Herbicide resistance mutant of AhALS2 gene and application of herbicide resistance mutant in peanut breeding

Abstract

The invention discloses a herbicide-resistant mutant of an AhALS2 gene and application of the herbicide-resistant mutant in peanut breeding. The mutant is subjected to site-specific mutagenesis on methionine at the 200th site of an amino acid sequence of AhALS2 protein to obtain isoleucine (namely M200I mutation). According to the invention, the herbicide-resistant peanut material with M200I mutation in the AhALS2-B gene and the AhALS2-A gene is respectively obtained by editing the AhALS2-B gene and the AhALS2-A gene. According to the invention, a brand new herbicide resistance site M200 in the peanut AhALS2 gene is successfully excavated and verified by using a base editing technology, and the M200I mutant is accurately created in peanuts by constructing a CBE-M200 editing carrier and has remarkable resistance to imidazolone herbicides. The invention not only provides a new germplasm and a core gene resource for peanut herbicide-resistant breeding, but also provides a new idea for cultivating herbicide-resistant varieties in other crops by using homologous editing.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and crop genetic breeding technology, specifically relating to a herbicide-resistant mutant of the AhALS2 gene and its application in flowering breeding. Background Technology

[0002] Peanuts are an important oilseed and cash crop globally, holding a significant position in my country's agricultural economy. Field weeds are one of the main biological stresses restricting high and stable peanut yields, and chemical weeding is currently the most efficient weed control method. However, my country currently lacks commercially available herbicide-resistant peanut varieties with independent intellectual property rights, as well as corresponding herbicide products. This, to some extent, limits the efficiency and effectiveness of weed control in peanut production, hindering the healthy development of the peanut industry. Therefore, cultivating new herbicide-resistant peanut germplasm suitable for my country's agricultural production needs is of great significance for improving peanut production technology and ensuring national food and oil security.

[0003] Acetolactate synthase (ALS) is a key enzyme in the first step of the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine) in plants. It is also the common target of five major classes of ALS inhibitor herbicides: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine salicylates, and sulfonamide carbonyl triazolinones. These herbicides inhibit the catalytic function of ALS by binding to its active site, leading to the inhibition of branched-chain amino acid synthesis and ultimately plant death.

[0004] Studies have shown that mutations in specific amino acid residues of ALS enzymes can alter their binding ability to ALS inhibitor herbicides, thereby conferring herbicide resistance to plants. In various plants such as Arabidopsis thaliana, rice, and maize, several conserved resistance-related sites have been identified, such as A122, P197, A205, D376, W574, S653, and G654 (all ALS amino acid numbers in this text correspond to the Arabidopsis thaliana ALS sequence, AT3G48560). Different amino acid substitutions at different sites or at the same position often lead to varying degrees of resistance to different types of ALS inhibitors. For example, mutations at the P197 site typically confer high resistance to sulfonylurea herbicides; mutations at the S653 and G654 sites mainly result in resistance to imidazolinone herbicides. The resistance resulting from simultaneous mutations at multiple key amino acid sites is additive.

[0005] In recent years, base editors (BEs) developed based on the CRISPR / Cas system, including the cytosine base editor (CBE) and the adenine base editor (ABE), have enabled precise base substitutions at specific sites in the genome without causing DNA double-strand breaks. This provides a powerful tool for screening novel and beneficial amino acid mutations in crops. Systematic editing of potential binding sites in ALS genes using base editors to discover new resistance mutations holds promise for breeding crop germplasm resources with new resistance types or stronger resistance.

[0006] The peanut genome contains two highly homologous copies of the ALS gene (AhALS2-A and AhALS2-B). Currently, the exploration of herbicide resistance sites in the peanut AhALS2 gene is insufficient, particularly research on precise mutations using cutting-edge base editing technology. Therefore, targeting the peanut AhALS2 gene with base editing technology to discover new herbicide resistance sites and cultivate new peanut germplasm with independent intellectual property rights and clearly defined resistance is of great value for promoting the advancement of peanut breeding technology and industrial development in my country. Summary of the Invention

[0007] In view of the problems of scarce peanut herbicide-resistant germplasm resources, low efficiency and high randomness of traditional mutation breeding in the existing technology, the present invention aims to provide a herbicide-resistant mutant of AhALS2 gene and its application in peanut breeding.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A herbicide-resistant mutant of the AhALS2 gene has a site-directed mutation of methionine to isoleucine at position 200 of the amino acid sequence of its AhALS2 protein, namely the M200I mutation; the M200I mutation exists in AhALS2-A and AhALS2-B, and the amino acid number corresponds to the amino acid sequence of the Arabidopsis thaliana ALS protein, with the gene accession number AT3G48560.

[0010] The nucleic acid molecule encoding the AhALS2 protein contains a nucleotide mutation that causes the amino acid at position 200 of the AhALS2 protein to change from methionine to isoleucine. In the AhALS2-B gene, this mutation corresponds to an ATG→ATA base substitution at position 579 of the open reading frame, and its nucleotide sequence is shown in SEQ ID NO.1, while the encoded amino acid sequence is shown in SEQ ID NO.2. In the AhALS2-A gene, this mutation corresponds to an ATG→ATA / ATC base substitution at position 585 of the open reading frame, and its nucleotide sequence is shown in SEQ ID NO.3, while the encoded amino acid sequence is shown in SEQ ID NO.4.

[0011] An sgRNA molecule for constructing the mutant, which targets the nucleotide sequence encoding methionine at position 200 of the peanut AhALS2 gene, wherein the target sequence of the sgRNA is: 5'-CAATCATGCGGCGGGGGACC-3'.

[0012] An annealing primer sequence for amplifying the sgRNA molecule is as follows:

[0013] sgRNA-M200-f: attgAATCATGCGGCGGGGGACC;

[0014] sgRNA-M200-r:aaacGGTCCCCCGCCGCATGATT.

[0015] An expression vector for a base-editing plant containing the sgRNA molecule, the expression vector having a cytosine base editor as its backbone and comprising an sgRNA expression cassette driven by the Arabidopsis U6 promoter and an nCas9-deaminase fusion protein expression cassette driven by a plant high-efficiency expression promoter.

[0016] A method for editing the herbicide-resistant mutant, characterized by comprising the following steps:

[0017] (1) Design and synthesize sgRNA that targets the nucleotide sequence corresponding to M200 of the AhALS2 gene;

[0018] (2) Constructing an expression vector for base-editing plants containing sgRNA;

[0019] (3) Transform the plant expression vector into peanut embryogenic callus;

[0020] (4) The transformed callus tissue was subjected to resistance screening and culture to obtain hygromycin-resistant callus lines;

[0021] (5) Extract genomic DNA from resistant callus lines, and identify the editing status of the AhALS2 gene target region by PCR amplification and sequencing, and screen for mutant lines with M200I mutation.

[0022] The application of the herbicide-resistant mutant in improving peanut resistance to ALS inhibitor herbicides.

[0023] Application of the herbicide-resistant mutant in herbicide-resistant flowering plants.

[0024] The present invention has the following beneficial effects:

[0025] 1. This invention, through bioinformatics analysis and base editing technology, has for the first time created the M200I mutant of the AhALS2 gene in peanut. Herbicide resistance identification showed that this mutant significantly improves peanut resistance to the imidazolinone herbicide (imidazolinone acetonide). This resistance site is reported for the first time in plants, enriching the library of available resistance mutation types for the ALS gene.

[0026] 2. This invention utilizes CBE base editing technology to directly introduce desired point mutations at specific sites in the peanut genome, achieving a precise transition from wild-type to resistant phenotype. This method avoids the randomness and time-consuming nature of traditional mutation breeding and can obtain mutant plants without exogenous transgenic components through backcrossing and other methods, making them easier to regulate and accepted by the market.

[0027] 3. The M200 locus of ALS is highly conserved in various crops such as Arabidopsis thaliana, peanut, maize, and rice. The M200I resistance mutation verified in peanut in this invention provides direct evidence and valuable allele resources for cultivating new herbicide-resistant germplasm in other crops using homologous gene editing, and has the potential for cross-species application.

[0028] 4. The CBE-M200 editing vector system constructed in this invention can efficiently achieve targeted editing in peanut cells. The obtained homozygous M200I mutant lines G3-30-1 and G3-122-1 have stable traits and clear resistance, and can be directly used as core parental materials for herbicide-resistant breeding, which can be used for subsequent new variety breeding work and can greatly accelerate the breeding process of herbicide-resistant peanut varieties. Attached Figure Description

[0029] Figure 1 A schematic diagram of the T-DNA region structure of the CBE-M200 plant expression vector for base editing.

[0030] Among them, AtU6: Arabidopsis U6 promoter; sgRNA scaffold: single-path guide RNA scaffold; 35S: cauliflower mosaic virus 35S promoter; Anc689: cytidine deaminase; nCas9(D10A): Cas9(D10A) nickase; UGI: uracil-DNA glycosylase inhibitor; NOS Ter: cinnamine synthase terminator; Hyg: hygromycin B resistance gene; RB / LB: T-DNA right / left boundary.

[0031] Figure 2 This is a Sanger sequencing peak diagram of target sites in some mutant strains of the T0 generation.

[0032] In this diagram, the target sequence is underlined, the original spacer adjacent motif (PAM) sequence is marked in blue, and any base mutations are marked with red arrows and text.

[0033] Figure 3 Phenotypic diagram of resistance to imidazole nicotinic acid in the T3 generation mutant line with homozygous genotype. Detailed Implementation

[0034] To make the technical solution of the present invention clearer, the present invention will be described in detail below with reference to embodiments and accompanying drawings. Experimental methods in the embodiments that do not specify specific conditions were operated under conventional conditions or manufacturer-recommended conditions; reagents and instruments not specified were all commercially available conventional products.

[0035] Example 1: Prediction of herbicide binding sites and sgRNA design for AhALS2 protein

[0036] 1. Binding site prediction: The structures of complexes between the resolved Arabidopsis thaliana ALS protein (gene accession number AT3G48560) and 13 different ALS inhibitor herbicides were retrieved and analyzed using the protein database (https: / / www.rcsb.org). Statistical analysis revealed that 30 amino acid residues are closely associated with the binding of herbicide molecules, specifically the following sites: G120, C121, A122, M124, T167, S168, Q195, V196, P197, R199, M200, I201, G202, A205, F206, Q207, K256, D257, Q260, M351, H352, D376, R377, G380, M490, M570, V571, W574, S653, and G654.

[0037] The M200 site was closely associated with herbicides in multiple analyzed complex structures. Through multiple sequence alignment, the amino acid sequence of Arabidopsis ALS was compared with that of peanut AhALS2-A (LOC112714609) and AhALS2-B (LOC112782285). It was determined that the corresponding site in the amino acid sequences of peanut AhALS2-A and AhALS2-B proteins is also methionine (Met, M). This indicates that the M200 site of the Arabidopsis ALS protein may be a highly conserved potential resistance site.

[0038] 2. sgRNA Design: Based on the gene sequences of AhALS2-A and AhALS2-B in the peanut reference genome (Arachis hypogaea Tifrunner.gnm2.J5K5Genomes), specific sgRNAs were designed targeting the codon (ATG) encoding the corresponding Arabidopsis M200 and its upstream and downstream sequences. To ensure editing efficiency and cover two homologous copies, the designed sgRNA target sequence was sgRNA-M200: 5′-CAATCATGCGGCGGGGGACC-3′ (SEQ ID NO.5). This target sequence is completely identical in AhALS2-A and AhALS2-B, and its original spacer neighbor motif (PAM) is TGG. The CBE editor can be used to edit the C·G base pairs within the target sequence, producing C·G to T·A base pair substitutions or C·G to G·C base pair substitutions, which may mutate ATG (Met) to ATA or ATC (Ile), i.e., the M200I mutation.

[0039] Example 2: Construction of a base editing vector targeting the AhALS2 M200 site

[0040] 1. Vector backbone linearization: The commercially available CBE base editing vector (Weimi Biotechnology (Jiangsu) Co., Ltd.) was digested with Bsa I restriction enzyme (Thermo Fisher Scientific). The digestion products were separated by 1% agarose gel electrophoresis, and the linearized vector fragments were recovered using a DNA purification kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0041] 2. sgRNA oligo annealing and ligation: Based on the designed target sequence, the corresponding annealing primers were designed: sgRNA-M200-f: 5′-attgAATCATGCGGCGGGGGACC-3′ (SEQ ID NO.6); sgRNA-M200-r: 5′-aaacGGTCCCCCGCCGCATGATT-3′ (SEQ ID NO.7).

[0042] The lowercase letters in the primer sequence are the sticky ends formed after annealing, which can be linked to the intermediate vector of sgRNA after enzyme digestion, while the uppercase letters are the target sequences.

[0043] The primers were dissolved to 10 OD using annealing buffer (Beijing Solarbio Science & Technology Co., Ltd.). 5 μL of each primer pair was mixed and annealed in a PCR instrument at 95 °C for 2 min, then cooled to 25 °C at a rate of 1 °C per minute. 1 μL of the annealed product was ligated into the linearized vector recovered in step 1 using T4 DNA ligase (Takara).

[0044] 3. Transformation and Positive Clone Identification: The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing the appropriate antibiotics, and incubated overnight at 37 °C. Single colonies were picked for shake culture, and the plasmid was extracted and verified by Sanger sequencing using primer U6-seq: 5′-gcctcttcgctattacgcca-3′ (SEQ ID NO.8). Plasmids with correct sequencing were successfully constructed as base-editing plant expression vectors, named CBE-M200. A schematic diagram of its T-DNA region structure is shown below. Figure 1 .

[0045] Example 3: Transformation of peanuts using the CBE-M200 recombinant vector and construction of mutants

[0046] 1. Induction and culture of peanut embryogenic callus

[0047] a. Take mature and plump seeds of Yuhua 9326, disinfect them with 75% ethanol for 1 min, soak them in 0.1% HgCl2 solution for 8 min, rinse them with sterile water 5-7 times, and soak them overnight.

[0048] b. In a clean bench, peel off the seed coat, remove the cotyledons, embryonic leaflets and hypocotyl, and inoculate the epicotyl onto MS induction medium (formulation shown in Table 1).

[0049] c. Culture it in the dark at 28 ℃, subculturing it every 3-4 weeks. After 3-4 subcultures, screen for materials that show embryogenic callus and continue culturing for 4-12 months to obtain embryogenic callus with good growth status that can be used for genetic transformation.

[0050] Table 1. MS induction medium stock solution formulation

[0051]

[0052] 2. Gene gun genetic transformation

[0053] a. Preparation of gold powder bullets: Weigh 60 mg of gold powder (particle size 1 μm) into a 2 mL centrifuge tube, and wash it sequentially with 1 mL of anhydrous ethanol by ultrasonication, 1 mL of anhydrous ethanol by vortexing, and 1 mL of sterile water by vortexing. After centrifugation and discarding the supernatant, resuspend the powder in 50% sterile glycerol to prepare a 60 mg / mL gold powder suspension, and store it at 4 ℃ for later use.

[0054] b. Plasmid encapsulation: Take 50 μL of gold powder suspension and add 10 μL of CBE-M200 plasmid (1 μg / μL), 50 μL of 2.5 M CaCl2, and 20 μL of 0.1 M spermidine sequentially, vortexing for 30 s after each addition. Centrifuge and discard the supernatant. Wash once with 70% ethanol and once with anhydrous ethanol, and finally resuspend in 60 μL of anhydrous ethanol.

[0055] c. Bombardment transformation: Spread the prepared peanut embryogenic callus tissue evenly in the center of MS medium. Use the GJ-1000 gene gun to bombard the callus according to the instructions.

[0056] d. Screening and proliferation: After bombardment, the callus tissue was cultured in the dark at 28 °C for 3 days, and then transferred to MS selection medium containing 20 mg / L hygromycin. After 1 month of selection culture, newly formed resistant callus tissue was picked and transferred to fresh MS medium containing hygromycin for subculture twice to obtain sufficient transformed callus material for subsequent analysis.

[0057] 3. Editing efficiency detection and mutant screening

[0058] a. Genomic DNA extraction: 144 independent callus lines were randomly selected from the hygromycin-resistant callus population transformed by CBE-M200, and genomic DNA was extracted from each line using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP350).

[0059] b. Target site PCR amplification: To detect the editing status of the two copies of AhALS2-A and AhALS2-B, specific primers were designed with the following sequences: AhALS2-A: forward primer AhALS2A-F: 5′-GGCGGCATATGAATGCTCTT-3′ (SEQ ID NO.9), reverse primer AhALS2A-R: 5′-AACCATTCCACAAGTGGCTGTA-3′ (SEQ ID NO.10);

[0060] AhALS2-B: Forward primer AhALS2B-F: 5′-ATGCGTACTAACTCAACATATGG-3′ (SEQ ID NO. 11), Reverse primer AhALS2B-R: 5′-ACAGACTAAGAATTAATCCGTGAC-3′ (SEQ ID NO. 12); PCR reaction system (50 μL): 100 ng genomic DNA template, 1× PrimeSTAR GXL buffer, 200 μM dNTPs, 0.3 μM each of forward and reverse primers, 1.25 U PrimeSTAR GXL DNA polymerase (Takara, catalog number: R050Q). Amplification program: 94℃ pre-denaturation for 30 s, followed by 30 cycles (98℃ denaturation for 10 s, 55℃ annealing for 15 s, 68℃ extension for 3.5 min), and a final extension at 72℃ for 5 min.

[0061] c. Sequencing and Mutation Identification: After purifying the PCR products, Sanger sequencing was performed using sequencing primer 991seq: 5′-TCCCTCTCCAACACCAAC-3′ (SEQ ID NO.13). The lines undergoing base editing were identified by analyzing the sequencing peak diagram.

[0062] d. Mutant acquisition: Sequencing results showed that the CBE-M200 editor achieved precise editing in multiple lines.

[0063] In the T0 generation line G3-30, an ATG→ATA mutation occurred at nucleotide 579 of the AhALS2-B copy open reading frame, resulting in a change of isoleucine (Ile, I) (M200I) at amino acid position 193 (corresponding to amino acid position 200 in Arabidopsis ALS) from Met. The nucleotide sequence after the mutation is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Sequencing peak diagrams are shown below. Figure 2 .

[0064] In the T0 generation line G3-122, an ATG→ATA / ATC bis-allelic mutation occurred at nucleotide 585 of the AhALS2-A copy open reading frame, resulting in an M200I mutation at amino acid 195 of AhALS2-A (corresponding to amino acid 200 of Arabidopsis ALS). The nucleotide sequence after the mutation is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4. Sequencing peak diagram is shown below. Figure 2 .

[0065] These lines were subsequently regenerated and self-crossed twice to obtain the genotype-homozygous T3 generation mutant lines G3-30-1 and G3-122-1.

[0066] Example 4: Herbicide resistance identification of AhALS2 M200I mutant

[0067] 1. Material preparation: Seeds of the homozygous mutant lines G3-30-1 (AhALS2-B M200I) and G3-122-1 (AhALS2-A M200I) obtained in Example 3 were selected, with wild-type Yuhua 9326 as a control.

[0068] 2. Planting and Treatment: Sow the seeds in nutrient pots filled with vermiculite and cultivate them under conventional greenhouse conditions. When the plants reach the 3-6 leaf stage, spray the plants evenly with an aqueous solution of imidazoline (IMI class herbicide) using a sprayer. The concentration of the active ingredient in the herbicide is 1 g ai / L (ai active ingredient).

[0069] 3. Phenotypic observation and recording: On days 0, 7, 14, 21 and 28 after herbicide application, observe and record the plant growth status and damage symptoms (such as chlorosis, wilting, growth inhibition, death, etc.), and take photos.

[0070] 4. Results Analysis

[0071] Figure 3 Phenotypic diagram of resistance to imidazole nicotinic acid in the T3 generation mutant line with homozygous genotype.

[0072] Figure 3 Figure A: Comparison of growth between wild-type Yuhua 9326 (WT) and mutant G3-30-1 after application of imidacloprid (1 g ai / L); Figure B: Comparison of growth between wild-type Yuhua 9326 (WT) and mutant G3-122-1 after application of imidacloprid (1 g ai / L). The numbers 0 d, 7 d, 14 d, 21 d, and 28 d below Figures A and B represent the number of days after herbicide application.

[0073] As can be seen, after herbicide application, wild-type Yuhua 9326 plants exhibited obvious damage symptoms, with stunted growth. In contrast, the lines G3-30-1 and G3-122-1 carrying the AhALS2 M200I mutation showed basically normal plant growth, with only a few leaves showing slight chlorosis, demonstrating significant herbicide resistance. This result proves that the AhALS2 protein M200I mutation can effectively confer resistance to imidazoline in peanuts.

[0074] In summary, this invention successfully identified and validated a novel herbicide resistance site, M200, in the peanut AhALS2 gene using base editing technology. By constructing the CBE-M200 editing vector, the M200I mutant was precisely created in peanut, exhibiting significant resistance to imidazolinone herbicides. This invention not only provides new germplasm and core gene resources for herbicide-resistant peanut breeding but also offers a new approach for cultivating herbicide-resistant varieties in other crops using homologous editing, demonstrating significant theoretical value and application prospects.

[0075] The following is a partial sequence from the text:

[0076] SEQ ID NO.1 (nucleotide sequence of the AhALS2-B gene mutant)

[0077]

[0078] SEQ ID NO.2 (Amino acid sequence of AhALS2 - B gene mutant)

[0079] MAATASKPSFPGFQSLQSSSTSSKQALTFTKFPNYPSSSSHTLRITCSLSNTNPKHNPKLSPPNASTAATSSPSVVVDNFVSRFAPNEPRKGADILVEALERQGVTDVFAYPGGASMEIHQALTRSSTIRNVLPRHEQGGVFAAEGYARSSGLAGVCIATSGPGATNLVSGLADALLDSVPLIAITGQVPRRIIGTDAFQETPIVEVTRSITKHAYLVLDVDDIPRIVNEAFFLAISGRPGPVLIDIPKDIQQQLAVPNWDQPVMLNAYMSRLPKAPNESYLEQIVRLLLESKKPVLYVGGGSLNASEELRRFVELTGVPVASTLMGLGSYPVGGENSLQMLGMHGTVYANYAVDKSDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKLPHVSVCGDLKLALSGINRILESRGVKGKLDFRAWREELNEQKLKFPLSYKTFGDDLIPPQHAIQVLDELTNGDAIISTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVVVDIDGDGSFMMNVQELATIRVENLPIKILLLNNQHLGMVVQWEDRFYKSNRAHTYLGDPSKENEIFPNMLHFADACGIPAARVTKKQDLREAIQKMLDTPGPYLLDVIVPHQEHVLPMIPANGSFEDVITEGDGRTKY

[0080] SEQ ID NO.3 (Nucleotide sequence of AhALS2 - A gene mutant)

[0081]

[0082] SEQ ID NO. 4 (Amino acid sequence of the AhALS2-A gene mutant)

[0083] MAATASKPSFPAFQSLQSSSTSSKQALTFTKFPNYPSSSSHTLRITCSLSNTNPNPKHNPKLSPPNASTAATSSPSVVGDNFVSRFAPNEPRKGADILVEALERQGVTDVFAYPGGASMEIHQALTRSSKIRNVLPRHEQGGVFAAEGYARSSGLAGVCIATSGPGATNLVSGLADALLDSVPLIAITGQVPRRIIGTDAFQETPIVEVTRSITKHAYLVLDVDDIPRIVNEAFFLAISGRPGPVLIDIPKDIQQQLAVPNWDQPVMLNAYMSRLPKAPNESYLEQIVRLLLESKKPVLYVGGGSLNASEELRRFVELTGVPVASTLMGLGSYPVGGENSLQMLGMHGTVYANYAVDKSDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKLPHVSVCGDLKLALSGINRILESRGVKGKLDFRAWREELNEQKLKFPLSYKTFGEDLIPPQHAIQVLDELTNGDAIISTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVVVDIDGDGSFMMNVQELATIRVENLPIKILLLNNQHLGMVVQWEDRFYKSNRAHTYLGDPSKENEIFPNMLHFADACGIPAARVTKKQDLREAIQKMLDTPGPYLLDVIVPHQEHVLPMIPANGSFEDVITEGDGRTKY

Claims

1. A herbicide-resistant mutant of the AhALS2 gene, characterized in that, The mutant has a site-directed mutation at position 200 of the methionine in its AhALS2 protein to isoleucine, i.e., the M200I mutation; the M200I mutation is present in AhALS2-A and AhALS2-B, and the methionine at position 200 corresponds to the amino acid sequence of the ALS protein of Arabidopsis thaliana, whose gene accession number is AT3G48560.

2. The herbicide-resistant mutant according to claim 1, characterized in that, The nucleic acid molecule encoding the AhALS2 protein contains a nucleotide mutation that causes the amino acid at position 200 of the AhALS2 protein to change from methionine to isoleucine. In the AhALS2-B gene, this mutation corresponds to an ATG→ATA base substitution at position 579 of the open reading frame, and its nucleotide sequence is shown in SEQ ID NO.1, while the encoded amino acid sequence is shown in SEQ ID NO.

2. In the AhALS2-A gene, this mutation corresponds to an ATG→ATA / ATC base substitution at position 585 of the open reading frame, and its nucleotide sequence is shown in SEQ ID NO.3, while the encoded amino acid sequence is shown in SEQ ID NO.

4.

3. An sgRNA molecule for constructing the mutant of claim 1, characterized in that, It targets the nucleotide sequence of the peanut AhALS2 gene that encodes methionine at position 200, and the target sequence of the sgRNA is: 5'-CAATCATGCGGCGGGGGACC-3'.

4. An annealing primer for amplifying the sgRNA molecule of claim 3, characterized in that, The annealing primer sequence is as follows: sgRNA-M200-f: attgAATCATGCGGCGGGGGACC; sgRNA-M200-r:aaacGGTCCCCCGCCGCATGATT.

5. An expression vector for a base-editing plant containing the sgRNA molecule of claim 3, characterized in that, The expression vector uses a cytosine base editor as its backbone and contains an sgRNA expression cassette driven by the Arabidopsis U6 promoter and an nCas9-deaminase fusion protein expression cassette driven by a plant high-efficiency expression promoter.

6. A method for editing the herbicide-resistant mutant of claim 1, characterized in that, Includes the following steps: (1) Design and synthesize sgRNA that targets the nucleotide sequence corresponding to M200 of the AhALS2 gene; (2) Constructing an expression vector for base-editing plants containing sgRNA; (3) Transform the plant expression vector into peanut embryogenic callus; (4) The transformed callus tissue was subjected to resistance screening and culture to obtain hygromycin-resistant callus lines; (5) Extract genomic DNA from resistant callus lines, and identify the editing status of the AhALS2 gene target region by PCR amplification and sequencing, and screen for mutant lines with M200I mutation.

7. The application of the herbicide-resistant mutant of claim 1 or 2 in improving peanut resistance to ALS inhibitor herbicides.

8. The application of the herbicide-resistant mutant of claim 1 in herbicide-resistant flowering plants.

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