Research On The Mutant Library Of Rice Grain Development Related Genes By CRISPR/Cas9 Technology

Rice is the staple food of more than half of the world’s population,and it is a model plant for gramineous crop research.The endosperm is the main part of rice seeds and also the main edible part,and its research is in line with the national strategic needs.With the completion of rice genome sequencing,genomics research has entered the era of functional genomics,and the creation of mutant libraries is a crucial link.In this study,based on the japonica rice variety Zhonghua 11,we used CRISPR/Cas9 technology to create a library of knockout mutants that specifically or preferentially express genes during rice grain development,and preliminarily analyzed the main agronomic and quality traits.In addition,we discovered a highly chalky mutant material.The main findings are as follows:1.Screened 310 specific or preferentially expressed genes during rice grain development,and 310 target genes were evenly distributed on 12 rice chromosomes.RNA-seq verified the expression characteristics of the selected genes,involving multiple biological processes or molecular functions,especially carbohydrate metabolism,indicating that rice endosperm development involves complex regulatory pathways,of which starch synthesis metabolism is the key process.2.Using CRISPR/Cas9 technology,2616 T0 transformants with 302 target genes were obtained,with an average mutation rate of 79.2%,and an average of 5.6 independent knockout transformants per target site.The Off Target tool was used to screen 15 target sites with high off-target probability,and sequencing analysis showed that none of the 15 target sites had off-target mutations.3.The analysis of the main agronomic and quality traits of the T1 generation mutants showed that compared with the wild-type parent,the grain shape,appearance,apparent amylose content,viscosity profile,and protein content of many mutants were significantly different.4.Compared with the wild-type parent,the plant height and main ear length of the high chalky mutant sg6772 have no obvious changes,the chalky grain rate and chalkiness are significantly increased,the grain thickness and thousand-grain weight are significantly reduced,and the taste and nutritional quality of rice are not obvious change.The starch granules in the endosperm cells of the mutant sg6772 are of different sizes,spherical,and loosely arranged.There are large gaps between the starch granules,resulting in a chalky phenotype.