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CN-121975825-A - Method for creating herbicide-resistant rice by gene editing OsEPSPS gene 3' UTR region

CN121975825ACN 121975825 ACN121975825 ACN 121975825ACN-121975825-A

Abstract

The invention relates to the technical field of gene editing, and particularly provides a method for creating herbicide-resistant rice by gene editing OsEPSPS gene 3'UTR region, wherein the method is to introduce-38/-13+12 bp double-allele deletion insertion mutation in OsEPSPS gene 3' UTR region, namely, the A in OsEPSPS gene initiation codon ATG is 0 site, two alleles are respectively located at 3363 to 3400 site deletion 38bp and located at 3377 to 3389 site deletion 13bp at the downstream thereof, and 12bp is inserted in the interval. The invention discovers that the rice can obtain the resistance to the EPSPS inhibitor herbicide by introducing the double allelic deletion insertion mutation, the resistance is identified in the T0 generation plant carrying the mutation, and the resistance can be stably inherited to the progeny plant, thereby having great application value for guaranteeing the safety of agricultural production and improving the use efficiency of the herbicide.

Inventors

  • ZHOU HUANBIN
  • REN BIN
  • YU MAN

Assignees

  • 中国农业科学院植物保护研究所

Dates

Publication Date
20260505
Application Date
20251222

Claims (8)

  1. The application of the OsEPSPS gene mutant in the rice EPSPS inhibitor herbicide is characterized in that the mutation type of the OsEPSPS gene mutant is any one of the following: 1) Introducing-38/-13+12 bp double-allele deletion insertion mutation in the 3' UTR region of OsEPSPS gene, namely taking A in the OsEPSPS gene start codon ATG as 0 position, deleting 38bp from 3363 to 3400 position on one allele positioned downstream of the start codon ATG, deleting 13bp from 3377 to 3389 position on the other allele positioned downstream of the start codon ATG and inserting 12bp in the interval; 2) Introducing a-38 bp/-38bp homozygous deletion mutation in a 3' UTR region of a OsEPSPS gene, namely taking A in an ATG of a OsEPSPS gene start codon as 0 position, deleting 38bp from 3363 to 3400 positions positioned downstream of the ATG of the start codon, and generating the deletion mutation on both alleles; 3) Introducing-13+12 bp/-13+12bp homozygously deleted insertion mutation in the 3' UTR region of OsEPSPS gene, namely taking A in the OsEPSPS gene initiation codon ATG as 0 position, deleting 13bp from 3377 to 3389 position positioned downstream of initiation codon ATG, inserting 12bp in the interval, and generating the deleted insertion mutation on both alleles; The nucleotide sequence of OsEPSPS gene is shown in SEQ ID NO.5, and the nucleotide sequence of insert 12bp is shown in SEQ ID NO. 13.
  2. 2. The use according to claim 1, wherein the herbicide is glyphosate.
  3. 3. The use according to claim 1, wherein the rice is of the type south japonica 46.
  4. 4. A method for making rice resistant to EPSPS inhibitor herbicide is characterized in that the-38/-13+12 bp double-allele deletion insertion mutation in claim 1 is introduced into the 3' UTR region of OsEPSPS gene in rice genome by gene editing technology, so as to obtain the rice resistant to EPSPS inhibitor herbicide.
  5. 5. The method of claim 4, wherein the gene editing is achieved by a CRISPR/Cas12i3 system.
  6. 6. The method according to claim 5, characterized in that it comprises the steps of: Step one, pHZLib-Cas12i3 vector is obtained; step two, a crRNA sequence or a target sequence for gene editing is obtained, wherein the crRNA sequence is shown as SEQ ID NO.6, and the target sequence is positioned at 34 th to 56 th positions in the sequence shown as SEQ ID NO. 6; Cloning the crRNA sequence into pHZLib-Cas12i3 vector to obtain pHZLib-Cas12i3-crRNA; And fourthly, converting pHZLib-Cas12i3-crRNA into agrobacterium, infecting rice callus, and screening the rice strain resisting the EPSPS inhibitor herbicide.
  7. 7. The method of claim 6, wherein the herbicide is glyphosate.
  8. 8. The method according to claim 6, wherein the rice is of the variety Nanjing 46.

Description

Method for creating herbicide-resistant rice by gene editing OsEPSPS gene 3' UTR 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 3' UTR region. 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 invention provides OsEPSPS gene mutants, the type of mutation of which OsEPSPS gene mutants is any of the following: 1) Introducing-38/-13+12 bp double-allele deletion insertion mutation in the 3' UTR region of OsEPSPS gene, namely taking A in the OsEPSPS gene start codon ATG as 0 position, deleting 38bp from 3363 to 3400 position on one allele positioned downstream of the start codon ATG, deleting 13bp from 3377 to 3389 position on the other allele positioned downstream of the start codon ATG and inserting 12bp in the interval; 2) Introducing a-38 bp/-38bp homozygous deletion mutation in a 3' UTR region of a OsEPSPS gene, namely taking A in an ATG of a OsEPSPS gene start codon as 0 position, deleting 38bp from 3363 to 3400 positions positioned downstream of the ATG of the start codon, and generating the deletion mutation on both alleles; 3) Introducing-13+12 bp/-13+12bp homozygously deleted ins