A mutant ALS polypeptide and its application

Abstract

The present invention provides a mutant ALS polypeptide and its application. Specifically, the present invention provides a mutant acetolactate synthase (ALS), wherein the mutant polypeptides include a mutant ALS1 and a mutant ALS3. The mutant ALS1 polypeptide is mutated at position 178 of SEQ ID No. 1 compared to the parent ALS1 polypeptide; and / or the mutant ALS3 polypeptide is mutated at position 172 of SEQ ID No. 2 compared to the parent ALS3 polypeptide. The mutated ALS polypeptide has strong tolerance to herbicides and has broad application prospects in the field of improving and cultivating plants resistant to ALS-inhibiting herbicides.

Description

[0001] This application claims priority to Chinese patent application CN202311361149.8, filed on October 20, 2023. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of agricultural genetic engineering, and in particular relates to herbicide-resistant genes, polypeptides and their application in plant breeding. Background Art

[0003] Acetolactate synthase (ALS) is a key enzyme in the synthesis of branched-chain amino acids. Many herbicides prevent the synthesis of branched-chain amino acids by inhibiting the activity of ALS enzyme in plants, thereby hindering DNA synthesis during cell division, and ultimately causing the plants to stop growing and gradually wither and die.

[0004] Currently, a variety of herbicides have been developed targeting ALS, including sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylamino-carbonyl compounds. Collectively, these compounds are referred to as ALS inhibitors. These herbicides boast strong selectivity, a broad spectrum of activity, low toxicity, and high efficacy, and are currently being widely used. While these herbicides kill weeds, they also harm crops that are generally not herbicide-resistant, limiting their potential for use. Furthermore, with extended herbicide use, an increasing number of weeds develop resistance to the herbicides, reducing their effectiveness, shortening their market life, and limiting their usefulness. Therefore, developing herbicide-resistant crop varieties is one approach to addressing these challenges, potentially expanding the scope of herbicide use and extending their useful life.

[0005] Currently, some ALS-resistant loci have been reported, but the resistance of the mutants and the range of herbicides they are applicable to are limited. Therefore, if crops with high herbicide resistance and a wide range of applications are to be cultivated, there is an urgent need to develop and improve tolerance systems to ALS-inhibiting herbicides.

[0006] CRISPR / Cas gene editing technology is an emerging genetic engineering technique in recent years. It uses guide RNA to cleave DNA. Various editing systems have been developed for different Cas systems, including Cas9, Cpf1, Cms1, C2c1, and C2c2. CRISPR / Cas editing technology can achieve three types of site-specific editing: The first is site-specific gene knockout. Under the guidance of a targeting RNA (gRNA), the Cas protein recognizes and cleaves the target site, creating a double-stranded DNA break. These breaks are typically repaired by non-homologous end joining (NHEJ), which can easily cause frameshift mutations that disrupt the gene. Site-specific knockout is highly efficient. The second method involves homologous replacement of the target sequence or site-specific insertion. When a double-stranded DNA break is created, homologous replacement or site-specific insertion can occur if a homologous repair template is nearby. The efficiency of homologous replacement is relatively low, and it decreases with the length of the sequence being replaced. The third method is single-base editing. Single-base editing is a gene editing method that uses the CRISPR / Cas system to target a deaminase to a specific site in the genome, thereby modifying a specific base. Integrating CRISPR technology can accelerate the screening of ALS-resistant peptides and improve crop tolerance to ALS inhibitors. This has important implications for expanding the use of herbicides and extending their shelf life. Summary of the Invention

[0007] The purpose of the present invention is to provide a mutant ALS polypeptide that can improve the resistance or tolerance of plants to ALS-inhibiting herbicides; the present invention also relates to biologically active fragments of the mutant ALS, polynucleotides encoding the protein or fragment and their uses.

[0008] On the one hand, the present invention provides a mutant polypeptide of acetolactate synthase (ALS), comprising a mutant ALS1 and a mutant ALS3; the mutant ALS1 has a mutation at the 178th amino acid corresponding to the amino acid sequence shown in SEQ ID No. 1 compared with the amino acid sequence of the parent ALS1; the mutant ALS3 has a mutation at the 172nd amino acid corresponding to the amino acid sequence shown in SEQ ID No. 2 compared with the amino acid sequence of the parent ALS3.

[0009] In another preferred embodiment, the mutant polypeptide is a herbicide resistance / tolerance polypeptide, especially a resistance / tolerance polypeptide to ALS inhibitor herbicides.

[0010] In another preferred embodiment, the amino acid at position 178 is mutated to a non-proline P, for example, S, A, V, G, Q, F, W, Y, D, N, E, K, M, T, C, R, H, L, I; preferably, S.

[0011] In another preferred embodiment, the amino acid at position 172 is mutated to a non-proline P, for example, S, A, V, G, Q, F, W, Y, D, N, E, K, M, T, C, R, H, L, I; preferably, S.

[0012] In another preferred example, the amino acid sequence of the parent ALS1 polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence shown in SEQ ID No. 1.

[0013] In another preferred example, the amino acid sequence of the parent ALS3 polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence shown in SEQ ID No. 2.

[0014] In another preferred embodiment, the amino acid sequence of the parent ALS1 is shown as SEQ ID No. 1.

[0015] In another preferred example, the amino acid sequence of the parent ALS3 is shown as SEQ ID No. 2.

[0016] In another preferred embodiment, the mutant polypeptide (herbicide-resistant polypeptide) is formed by mutation of the polypeptide shown in SEQ ID No. 1.

[0017] In another preferred embodiment, the mutant polypeptide (herbicide-resistant polypeptide) is formed by mutation of the polypeptide shown in SEQ ID No. 2.

[0018] In another preferred embodiment, except for the above mutation, the rest of the amino acid sequence of the mutant polypeptide is identical or substantially identical to the sequence shown in SEQ ID No. 1.

[0019] In another preferred embodiment, except for the above mutation, the remaining amino acid sequence of the mutant polypeptide is identical or substantially identical to the sequence shown in SEQ ID No. 2.

[0020] In another preferred embodiment, the substantially identical amino acids are different in at most 50 (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acids, wherein the differences include substitutions, deletions, or additions of amino acids, and the mutant protein has herbicide tolerance activity (ALS inhibitor herbicides).

[0021] Those skilled in the art will appreciate that protein structure can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into a protein's amino acid sequence without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the substituted residue, i.e., a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where one amino acid is replaced with another amino acid from the same group, fall within the scope of the present invention, as long as the substitution does not inactivate the biological activity of the protein. Therefore, the proteins of the present invention may contain one or more conservative substitutions in their amino acid sequences, preferably generated by substitutions according to Table 1. Furthermore, the present invention also encompasses proteins containing one or more other non-conservative substitutions, as long as such non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention. Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A "non-essential" amino acid residue is an amino acid residue that can be changed (deleted, substituted or replaced) without changing the biological activity, while an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can be made in the non-conserved regions of ALS. Generally speaking, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants can have fewer conservative or non-conservative changes in conserved regions.

[0022] It is well known in the art that one or more amino acid residues can be altered (substituted, deleted, truncated, or inserted) from the N- and / or C-terminus of a protein while retaining its functional activity. Thus, proteins in which one or more amino acid residues are altered from the N- and / or C-terminus of an ALS protein while retaining its desired functional activity are also within the scope of the present invention. These alterations may include those introduced by modern molecular methods such as PCR, which involves PCR amplification of a protein coding sequence by altering or extending the amino acid coding sequence by including the amino acid coding sequence in the oligonucleotides used in the PCR amplification.

[0023] It will be appreciated that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of ALS proteins can be prepared by mutations in the DNA. These can also be accomplished by other forms of mutagenesis and / or by directed evolution, for example, using known mutagenesis, recombination, and / or shuffling methods, in combination with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0024] Those skilled in the art will appreciate that these minor amino acid changes in the ALS proteins of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, lesser effects can be expected.

[0025] Those skilled in the art can identify the essential amino acids of the ALS protein using methods known in the art, such as site-directed mutagenesis, protein evolution, or bioinformatics analysis. The catalytic domain, active site, or other functional domains of the protein can also be determined through physical structural analysis, such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutation of amino acids at putative key sites.

[0026] Table 1

[0027]

[0028]

[0029] In another preferred embodiment, the parent ALS1 and parent ALS3 polypeptides are derived from monocotyledonous plants and / or dicotyledonous plants.

[0030] In another preferred embodiment, the parent ALS1 and parent ALS3 polypeptides are derived from one or more plants selected from the following groups: Gramineae, Leguminosae, Chenopodiaceae, and Cruciferae.

[0031] In another preferred embodiment, the parent ALS1 and parent ALS3 polypeptides are derived from one or more plants selected from the following group: Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0032] In another preferred embodiment, the parent ALS1 and parent ALS3 polypeptides are derived from soybeans.

[0033] In another aspect, the present invention provides a polynucleotide encoding the mutant polypeptide.

[0034] In another preferred embodiment, the polynucleotide is selected from the following group: genomic sequence, cDNA sequence, RNA sequence, or a combination thereof.

[0035] In another preferred embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0036] In another preferred embodiment, the polynucleotide further contains a regulatory element operably linked thereto.

[0037] In another preferred embodiment, the polynucleotide further contains auxiliary elements flanking the ORF of the mutant polypeptide selected from the following groups: a signal peptide, a secretory peptide, a tag sequence (such as 6×His), a nuclear localization signal or a combination thereof.

[0038] In another preferred example, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the mutant polypeptide.

[0039] In another preferred embodiment, the promoter is selected from the following group: a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.

[0040] In another aspect, the present invention provides a nucleic acid construct comprising the polynucleotide and a regulatory element operably linked thereto.

[0041] In another preferred embodiment, the regulatory element is selected from one or more of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, and marker gene.

[0042] On the other hand, the present invention also provides a vector comprising a nucleic acid sequence encoding the mutant ALS polypeptide of the present invention. Preferably, the vector further comprises an expression control element operably linked to the nucleic acid sequence.

[0043] In another preferred embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector or an integration vector.

[0044] In another preferred embodiment, the vector may be a vector for editing the endogenous ALS gene of the host cell.

[0045] In another preferred embodiment, the expression vector further contains at least one replication origin to achieve self-replication.

[0046] In another preferred embodiment, the vector may be a vector which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated.

[0047] The vector can be a plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art.

[0048] Preferably, the vector in the present invention is a plasmid.

[0049] In another aspect, the present invention provides a host cell, wherein the host cell comprises the nucleic acid construct or the polynucleotide is integrated into its genome.

[0050] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, an animal cell, or a plant cell.

[0051] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0052] In another preferred embodiment, the plants include angiosperms and gymnosperms.

[0053] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0054] In another preferred embodiment, the plants include herbaceous plants and woody plants.

[0055] In another preferred embodiment, the plants include Arabidopsis, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0056] On the other hand, the present invention provides an editing vector system, which comprises one or more vectors, and the one or more vectors at least contain a guide sequence targeting the parent ALS. The guide sequence contains a nucleotide sequence of part of the parental ALS, preferably contains at least 15bp of ALS nucleotide sequence, and more preferably includes at least 20bp of ALS nucleotide sequence. In one embodiment, the editing vector system also includes a gene editing enzyme. The gene editing enzyme includes nucleases of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Tanscription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease) editing tools.

[0057] Preferably, the gene editing enzyme is a Cas protein, also known as CRISPR enzyme or Cas effector protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein.

[0058] Preferably, the Cas protein is operably linked to a first regulatory element.

[0059] In other embodiments, the gene editing enzyme is a Cas12 protein, for example, Cas12a, Cas12b, or Cas12i, and the vector further includes a unidirectional repeat sequence (DirectRepeat) that specifically binds to the Cas12 protein. After the unidirectional repeat sequence is operably connected to the guide sequence, a guide guide sequence (gRNA) is formed. The guide sequence of the gRNA is shown in SEQ ID No.5 or SEQ ID No.6; preferably, the gRNA is operably connected to the second regulatory element.

[0060] Such regulatory elements include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0061] Preferably, the editing vector system further comprises a base editing element, and the base editing element is selected from adenine deaminase and / or cytosine deaminase.

[0062] Preferably, the base editing element is selected from Anc689 deaminase, evoFENRY deaminase, APOBEC deaminase or TadA deaminase.

[0063] In one embodiment, the editing vector further comprises resistance genes for easy screening, wherein the resistance genes include hyg, bar, kana, rif, spec, and amp, and the resistance genes are well known to those skilled in the art.

[0064] Preferably, the Cas protein is selected from dCas12i or other nuclease-inactivated Cas12i proteins. Wherein dCas12i represents Cas12i with a D619A mutation. Preferably, the amino acid sequence of the wild-type Cas12i protein is shown in SEQ ID No. 4.

[0065] In another aspect, the present invention provides a gene editing reagent capable of producing the aforementioned mutant polypeptide in plants; the gene editing reagent comprises a CRISPR / Cas protein and a guide RNA (gRNA), wherein the gRNA can target endogenous ALS in plants; and optionally, the gene editing reagent further comprises a base editing element selected from adenine deaminase and / or cytosine deaminase. Preferably, the base editing element is cytosine deaminase.

[0066] Preferably, the base editing element is selected from Anc689 deaminase, evoFENRY deaminase, APOBEC deaminase or TadA deaminase. Preferably, the base editing element is Anc689 deaminase.

[0067] In another embodiment, the gene editing reagent includes the above-mentioned editing vector system.

[0068] In another aspect, the present invention provides a reagent that can be used to improve the herbicide resistance or tolerance of plant cells, plant tissues or plants, and the reagent contains the mutant polypeptide or nucleotide encoding the mutant polypeptide according to the present invention.

[0069] On the other hand, the present invention provides the use of the above-mentioned mutant polypeptide, or the above-mentioned polynucleotide, or the above-mentioned nucleic acid construct, or the above-mentioned host cell in preparing plants with resistance / tolerance to ALS-inhibiting herbicides, or in reagents or kits for preparing plants with resistance / tolerance to ALS-inhibiting herbicides.

[0070] In another preferred embodiment, the ALS-inhibiting herbicide is selected from the following group: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl (thio) benzoates, sulfonylamino-carbonyl or a combination thereof.

[0071] In another preferred embodiment, the ALS-inhibiting herbicide is a triazolinone herbicide.

[0072] In another preferred embodiment, the triazolinone herbicide is selected from the group consisting of flucarbazone-sodium, propoxycarbazone-sodium, thiencarbazone-methyl, or a combination thereof.

[0073] In another preferred embodiment, the ALS-inhibiting herbicide is fluazifop-methyl.

[0074] In another preferred embodiment, the tolerance of the mutant polypeptide to the maximum ALS-inhibiting herbicide concentration is increased by at least 1.5 times, preferably at least 2 times, preferably at least 3 times, preferably at least 4 times, preferably at least 5 times, preferably at least 6 times, and preferably at least 10 times compared to the parent ALS polypeptide.

[0075] In another preferred embodiment, the maximum tolerance concentration of the plant containing the mutant polypeptide to the ALS-inhibiting herbicide is at least 2 times higher than that of the parent plant, preferably 3 times higher, preferably 4 times higher, preferably 5 times higher, preferably 6 times higher, preferably 7 times higher, preferably 8 times higher, preferably 10 times higher, preferably 15 times higher, preferably 20 times higher, and preferably 30 times higher.

[0076] In another preferred embodiment, the mutant ALS polypeptide confers a plant with a yield of at least 0.01 mg L -1 , preferably at least 0.05 mg L -1 , preferably at least 0.1 mg L -1 , preferably to 0.5 mg L -1 , preferably at least 1 mg L -1 , preferably at least 10 mg L -1 , preferably at least 5 mg L -1 , preferably at least 10 mg L -1 -1000mg L -1 , preferably at least 100-1000 mg L -1Tolerance of ALS-inhibiting herbicides at different concentrations.

[0077] In another preferred embodiment, the plant is a monocotyledonous plant and / or a polycotyledonous plant.

[0078] In another preferred embodiment, the plant is selected from one or more of the following groups: Poaceae, Leguminosae, Chenopodiaceae, and Cruciferae.

[0079] In another preferred embodiment, the plant is selected from one or more of the following groups: Arabidopsis, rice, tobacco, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, and strawberry.

[0080] In another preferred embodiment, the plant is soybean.

[0081] In another aspect, the present invention provides a plant cell, plant seed, plant tissue, plant part or plant comprising the mutant polypeptide, or the polynucleotide, or the nucleic acid construct, or the host cell.

[0082] In another aspect, the present invention provides a method for preparing the mutant polypeptide, comprising the steps of: (a) culturing a host cell containing the mutant polypeptide under conditions suitable for expression, thereby expressing the mutant polypeptide; and, optionally, (b) isolating the mutant polypeptide.

[0083] In another aspect, the present invention provides a method for conferring resistance / tolerance to ALS-inhibiting herbicides on plants or a method for preparing plants with resistance / tolerance to ALS-inhibiting herbicides, the method comprising the step of performing gene editing on the plants using the above-mentioned gene editing reagent.

[0084] In another aspect, the present invention provides a method for conferring resistance or tolerance to ALS-inhibiting herbicides on plants or a method for preparing plants with resistance / tolerance to ALS-inhibiting herbicides, the method comprising the step of introducing the ALS mutant polypeptide into plant cells, plant tissues, plant parts or plants.

[0085] In another preferred embodiment, the method includes the step of expressing the mutant polypeptide in a plant cell, plant tissue, plant part or plant, for example, expressing the mutant polypeptide through an expression vector, or integrating the polynucleotide encoding the mutant polypeptide into the plant genome for expression.

[0086] In another preferred embodiment, the method includes natural mutation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), and gene editing.

[0087] In another preferred embodiment, the above method comprises the following steps:

[0088] (1) Providing Agrobacterium carrying an expression vector, wherein the expression vector contains a DNA coding sequence for the mutant polypeptide or an active fragment thereof;

[0089] (2) contacting plant cells, plant tissues, or plant parts with the Agrobacterium of step (1), thereby transferring the DNA coding sequence of the mutant polypeptide or its active fragment into the plant cells and integrating it into the chromosomes of the plant cells; and

[0090] (3) Selecting a plant cell into which the DNA coding sequence of the mutant polypeptide or its active fragment has been transferred.

[0091] In another preferred embodiment, the method comprises the step of mutating the endogenous ALS of the plant to introduce the mutant polypeptide.

[0092] In another preferred embodiment, the method comprises the steps of mutating the endogenous ALS nucleotide sequence of the plant and expressing the mutated polypeptide to introduce the mutated polypeptide.

[0093] In another preferred embodiment, the method includes natural mutation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), and gene editing.

[0094] In another preferred embodiment, the method comprises the following steps:

[0095] (1) Introducing expression vectors containing gene editing tools into plant cells, plant tissues, or plant parts;

[0096] (2) allowing a gene editing tool to act on its endogenous ALS coding sequence and causing mutation at the above-mentioned mutation sites corresponding to SEQ ID No. 1 and / or SEQ ID No. 2;

[0097] (3) Screening for mutant plant cells, plant tissues, and plant parts;

[0098] (4) Isolating the gene editing tool.

[0099] In another preferred embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0100] On the other hand, the present invention provides a plant with herbicide resistance / tolerance, which contains one or more of the mutant ALS, the polynucleotide encoding the mutant ALS, the fusion protein, the vector and the nucleic acid construct; or the polynucleotide is integrated into the plant genome.

[0101] In another aspect, the present invention provides a plant cell, plant tissue, plant part, or plant that is tolerant to ALS-inhibiting herbicides or resistant / tolerant to ALS-inhibiting herbicides, wherein the plant cell, plant tissue, plant part, or plant contains the mutant polypeptide or its polynucleotide sequence.

[0102] In another aspect, the present invention provides a method for improving plants, the method comprising the steps of:

[0103] (a) providing a plant cell, and modifying the plant cell by genetic engineering so that the plant cell expresses the ALS-inhibiting herbicide-resistance polypeptide;

[0104] and (b) regenerating the plant cell of step (a) into a plant.

[0105] In another preferred embodiment, the improved plant comprises increasing the plant's resistance / tolerance to ALS-inhibiting herbicides.

[0106] In another aspect, the present invention provides a method for identifying or selecting transformed plant cells, plant tissues, plants or parts thereof, comprising: (i) providing a transformed plant cell, plant tissue, plant or part thereof, wherein the transformed plant cell, plant tissue, plant or part thereof comprises the polynucleotide shown or a variant or derivative thereof, wherein the polynucleotide encodes a mutant polypeptide used as a selectable marker, and wherein the transformed plant cell, plant tissue, plant or part thereof may comprise another isolated polynucleotide portion comprising; (ii) contacting the transformed plant cell, plant tissue, plant or part thereof with at least one ALS-inhibiting herbicide; (iii) determining whether the plant cell, plant tissue, plant or part thereof is affected by the inhibitory herbicide; and (iv) identifying or selecting the transformed plant cell, plant tissue, plant or part thereof.

[0107] Another aspect of the present invention provides a method for controlling unwanted vegetation at a plant cultivation site, the method comprising:

[0108] (1) providing a plant comprising the mutant polypeptide, the polynucleotide, the nucleic acid construct, or the host cell, or providing the obtained plant;

[0109] (2) Cultivating the plant of step (1) and applying an effective amount of an ALS-inhibiting herbicide to the cultivation site.

[0110] In one embodiment, the unwanted plants are weeds.

[0111] In another aspect, the present invention also provides a method for controlling weed growth near plants, comprising:

[0112] a) providing the above-mentioned herbicide-resistant plants;

[0113] b) applying an effective amount of a herbicide to the plants and weeds in the vicinity of the plants, thereby controlling the weeds in the vicinity of the plants.

[0114] General Definition

[0115] Unless otherwise defined in this application, the scientific terms or professional terms used in the present invention have the meanings understood by those skilled in the art. When the meanings understood by those skilled in the art conflict with the meanings defined in this application, the meanings defined in this application shall prevail.

[0116] As used herein, the term "AxxB" indicates that the amino acid A at position xx is changed to amino acid B. For example, "P178S" indicates that the amino acid P at position 178 is mutated to S, and so on. For double or multiple mutations, each mutation is separated by " / " or "+". For example, ALS1(P178S) / ALS3(P172S) indicates that P at position 178 of the ALS1 amino acid sequence is replaced by S, and P at position 172 of the ALS3 amino acid sequence is replaced by S.

[0117] As used herein, the term "ALS" refers to acetolactate synthase, a key enzyme in the synthesis of branched-chain amino acids.

[0118] As used herein, the terms "ALS inhibitor," "ALS herbicide," "ALS-inhibiting herbicide," and "ALS-inhibiting herbicide" are used interchangeably and refer to substances that have herbicidal activity on their own or in combination with other herbicides and / or additives that modify their effects, and that act by inhibiting ALS. Substances that can act as herbicides by inhibiting ALS are well known in the art and include sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl (thio) benzoates, and sulfonylamino-carbonyl triazolinones.

[0119] Sulfonylurea herbicides include: Amidosulfuron, Bensulfuron-methyl, Chlorimuron-ethyl, Chlorsulfuron, Cinosulfuron, Flucetosulfuron, Flazasulfuron, Sulfometuron-methyl, Thifensulfuron methyl), Tribenuron-methyl, Prosulfuron, Sulfosulfuron, Primisulfuron-methyl, Foramsulfuron; Imidazolinone herbicides include: Imazamethabenz-methyl, Imazapyr, Imazamox, Imazaquin, Imazapic, Imazethapyr; Triazolopyrimidine herbicides include: Triafamone, Cloransulam-methy, Diclosulam, Diclosulam (Florasulam), Flumetsulam, Metosulam, Penoxsulam, Pyroxsulam; Pyrimidinyl (thio) benzoates herbicides include: Bispyribac-sodium, Pyribenzoxim, Pyriftalid, Pyriminobac-methyl, Pyrithiobac-sodium; Sulfonylamino-carbonyl herbicides include: Flucarbazone-sodium, Propoxycarbazone-sodium, Thiencarbazone-methyl, etc.Preferably, the herbicide is a triazolinone; more preferably, the herbicide is fluazifop-methyl; the herbicide can be used to control unwanted plants (such as weeds) before emergence, after emergence, before planting and at planting, taking into account the type of crops or weeds to be applied.

[0120] The term "effective amount" or "effective concentration" means an amount or concentration that is sufficient to kill similar parent (or wild type) plants, plant tissues, plant cells or host cells or inhibit their growth, but the amount does not kill the herbicide-resistant plants, plant tissues, plant cells and host cells of the present invention or does not seriously inhibit their growth. Generally, the effective amount of a herbicide is the amount routinely used to kill the target weeds in agricultural production systems. This amount is known to those of ordinary skill in the art. The herbicides of the present invention show herbicidal activity when applied directly to plants or to the location of plants at any growth stage or before planting or emergence. The observed effect depends on the plant species to be controlled, the growth stage of the plant, the application parameters of the dilution and the spray droplet size, the particle size of the solid component, the environmental conditions during use, the specific compound used, the specific adjuvant and carrier used, the soil type, etc., and the amount of the chemical applied. As known in the art, these factors and other factors can be adjusted to promote non-selective or selective herbicidal effects.

[0121] The term "parent nucleotide or polypeptide" refers to a nucleic acid molecule or polypeptide (protein) that can be found in nature, including wild-type nucleic acid molecules or proteins (polypeptides) that have not been artificially modified, and may also include nucleic acid molecules or proteins (polypeptides) that have been artificially modified but do not contain the content of the present invention. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be deduced from the nucleotide sequence. The "parent plant" is a plant containing the parent nucleotide or polypeptide. The "parent nucleotide or polypeptide" can be extracted from the parent plant according to techniques well known to those skilled in the art, or obtained by chemical synthesis. The amino acid sequence of the parent ALS polypeptide is shown in SEQ ID No.1 or SEQ ID No.2.

[0122] The "tolerance" or "resistance" mentioned in the present invention refers to the ability of the ALS protein or cells, tissues or plants containing the protein to withstand herbicides while maintaining enzyme activity or viability or plant growth, which can generally be characterized by parameters such as the amount or concentration of the herbicide used. Furthermore, the ALS enzyme with "enhanced tolerance to ALS-inhibiting herbicides" or "enhanced resistance to ALS-inhibiting herbicides" mentioned in the present invention refers to an ALS enzyme that, under the same conditions as the parent ALS enzyme, exhibits a maximum tolerance concentration that is at least 2-30 times higher than that of the parent ALS enzyme. The plant with "enhanced tolerance to ALS-inhibiting herbicides" or "enhanced resistance to ALS-inhibiting herbicides" refers to a plant whose tolerance or resistance to the ALS-inhibiting herbicide is improved compared to a plant containing the parent ALS gene, and whose tolerance concentration is at least 2-30 times higher than that of the parent plant. The optimal degree of improved "tolerance" or "resistance" described in the present invention is that at the same herbicide dosage or concentration, unwanted plants can be reduced, inhibited or killed without affecting the growth or viability of plants containing the mutant protein of the present invention.

[0123] The "imparting resistance or tolerance to ALS-inhibiting herbicides on plants" mentioned in the present invention includes the following: when the parent plant has no resistance or tolerance to ALS-inhibiting herbicides, or the parent plant has a certain or low tolerance to ALS-inhibiting herbicides (at the same herbicide concentration), by introducing the mutant polypeptides described in the present invention or nucleotides encoding the mutant polypeptides into the plant, a certain degree of herbicide resistance or tolerance is imparted to the non-resistant plant, thereby improving the tolerance of the plant with a certain or low tolerance to the herbicide.

[0124] The terms "protein", "polypeptide" and "peptide" are used interchangeably in the present invention and refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis. The term "mutant protein" or "mutant protein" refers to a protein that has one or more amino acid residue substitutions, insertions, deletions and / or additions compared to the amino acid sequence of the parent protein. As used herein, the terms "herbicide-resistant polypeptide", "mutated ALS polypeptide", "mutant ALS polypeptide", "mutant ALS protein", "mutant ALS enzyme", "mutant protein", "mutant polypeptide", "polypeptide of the present invention" and the like are used interchangeably.

[0125] The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0126] The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

[0127] The term "mutant protein" or "mutant protein" refers to a protein that has one or more amino acid residue substitutions, insertions, deletions and / or additions compared to the amino acid sequence of a parent protein.

[0128] In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0129] The terms "polynucleotide," "nucleotide sequence," "nucleic acid sequence," "nucleic acid molecule," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.

[0130] As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0131] In the present invention, "host organism" should be understood as any unicellular or multicellular organism into which mutant ALS protein encoding nucleic acid can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells and plants, etc.

[0132] The term "regulatory element," also known as a "regulatory element," as used herein, is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a temporally dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0133] As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0134] A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" proteins for import into the cell nucleus via nuclear transport. That is, proteins with an NLS are transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the protein surface. Exemplary NLSs include, but are not limited to, NLSs from the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

[0135] The term "vector" refers to a vector that contains elements that allow the vector to be integrated into the host cell genome or to replicate autonomously within the cell independently of the genome. The vector may contain any elements that ensure self-replication. It usually carries genes that are not part of the central metabolism of the cell and is usually in the form of double-stranded DNA. The choice of vector usually depends on the compatibility of the vector with the host cell into which the vector is to be introduced. If a vector is used, the choice of vector depends on methods well known to those skilled in the art for transforming host cells. For example, a plasmid vector can be used.

[0136] The term "plant" is to be understood as meaning any differentiated multicellular organism capable of photosynthesis, including crop plants, in particular monocotyledonous or dicotyledonous plants, at any stage of maturity or development, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., cantaloupe, watermelon, Crenshaw melon, honeydew melon, cantaloupe), oilseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, collard greens, Chinese cabbage, bok choy), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, bulb onion , rutabagas, eggplant (also known as eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries ), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pome fruits, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, meadowsweet, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), Oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, industrial hemp, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, as well as trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0137] The term "unwanted plants" is understood to mean plants that interfere with the normal growth of desired plants (e.g., crops) and have no practical or application value, and may include weeds, such as dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, and These include sedge, lycoris, cycad, dandelion, iris, iris, sedge, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family, sedge family,Monocotyledonous weeds include, but are not limited to, weeds of the genera Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, and Cyperus. The undesirable plants may include plants of the genera Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. The undesirable plants may also include plants other than the desired cultivated plants, such as parts of soybeans or small amounts of crops naturally growing in soybean cultivation areas.

[0138] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue culture, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0139] In the present invention, "plant cell" is understood to be any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0140] In the present invention, the term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. The gene editing tools referred to in CRISPR technology include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, etc.). The gene editing tools referred to in TALEN technology are restriction enzymes that can cut specific DNA sequences, which include a TAL effector DNA binding domain and a DNA cleavage domain. The gene editing tools referred to in ZFN technology are also restriction enzymes that can cut specific DNA sequences, which include a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that by constructing the nucleotides encoding the gene editing tools and other regulatory elements into a suitable vector and then transforming the cells, the editing of the genome in the cell can be achieved. The types of editing include gene knockout, insertion, and base editing.

[0141] In the present invention, the term "maximum tolerance concentration" refers to the herbicide concentration that acetolactate synthase (ALS) can withstand when the herbicide is applied and can still substantially maintain its catalytic activity and / or does not affect the normal growth of the plant.

[0142] The term "homology" or "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. Therefore, the compositions and methods of the present invention also include homologs of the nucleotide sequences and polypeptide sequences of the present invention. "Homology" can be calculated by known methods including, but not limited to, Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M. and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton, NJ (1993). Stockton Press, New York (1991).

[0143] Specific amino acid positions (numbers) within the proteins of the present invention are determined by aligning the amino acid sequence of the target protein with SEQ ID No. 1 or SEQ ID No. 2 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. The default parameters for the ClustalW2 (1.82) algorithm are preferably used: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used to determine the position of specific amino acids in the protein of the present invention by aligning the amino acid sequence of the protein with SEQ ID No. 1 or SEQ ID No. 2 using default parameters suitable for multiple alignment (gap opening penalty: 10 log gap extension penalty 0.05).

[0144] It should be understood that the amino acid numbering in the mutant proteins of the present invention is based on SEQ ID No. 1 or SEQ ID No. 2. When a specific mutant protein has a sequence homology of 70% or more with the sequence shown in SEQ ID No. 1 or SEQ ID No. 2, the amino acid numbering of the mutant protein may be misplaced relative to the amino acid numbering of SEQ ID No. 1, such as a misplacement of 1-5 amino acid positions toward the N-terminus or C-terminus. Using conventional sequence alignment techniques in the art, those skilled in the art will generally understand that such misplacement is within a reasonable range, and it should not be considered that a mutant protein with a homology of 80% (e.g., 90%, 95%, 98%) and having the same or similar herbicide tolerance activity is not within the scope of the mutant proteins of the present invention due to a misplacement in amino acid numbering.

[0145] In the present invention, the parent acetolactate synthase (ALS) protein can be derived from any plant, particularly the aforementioned monocotyledonous or dicotyledonous plants. Prior art literature has disclosed the protein sequences and coding sequences of parent (e.g., wild-type) acetolactate synthases from several sources, and these prior art literatures are incorporated herein by reference.

[0146] Preferably, the parent acetolactate synthase (ALS) protein of the present invention is derived from soybean. More preferably, the parent acetolactate synthase protein has the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0147] The present invention also includes the mutant polypeptide (protein) and its active fragments, variants, derivatives and analogs, including substances produced by any substitution, mutation or modification of the protein described below.

[0148] The main advantages of the present invention are:

[0149] The present invention screens out an ALS mutant polypeptide with high resistance to ALS inhibitor herbicides, which can be used to prepare plants that are resistant or tolerant to ALS inhibitor herbicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 The wild-type plants, ALS1(P178S) single mutant plants, ALS3(P172S) single mutant plants, ALS4(P180S) single mutant plants and ALS1(P178S) / ALS3(P172S) double mutant plants were sprayed with 210 mg L -1 The plants showed results 7 days after the addition of fluazifop-methyl.

[0151] Figure 2 .Plant performance results of wild-type plants, ALS1(P178S) single mutant plants, ALS3(P172S) single mutant plants and ALS1(P178S) / ALS3(P172S) double mutant plants after spraying with different concentrations of fluazifop-methyl.

[0152] Figure 3 Comparison of plant height, number of nodes, number of pods, number of tillers, 100-grain weight, and yield per mu between wild-type plants and ALS1(P178S) / ALS3(P172S) double mutant plants after spraying with different concentrations of fluazifop-butyl. DETAILED DESCRIPTION

[0153] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0154] The present invention is further explained in conjunction with the examples of the following experiments. All methods and operations described in these embodiments are provided by way of example and should not be construed as restrictive. The method for the operation of relevant DNA can be referred to Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel FM Greene Publishing Associates and Wiley Interscience, 1989, Molecular Cloning, T.Maniatis et al., 1982, or Sambrook J. and Russell D., 2001, Molecular Cloning: alaboratory manual, version 3.

[0155] Example 1. Construction of base editing vector and screening of herbicide-resistant mutation sites

[0156] 1. Construction of a base editor targeting the endogenous ALS gene in soybean

[0157] Base editors can achieve C / G->T / A (CBE) base conversion within a certain sequence window. This paper uses the Anc689 BE4max-dCas12i base editor as a vector, designs gRNA in the soybean endogenous ALS gene, and clones it into the Anc689 BE4max-dCas12i vector to form a base editor targeting the soybean endogenous ALS gene. In this example, Cas12i is Cas12f.4 from CN111757889B, with the amino acid sequence shown in SEQ ID No. 4; dCas12i is Cas12i with the D619A mutation, which inactivates its nuclease activity.

[0158] The soybean genome contains four ALS homologous genes: GmALS1, GmALS2, GmALS3, and GmALS4. GmALS1, GmALS3, and GmALS4 were selected as targets for herbicide-resistant soybean. Using the BE4max-dCas12i3 system, the C residues at codon P178 in GmALS1, P172 in GmALS3, and P180 in GmALS4 were converted to T residues, resulting in the mutation of proline P to serine S at positions 178, 172, and 180 in GmALS1, GmALS3, and GmALS4, respectively. The Genebank IDs for GmALS1, GmALS3, and GmALS4 are KC254825.1, NP_001341804.1, and KC254824.1, respectively, and their amino acid sequences are SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively.

[0159] Amino acid sequence of GmALS1:

[0160] MAATASRTTRFSSSSSHPTFPKRITRSTLPLSHQTLTKPNHALKIKCSISKPP

[0161] TAAPFTKEAPTTEPFVSRFASGEPRKGADILVEALERQGVTTVFAYPGGAS

[0162] MEIHQALTRSAAIRNVLPRHEQGGVFAAEGYARSSGLPGVCIATSGPGATN

[0163] LVSGLADALMDSVPVVAITGQVPRRMIGTDAFQETPIVEVSRSITKHNYLIL

[0164] DVDDIPRVVAEAFFVATSGRPGPVLIDIPKDVQQQLAVPNWDEPVNLPGY

[0165] LARLLPRPPAEAQLEHIVRLIMEAQKPVLYVGGGSLNSSAELRRFVELTGIPV

[0166] ASTLMGLGTFPIGDEYSLQMLGMHGTVYANYAVDNSDLLLAFGVRFDDR

[0167] VTGKLEAFASRAKIVHIDIDSAEIGKNKQAHVSVCADLKLALKGINMILEE

[0168] KGVEGKFDLGGWREEINVQKHKFPLGYKTFQDAISPQHAIEVLDELTNGD

[0169] AIVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVA

[0170] NPGAVVVDIDGDGSFIMNVQELATIRVENLPVKILLLNNQHLGMVVQWED

[0171] RFYKSNRAHTYLGDPSSESEIFPNMLKFADACGIPAARVTKKEELRAAIQRMLDTPGPYLLDVIVPHQEHVLPMIPSNGSFKDVITEGDGRTRY(SEQ ID NO.1);

[0172] Amino acid sequence of GmALS3:

[0173] MAAITAPKAAFSVLPSSSHSPNPFVRFAIPYSPHHSQRRSLRISSALSDATTK

[0174] SSTAAAEAFASRFGLDEPRKGADILVEALERQGVTDVFAYPGGASMEIHQ

[0175] ALTRSSSIRNVLPRHEQGGVFAAEGYARSSGLPGVCIATSGPGATNLVSGL

[0176] ADALLDSVPLVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVDDI

[0177] PRIVNEAFFLATSGRPGPVLIDIPKDIQQQLAIPNWDQPIRLPGYTSRLPKSP

[0178] NEKHLELIVRLVMESKKPVLYVGGGCLNSSEELRRFVELTGVPVASTLMG

[0179] LGAYPIADDNSLQMLGMHGTVYANYAVDRADLLLAFGVRFDDRVTGKL

[0180] EAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALKGINRVLESRGVAG

[0181] KLDFRGWREELNEQKRRFPLSYKTFEKEISPQYAIQVLDELTNGEAIVSTG

[0182] VGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAV

[0183] VVDIDGDGSFMMNVQELATIKVEKLPVKILLLNNQHLGMVVQWEDRFYK

[0184] SNRAHTYLGDPSNENAIYPNMLKFADACGIPAARVTKKEDLRAAIQKMLETPGPYLLDVIVPHQEHVLPMIPSNGTFQDVITEGDGRTSY (SEQ ID NO.2);

[0185] Amino acid sequence of GmALS4:

[0186] MAATTAPKPAFTALPSSSSSSSQKPFLRLALQFPSLPNSSYHSQRPSLKISSA

[0187] LSDATAKTTTAAAAEDFVSRFGLEEPRKGADILVEALERQGVTDVFAYPG

[0188] GASMEIHQALTRSASIRNVLPRHEQGGVFAAEGYARSSGIPGVCIATSGPG

[0189] ATNLVSGLADAMLDSVPLVAITGQVPRRMIGTDAFQETPIVEVTRSVTKH

[0190] NYLVLDVDDIPRIVNEAFFLATSGRPGPVLIDIPKDIQQQFAIPNWDQPIRLP

[0191] GYMSRLPKSPNENHLELIVRLVMESKKPVLYVGGGCLNSSEELRRFVELT

[0192] GVPVASTLMGLGAYPIADENSLQMLGMHGTVYANYAVDKADILLAFGVR

[0193] FDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALKGINH

[0194] MLESRGVGGKLDFRGWREELNEQKRRFPLSYKTFEDEISPQYAIQVLDELT

[0195] NGDAIVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVVVDIDGDGSFIMNVQELATIKVEKLPVKILLLNNQHLGMVVQWEDRFYKSNRAHTYLGDPSNENAIFPDMLKFADACGIPAARVTKKEDLRAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSNGTFQDVITEGDGRTSY(SEQ ID NO.3);

[0196] Amino acid sequence of Cas12i:

[0197]

[0198] The gRNA sequences targeting the soybean ALS gene are shown in the following table:

[0199] gRNA number guide-PAM sequence (5'-3') Target SEQ ID NO. gRNA1 CCAGGTCCCCCGCCGGATG GmALS1 5 gRNA2 CCAGGTCCCCCGCCGCATG GmALS3, 4 6

[0200] 2. Genetic transformation of soybean and screening and identification of herbicide-resistant plants

[0201] The above-constructed base editors were transformed into soybeans through Agrobacterium, and we successfully obtained ALS1 (P178S) single mutant plants (proline P at position 178 of GmALS1 mutated to serine S), ALS3 (P172S) single mutant plants (proline P at position 172 of GmALS3 mutated to serine S), ALS4 (P180S) single mutant plants (proline P at position 180 of GmALS4 mutated to serine S), and ALS1 (P178S) / ALS3 (P172S) double mutant plants (proline P at position 178 of GmALS1 mutated to serine S, and proline P at position 172 of GmALS3 mutated to serine S).

[0202] To evaluate the herbicide resistance potential of the mutant plants, we obtained stable homozygotes without transgenes in the T1 generation. Wild-type plants, ALS1(P178S) single mutant plants, ALS3(P172S) single mutant plants, ALS4(P180S) single mutant plants, and ALS1(P178S) / ALS3(P172S) double mutant plants at the three-leaf stage in the greenhouse were sprayed with flucarbazone-sodium at a concentration of 210 mgL-1. Seven days after spraying, the results were as follows: Figure 1 As shown, the wild-type plants and ALS4 (P180S) single mutant plants suffered severe herbicide damage, with yellowing and drying of leaves; while the ALS1 (P178S) single mutant plants, ALS3 (P172S) single mutant plants and ALS1 (P178S) / ALS3 (P172S) double mutant plants did not show obvious herbicide damage, and their leaves showed normal green color and morphology. Figure 1 In the figure, WT indicates wild-type plants, als1 indicates ALS1(P178S) single mutant plants, als3 indicates ALS3(P172S) single mutant plants, als4 indicates ALS4(P180S) single mutant plants, and als1 / als3 indicates ALS1(P178S) / ALS3(P172S) double mutant plants.

[0203] To evaluate the herbicide resistance of ALS1(P178S) single mutant plants, ALS3(P172S) single mutant plants, and ALS1(P178S) / ALS3(P172S) double mutant plants, wild-type plants and the mutant plants at the three-leaf stage were sprayed with different concentrations of fluazifop-methyl. Figure 2 As shown, 15 and 30 days after spraying, wild-type plants -1 At doses above 420 mg L -1 The high dose showed slight phytotoxicity, and at 140 and 280 mg L -1 ALS3(P172S) single mutant plants grew normally at 140mg L -1 Normal growth without phytotoxicity at the dose of 280mg L -1 At the dosage of 420mg L -1 The ALS1(P178S) / ALS3(P172S) double mutant plants showed obvious phytotoxicity and plant wilting at the herbicide concentrations of 140, 280 and 420 mg L -1 The plants can grow normally and have no phytotoxicity at any dosage. Figure 2 In the figure, WT indicates wild-type plants, als1 indicates ALS1(P178S) single mutant plants, als3 indicates ALS3(P172S) single mutant plants, als4 indicates ALS4(P180S) single mutant plants, and als1 / als3 indicates ALS1(P178S) / ALS3(P172S) double mutant plants.

[0204] These results indicate that both the P178S mutation in GmALS1 and the P172S mutation in GmALS3 can enhance the herbicide resistance of plants; and the simultaneous mutation of the P178S mutation in GmALS1 and the P172S mutation in GmALS3 can produce a synergistic effect of herbicide resistance, which is significantly stronger than the effect of the single mutation of P178S in GmALS1 and the single mutation of P172S in GmALS3; the P180S mutation in GmALS4 cannot enhance herbicide resistance.

[0205] In terms of other agronomic traits, compared with wild-type plants, there were no significant differences in plant height, 100-grain weight and yield among ALS1 (P178S) single mutant plants, ALS3 (P172S) single mutant plants and ALS1 (P178S) / ALS3 (P172S) double mutant plants.

[0206] In addition, the effects of herbicides on wild-type plants and ALS1(P178S) / ALS3(P172S) double mutant plants were evaluated. Different concentrations of fluazifop-methyl were applied in the field. Figure 3 The wild-type plants were treated with 280 mg L -1 When the ALS1(P178S) / ALS3(P172S) double mutant plants were treated with 280mg L -1 of fluazifop-methyl, 420mg L -1 When flucarbazone was applied or no flucarbazone was applied, the traits such as plant height, mainstalk nod number, pod number, tiller number, hundred-kernel weight and yield per mu had no significant differences from those of the wild type plants not applied with flucarbazone. Figure 3 In the figure, WT indicates wild-type plants, and als1 / als3 indicates ALS1(P178S) / ALS3(P172S) double mutant plants.

[0207] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A mutant polypeptide of acetolactate synthase (ALS), characterized in that: The mutant polypeptides include mutant ALS1 and mutant ALS3; Compared with the amino acid sequence of the parent ALS1, the mutant ALS1 has a mutation at amino acid position 178 corresponding to the amino acid sequence shown in SEQ ID No. 1, and the amino acid sequence of the parent ALS1 is shown in SEQ ID No. 1; Compared with the amino acid sequence of the parent ALS3, the mutant ALS3 has a mutation at amino acid position 172 corresponding to the amino acid sequence shown in SEQ ID No. 2, and the amino acid sequence of the parent ALS3 is shown in SEQ ID No. 2; The amino acids at positions 178 and 172 are mutated to S; The parent ALS1 and the parent ALS3 are derived from soybean.

2. A polynucleotide encoding the mutant polypeptide according to claim 1.

3. A nucleic acid construct, characterized in that The nucleic acid construct contains the polynucleotide according to claim 2.

4. The nucleic acid construct according to claim 3, characterized in that The nucleic acid construct also contains regulatory elements operably linked thereto.

5. The nucleic acid construct according to claim 4, characterized in that The regulatory element is selected from one or any combination of the following groups: enhancer, transposon, promoter, terminator, and marker gene.

6. A host cell, which is a non-plant cell, characterized in that: The host cell comprises the nucleic acid construct according to any one of claims 3 to 5 or the polynucleotide according to claim 2 is integrated into its genome.

7. A gene editing reagent, characterized in that The gene editing reagent is capable of producing the mutant polypeptide of claim 1 in a plant; the gene editing reagent comprises a CRISPR / Cas protein and a gRNA, and the gRNA can target endogenous ALS in the plant; The plant is soybean.

8. The gene editing reagent according to claim 7, characterized in that The gene editing reagent further comprises a base editing element selected from adenine deaminase and / or cytosine deaminase.

9. Use of the mutant polypeptide according to claim 1, or the polynucleotide according to claim 2, or the nucleic acid construct according to any one of claims 3 to 5, or the host cell according to claim 6 in preparing a plant resistant / tolerant to an ALS-inhibiting herbicide, or in a reagent or kit for preparing a plant resistant / tolerant to an ALS-inhibiting herbicide; The plant is soybean.

10. The use according to claim 9, characterized in that The ALS-inhibiting herbicide is selected from sulfonylureas, imidazolinones, triazole pyrimidines, pyrimidine salicylates, triazolinones, or a combination thereof.

11. A method for conferring resistance / tolerance to an ALS-inhibiting herbicide on a plant or a method for preparing a plant resistant / tolerant to an ALS-inhibiting herbicide, the method comprising the step of gene editing the plant using the reagent according to any one of claims 7 to 8; The plant is soybean.

12. A method for conferring resistance / tolerance to an ALS-inhibiting herbicide on a plant or a method for preparing a plant having resistance / tolerance to an ALS-inhibiting herbicide, the method comprising the step of introducing the mutant polypeptide of claim 1 into a plant cell, plant seed, plant tissue, plant part or plant; the plant is soybean.

13. The method according to claim 12, characterized in that The method comprises the step of expressing the mutant polypeptide of claim 1 in plant cells, plant seeds, plant tissues, plant parts or plants.

14. The method according to claim 12, characterized in that The method comprises the step of mutating the endogenous ALS of the plant to thereby introduce the mutant polypeptide according to claim 1 .

15. A method of controlling unwanted vegetation at a plant cultivation site, the method comprising: (1) Providing a plant comprising the mutant polypeptide according to claim 1, or the polynucleotide according to claim 2, or the nucleic acid construct according to any one of claims 3 to 5, or the host cell according to claim 6, or providing a plant obtained by the method according to any one of claims 11 to 14; (2) cultivating the plant of step (1) and applying an ALS-inhibiting herbicide to the cultivation site; The plant is soybean.

16. The method according to any one of claims 11 to 15, characterized in that: The ALS-inhibiting herbicide is selected from sulfonylureas, imidazolinones, triazole pyrimidines, pyrimidine salicylates, triazolinones, or a combination thereof.

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