Quality Gene OsGBSSⅠ Promoter Editing Using CRISPR/Cas12a System In Rice

"High quality and high yield"has always been the most important goal of rice breeding.The quality of rice is mainly determined by the composition,ratio and structure of amylose and amylopectin.The synthesis of amylose is mainly controlled by OsGBSSⅠ.Regulating the expression of OsGBSSⅠcan change the amylose content in the endosperm,so as to achieve the purpose of quality breeding.In this study,based on the CRISPR-Cas12a gene editing technology,the promoter of Nipponbare OsGBSSⅠgene was edited at multiple target sites to create mutant materials with different amylose content,providing new ideas and examples of rice quality improvement.The specific research content and results of the full text are as follows:1.For the rice OsGBSSⅠgene,12 crRNAs were designed and two directed editing vectors based on the CRISPR-Cas12a system were constructed,namely p ZJP080 and p ZJP081（containing 6 crRNAs respectively）.Using Agrobacterium-mediated genetic transformation of rice,p ZJP080 vector and p ZJP081 vector were used to obtain 6 independent regenerated calluses,totaling 30 and 27,totaling 135,respectively.The transgene positive test results showed that the positive rate of p ZJP080vector was 93%,and the positive rate of p ZJP081 vector was 100%.2.Using PCR-SSCP to analyze the mutation efficiency of crRNA single target site,the results show that:OsGBSSⅠ-crRNA04 has the lowest mutation efficiency（36%）;OsGBSSⅠ-crRNA11 has the highest mutation efficiency（87%）,OsGBSSⅠ-crRNA01site mutation efficiency is 47%;OsGBSSⅠ-crRNA02/06 site mutation efficiency is64%;OsGBSSⅠ-crRNA03/07 site mutation efficiency is 53%;OsGBSSⅠ-crRNA05site mutation efficiency is 47%;OsGBSSⅠ-crRNA08 site mutation efficiency was 59%;OsGBSSⅠ-crRNA09 site mutation efficiency was 60%;OsGBSSⅠ-crRNA10 site mutation efficiency was 77%.The mutation efficiency of OsGBSSⅠ-crRNA12 site was67%.Among them,the efficiency of one crRNA site mutation is 3.03%;the efficiency of two crRNA site mutations is 9.09%;the efficiency of three crRNA site mutations is18.18%,and the efficiency of four crRNA site mutations is 9.09%;The mutation efficiency of five crRNA sites was 36.36%;the efficiency of six crRNA site mutations was 24.24%.Using PCR agarose electrophoresis to screen and identify large fragment deletion mutants,the results showed that the efficiency of large fragment deletion in p ZJP081 vector mutant material was 41%.3.Using SSCP and Sanger sequencing methods to identify mutant T1 generation plants,45 homozygous mutants of different genotypes were obtained.Gene mutations are mainly insertions and deletions of 1-40 bp at each crRNA,and there are 16 large deletion genotypes of 100 bp or more.4.Determine the amylose content（AC）and gel consistency（GC）of homozygous mutant plants.The results showed that the AC content of the mutant materials decreased to varying degrees due to mutations of different genotypes.There were 8 mutant materials with AC between 16%and 18%,and 27 mutant materials with AC between14%and 14%.Among 16%,there are 5 mutant materials whose AC changes are between 12%and 14%;there are 5 mutant materials whose AC changes are between 8%and 12%,which meets the standard of high-quality soft rice;there are even 6 The AC content of the large-segment deletion mutant material is less than 5%,which is equivalent to the AC content of the mutant mutant of the coding region.The GC length change of the mutant material showed an increase in the GC length as the AC decreased,and the change between AC and GC was inversely proportional.5.Part of the mutant materials were selected for gene expression analysis.The results showed that the AC content of mutants decreased with the decrease of OsGBSSⅠgene expression.At the same time,the KI-I₂staining and chalkiness observation were performed on the seeds of this part of the mutant material.The results also showed that as the AC content decreased,the transparency of the seeds gradually decreased,and the endosperm stained with KI-I2 also showed a result of gradually lightening color.Unanimous.