Search

CN-122012441-A - Herbicide resistance mutant of AhALS gene and application of herbicide resistance mutant in peanut breeding

CN122012441ACN 122012441 ACN122012441 ACN 122012441ACN-122012441-A

Abstract

The invention discloses a AhALS gene herbicide resistance mutant and application thereof in peanut breeding, wherein the mutant is subjected to site-directed mutation of methionine at the 200 th site of an amino acid sequence of AhALS protein into isoleucine (namely M200I mutation). Herbicide resistant peanut materials in which the M200I mutation occurred in AhALS-B gene and AhALS-a gene were obtained by editing, respectively. The invention successfully excavates and verifies a brand new herbicide resistance site M200 in the peanut AhALS gene by utilizing a base editing technology, and accurately creates an M200I mutant in the peanut by constructing a CBE-M200 editing carrier, wherein the mutant has obvious resistance to imidazolinone herbicides. The invention not only provides new germplasm and core gene resources for peanut herbicide-resistant breeding, but also provides a new idea for cultivating herbicide-resistant varieties in other crops by utilizing homologous editing.

Inventors

  • ZHANG XINYOU
  • DONG WENZHAO
  • XUE LULU
  • SHI LEI
  • ZHAO HUANHUAN
  • LI XIAONA
  • FANG YUANJIN
  • DAI XIAODONG
  • GAO WEI
  • QIN LI

Assignees

  • 河南省农业科学院
  • 神农种业实验室

Dates

Publication Date
20260512
Application Date
20260228

Claims (8)

  1. 1. Herbicide-resistant mutant of the AhALS gene, characterized in that it has site-directed mutation of methionine AT position 200 of the amino acid sequence of the AhALS protein to isoleucine, the M200I mutation, which is present in AhALS-A and AhALS-B, the methionine AT position 200 corresponding to the amino acid sequence of the ALS protein of Arabidopsis Arabidopsis thaliana, with the gene accession number AT3G48560.
  2. 2. The herbicide resistance mutant according to claim 1, characterized in that the nucleic acid molecule encoding the AhALS protein comprises a nucleotide mutation which results in the conversion of amino acid 200 of the AhALS protein from methionine to isoleucine, in the AhALS-B gene the mutation corresponds to an atg→ata base substitution at nucleotide 579 of the open reading frame, the nucleotide sequence of which is shown in SEQ ID No.1, the encoded amino acid sequence of which is shown in SEQ ID No.2, and in the AhALS-a gene the mutation corresponds to an atg→ata/ATC base substitution at nucleotide 585 of the open reading frame, the nucleotide sequence of which is shown in SEQ ID No.3, the encoded amino acid sequence of which is shown in SEQ ID No. 4.
  3. 3. A sgRNA molecule for constructing the mutant of claim 1, wherein the target sequence of the sgRNA is 5'-CAATCATGCGGCGGGGGACC-3', and the nucleotide sequence of the mutant is a nucleotide sequence encoding methionine at 200 of peanut AhALS gene.
  4. 4. An annealing primer for amplifying the sgRNA molecule of claim 3, wherein the annealing primer sequence is: sgRNA-M200-f:attgAATCATGCGGCGGGGGACC; sgRNA-M200-r:aaacGGTCCCCCGCCGCATGATT。
  5. 5. An expression vector of a base editing plant containing the sgRNA molecule of claim 3, wherein the expression vector uses a cytosine base editor as a skeleton, and comprises an sgRNA expression cassette driven by an arabidopsis U6 promoter and a nCas-deaminase fusion protein expression cassette driven by a plant efficient expression promoter.
  6. 6. A method of editing the herbicide resistance mutant of claim 1, comprising the steps of: (1) Designing and synthesizing sgRNA of a nucleotide sequence corresponding to a target AhALS gene M200; (2) Constructing an expression vector of a base editing plant containing sgRNA; (3) Transforming the plant expression vector into peanut embryogenic callus; (4) Performing resistance screening and culture on the transformed callus to obtain hygromycin resistance callus lines; (5) Extracting genomic DNA of the resistant callus line, identifying the editing condition of AhALS gene target area through PCR amplification and sequencing, and screening to obtain the mutant line with M200I mutation.
  7. 7. Use of the herbicide-resistant mutant of claim 1 or 2 to increase resistance of peanuts to ALS inhibitor herbicides.
  8. 8. Use of the herbicide-resistant mutant of claim 1 in herbicide-resistant peanut breeding.

Description

Herbicide resistance mutant of AhALS gene and application of herbicide resistance mutant in peanut breeding Technical Field The invention belongs to the technical field of plant genetic engineering and crop genetic breeding, and particularly relates to a AhALS gene herbicide resistance mutant and application thereof in peanut breeding. Background Peanuts are globally important oil plants and cash crops, and play an important role in agricultural economy in China. The field weeds are one of main biotic stress for restricting the high and stable yield of peanuts, and chemical weeding is the most efficient weed prevention and control means at present. However, commercial herbicide-resistant peanut varieties and matched herbicide products with independent intellectual property rights are not available in China, so that the efficiency and benefit of weed control in the peanut production process are limited to a certain extent, and the healthy development of the peanut industry is restricted. Therefore, the cultivation of new herbicide-resistant peanut germplasm suitable for agricultural production requirements in China has important significance for improving the technical level of peanut production and guaranteeing the national grain and oil safety. Acetolactate synthase (Acetolactate synthase, ALS) is a key enzyme in the first step of biosynthesis of branched-chain amino acids (valine, leucine, isoleucine) in plants, and is also a combined action target of five types of ALS inhibitor herbicides such as sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine salicylates, and sulfonylaminocarbonyl triazolinones. The herbicide can inhibit the catalytic function of ALS enzyme by combining with the active center of the ALS enzyme, so that the synthesis of branched chain amino acid is blocked, and the death of plants is caused. Studies have shown that mutations in specific amino acid residues of the ALS enzyme alter its ability to bind to ALS inhibitor herbicides, thereby conferring herbicide resistance to plants. In a variety of plants such as Arabidopsis, rice, maize, and the like, a number of conserved sites associated with resistance have been identified, such as A122, P197, A205, D376, W574, S653, and G654, and the like (ALS amino acid numbers correspond to Arabidopsis ALS sequences throughout, AT3G 48560). Different amino acid substitution patterns at different sites or at the same position often result in different levels of resistance to different types of ALS inhibitors. For example, mutations at the P197 position generally confer greater resistance to sulfonylurea herbicides, and mutations at the S653 and G654 positions result in resistance to imidazolinone herbicides. Resistance resulting from simultaneous mutation of multiple key amino acid sites is additive. In recent years, base Editors (Base Editors, BEs) developed based on CRISPR/Cas systems, including cytosine Base Editors (Cytosine Base Editor, CBE) and adenine Base Editors (Adenine Base Editor, ABE), are capable of achieving precise Base substitution at specific genomic sites without causing DNA double strand breaks, providing a powerful tool for screening novel, beneficial amino acid mutations in crops. The potential binding sites of the ALS gene are systematically edited by using a base editor, new resistance mutations are mined, and crop germplasm resources with new resistance types or stronger resistance are expected to be cultivated. The peanut genome contains two highly homologous copies of the ALS gene (AhALS-A and AhALS-B). At present, the herbicide-resistant locus of the peanut AhALS gene is not fully excavated, and particularly, the research on precise mutation by utilizing a leading-edge base editing technology is more lacking. Therefore, the base editing technology is utilized to target the peanut AhALS2 gene, a new herbicide resistance site is excavated, and new peanut germplasm with autonomous intellectual property and definite resistance is cultivated, so that the method has important value for promoting the progress of peanut breeding technology and industrial development in China. Disclosure of Invention Aiming at the problems of shortage of herbicide-resistant germplasm resources of peanuts, low traditional mutation breeding efficiency, high randomness and the like in the prior art, the invention aims to provide a AhALS gene herbicide-resistant mutant and application thereof in peanut breeding. In order to achieve the above purpose, the invention adopts the following technical scheme: A herbicide resistance mutant of AhALS gene, in which methionine AT 200 th position of amino acid sequence of AhALS protein is site-directed mutated to isoleucine, namely M200I mutation, wherein the M200I mutation exists in AhALS-A and AhALS-B, the amino acid number corresponds to the amino acid sequence of ALS protein of Arabidopsis thaliana (Arabidopsis thaliana), and the gene accession number is AT3G48560. The nucleic acid molecule for cod