Genome Editing For Target Gene Mutations In Rice Important Genes Using The CRISPR/Cas9 System

Rice is the world's consumption of the largest number of food crops, with the increase of the world population, Rice's production and the improvement of the rice's quality had increasingly become the focus of plant breeders. Traditional breeding method were based on cross breeding, which was time-consuming and was relied on the experience of breeders, it's difficult to meet the demand of directional improved properties; Technical means of physical, chemical, aerospace and biological mutagenesis difficult to achieve site-directed mutagenesis; Genetically modified method had not been widely recognized, Traditional site-directed mutagenesis techniques technical process complex and difficult to widely,so it's urgent need to establish safe and efficient target-gene modification process, to implement varieties' inprovements directly, and to provide new germplasm which was available for the utilization of breeding. In recent years, genome fixed-point editing techniques, represented by the CRISPR/Cas9, had become the focus in the plant breeding techniques, which provided a safe and efficient new way for rice germplasm innovation. CRISPR/Cas9 had obvious advantages: it only need to design 20 bases of nucleotide sequence which paired with target sequence. It's simple operation, short experiment period, low cost. At present this technology had been widely used in the microbial and animal and plant gene editing, But the application of CRISPR/Cas9 system in the research and rice gene editing is not enough.This research used CRISPR/Cas9 technology to designated edit and control the gene of rice's 1000-grain weight TGW6 and rice chalk gene chalk5, we had preliminary set up a rice gene fixed-point editing technology platform based on the CRISPR/Cas9 system and a stable and reliable key agronomic traits of rice gene mutation technique system. It's hopeful to creat a number of important value non-transgenic rice germplasm and a bath of rice mutants for basic research, which laied the foundation for major new rice varieties breeding and basic research, and it had important theoretical and practical significance. The main IV research results are as follows:?1?To construct four targets dual expression vectors successfully, which is p LY CRISPR/Cas9-TGW6,p LY CRISPR/Cas9-Chalk5-1 and CRISPR/Cas9-Chalk5-2 respectively. By Agrobacterium mediated genetic transformation in rice mature embryo, a certain number of tissue culture plantlets' T₀ generation of rice H447, S95 B and 02428 had gained.?2?The targeting effect test of tissue culture plantlets' T₀ generation showed that the same target's mutation efficiency in different rice and gene was not very consistent, and the four targets not all had mutations. The mutation types of gene DNA sequence were mainly base deletion, mismatch and insertion, which was given priority to with base deletion and mismatch.? 3? we transformed p LY CRISPR/Cas9-TGW6 vector into indica rice S95 B, transformed p LY CRISPR/Cas9-Chalk5-1 vector into indica rice S95 B and H447, transformed p LY CRISPR/Cas9-Chalk5-2 vector into Japonica rice 02428, separately. Tissue culture seedlings were obtained successfully, and the transgenic T₀ generation positive plants were 18, 13, 8 and 10 strains, respectively. Among them, there were respectively 18, 13, 7 and 9 mutant plants. The mutation rate was 87.5-100%. There were respectively 6, 4, 4 and 5 of double allelic mutation and the occurrence of allelic mutations were respectively 11, 9, 3 and 4 plants, and we also found two chimeric plants.?4?We identified four mutant strains with the corresponding phenotype, the results showed that the mutation effects of mutated offspring was obviously different, the corresponding characters had not been consistent changed. The phenotypic identification of 1000 grain weight of mutant plants of indica rice S95 B which transformed p LY CRISPR/Cas9-TGW6 vector showed little change compared with the control, Only one was greater than 7.09%, and the others were lower than the control group. The results of the identification of the chalkiness characters of indica rice S95 B and H447 which transformed p LY CRISPR/Cas9-Chalk5-1 vector were different, the chalkiness of S95 B mutant rice reached 28.91%, the chalkiness of H447 mutant rice occurred polarization situation, 19.44% ? The chalkiness trait of the mutant japonica rice 02428 which transformed p LY CRISPR/Cas9-Chalk5-2 vector was reduced, maximum reached 61.29%.?5?we preliminary established a set of technology system of fixed point editing rice gene, which based on CRISPR/Cas9 system, and we had standardized and improved the technology flow of rice mutant gene identification and phenotype identification. The technology platform of fixed point editing rice gene based on CRISPR/Cas9 system was preliminarily built.