CN-121495992-B - Method for creating herbicide-resistant rice by gene editing OsEPSPS gene intron region

Abstract

The invention relates to the technical field of gene editing, and provides a method for creating herbicide-resistant rice by gene editing OsEPSPS gene intron regions, which comprises the steps of introducing a WT/-22bp monoallelic deletion mutation or a WT/-32+6bp monoallelic deletion insertion mutation into OsEPSPS gene intron regions, namely, taking A in an ATG (ATG) of a OsEPSPS gene initiation codon as 0 position, deleting 22bp from 771 to 792 positions positioned at the downstream of one allele or deleting 32bp from 775 to 806 positions positioned at the downstream of one allele, and inserting 6bp in the interval, wherein the other allele is unchanged. The invention discovers that the resistance to EPSPS inhibitor herbicides can be obtained for rice by introducing the mutation, the resistance is identified in T0 generation plants carrying the mutation, and the resistance can be stably inherited to progeny plants, thereby having great application value for improving the herbicide use efficiency.

Inventors

• ZHOU HUANBIN
• YU MAN
• YAN FANG
• REN BIN

Assignees

• 三亚中国农业科学院国家南繁研究院
• 中国农业科学院植物保护研究所

Dates

Publication Date

20260512

Application Date

20260114

Claims (2)

1. The application of the OsEPSPS gene mutant in herbicide glyphosate resisting EPSPS inhibitor of rice is characterized in that the mutation type of the OsEPSPS gene mutant is that-22 bp/-22bp homozygous deletion mutation is introduced into OsEPSPS gene intron region, namely, 22bp is deleted from 771-792 positions positioned downstream of initiation codon ATG by taking A in initiation codon ATG of OsEPSPS gene as 0 position, the homozygous deletion mutation occurs on both alleles, the nucleotide sequence of OsEPSPS gene is shown as SEQ ID NO.5, and the rice variety is Nanjing 46.
2. 2. A method for making rice resistant to EPSPS inhibitor herbicide glyphosate is characterized in that the method is to obtain the rice resistant to EPSPS inhibitor herbicide glyphosate by utilizing the homozygous deletion mutation of-22 bp/-22bp in claim 1, wherein the rice is of a variety of Nanjing 46.

Description

Method for creating herbicide-resistant rice by gene editing OsEPSPS gene intron region Technical Field The invention belongs to the fields of plant gene editing and crop molecular breeding, and relates to a method for creating herbicide-resistant rice by gene editing OsEPSPS gene intron regions. Background In the production process, grass damage is one of important factors influencing the yield and quality of rice, and is more easily restricted by grass damage particularly in the direct seeding cultivation process of rice. Chemical control is currently a relatively cost effective weeding method in view of the current reduction in agricultural labor (manual weeding) and the ongoing development of mechanical weeding. The non-selective herbicide has a lethal effect on all green plants, has a simple using method, is unsuitable for use and is easy to generate phytotoxicity on rice, and the selective herbicide is effective on specific weeds, but has high requirements on application time and mode, and is tedious to use and high in cost. Therefore, the cultivation of non-selective herbicide resistant rice can help to improve the chemical control effect of weeds in rice fields and simplify control measures, and finally, the yield and quality of rice are improved. Glyphosate (Glyphosate), which is a broad-spectrum, low-toxicity and contact-killing herbicide, is widely used for weed control in agricultural production. Glyphosate resistant transgenic crops are also the most important traits in current transgenic crops. Glyphosate specifically binds to and inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS), a key enzyme of the shikimate pathway, thereby blocking the synthesis of aromatic amino acids, resulting in hindered protein synthesis, inhibited growth and development, and ultimately leading to plant death. At present, two main modes of creating glyphosate-resistant crops are that a transgenic crop with glyphosate resistance is obtained by expressing an EPSPS which is derived from microorganisms and is insensitive to glyphosate in the crop body, but the application of the transgenic crop needs strict and complicated safety evaluation and approval, and the other mode is that the EPSPS protein resistance of glyphosate-resistant weeds is changed (P171S, TIPS and the like), and the gene editing technology is utilized to accurately edit the endogenous EPSPS gene coding region of the crop to obtain the glyphosate-resistant gene editing crop, but the protein sequence is changed and the protein function is influenced, so that the adverse effect on the growth and development of the crop is caused. The current generation of glyphosate-resistant crops still faces the problems of lack of heterologous resistance genes and poor resistance after mutation of endogenous resistance genes, and more glyphosate-resistant gene resources are excavated, so that the method has very important significance for cultivating the glyphosate-resistant crops. The study is based on a CRISPR/Cas12i3 gene editing system, performs directional editing on a non-coding regulatory region of a rice OsEPSPS gene, screens and identifies novel rice germplasm with stable EPSPS inhibitor herbicide resistance. The research not only expands the creating way of herbicide-resistant rice, but also provides important reference for non-coding region function research and precise molecular breeding. Disclosure of Invention The invention aims to create a new rice germplasm which is resistant to glyphosate and has no influence on growth and development by editing a non-coding regulatory region of a rice endogenous OsEPSPS gene by utilizing a CRISPR/Cas12i3 system, provide a brand new technical scheme and gene resources for developing safe and efficient herbicide-resistant rice varieties, and finally realize light simplification, cost reduction and synergy of rice field weed management. In order to achieve the above purpose, the present invention provides the following technical solutions: In a first aspect the present invention provides a mutant of OsEPSPS genes, the type of mutation of the OsEPSPS gene mutant being any of the following: 1) Introducing WT/-22bp monoallelic deletion mutation in OsEPSPS gene intron, namely deleting 22bp from 771 to 792 of one allele with A in OsEPSPS gene initiation codon ATG as 0 position and maintaining the other allele unchanged; 2) Introducing-22 bp/-22bp homozygous deletion mutation into OsEPSPS gene intron region, namely taking A in OsEPSPS gene start codon ATG as 0 position, deleting 22bp from 771 to 792 positions positioned downstream of start codon ATG, and making said homozygous deletion mutation on both alleles; 3) Introducing WT/-32+6bp monoallelic deletion insertion mutation into OsEPSPS gene intron region, namely taking A in OsEPSPS gene initiation codon ATG as 0 position, deleting 32bp from 775 to 806 positions positioned downstream of initiation codon A