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CN-121975857-A - Method for creating haloxyfop-R-methyl herbicide-resistant rice by gene editing OsACCase gene promoter region

CN121975857ACN 121975857 ACN121975857 ACN 121975857ACN-121975857-A

Abstract

The invention relates to the technical field of gene editing, and particularly provides a method for creating haloxyfop-R-methyl herbicide-resistant rice by gene editing OsACCase gene promoter regions. The method constructs a crRNA plasmid library covering OsACCase gene promoter region based on CRISPR/LbCas a system, converts rice callus through agrobacterium mediation, through resistance screening, haloxyfop-R-methyl herbicide screening and molecular identification, the haloxyfop-R-methyl resistant rice line is obtained. The strain generates-4 bp/-5bp double-allele deletion mutation in 1865 position region upstream of OsACCase gene initiation codon ATG, and the homozygote mutant strain and the double-allele deletion mutant strain can normally grow after being sprayed with 6 times field recommended dose of haloxyfop-R-methyl. According to the invention, herbicide resistance is realized by editing the gene promoter region, the growth defect of mutation of the coding region is avoided, the breeding technology is enriched, and the weed control is assisted.

Inventors

  • LIU YUQING
  • WANG XIAONING
  • ZHOU HUANBIN
  • YAN FANG
  • REN BIN
  • LI XUEQI

Assignees

  • 海南省农业科学院三亚研究院(海南省实验动物研究中心)
  • 海南省农业科学院粮食作物研究所
  • 三亚中国农业科学院国家南繁研究院

Dates

Publication Date
20260505
Application Date
20260403

Claims (6)

  1. The application of an OsACCase gene mutant in rice anti-ACCase inhibitor herbicide haloxyfop-R-methyl is characterized in that the mutation type of the OsACCase gene mutant is any one of the following: 1) Introducing-4 bp/-5bp double-allele deletion mutation in OsACCase gene promoter region, namely taking A in OsACCase gene start codon ATG as 0 position, deleting 4bp in 1865-1868 positions on one allele located upstream of start codon ATG, deleting 5bp in 1865-1869 positions on the other allele located upstream of start codon ATG; 2) Introducing-5 bp/-5bp homozygous deletion mutation in OsACCase gene promoter region, namely taking A in OsACCase gene initiation codon ATG as 0 position, deleting 5bp in 1865-1869 positions positioned upstream of initiation codon ATG, and making said deletion mutation on both alleles; The nucleotide sequence of OsACCase gene is shown in SEQ ID No. 1.
  2. 2. The use according to claim 1, wherein the rice is of the variety Kitaake.
  3. 3. A method for making rice resistant to ACCase inhibitor herbicide haloxyfop-R-methyl is characterized in that-4 bp/-5bp double-allele deletion mutation in claim 1 is introduced into OsACCase gene promoter region in rice genome by gene editing technology, so as to obtain rice resistant to ACCase inhibitor herbicide haloxyfop-R-methyl.
  4. 4. The method of claim 3, wherein the gene editing is accomplished by a CRISPR/LbCas a system.
  5. 5. The method according to claim 4, characterized in that it comprises the steps of: Firstly, synthesizing a LbCas a sequence optimized by rice codons, replacing Cas9-NG genes in a pUbi Cas9-NG vector to obtain a pUbi LbCas a vector, simultaneously synthesizing a crRNA expression frame fragment, fusing with a pENTR4 vector skeleton to obtain a pENTR4 crRNA vector, integrating the crRNA expression frame into the pUbi LbCas a vector through LR cloning reaction to obtain a pUbi LbCas a-crRNA vector, cloning the ccdB expression frame into the pUbi LbCas a-crRNA vector digested by BsaI to obtain a pHZLib-LbCas a vector; step two, a crRNA sequence for gene editing is obtained, wherein the crRNA sequence is shown as SEQ ID No. 11; Step three, replacing ccdB gene on pHZLib-LbCas a vector with the crRNA, so that the crRNA sequence is cloned into pHZLib-LbCas a vector to obtain pHZLib-LbCas12a-OsACCasecrRNA; And fourthly, converting pHZLib-LbCas12a-OsACCasecrRNA into agrobacterium, infecting rice callus, and screening the rice strain resisting the ACCase inhibitor herbicide.
  6. 6. The method according to claim 5, wherein the rice is of a variety Kitaake.

Description

Method for creating haloxyfop-R-methyl herbicide-resistant rice by gene editing OsACCase gene promoter region Technical Field The invention belongs to the fields of plant gene editing and crop molecular breeding, and relates to a method for creating haloxyfop-R-methyl herbicide resistant rice by gene editing OsACCase gene promoter regions. Background The rice is a core ration crop and an important economic crop in China, and has irreplaceable strategic positions in the aspect of guaranteeing national grain safety. Weeds are used as main pests in the rice field ecological system, seriously interfere the growth and development process of rice, and reduce the yield and quality. Especially in recent years, with the rapid transformation of agricultural production in China to the mechanized and intelligent directions, the community structure of paddy field weeds is remarkably changed, and the severe situations of rapid increase of the types of resistant weeds, rapid population succession rate and annual expansion of the occurrence area are presented, so that new challenges and tasks are brought to comprehensive prevention and control of paddy field weeds. Acetyl-coa carboxylase (actyl-CoA carboxylase, ACCase) is a key rate-limiting enzyme in plant chloroplasts that regulates the de novo synthesis pathway of fatty acids, which catalyzes the initial step of the synthesis process. The herbicide taking ACCase as an action target has wide application in preventing and killing gramineous weeds in broadleaf crop fields, wherein the haloxyfop-R-methyl has become one of core agents for preventing and controlling broadleaf crop fields by virtue of the remarkable advantages of wide preventing and killing spectrum, stable preventing and controlling effect, suitability for various production scenes and the like. But is limited by rice sensitivity, the agent is not registered and applied in rice production at present, and ACCase inhibitor herbicides which are approved to be used in the current rice production are only three types of cyhalofop-butyl, metamifop and fenpropion, so that the prevention and control diversified requirements of paddy weeds, especially resistant weeds, are difficult to meet, and the prevention and control bottleneck is increasingly outstanding. Under the background, the cultivation of the rice variety resisting the ACCase inhibitor herbicide is particularly urgent, the cultivation of the haloxyfop-R-methyl-resistant rice can break through the selection limitation of the existing medicines, the high-efficiency herbicide can be applied to the prevention and control of paddy field weeds, the problems of spread of the resistant weeds, poor prevention and control effects and the like can be solved more pertinently, and the high-efficiency herbicide has important practical significance and application value in improving the comprehensive prevention and control capability of paddy field weeds, ensuring the high and stable yield of rice and building the national grain safety defense line. Aiming at the cultivation of crops against ACCase inhibitor herbicides, the current core technical path is mainly to carry out site-directed mutation on the gene coding region of target protein ACCase by molecular biology techniques such as gene editing and the like, induce the change of the coded amino acid sequence, further reduce the binding affinity of protein and herbicide, avoid the interference of the herbicide on the normal physiological metabolism of the crops and finally lead the crops to obtain herbicide resistance. However, ACCase protein mutations often accompany altered enzymatic activity, which in turn leads to undesirable phenotypes such as defective growth and development in crops. Therefore, the method for excavating more haloxyfop-R-methyl resistant gene resources has very important significance for cultivating haloxyfop-R-methyl resistant crops. The study is based on a CRISPR/LbCas a gene editing system, and performs directed editing aiming at a non-coding regulatory region of a rice OsACCase gene, so that new rice germplasm with ACCase inhibitor herbicide resistance is successfully screened and identified. The research not only enriches and expands the technological path of herbicide-resistant rice, but also provides important reference for functional analysis of plant non-coding regions and precise molecular breeding. Disclosure of Invention The invention aims to create a novel rice germplasm which is resistant to haloxyfop-R-methyl herbicide and is not influenced in growth and development by utilizing a CRISPR/LbCas a system and editing a non-coding regulatory region of a rice endogenous OsACCase gene, thereby providing a brand new technical scheme and gene resources for developing safe and efficient herbicide-resistant rice varieties and finally realizing light simplification, cost reduction and synergy of rice field weed management. In order to achieve the above purpose, the present invention provides