risk of resistance development is the core advantage of these products, contributing to their leading growth. Based on their favorable characteristics, the HPPD inhibitor herbicides still possess significant market growth potential. However, HPPD inhibitor herbicides are mostly used in corn and wheat fields, and due to crop safety concerns, they have not been widely applied to areas where cash crops such as peanuts, soybeans, rice, and sorghum are grown. Furthermore, some highly active HPPD inhibitors still under development have not been further developed due to a lack of crop safety. Therefore, developing HPPD inhibitor-resistant crops is of great significance for the application of HPPD inhibitor herbicides and the cultivation of cash crops.

[0004] HIS1 and HSL can detoxify HPPD inhibitors and can be used to develop HPPD-resistant herbicide crops. Currently, several mutant proteins capable of degrading HPPD inhibitors have been reported in this field, such as those with high metabolic activity. Axis HSL1 mutants are well-documented, but other HSL subtypes and HSL mutants from other species are rarely reported and exhibit weak metabolic effects against HPPD inhibitors. Our research team previously developed... Axis HSL4, Sat HSL1, Change HSL1A, ChangeThe HSL1B mutant protein also exhibits relatively weak catalytic activity in metabolizing HPPD inhibitors. Therefore, obtaining HSL proteins with significantly enhanced metabolic activity against HPPD inhibitors through modification, and developing new HPPD-resistant herbicide crops based on these proteins, has significant theoretical and economic value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing HPPD inhibitor herbicides, such as poor degradation effect and harm to crop safety.

[0006] To achieve the above objectives, a first aspect of the present invention provides an HSL mutant, said mutant being selected from... Axis HSL4 enzyme, Sat HSL1 enzyme, Change HSL1A enzyme, Hv HSL6D enzyme and Change A mutant of at least one of the HSL1B enzymes; Axis The amino acid sequence of the HSL4 enzyme is shown in SEQ ID NO. 1; Sat The amino acid sequence of the HSL1 enzyme is shown in SEQ ID NO. 2; Change The amino acid sequence of the HSL1A enzyme is shown in SEQ ID NO. 3; Hv The amino acid sequence of the HSL6D enzyme is shown in SEQ ID NO. 4; Change The amino acid sequence of the HSL1B enzyme is shown in SEQ ID NO. 5; The mutation site of the mutant includes at least one of the following: a: At least three of the positions 142, 207, 303, and 334 of SEQ ID NO. 1 are mutated; b: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO. 2; c: At least six of the following positions in SEQ ID NO. 3 are mutated: positions 142, 207, 301, 338, 334, 120, 205, 303, and 341. d: Mutations occur at positions 147, 212, 304, 341, 337, and 214 of SEQ ID NO. 4; e: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO. 5.

[0007] A second aspect of the present invention provides a gene encoding the mutant, the gene having a nucleotide sequence capable of encoding the amino acid sequence of the mutant described in the first aspect.

[0008] A third aspect of the present invention provides a recombinant vector containing the gene described in the second aspect.

[0009] A fourth aspect of the present invention provides a transgenic cell containing the recombinant vector described in the third aspect.

[0010] A fifth aspect of the present invention provides a composition comprising the HSL mutant described in the first aspect as an active ingredient.

[0011] The sixth aspect of the present invention provides the use of any one of the HSL mutants described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, the transgenic cells described in the fourth aspect, and the composition described in the fifth aspect in improving crop resistance to herbicides.

[0012] A seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, causing the target plant to express the HSL mutant described in the first aspect, thereby acquiring resistance to herbicides.

[0013] The eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring a recombinant vector containing the third aspect into a target plant by hybridization, transfer, or backcrossing, so that the target plant expresses the HSL mutant of the first aspect to obtain herbicide resistance.

[0014] A ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HIS1 / HSL gene of a target plant by means of a CRISPR / Cas gene editing method, so that the target plant expresses the HSL mutant described in the first aspect, thereby obtaining resistance to herbicides.

[0015] Compared with existing technologies, the HSL mutant and its encoding gene with highly efficient catalytic activity against HPPD inhibitors provided by this invention have at least the following advantages: By transferring the gene provided by this invention into target plants, the target plants can acquire resistance to HPPD inhibitors (herbicides). Therefore, the HSL mutant and its encoding gene provided by this invention can be used to cultivate herbicide-resistant plants.

[0016] Other features and advantages of the present invention will be described in detail in the following detailed description section.

[0017] Explanation of reference numerals in the attached figures Figure 1 yes Axis Figure showing the results of the Topramezone resistance test on HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana; Figure 2 yes Axis Figure showing the results of the resistance test of HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana to Benzobicyclon-OH; Figure 3 yes Sat Figure showing the results of the resistance test of HSL1-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Benzobicyclon-OH; Figure 4 yes Change Figure showing the results of the resistance test of transgenic Arabidopsis thaliana HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I to Benzobicyclon-OH; Figure 5 yes Change Figure showing the results of the Tefuryltrione resistance test in HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic Arabidopsis thaliana; Figure 6 yes Hv Figure showing the results of the resistance test of transgenic Arabidopsis thaliana HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R to Benzobicyclon-OH; Figure 7 yes Hv Figure showing the results of the Tefuryltrione resistance test in HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic Arabidopsis thaliana; Figure 8 yes Change Figure showing the results of the Mesotrione resistance test in HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana; Figure 9 yes Change Figure showing the results of the resistance test of HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Tefrulyltrione; Figure 10 yes ChangeFigure 1 shows the results of the resistance test of HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Benzobicyclon-OH. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] As previously stated, the first aspect of this invention provides an HSL mutant, the mutant being selected from... Axis HSL4 enzyme, Sat HSL1 enzyme, Change HSL1A enzyme, Hv HSL6D enzyme and Change A mutant of at least one of the HSL1B enzymes; Axis The amino acid sequence of the HSL4 enzyme is shown in SEQ ID NO. 1; Sat The amino acid sequence of the HSL1 enzyme is shown in SEQ ID NO. 2; Change The amino acid sequence of the HSL1A enzyme is shown in SEQ ID NO. 3; Hv The amino acid sequence of the HSL6D enzyme is shown in SEQ ID NO.4; Change The amino acid sequence of the HSL1B enzyme is shown in SEQ ID NO. 5; the mutation site of the mutant includes at least one of the following: a: At least three of the positions 142, 207, 303, and 334 of SEQ ID NO. 1 are mutated; b: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO. 2; c: At least six of the following positions in SEQ ID NO. 3 are mutated: positions 142, 207, 301, 338, 334, 120, 205, 303, and 341. d: Mutations occur at positions 147, 212, 304, 341, 337, and 214 of SEQ ID NO. 4; e: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO. 5.

[0020] Proteins derived by those skilled in the art from the amino acid sequences disclosed in this invention using techniques known in the art, such as substitution, deletion, or addition of one or more amino acids, whose enzyme activity remains unchanged, should fall within the scope of protection of this invention. Unchanged enzyme activity means that, under the same assay conditions, the percentage (relative activity) between the enzyme activity of the protein derived from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5 and the enzyme activity of the HSL mutant is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0021] Preferably, the amino acid sequence of the mutant includes at least one of the following amino acid sequences: X1: The amino acid sequence derived from the mutation of tyrosine at position 142 to histidine, isoleucine at position 207 to phenylalanine, and phenylalanine at position 303 to serine in the amino acid sequence shown in SEQ ID NO. 1. X2: The amino acid sequence derived from the mutation of tyrosine at position 142 to histidine, isoleucine at position 207 to phenylalanine, phenylalanine at position 303 to serine, and leucine at position 334 to arginine in the amino acid sequence shown in SEQ ID NO. 1. X3: The amino acid sequence derived from the mutation of glutamine at position 140 to histidine, tyrosine at position 205 to phenylalanine, lysine at position 336 to phenylalanine, and leucine at position 332 to arginine in the amino acid sequence shown in SEQ ID NO. 2. X4: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, and methionine at position 120 is mutated to isoleucine. X5: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, and isoleucine at position 341 is mutated to phenylalanine. X6: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, and isoleucine at position 341 is mutated to phenylalanine. X7: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, glycine at position 303 is mutated to serine, and isoleucine at position 341 is mutated to phenylalanine. X8: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO.4: asparagine at position 147 is mutated to histidine, glutamine at position 212 is mutated to phenylalanine, valine at position 304 is mutated to leucine, histidine at position 341 is mutated to phenylalanine, methionine at position 337 is mutated to arginine, and isoleucine at position 214 is mutated to arginine. X9: The amino acid sequence derived from the mutation of glutamine at position 140 to histidine, tyrosine at position 205 to phenylalanine, lysine at position 336 to phenylalanine, and leucine at position 332 to arginine in the amino acid sequence shown in SEQ ID NO. 5.

[0022] According to a preferred embodiment 1 of the present invention, the mutant is a mutant in which tyrosine at position 142 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, and phenylalanine at position 303 is mutated to serine; the present invention names this mutant as Axis HSL4-Y142H / I207F / F303S.

[0023] Axis The amino acid sequence of HSL4-Y142H / I207F / F303S (SEQ ID NO. 15) is shown below: MEGSMEDVRSTLLVQELAGMRSKSVPRQYIVQQEDQPTIAATASFPIVDLGRLSQPDGDANEAVKLRQAMESWGLFMVTNHGIEDALMDNVMNVSREFFQQHLGEKQKYTNLIDGKHFQLEGYGNDQVKSDTQILDWLDRLHLKVDPADERNLSVWPKHPESFRDVLDEFLIKCDGV KNSLPSMAKLLKLNEDYFVRQFSDRPTTFARFNYYPQCPRPDLVYGMKPHSDATILTILMVDNDVGGLQVLKDGVWYDVPTKPHTLLINLGDHMEIMSNGIFKSSVHRVMTNPEKERISVVLFYSMNLEKEIEPALELIDERHPARYKRVKIMDYLAGLFEHFLQGTRVIDTVKI.

[0024] According to a preferred embodiment 2 of the present invention, the mutant is a mutant in which tyrosine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 303 is mutated to serine, and leucine at position 334 is mutated to arginine; the present invention names this mutant as... Axis HSL4-Y142H / I207F / F303S / L334R.

[0025] Axis The amino acid sequence of HSL4-Y142H / I207F / F303S / L334R (SEQ ID NO. 16) is shown below: MEGSMEDVRSTLLVQELAGMRSKSVPRQYIVQQEDQPTIAATASFPIVDLGRLSQPDGDANEAVKLRQAMESWGLFMVTNHGIEDALMDNVMNVSREFFQQHLGEKQKYTNLIDGKHFQLEGYGNDQVKSDTQILDWLDRLHLKVDPADERNLSVWPKHPESFRDVLDEFLIKCDGV KNSLLPSMAKLLKLNEDYFVRQFSDRPTTFARFNYYPQCPRPDLVYGMKPHSDATILTILMVDNDVGGLQVLKDGVWYDVPTKPHTLLINLGDHMEIMSNGIFKSSVHRVMTNPEKERISVVLFYSMNLEKEIEPALELIDERHPARYKRVKIMDYRAGLFEHFLQGTRVIDTVKI.

[0026] According to a preferred embodiment 3 of the present invention, the mutant is a mutant in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, lysine at position 336 is mutated to phenylalanine, and leucine at position 332 is mutated to arginine; the present invention names this mutant as... Sat HSL1-Q140H / Y205F / K336F / L332R.

[0027] Sat The amino acid sequence of HSL1-Q140H / Y205F / K336F / L332R (SEQ ID NO. 17) is shown below: MAGESWKVPTPVKDLAALVEEPPSQFVQREEDRPGSLMLAADMPDPLPIVDLDKMSTADEATKLRSALQTWGLFLATNHGIDVSLMEDLMKASREFFNQPLQERQKYSNLREGTRFQLEGYGSDPVIAQDHILDWSDRLHLKVEPEDERNLAQWPKHPESFRDLLHEYATKTKTVM VKILRAMAKTLELDEEDFIDQIGGRPQAFARFNYYPPCPRPELVLGIKAHSDGPLLTVLLVDREVGGLQIQRENKWFNVPSIPHALVINLGDSLEIMSNGIFKSPVHRVVTNAEKERISLAMLYAVQRDNVLEPAPGLLDEKRPAKYRRITEAHFREGVFEHFSKGMRMIETLKI.

[0028] According to a preferred embodiment 4 of the present invention, the mutant is a mutant in the amino acid sequence shown in SEQ ID NO. 3 where glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, and methionine at position 120 is mutated to isoleucine; the present invention names this mutant as... Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I.

[0029] Change The amino acid sequence (SEQ ID NO.18) of HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I is shown below: MADESWRVPPTPVQELAAGVVEPPTQFVLQEQDRPGSGTLLFATDMPEPIPVVDLSRLAAADEASKLRSALETWGLFLVTKHGIEASLMDDVMAASRDFFYQPLEAKQEYSNLIGGKRFQIEGYGNDMVKSKDQILDWQDRLHLRVEPQDERNLAYWPKHPDSFRDLLEKYASKTKIV RNKVLRAMGKTLELGEDYFISQIGDRASAFARFNYYPPCPRPDLVFGIKPHSDGGAVTILLVDKDVGGLQVQKDGVWYTVPSMPHTLLVNLGDSMEIMNNGIFKSPVHRVVTNAEKERLSLAMLYGVEGQRVLEPALGLLGEERPARYRKIMASDYRIGLFQGIAEGQRFIETLKI.

[0030] According to a preferred embodiment 5 of the present invention, the mutant is a mutant in the amino acid sequence shown in SEQ ID NO. 3 where glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, and isoleucine at position 341 is mutated to phenylalanine; the present invention names this mutant as... Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / I341F.

[0031] Change The amino acid sequence (SEQ ID NO. 19) of HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / I341F is shown below: MADESWRVPPTPVQELAAGVVEPPTQFVLQEQDRPGSGTLLFATDMPEPIPVVDLSRLAAADEASKLRSALETWGLFLVTKHGIEASLMDDVMAASRDFFYQPLEAKQEYSNLIGGKRFQIEGYGNDMVKSKDQILDWQDRLHLRVEPQDERNLAYWPKHPDSFRDLLEKYASKTKIV RNKVLRAMGKTLELGEDYFISQIGDRASAFARFNYYPPCPRPDLVFGIKPHSDGGAVTILLVDKDVGGLQVQKDGVWYTVPSMPHTLLVNLGDSMEIMNNGIFKSPVHRVVTNAEKERLSLAMLYGVEGQRVLEPALGLLGEERPARYRKIMASDYRIGLFQGFAEGQRFIETLKI.

[0032] According to a preferred embodiment 6 of the present invention, the mutant is a mutant in the amino acid sequence shown in SEQ ID NO. 3 where glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, and isoleucine at position 341 is mutated to phenylalanine; the present invention names this mutant as... Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / I341F.

[0033] Change The amino acid sequence (SEQ ID NO. 20) of HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / I341F is shown below: MADESWRVPPTPVQELAAGVVEPPTQFVLQEQDRPGSGTLLFATDMPEPIPVVDLSRLAAADEASKLRSALETWGLFLVTKHGIEASLMDDVMAASRDFFYQPLEAKQEYSNLIGGKRFQIEGYGNDMVKSKDQILDWQDRLHLRVEPQDERNLAYWPKHPDSFRDLLEKYASKTKIV RNKVLRAMGKTLELGEDYFISQIGDRAPAFARFNYYPPCPRPDLVFGIKPHSDGGAVTILLVDKDVGGLQVQKDGVWYTVPSMPHTLLVNLGDSMEIMNNGIFKSPVHRVVTNAEKERLSLAMLYGVEGQRVLEPALGLLGEERPARYRKIMASDYRIGLFQGFAEGQRFIETLKI.

[0034] According to a preferred embodiment 7 of the present invention, the mutant is a mutant in the amino acid sequence shown in SEQ ID NO. 3 where glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, glycine at position 303 is mutated to serine, and isoleucine at position 341 is mutated to phenylalanine; the present invention names this mutant as... Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / G303S / I341F.

[0035] Change The amino acid sequence (SEQ ID NO. 21) of HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / G303S / I341F is shown below: MADESWRVPPTPVQELAAGVVEPPTQFVLQEQDRPGSGTLLFATDMPEPIPVVDLSRLAAADEASKLRSALETWGLFLVTKHGIEASLMDDVMAASRDFFYQPLEAKQEYSNLIGGKRFQIEGYGNDMVKSKDQILDWQDRLHLRVEPQDERNLAYWPKHPDSFRDLLEKYASKTKIV RNKVLRAMGKTLELGEDYFISQIGDRAPAFARFNYYPPCPRPDLVFGIKPHSDGGAVTILLVDKDVGGLQVQKDGVWYTVPSMPHTLLVNLGDSMEIMNNGIFKSPVHRVVTNAEKERLSLAMLYSVEGQRVLEPALGLLGEERPARYRKIMASDYRIGLFQGFAEGQRFIETLKI.

[0036] According to a preferred embodiment 8 of the present invention, the mutant is a mutant in which asparagine at position 147 is mutated to histidine, glutamine at position 212 is mutated to phenylalanine, valine at position 304 is mutated to leucine, histidine at position 341 is mutated to phenylalanine, methionine at position 337 is mutated to arginine, and isoleucine at position 214 is mutated to arginine; the present invention names this mutant as... Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R.

[0037] Hv The amino acid sequence (SEQ ID NO.22) of HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R is shown below: MASYDQQFKILEVPPIVQELVGAGVKEPPSQYVLPEQYRPAAAAVSEMPEPIPIIDLSRLSAGSAEEFDKLRSALENWNLFLAVGHGMEPSFLAEAMKATREFFNLSIEEKQKYSNIVGGEKMGMDGYGNDMVVKENQVLDWNDRLHLLVEPESLRTYRLWPTQPPSFRDVLCEYTVR CKAATNIVIRNMAKMLNLQEEHLVNMIGDNSITFARFNYYPQCPRPDHVLGLKAHTDGSIITVNFADAEGLQLERNGVWYNVPIVPNALVMNIGDIMEILSNGFFKSLVHRVVTNAEKERLSLVLLYTLELETQLEPVSELVDDKRPARYMKIKLNDYREKYFDTYATGTLAIDGVKI.

[0038] According to a preferred embodiment 9 of the present invention, the mutant is a mutant in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, lysine at position 336 is mutated to phenylalanine, and leucine at position 332 is mutated to arginine; the present invention names this mutant as Change HSL1B-Q140H / Y205F / K336F / L332R.

[0039] Change The amino acid sequence of HSL1B-Q140H / Y205F / K336F / L332R (SEQ ID NO. 23) is shown below: MAVESWTVPTPVKDLAALVDEPPSRFVQREEHRPGSLMLAADMPDPLPIVDLNKLSTADEAAKLRSALQTWGLFLATNHGIDASLMEDLMEASREFFHQPLQERQKYSNLREGTRFQLEGYGSDPVVAQDHILDWNDRLHLKVEPEDERSLAQWPKYPESFRDLLHEYASKTKSMR DRILRAMAKILELDEEEFIKQLGASPQAFARFNYYPPCPPRPELVLGIKAHSDGPVLTVLLVDREVGGLQVQRENTWFNVPFVPHTLVINLGDSLEIMSNGIFKSPVHRVVTNAEKERISLAMLYAVERDNVLQPAAGLLDEKRPARYRRITEADFREGVFEHFSKGIRMIETLKI.

[0040] This invention does not impose any special requirements on the synthesis method of the amino acids described above. It can be carried out in a manner known in the art. For example, the HSL mutant described above can be obtained by artificial synthesis, or its encoding gene can be synthesized first and then obtained through biological expression.

[0041] This invention does not have special requirements regarding the selection of amino acid side chain functional groups. The HSL mutants described above can also be chemically modified, such as by acetylation, formylation, phosphorylation, sulfonation, ubiquitination, and glycosylation of specific amino acids in the protein.

[0042] This invention does not impose any special requirements on the selection of gene expression regulatory elements. With knowledge of the amino acid sequence of the HSL mutant described in this invention, those skilled in the art can design and select appropriate elements, such as promoters, terminators, enhancers, regulatory sequences, and inducible elements, based on well-known biological knowledge, to assist in the transcription of the coding gene of the aforementioned HSL mutant, or to improve expression efficiency and expression level.

[0043] This invention does not have special requirements for the selection of purification tags. Common tags in the art can be used to modify the mutants, such as by attaching tags (e.g., at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc) to the amino and / or carboxyl ends of the mutants. These tags do not affect the activity of the mutants of this invention. In practical applications, the addition of tags can be selected based on requirements.

[0044] As previously stated, a second aspect of the present invention provides a gene encoding the mutant, the gene having a nucleotide sequence capable of encoding the amino acid sequence of the mutant described in the first aspect.

[0045] Preferably, the nucleotide sequence of the gene includes at least one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.6; (2) The nucleotide sequence as shown in SEQ ID NO.7; (3) The nucleotide sequence shown in SEQ ID NO.8; (4) The nucleotide sequence shown in SEQ ID NO.9; (5) The nucleotide sequence shown in SEQ ID NO.10; (6) The nucleotide sequence shown in SEQ ID NO.11; (7) The nucleotide sequence shown in SEQ ID NO.12; (8) The nucleotide sequence shown in SEQ ID NO.13; (9) The nucleotide sequence shown in SEQ ID NO.14.

[0046] The nucleotide sequence represented by SEQ ID NO.6 of this invention is shown below:

[0047] The nucleotide sequence represented by SEQ ID NO.7 of this invention is shown below:

[0048] The nucleotide sequence represented by SEQ ID NO.8 of this invention is shown below:

[0049] The nucleotide sequence represented by SEQ ID NO.9 of this invention is shown below:

[0050] The nucleotide sequence shown in SEQ ID NO.10 of this invention is as follows:

[0051] The nucleotide sequence shown in SEQ ID NO.11 of this invention is as follows:

[0052] The nucleotide sequence represented by SEQ ID NO.12 of this invention is shown below:

[0053] The nucleotide sequence shown in SEQ ID NO.13 of this invention is as follows:

[0054] The nucleotide sequence shown in SEQ ID NO.14 of this invention is as follows:

[0055] Those skilled in the art can reasonably deduce the corresponding nucleotide sequence from the amino acid sequence disclosed in this invention based on the known codon tables. Therefore, this invention does not impose a unique limitation on the specific base combination of the nucleotide sequence. The nucleotide sequence includes, but is not limited to, any nucleotide sequence that can encode the same amino acid sequence and meets the requirements of conventional genetic engineering operations.

[0056] Nucleotide sequences that have at least 90% identity with the nucleotide sequences disclosed in this invention, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, should also fall within the protection scope of this invention.

[0057] The present invention does not have any special requirements for the synthesis method of nucleotide sequences, and can be carried out in a manner known in the art. For example, the nucleotide sequences provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis.

[0058] Based on the knowledge of the mutants and nucleotide sequences described above in this invention, those skilled in the art can easily identify the gene encoding the mutants described in this invention. For example, site-directed mutagenesis can be performed on the nucleotide sequence shown in SEQ ID NO. 6 to obtain the gene encoding the mutant. The site-directed mutagenesis methods used include, but are not limited to, ZFN site-directed mutagenesis, TALEN site-directed mutagenesis, and / or CRISPR-Cas9 and other genomic site-directed mutagenesis methods.

[0059] As previously described, a third aspect of the present invention provides a recombinant vector containing the gene described in the second aspect.

[0060] This invention does not impose any particular limitation on the choice of vector. Any vector existing in the art that can be used for protein expression is applicable to this invention, such as various commercially available plasmids, granules, bacteriophages, and retroviruses, which can be selected according to specific circumstances. For example, pGWC, pB2GW7.0, or pET-28a can be used. This invention does not impose any particular limitation on the construction method of the recombinant vector; it can be constructed using any method existing in the art. This invention also does not impose any particular limitation on the steps involved in constructing the vector.

[0061] By way of example, the present invention inserts the gene into the vector pET-28a to form the recombinant vector by means of enzyme digestion and ligation.

[0062] As previously described, a fourth aspect of the present invention provides a transgenic cell containing the recombinant vector described in the third aspect.

[0063] This invention does not impose any particular limitations on gene transfer technology. The recombinant vector can be transformed, transduced, or transfected into host cells using methods known in the art, such as protoplast transformation, chemical transformation, alkali metal ion transformation, and electrotransformation. The host cell can be a prokaryotic cell or a eukaryotic cell, whichever is appropriate depending on the specific circumstances.

[0064] As previously described, a fifth aspect of the present invention provides a composition comprising the HSL mutant described in the first aspect as an active ingredient.

[0065] The composition of the present invention contains the mutant described in the first aspect of the present invention as an active ingredient, and may also contain solvents (such as glycerol, sugars and protein protectants such as protease inhibitors) known to those skilled in the art.

[0066] As previously stated, the sixth aspect of the present invention provides the use of any one of the HSL mutants described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, the transgenic cells described in the fourth aspect, and the composition described in the fifth aspect in improving crop resistance to herbicides.

[0067] Preferably, the herbicide is an HPPD inhibitor.

[0068] Preferably, the herbicide is at least one of triketone compounds and pyrazole compounds.

[0069] Preferably, the herbicide is at least one selected from mesotrione, benzobicyclon-OH, tefuryltrione, sulcotrione, Y18024, methylquinalazine (Y13287), toprazolezone, and pyraclostrobin precursor (Y16550), with the following molecular structural formula:

[0070] As previously stated, a seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, thereby causing the target plant to express the HSL mutant described in the first aspect, in order to obtain resistance to herbicides.

[0071] As previously stated, the eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring a recombinant vector containing the third aspect into a target plant by hybridization, transfer, or backcrossing, so that the target plant expresses the HSL mutant of the first aspect to obtain herbicide resistance.

[0072] As previously stated, the ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HIS1 / HSL gene of a target plant by means of a CRISPR / Cas gene editing method, so that the target plant expresses the HSL mutant described in the first aspect, thereby obtaining resistance to herbicides.

[0073] Preferably, the target plant described in the eighth and ninth aspects of the present invention is selected from at least one of rice, sorghum, barley, and maize.

[0074] This invention does not impose special requirements on the method of constructing transgenic plants. Target plants containing HSL mutants can be constructed using any method among transgenic technology and gene editing technologies such as CRISPR / Cas. Alternatively, the plants can be used as parental material to hybridize with other superior plant varieties and then backcross to further transfer herbicide resistance traits to other target plant varieties.

[0075] This invention does not impose special requirements on the transgenic method, which includes direct or indirect transformation methods. Direct transformation methods include chemical induction, liposome transformation, gene gun transformation, electroporation, and microinjection. Indirect transformation methods include Agrobacterium-mediated transformation and virus-mediated transformation.

[0076] In this invention, the term "plant" has the broadest meaning, and examples of plants include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones and plant parts used for asexual reproduction (e.g., cuttings, layerings, young shoots, rhizomes, underground stems, clumps, root necks, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.).

[0077] The term "plant" further encompasses the whole plant, plant parents and offspring, and plant parts, including seeds, branches, stems, leaves, roots (including tubers), flowers, florets, fruits, fleshy stems, peduncles, 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 the plant, as well as tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores. All of the above-mentioned components include the target gene / nucleic acid provided in this invention.

[0078] The plants particularly applicable to the methods of this invention include all plants belonging to the superfamily Plantae, especially monocots and dicots, including food crops, forage or hay legumes, ornamental plants, trees or shrubs.

[0079] According to a preferred embodiment of the invention, the plant is a crop plant. Examples of crop plants include, in particular, rice, wheat, corn, sorghum, barley, soybean, sunflower, rapeseed, alfalfa, cotton, tomato, potato, or tobacco.

[0080] More preferably, the crops are rice, sorghum, barley, and corn.

[0081] In the following examples, unless otherwise specified, room temperature refers to 25±2℃; the experimental methods used are all conventional methods; and the materials and reagents involved are all commercially available.

[0082] The formula for Luria-Bertani (LB) medium is: 5 g yeast extract, 10 g NaCl, 10 g peptone, diluted to 1000 mL with distilled water, and sterilized at 121°C for 20 min.

[0083] Preparation method of 50×TAE solution: Weigh 242 g of Tris and 18.6 g of EDTA into a 1 L beaker, add about 800 mL of deionized water, stir, add 57.1 mL of glacial acetic acid, and after fully dissolving, adjust the pH value to 8.3 with NaOH, then add deionized water to make up to 1 L and store at room temperature (25℃).

[0084] Preparation method of 1×TAE solution: Take 20 mL of 50×TAE solution and add deionized water to make up to 1 L.

[0085] Example 1 Synthesis of nucleotide sequences encoding genes The following encoded genes were all synthesized by Wuhan Baijiesi Biotechnology Co., Ltd. based on the nucleotide sequences provided in this invention.

[0086] In this invention, the correspondence between the nucleotide sequence of the gene and the HSL mutant or wild-type HSL encoded by the gene is shown in Table 1.

[0087] Table 1

[0088] Example 2 Construction of recombinant vectors (1) Amplification of the target gene and vector Five HSL wild-type nucleotide sequence fragments were synthesized by Wuhan Baijies Biotechnology Co., Ltd. based on the amino acid sequences of SEQ ID NO.1-SEQ ID NO.5, after codon optimization. Gene fragments of the HSL mutant were also synthesized by Wuhan Baijies Biotechnology Co., Ltd. based on the complete sequences of SEQ ID NO.6-SEQ ID NO.14. Using the synthesized products as templates, the target gene fragments were amplified by PCR. The PCR reaction system, PCR program settings, and primers used are shown in Tables 2, 3, and 4 below. Table 2

[0089] Table 3

[0090] The extension time at 72 °C depends on the specific fragment length and the efficiency of the enzyme used. Steps 2-4 in the above procedure are repeated for 25 cycles.

[0091] Table 4

[0092] (2) Nucleic acid agarose gel electrophoresis and gel recovery of PCR products a. Weigh 1 g of agarose into an Erlenmeyer flask, add 100 mL of 1×TAE solution, heat in a microwave oven until the agarose particles are completely dissolved, cool until it is not hot to the touch, add 5 µL of ethidium bromide solution, mix well, and pour into a gel casting tank with a comb already placed in it, and let it cool and solidify. b. Before use, remove the comb, put the gel into the electrophoresis tank, add an appropriate amount of 1×TAE solution to the electrophoresis tank; add the sample into the well, cover the electrophoresis tank and start electrophoresis, and stop electrophoresis when the bromophenol blue moves to 1 / 3 of the way from the bottom. c. Cut the target band and place it in a clean 1.5 mL EP tube for gel recovery; the gel recovery kit was purchased from TIANGEN (catalog number DP209); dissolve each 100 mg of gel in 300 µL of sol solution PN (provided in the kit) and incubate in a 50 ℃ water bath, constantly inverting the tube to mix and help the gel dissolve. d. Add the solution from c to the pretreated adsorption column (provided by the kit), let it stand at room temperature for 5 min to allow it to fully bind, and then centrifuge at 12000 rpm for 1 min; e. Pour out the liquid in the collection tube and repeat step d once to increase the product recovery rate; f. Add 500 µL of wash solution PW (provided in the kit) to the purification column and centrifuge at 12000 rpm for 1 min; repeat this step once. g. Centrifuge the adsorption column at 13000 rpm for 2 min to remove as much PW solution as possible; h. Place the purification column into a new 1.5 mL EP tube, heat at 65°C for 10 min, and allow the PW solution to evaporate completely; i. Add 50 µL of elution solution (provided in the kit) to the center of the adsorption column, place on a 65°C heating plate for 2 min, and centrifuge at 13000 rpm for 2 min to elute the DNA; repeat this step once. j. Finally, the recovered PCR products were stored at -20 ℃.

[0093] (3) Enzyme digestion reaction After electrophoresis confirmed the correct DNA fragment size, the pET-28a vector (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11905ES03) and PCR products were subjected to double enzyme digestion. BamHI endonuclease (catalog number 15003ES76) and XhoI endonuclease (catalog number 15034ES76) were both purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., and digestion solutions were included with each purchase. The digestion system is shown in Table 5. Table 5

[0094] After incubation at 37 °C for 3 h, electrophoresis was performed and the enzyme was recovered using a gel extraction kit. To improve the efficiency of vector digestion, an additional 1.5 µL of restriction endonuclease was added and the digestion time was extended by 5 h.

[0095] (4) Connection reaction The recovered gene fragments were ligated according to the following system. The ligation reagent Solution 1 (containing T4 DNA ligase and matching buffer) was purchased from New England BioLabs (China) (catalog number #M0202S), and then incubated at 16℃ for at least 5 hours. The ligation system is shown in Table 6. Table 6

[0096] (5) Transformation After ligation, the ligation product was transformed into E. coli JM109 competent cells (purchased from Promega, product number ST1105) and cultured. The specific steps are as follows: a. Take out a tube of competent cells and place it on ice to thaw for 10 minutes; b. Add 2.5 µL of the ligation product to the competent cells, mix well, and incubate on ice for 30 min; c. Heat shock at 42℃ for 90 s, then place on ice for 2 min; d. Add 200 µL of Luria-Bertani (LB) medium (antibiotic-free) to the tube and incubate at 37°C and 220 rpm for 30 min; bring the solid LB plate containing 50 μg / mL kanamycin sulfate to room temperature for preheating. e. Take out the bacterial culture and spread it evenly on a solid LB agar plate containing 50 μg / mL kanamycin sulfate, and incubate it in a constant temperature incubator at 37℃ for 16 h.

[0097] (6) Sequencing Some single clones were randomly selected from the plates cultured for 16 h and transferred to a sterile plate. The plates were then cultured at 37°C for 5 h and sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. After the sequencing results were returned, the sequences were compared to confirm the correct clones.

[0098] Example 3 Expression and purification of target proteins (1) Construction of expression strain The correctly sequenced plasmids were transformed into E. coli BL21(DE3) cells (purchased from Kangti Life Technology Co., Ltd., product number KTSM109) using the same transformation method as in Example 2.

[0099] (2) Expression First, perform a small-scale protein extraction to explore the optimal expression conditions and purification strategy. Once the conditions are determined, proceed with large-scale extraction. The process is as follows: a. Pick a single colony from a plate that has been cultured for 16 h and add it to 100 mL of LB medium containing 50 μg / mL kanamycin sulfate. Incubate at 37 °C and 220 rpm for 5 h. Obvious turbidity was observed. b. Dilute the inoculum from step a in a 1:100 volume ratio to 2000 mL of LB medium (containing 50 μg / mL kanamycin sulfate) and incubate at 37 °C and 220 rpm for 3 h; wait for OD... 600 When the value reached 0.7, the temperature of the bacterial culture was lowered to 20℃ and isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.2mM) was added for induction, and the induction time was 14 h. c. Centrifuge at 4℃ and 4000 rpm for 10 min to collect the bacteria for protein purification.

[0100] (3) Purification a. Resuspend the collected *E. coli* cells in Lysis Buffer (20 mM Hepes, 150 mM NaCl, pH 7.0). Mix the cell lysis solution with LB broth of the collected *E. coli* cells at a volume ratio of 30 mL: 1 L. Stir on a magnetic stirrer for 20 min to ensure homogeneity. During this time, add 75 µL of 100 mM serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), 60 µL of 40 mg / mL lysozyme for lysing bacterial cell walls, 60 µL of 1 mg / mL deoxyribonuclease I for degrading nucleic acids, and 15 µL of 4 M cofactor MgCl2 to 60 mL of the bacterial suspension. Then, sonicate to disrupt the cells. b. After sonication, centrifuge at 13000 rpm and 4℃ for 1 h, collect the supernatant for affinity chromatography; protein purification is performed in a constant temperature room at 4℃. c. First, pour the supernatant after centrifugation into the prepared Ni column for thorough binding; then wash with 50 mL of 10 mM imidazole solution and 50 mL of 30 mM imidazole solution to remove impurities; finally, elute the target protein with 20 mL of 250 mM imidazole solution; then analyze the results by SDS-PAGE, where the imidazole solution contains 20 mM Hepes in addition to imidazole. d. Dilute the affinity chromatography-resolved protein and load it onto an ion exchange column. After loading, elute with a linear gradient (NaCl from 0 to 500 mM) using a NaCl-free buffer (20 mM Hepes, pH 7.0) and a NaCl-containing buffer (20 mM Hepes, 1 M NaCl, pH 7.0). This step is performed using a protein purification instrument. Perform SDS-PAGE analysis on the results, and combine and concentrate proteins with good properties and purity for the next step of molecular sieve chromatography. e. Before loading the molecular sieve chromatography sample, centrifuge at 13000 rpm for 5 min to remove some of the precipitated protein and impurities. Then, use a syringe to transfer the sample into the loading loop. Next, elute the protein with elution buffer (100 mM NaCl, 20 mM Hepes, pH 7.0), collecting 0.5 mL of sample from each tube. Then, analyze the protein using SDS-PAGE. Combine the proteins that need to be stored, then aliquot and freeze. For proteins used to measure activity, add glycerol to make the glycerol volume 25% of the total solution volume before storage.

[0101] Test Example 1 Catalytic activity study of HSL mutants against HPPD inhibitors Under the premise of ensuring that the concentrations of all proteins and the initial concentrations of HPPD inhibitors are the same, each protein is reacted with the HPPD inhibitor separately. After the reaction, the residual concentration of HPPD inhibitor is determined by high-performance liquid chromatography (HPLC), which reflects the catalytic rate of each protein on the HPPD inhibitor. The specific test method steps are as follows: a. Preparation of buffer solutions, substrates, and cofactors: HEPES assay buffer: First, prepare a 1 M stock solution, adjust its pH to 7.0 with sodium hydroxide, dilute to a concentration of 20 mM before use, and filter through a 0.22 μm filter membrane; Preparation of substrate (i.e. HPPD inhibitor): First, prepare a 10 mM stock solution with DMSO, and then dilute it with HEPES activity assay buffer before use. Preparation of sodium ascorbate: Prepare a 20 mM solution with deionized water and store at -80 ℃. Preparation of ferrous sulfate: Prepare a 1 mM solution with deionized water and store at -80 ℃. Preparation of α-ketoglutaric acid: Prepare a concentration of 50 mM with deionized water and store at -80 °C. b. During the test, first mix 150 μL of 20 mM HEPES activity assay buffer, 20 μL of 500 μM substrate, 8 μL of 20 mM sodium ascorbate, 8 μL of 1 mM ferrous sulfate and 4 μL of 50 mM α-ketoglutarate, and finally add 10 μL of 10 mg / mL enzyme, for a total volume of 200 μL, and react at 30 ℃ for 10 h. c. Add 20 μL of 10% trifluoroacetic acid to terminate the reaction, centrifuge at 12000 g for 10 min, and take 180 μL to add to the sample vial for testing; d. When analyzing samples using HPLC, the sample loading volume is 60 μL. The area of ​​the remaining substrate absorption peak at 286 nm is measured, and the amount of remaining substrate is calculated based on the substrate standard curve (i.e., control) to evaluate the catalytic activity of each protein. Each sample is analyzed three times.

[0102] e. Determination of the standard curve of the substrate (HPPD inhibitor): Using the same reaction system as above (but without enzyme), different substrate concentrations were set, such as 0.1 μM, 0.5 μM, 1 μM, 10 μM, and 50 μM, to obtain the relationship between substrate concentration and peak area.

[0103] This test case examined representative HPPD inhibitor herbicides: mesotrione, benzobicyclon-OH (BBC-OH), tefuryltrione, sulcotrione, Y18024, methylquinalazine (Y13287), toprazole, and pyraclostrobin precursor (Y16550). The catalytic effects of the aforementioned HSL mutant proteins on these HPPD inhibitors were also tested.

[0104] Control: This refers to adding 50 μM of substrate to the reaction system without adding enzyme, and treating the remaining amount of substrate as the amount when the substrate is not catalyzed.

[0105] Sample: Add 50 μM of substrate to the enzyme reaction system and observe the remaining peak area of ​​the substrate at the same retention time to determine the catalytic efficiency of the enzyme on the substrate (i.e., HPPD inhibitor).

[0106] To compare the catalytic efficiency of wild-type and mutant strains against HPPD inhibitors, the protein concentrations of wild-type and mutant strains were adjusted to be consistent during the test.

[0107] The substrate concentration (i.e. HPPD inhibitor) in the control was 50 μM. The remaining substrate peak area of ​​each sample was substituted into the substrate standard curve to calculate the remaining substrate concentration in each sample. The specific experimental results are shown in Table 7.

[0108] As can be seen from Table 7, each mutant showed a significant improvement in the degradation effect of specific HPPD inhibitors compared to its wild type.

[0109] Axis HSL4-Y142H / I207F / F303S and Axis HSL4-Y142H / I207F / F303S / L334R compared to wild type Axis HSL4 significantly enhances the degradation effect of BBC-OH, and it is worth noting that... Axis HSL4-Y142H / I207F / F303S simultaneously significantly enhances the metabolic activity of pyrazole HPPD inhibitors Topramezone and Y16550.

[0110] Sat HSL1-Q140H / Y205F / K336F / L332R compared to wild type Sat HSL1 significantly improves the degradation effect of BBC-OH and slightly improves the degradation effect of Tefryltrione.

[0111] Compared to the wild type, Change All four mutants of HSL1A can completely metabolize BBC-OH, among which Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / I341F, Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / I341F and Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / G303S / I341F can significantly enhance the metabolic activity of mesotrione.

[0112] Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R exhibits broad-spectrum metabolic activity, significantly enhancing the metabolic activity of mesotrione, sulcotrione, tefruryltrione, and BBC-OH, and slightly enhancing the metabolic activity of Y18024, Y13287, Y16550, and Topramezone.

[0113] Change HSL1B-Q140H / Y205F / K336F / L332R significantly enhances the metabolic activity of mesotrione and tefuryltrione, and slightly enhances the metabolic activity of BBC-OH.

[0114] Table 7: Degradation of representative HPPD inhibitors by HSL mutants

[0115] Note: The lower the residual concentration, the better the degradation effect. The reaction conditions in Table 6 are: reaction temperature 30 ℃, reaction time 10 h, substrate (compound) concentration of 50 μM, and protein concentration of 0.5 mg / mL.

[0116] Test Example 2: Enzyme Kinetics Study of HSL Mutants Under the premise of ensuring that the concentrations of all proteins are the same, high-performance liquid chromatography (HPLC) was used to test the initial reaction rates of mutant proteins catalyzing different concentrations of HPPD inhibitors. The slope and intercept of the linearized Michaelis–Menten equation were calculated to obtain... K m and k cat .

[0117] The specific testing method steps are as follows: a. The preparation of buffer solution, substrate, and cofactor is the same as in Test Example 1. b. During the test, the activity test buffer (final concentration 20 mM), substrate (gradient concentrations obtained from preliminary experiments to ensure the maximum substrate reaction amount is below 10%), sodium ascorbate (final concentration 2 mM), ferrous sulfate (final concentration 100 μM), and α-ketoglutarate (final concentration 1 mM) were mixed first, and then the enzyme (enzyme concentration obtained from preliminary experiments to ensure the maximum substrate reaction amount is below 10%) was added. The total volume was 200 μL, and the reaction was carried out at 30 ℃ for 10 min to 30 min (reaction time obtained from preliminary experiments to ensure the maximum substrate reaction amount is below 10%). The reaction time, substrate concentration gradient, and enzyme concentration during the test are shown in Table 8.

[0118] Table 8

[0119] c. Add 20 μL of 10% trifluoroacetic acid (using deionized water as solvent) to terminate the reaction, centrifuge at 12000 g for 10 min, and take 180 μL of the supernatant to add to the sample vial for testing; d. When using HPLC to detect the sample, the sample loading volume is 60 μL. The area of ​​the remaining substrate absorption peak at 286 nm is detected, and the amount of substrate consumed in different reactions is calculated based on the substrate standard curve (i.e., control). Each sample is repeated three times.

[0120] e. The initial rate was determined using a standard curve, and enzyme kinetic parameters were obtained by plotting the Michaelis-Menten equation. K m and k cat .

[0121] This test case examined the enzyme kinetic parameters of the HSL mutant, which exhibits high metabolic activity against HPPD inhibitors and was measured in Test Case 1, catalyzing HPPD inhibitor herbicides. K m and k cat The details are as follows: Axis HSL4-Y142H / I207F / F303S against benzobicyclon-OH technical grade; Sat HSL1-Q140H / Y205F / K336F / L332R against Benzobicyclon-OH; Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I versus Benzobicyclon-OH; Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / G303S / I341F Mesotrione and Benzobicyclon-OH; Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R is effective against Benzobicyclon-OH, Mesotrione, and Tefrulyltrione. Change HSL1B-Q140H / Y205F / K336F / L332R is effective against Benzobicyclon-OH, Mesotrione, and Tefrulyltrione.

[0122] The Michaelis–Menten equation describes the relationship between the rate of an enzyme-catalyzed reaction and the substrate concentration.

[0123] in: v It is the reaction rate (initial rate).

[0124] V max This is the maximum reaction rate (at which point the enzyme is fully saturated with the substrate, and the reaction rate no longer increases with increasing substrate concentration).

[0125] [S] is the concentration of the substrate.

[0126] K m This is the Michaelis constant, which represents the enzyme's affinity for its substrate. Low Michaelis constant... K m The value indicates that the enzyme has a high affinity for the substrate.

[0127]

[0128] By reaction rate v Plotting [S] on the ordinate and substrate concentration [S] on the abscissa yields the Michaelis-Menten curve. V max The substrate concentration value corresponding to 1 / 2 of the value is the Michaelis constant. K m The rate at which an enzyme-catalyzed reaction proceeds is called the catalytic constant. k cat This represents the number of times each enzyme molecule catalyzes the conversion of the substrate into the product per unit time. It is calculated using the following formula:

[0129] In addition, the catalytic efficiency of enzymes on substrates is k cat / K m The value is calculated, and the larger the value, the stronger the enzyme's catalytic efficiency on the substrate. Therefore, it is often used to measure the ability of an enzyme to catalyze its substrate.

[0130] Table 9 shows the results obtained using the above methods. K m 、k cat And calculation k cat / K m Enzyme kinetic parameters.

[0131] Table 9: Enzyme kinetic parameters of HSL mutants against representative HPPD inhibitors

[0132] Note: ND indicates that the catalytic activity is too weak to determine the Michaelis constant.

[0133] Axis HSL4-Y142H / I207F / F303S compared to wild type Axis HSL4 significantly enhances both substrate affinity and catalytic rate for Benzobicyclon-OH, exhibiting detectable catalytic activity.

[0134] Sat HSL1-Q140H / Y205F / K336F / L332R compared to wild type Sat HSL1 significantly improves both substrate affinity and catalytic rate for Benzobicyclon-OH, resulting in a markedly enhanced catalytic activity.

[0135] Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I compared to the wild type Change HSL1A significantly enhances substrate affinity and catalytic rate for benzobicyclon-OH. Further, a nine-fold mutant (ZmHSL1A-Q142H / I207F / F301L / R338F / I334R / M120I / S205P / G303S / I341F) was obtained, exhibiting detectable catalytic activity for both benzobicyclon-OH and mesotrione. Among these, the six-fold mutant (ZmHSL1A-Q142H / I207F / F301L / R338F / I334R / M120I) showed superior catalytic rate and overall catalytic efficiency for benzobicyclon-OH compared to the nine-fold mutant, while the nine-fold mutant exhibited better catalytic activity for mesotrione.

[0136] Hv Compared to the wild type, HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R Hv HSL6D significantly improved the catalytic rates of Benzobicyclon-OH, Mesotrione, and Tefrulyltrione, exhibiting high overall catalytic efficiency, with the best catalytic effect on Mesotrione.

[0137] Change HSL1B-Q140H / Y205F / K336F / L332R compared to wild type ChangeHSL1B significantly improves the catalytic efficiency for Benzobicyclon-OH, Mesotrione, and Tefryltrione. Among them, it has the most outstanding catalytic effect for Benzobicyclon-OH, which combines higher catalytic rate and substrate affinity.

[0138] Test Example 3: Herbicide Resistance Study of HSL Mutant Transgenic Arabidopsis thaliana To verify the resistance of the mutant protein to HPPD herbicide in plants, the HSL gene was introduced into wild-type Arabidopsis thaliana from Colombia via Agrobacterium tumefaciens, and the albinism of the transgenic Arabidopsis thaliana on culture media containing different concentrations of HPPD inhibitors was detected.

[0139] The specific testing steps are as follows: a. Axis The full length of HSL4 (SEQ ID NO. 1) and its mutant (SEQ ID NO. 15), Sat The full length of HSL1 (SEQ ID NO. 2) and its mutant (SEQ ID NO. 17), Change The full length of HSL1A (SEQ ID NO. 3) and its mutant (SEQ ID NO. 18), Hv The full-length HSL1 (SEQ ID NO. 4) and its mutant (SEQ ID NO. 22) and Change The full-length cDNA sequences of HSL1B (SEQ ID NO. 5) and its mutant (SEQ ID NO. 23), after codon optimization, were fused to a repetitive cauliflower mosaic virus 2×35S promoter to drive its sustained expression. Plasmid construction and Arabidopsis transformation were performed by Wuhan Aidijing Biotechnology Co., Ltd. Transformed T1 seeds were screened on 1 / 2 MS solid medium supplemented with glufosinate (20 mg / L). Resistant seedlings were transferred to soil to obtain homologous T3 plants for subsequent functional analysis.

[0140] Preparation of 1 / 2 MS medium: Weigh 39.45 g of 1 / 2 MS medium powder (purchased from Solarbio, catalog number M8525), dissolve it in 1000 mL of distilled water by heating, and autoclave at 118 ℃ for 30 minutes.

[0141] Preparation of 20 mg / L glufosinate: Measure 40 µL of 10% glufosinate aqueous solution (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number A614229-0100) and add it to 200 mL of sterilized 1 / 2 MS medium. Mix thoroughly and set aside.

[0142] b. During testing, the HPPD inhibitor of the appropriate concentration was first diluted with sterile water, and 1 / 2 MS medium was added to bring the final concentration of HPPD inhibitor to 1 nM to 10 µM. The solution was then poured into 9 cm diameter quarter plates and allowed to solidify. Finally, seeds of Colombian wild-type Arabidopsis thaliana, HSL wild-type transgenic Arabidopsis thaliana, HSL mutant transgenic line 1 transgenic Arabidopsis thaliana, and HSL mutant transgenic line 2 transgenic Arabidopsis thaliana were evenly sprinkled in four areas on the same plate, respectively.

[0143] Preparation of test compounds: First, prepare a 10 mM stock solution using DMSO, then dilute with sterile water before use. c. Invert the culture medium plates containing Arabidopsis seeds and place them in a 4 ℃ refrigerator for 48 h. Then transfer them to a 24 ℃ light incubator and culture them upright for 10 days. Observe the growth and albinism of the Arabidopsis plants.

[0144] Figure 1 for Axis Results of Topramezone resistance test in HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana, top left. Axis HSL4 genetically modified, top right Colombian wild type, bottom left Axis HSL4-Y142H / I207F / F303S transgenic line 1, lower right Axis HSL4-Y142H / I207F / F303S transgenic line 2 transgenic. From... Figure 1 It can be seen from this that Axis HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana is resistant to 0.032 µM Topramezone and is compatible with... Axis HSL4 showed no significant difference, but was slightly better than the Colombian wild type.

[0145] Figure 2 for Axis Results of resistance test of HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana to Benzobicyclon-OH, top left. Axis HSL4 genetically modified, top right Colombian wild type, bottom left Axis HSL4-Y142H / I207F / F303S transgenic line 1, lower right Axis HSL4-Y142H / I207F / F303S transgenic line 2 transgenic. From... Figure 2 It can be seen from this that Axis HSL4-Y142H / I207F / F303S transgenic Arabidopsis thaliana is resistant to 0.064 µM Topramezone and is compatible with... Axis HSL4 showed no significant difference, but was slightly better than the Colombian wild type.

[0146] Figure 3 for Sat Results of resistance test of HSL1-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Benzobicyclon-OH, top left. Sat HSL1 transgenic, top right Colombian wild-type, bottom left Sat HSL1-Q140H / Y205F / K336F / L332R transgenic line 1, lower right Sat HSL1-Q140H / Y205F / K336F / L332R transgenic line 2. From... Figure 3 It can be seen from this that Sat The HSL1-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana exhibits resistance to 0.5 µM benzobicyclon-OH, significantly superior to... Sat HSL1 and Colombian wild type.

[0147] Figure 4 for Change Results of resistance test of transgenic Arabidopsis thaliana HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I to Benzobicyclon-OH, top left. Change HSL1A transgenic, top right Colombian wild-type, bottom left Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic line 1, lower right Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic line 2 transgenic. From Figure 4 It can be seen from this that Change The HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic Arabidopsis thaliana is resistant to 0.064 µM benzobicyclon-OH and... Change HSL1A showed no significant difference, but was slightly better than the Colombian wild type.

[0148] Figure 5 for Change Results of the resistance test of HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic Arabidopsis thaliana to Tefrulyltrione, top left. ChangeHSL1A transgenic, top right Colombian wild-type, bottom left Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic line 1, lower right Change HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic line 2 transgenic. From Figure 5 It can be seen from this that Change The HSL1A-Q142H / I207F / F301L / R338F / I334R / M120I transgenic Arabidopsis thaliana is resistant to 0.032 µM of Tefuryltrione and... Change HSL1A showed no significant difference, but was slightly better than the Colombian wild type.

[0149] Figure 6 for Hv Results of resistance test of transgenic Arabidopsis thaliana HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R to Benzobicyclon-OH, top left. Hv HSL6D transgenic, top right Colombian wild-type, bottom left Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic line 1, lower right Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic line 2. From Figure 6 It can be seen that, Hv The HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic Arabidopsis thaliana is resistant to 0.128 µM benzobicyclon-OH, which is superior to... Hv HSL6D and Colombian wild type.

[0150] Figure 7 for Hv Results of the resistance test of HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic Arabidopsis thaliana to Tefuryltrione, top left. Hv HSL6D transgenic, top right Colombian wild-type, bottom left Hv HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic line 1, lower right HvHSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic line 2. From Figure 7 It can be seen that, Hv The HSL6D-N147H / Q212F / V304L / H341F / M337R / I214R transgenic Arabidopsis thaliana exhibits resistance to 0.64 µM tefrost, significantly superior to... Hv HSL6D and Colombian wild type.

[0151] Figure 8 for Change Results of the Mesotrione resistance test in HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana, top left. Change HSL1B transgenic, upper right Colombian wild-type, lower... Change HSL1B-Q140H / Y205F / K336F / L332R transgenic. From... Figure 8 It can be seen that, Change The HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana exhibits resistance to 0.5 µM mesotrione, significantly superior to... Change HSL1B and Colombian wild type.

[0152] Figure 9 for Change Results of the resistance test of HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Tefrulyltrione, top left. Change HSL1B transgenic, top right Colombian wild-type, bottom left. Change HSL1B-Q140H / Y205F / K336F / L332R transgenic line 1, bottom right. Change HSL1B-Q140H / Y205F / K336F / L332R transgenic line 2. From Figure 9 It can be seen that, Change The HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana exhibits resistance to 0.256 µM tefrost, significantly superior to... Change HSL1B and Colombian wild type.

[0153] Figure 10 for Change Results of resistance test of HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana to Benzobicyclon-OH, top left. ChangeHSL1B transgenic, top right Colombian wild-type, bottom left. Change HSL1B-Q140H / Y205F / K336F / L332R transgenic line 1, bottom right. Change HSL1B-Q140H / Y205F / K336F / L332R transgenic line 2. From Figure 10 It can be seen that, Change The HSL1B-Q140H / Y205F / K336F / L332R transgenic Arabidopsis thaliana exhibits resistance to 0.256 µM benzobicyclon-OH, significantly superior to... Change HSL1B and Colombian wild type.

[0154] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An HSL mutant, characterized in that, The mutant is selected from Os HSL4 enzyme, Sb HSL1 enzyme, Zm HSL1A enzyme, Hv HSL6D enzyme and Zm A mutant of at least one of the HSL1B enzymes; Os The amino acid sequence of the HSL4 enzyme is shown in SEQ ID NO. 1; Sb The amino acid sequence of the HSL1 enzyme is shown in SEQ ID NO. 2; Zm The amino acid sequence of the HSL1A enzyme is shown in SEQ ID NO. 3; Hv The amino acid sequence of the HSL6D enzyme is shown in SEQ ID NO. 4; Zm The amino acid sequence of the HSL1B enzyme is shown in SEQ ID NO. 5; the mutation site of the mutant includes at least one of the following: a: At least three of the positions 142, 207, 303, and 334 of SEQ ID NO. 1 are mutated; b: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO. 2; c: At least six of the following positions in SEQ ID NO. 3 are mutated: positions 142, 207, 301, 338, 334, 120, 205, 303, and 341. d: Mutations occur at positions 147, 212, 304, 341, 337, and 214 of SEQ ID NO. 4; e: Mutations occur at positions 140, 205, 336, and 332 of SEQ ID NO.

5.

2. The HSL mutant according to claim 1, characterized in that, The amino acid sequence of the mutant includes at least one of the following amino acid sequences: X1: The amino acid sequence derived from the mutation of tyrosine at position 142 to histidine, isoleucine at position 207 to phenylalanine, and phenylalanine at position 303 to serine in the amino acid sequence shown in SEQ ID NO.

1. X2: The amino acid sequence derived from the mutation of tyrosine at position 142 to histidine, isoleucine at position 207 to phenylalanine, phenylalanine at position 303 to serine, and leucine at position 334 to arginine in the amino acid sequence shown in SEQ ID NO.

1. X3: The amino acid sequence derived from the mutation of glutamine at position 140 to histidine, tyrosine at position 205 to phenylalanine, lysine at position 336 to phenylalanine, and leucine at position 332 to arginine in the amino acid sequence shown in SEQ ID NO.

2. X4: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, and methionine at position 120 is mutated to isoleucine. X5: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, and isoleucine at position 341 is mutated to phenylalanine. X6: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, and isoleucine at position 341 is mutated to phenylalanine. X7: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO. 3: glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, arginine at position 338 is mutated to phenylalanine, isoleucine at position 334 is mutated to arginine, methionine at position 120 is mutated to isoleucine, serine at position 205 is mutated to proline, glycine at position 303 is mutated to serine, and isoleucine at position 341 is mutated to phenylalanine. X8: The amino acid sequence derived from the following mutations in the amino acid sequence shown in SEQ ID NO.4: asparagine at position 147 is mutated to histidine, glutamine at position 212 is mutated to phenylalanine, valine at position 304 is mutated to leucine, histidine at position 341 is mutated to phenylalanine, methionine at position 337 is mutated to arginine, and isoleucine at position 214 is mutated to arginine. X9: The amino acid sequence derived from the mutation of glutamine at position 140 to histidine, tyrosine at position 205 to phenylalanine, lysine at position 336 to phenylalanine, and leucine at position 332 to arginine in the amino acid sequence shown in SEQ ID NO.

5.

3. A gene encoding the HSL mutant according to any one of claims 1-2, characterized in that, The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the mutant described in claim 1 or 2; Preferably, the nucleotide sequence of the gene includes at least one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.6; (2) The nucleotide sequence shown in SEQ ID NO.7; (3) The nucleotide sequence as shown in SEQ ID NO.8; (4) The nucleotide sequence shown in SEQ ID NO.9; (5) The nucleotide sequence shown in SEQ ID NO.10; (6) The nucleotide sequence shown in SEQ ID NO.11; (7) The nucleotide sequence shown in SEQ ID NO.12; (8) The nucleotide sequence shown in SEQ ID NO.13; (9) The nucleotide sequence shown in SEQ ID NO.

14.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 3.

5. A transgenic cell, characterized in that, The transgenic cell contains the recombinant vector as described in claim 4.

6. A composition, characterized in that, The composition contains the HSL mutant as described in any one of claims 1-2 as an active ingredient.

7. The use of the HSL mutant according to any one of claims 1-2, the gene according to claim 3, the recombinant vector according to claim 4, the transgenic cell according to claim 5, and the composition according to claim 6 in improving crop resistance to herbicides; Preferably, the herbicide is an HPPD inhibitor; Preferably, the herbicide is at least one of triketone compounds and pyrazole compounds; Preferably, the herbicide is at least one of nicosulfuron, bicyclosulfuron technical grade, furazolidone, sulfuron, Y18024, methylquinallon, benzoxazolone, and pyraclostrobin precursor.

8. A method for improving crop herbicide resistance, characterized in that, The method includes: transferring the recombinant vector of claim 4 into a target plant, causing the target plant to express the HSL mutant of claim 1 or 2, so as to obtain resistance to herbicides.

9. A method for improving crop herbicide resistance, characterized in that, The method includes: transferring the recombinant vector of claim 4 into a target plant through hybridization, transfer, or backcrossing, so that the target plant expresses the HSL mutant of claim 1 or 2 to obtain resistance to herbicides; Preferably, the target plant is selected from at least one of rice, sorghum, barley, and corn.

10. A method for improving crop herbicide resistance, characterized in that, The method includes: modifying the HIS1 / HSL gene of the target plant using a CRISPR / Cas gene editing method, so that the target plant expresses the HSL mutant as described in claim 1 or 2, in order to obtain resistance to herbicides; Preferably, the target plant is selected from at least one of rice, sorghum, barley, and corn.

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