Genome Editing Based On CRISPR/Cas9 System And Study Of Genetic Transformation Technology In Rice

Rice is one of the most important food crops,rice food security is facing severe challenges currentbecause of the increased population,the reduced arable land,the environmental pollution and the frequently extreme natural disasters.Traditional breeding techniques rely on cross breeding,it can't satify the characteristics of directional improvement and the mutagenesisbreeding was difficult to get targeted mutation.Although the artificial transformation of rice genes at the genome level is of great significance to crop improvement,but all along,scientists have not found an effective plant genome editing technology.In recent years,the latest discovery of C RISPR / Cas9 technology with s imple and efficient features is widely used in rice and other crops in the genome editing.Conventional rice genetic transformation is generally Agrobacterium-mediated method,the foreign genetic material is integrated into the recipient genome by the Ti plasmid.However,the Agrobacterium-mediated method is highly selective for genotypes,and the method requires a complex set of artificial tissue culture processes,which increa ses the complexity of genetic transformation.In C hina,pollen tube pathwayand ear stem injectionhad the successful example of genetic transformation in rice.Genetic transformation of pollen tube pathway and ear stem injection based on C RISPR / Cas9 do not require external DNA to be integrated into the genome to play a fixed-point editing effect.Therefore,the binding energy of exogenous DNA and C RISPR / Cas9 can create a new genetic transformation system.In this study,we used the C RISPR / Cas9 technique to edit the rice gene tms5 and SBE3,and to elucidate the genetic transformation efficiency of Agrobacterium tumefaciens-mediated method,pollen tube pathway and ear stem injection.Attempts to establish a pollen tube pathway and aear stem injection genetic transformation system based on CRISPR / Cas9 technology in rice.The results are as follows:1 ? The pYLCRISPR / Cas9-SBE3 single target and the pYLCRISPR / Cas9-tms5four-target binary knockout vectors were successfully constructed.The rice callus was transformed by Agrobacterium tumefaciens,and the rice plants were introduced by pollen tube pathway and ear stem injection.2?10 strains of pYLCRISPR / Cas9-tms5 knockout plants were obtained by screening,and the transformation rate was 61.53%.12 strains of pYLC RISPR / Cas9-tms5 knocked plants were obtained by pollen tube pathway,and the transformation rate was 6%.3?By analyzing the mutant types of knockout plants,two of the 10 strains of pYLC RISPR / Cas9-tms5 knockout plants were homozygous mutations,the mutation rate was 20%,the 7 strains were heterozygous mutations,mutations rate was 70%,1 strain was double allele mutation,mutation rate was 10%,the target of Mutation efficiency analysis showed that the mutation rate T1(61.54%)= T2(61.54%)> T3(53.85%)> T4(15.38%).p YLC RISPR / Cas9-tms5 through pollen tube pathway knockout plants were 12 strains,3 strains were integrated into the chromosome,accounting for 25%,not integrated into the chromosome were 9 strains,accounting for 75%.There were 9 heterozygous mutations,the mutation rate was 75%,the double allele had 3 strains,the mutation rate was 25%,the mutant rate was T2(66.67%)> T3(33.33%)> T1(11.11%)= T4(11.11%).This paper first validated the reliability of genetic transformation of pollen tube pathway based on CRISPR / Cas9 technology,which can be used as a new genetic transformation technique in CRISPR / Cas9 system.