Target Rice BADH2 And GL7NR Gene Modification By CRISPR/Cas9 And Genetic Analysis

In order to improve the length of the northeast rice and popularize the fragrant rice,the paper researched on the gene that regulate the size and fragrance of the northeast rice.BADH2,a gene regulating its fragrance,and GL7,a negative regulatory gene of the length gene GL7NR,were cloned.CRISPR/Cas9,a novel gene editing technology that appeared in recent years,was used to directed edit BADH2 and GL7NR agrobacterium with target gene was transfected into rice callus and a series of transgenic plants were produced.By traditional PCR and sequencing techniques,the target site exists one or more replacement or insertion.At the same time,through the CRISPR mutation site prediction tool to detect whether there is a phenomenon of off-target.The research perhaps provides some theoretical guidance for the improvement of fine agronomic characteristic of rice.The main results are as follows:(1)The recombinant vector was electro-transformed into Agrobacterium tumefaciens.The engineering Agrobacterium strain with recombinant plasmid contained pCAMBIA1301-fgr-sgRNA-Cas9 and pCAMBIA1301-g17nr-sgRNA-Ag9 identified by bioinformatics and PCR.(2)Agrobacterium-mediated genetic transformation of pCAMBIA1301-fgr-sgRNA-Cas9 recombinant Agrobacterium was carried out in four different rice cultivars.The pCAMBIA1301-gl7nr-sgRNA-Cas9 recombinant Agrobacterium was transferred into YYG-22 and then the positive strains of transgenic rice were detected by conventional PCR.There are 14 strains,21 strains of YYG-10 and 15 strains of YYG-22.(3)Extract rice DNA and use the conventional PCR and sequencing techniques to detect the mutation sites in rice varieties.The results showed that there were 4 strains of early BZ in Song B12 and the mutation rate was 22.2%.the rate of mutation was 35.7%in 5 strains of early B12.The mutation rate of YYG-10 was 23.8%and the number of YYG-22 was 4 and the mutation rate was 26.6%.(4)Genotype and phenotypic analysis of fragrance rice T1 generation:Total of 25 plants in T,were fragrant.,13 of them have no hpt mark.Twenty strains have been tested,fifteen of them are T base insertion,five of them are C base insertion.(5):Genotype and phenotypic analysis of grain length rice T1 generation:There was no significant difference in seed length between mutant and non-mutant.Eight plants with longer grain lengths were detected and two of them were changed.Four of them were homozygous for T-base insertion and two of them have no changed.