CN-121160690-B - Method for rapidly screening high-efficiency gRNA of alfalfa

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for rapidly screening high-efficiency gRNA of alfalfa. The method comprises the steps of carrying out enzymolysis extraction and purification on alfalfa seedling cotyledons to obtain protoplasts, introducing a gene editing vector containing gRNA to be screened into the protoplasts, culturing for 30 hours to obtain the protoplasts after gene editing, extracting genome DNA of the protoplasts after gene editing, amplifying nucleotide sequences of target sites of the gRNA to be screened by PCR, carrying out high-throughput sequencing, and screening out high-efficiency gRNA. The method solves the problems of long evaluation period and low efficiency of the gRNA target gene editing system in the prior art.

Inventors

• ZHANG KUN
• JIA LEI
• YU AILING
• DU MIN
• CONG LILI
• YUAN FENG
• LIU YALING
• JIA SHIZHEN
• WANG ZENGYU
• Gong xiaonan

Assignees

• 青岛农业大学
• 内蒙古草业技术创新中心有限公司

Dates

Publication Date

20260505

Application Date

20251118

Claims (1)

1. 1. A method for rapidly screening high-efficiency gRNA of alfalfa is characterized by comprising the following steps: S1, sterilizing alfalfa seeds, inoculating the alfalfa seeds on a culture medium, culturing the alfalfa seeds in dark at 25 ℃ for 4 days after vernalization to obtain seedlings, crushing cotyledons of the seedlings, carrying out enzymolysis by using cellulase and eductase, uniformly mixing and filtering the obtained products to obtain filtrate, and centrifuging and purifying the filtrate to obtain protoplasts at 150 g; the enzymolysis condition is 25 ℃ and 12 hours; S2, re-suspending the protoplast to obtain protoplast suspension, mixing the protoplast suspension with the concentration of 1X 10 5 g -1 FW, a gene editing carrier solution and a PEG solution, standing at 25 ℃ to induce transformation for 15min, stopping reaction, centrifuging, re-suspending, and culturing at 31 ℃ for 30h under dark condition to obtain the protoplast after gene editing, wherein the gene editing carrier comprises gRNA to be screened; the promoter of the gene editing vector is MsU6-38 promoter derived from alfalfa, and the nucleotide sequence of the MsU6-38 promoter is shown as SEQ ID NO. 1; The volume ratio of the protoplast suspension to the gene editing carrier to the PEG solution is 10:1:11; S3, extracting genome DNA of the protoplast after gene editing, amplifying nucleotide sequences of target sites of the gRNAs to be screened by PCR, performing high-throughput sequencing, and analyzing editing efficiency of different gRNAs, thereby screening the gRNAs with highest editing efficiency; the nucleotide sequence of the gRNA with highest editing efficiency is shown in SEQ ID NO. 13.

Description

Method for rapidly screening high-efficiency gRNA of alfalfa Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a method for rapidly screening high-efficiency gRNA of alfalfa. Background Alfalfa, one of the most important leguminous grasses worldwide, is widely planted for its high protein content, nitrogen fixation capacity and environmental suitability. However, traditional breeding cycle of alfalfa is long, time cost and economic cost are high, biological breeding researches such as gene function research, gene editing and the like of alfalfa are more and more concerned in recent years, and correspondingly, technical means of related research of alfalfa are more and more demanded. After successful application of CRISPR/Cas9 technology to plants, herbaceous plants also successively establish gene editing systems and apply them to gene function research and biological breeding. Because of the different genomes of different species, the gene editing means requires different conditions when applied to different species. Alfalfa is a highly heterozygous autotetraploid plant, the genetic background is very complex, and the editing difficulty is high. Currently, as multiple alfalfa genomes are deciphered, the gene editing technology architecture of CRISPR/Cas9 is gradually established. The breakthrough progress lays a foundation for molecular breeding of alfalfa, so that researchers can introduce mutation into four alleles of alfalfa at the same time, and mutants with obvious phenotypic change are created. The transient plant protoplast transformation system is an efficient gene function research tool, and can realize rapid and transient gene expression analysis by introducing exogenous DNA or RNA into the plant protoplast after wall removal. The method can effectively avoid the regeneration disorder, directly utilizes the dissociated cells to carry out gene operation, and simultaneously has the advantages of finishing expression verification and high-throughput screening within 24-48 hours of experimental period. Numerous studies have shown that protoplasts can provide an ideal in situ validation platform for gRNA screening. Compared with the traditional leaf disc method, the protoplast transformation cycle is short and only needs 2-3 d, so that transient expression detection can be realized, and the problem of chimerism of regenerated plants is avoided. In theory, the efficient gRNA sequence can be rapidly screened by separating plant protoplasts, introducing a CRISPR/Cas9-gRNA complex and analyzing the editing efficiency of target sites through high-throughput sequencing, but the technical means is not reported in alfalfa at present. The alfalfa gene editing still faces multiple technical challenges at present, such as low genetic transformation efficiency, difficult multiallelic editing, low gRNA screening efficiency, complex regenerated plant genotypes and the like. Of particular note, the design and screening of grnas is the "command hub" of the entire CRISPR/Cas9 system, the efficiency of which directly affects the success rate of gene editing. Therefore, the development of an efficient gRNA screening method has important significance for promoting alfalfa gene editing breeding. Disclosure of Invention Aiming at the technical problems, the invention provides a method for rapidly screening high-efficiency gRNA of alfalfa. The invention adopts the following technical scheme: The invention provides a method for rapidly screening alfalfa high-efficiency gRNA, which comprises the following steps: s1, sterilizing alfalfa seeds, inoculating the alfalfa seeds on a culture medium, carrying out dark culture for 4d after vernalization, crushing seedling cotyledons, carrying out enzymolysis by using cellulase and educing enzyme, uniformly mixing and filtering the obtained product to obtain filtrate, centrifuging and purifying the filtrate to obtain protoplast, S2, resuspending the protoplast to obtain protoplast suspension, mixing the protoplast suspension with the concentration of 1X 10 5g-1 FW, a gene editing carrier solution and a PEG solution, standing for induction and transformation for 15min, terminating the reaction, centrifuging, resuspending, and culturing for 30h under dark condition to obtain the protoplast after gene editing, wherein the gene editing carrier comprises gRNA to be screened; The volume ratio of the protoplast suspension to the gene editing carrier to the PEG solution is 10:1:11; S3, extracting genome DNA of the protoplast after gene editing, amplifying nucleotide sequences of target sites of the gRNAs to be screened by PCR, performing high-throughput sequencing, and analyzing editing efficiency of different gRNAs, thereby screening the gRNAs with highest editing efficiency. According to the method for rapidly screening the high-efficiency gRNA of the alfalfa, firstly, the seedling cotyledons obtained by dark culture for 4d are s