Establishment Of Genetic Transformation And CRISPR/Cas9 Gene Editing System Of Chinese Cabbage

Chinese cabbage(Brassica rapa L.ssp.Pekinensis)is a common economical agricultural product in the field of crops,especially in Asia,such as China,Japan and South Korea.With the development of genetic engineering technology,Agrobacterium-mediated genetic transformation system and gene editing technology have been continuously applied in the process of Chinese cabbage breeding,which has accelerated the breeding process of Chinese cabbage and improved the cabbage traits.It laid the experimental foundation for the study of Chinese cabbage genetic breeding.This study establishes a stable and efficient Chinese cabbage genetic transformation system,and the CRISPR/Cas9 system is successfully applied to Chinese cabbage gene editing.(1)The Chinese cabbage?C-24?inbred line is selected as the test material,and the Brassica gene Bra A1000785 overexpression vector is constructed.Agrobacterium mediated method is used to screen for factors such as Agrobacterium status,Agrobacterium liquid concentration,AS,infestation time and ultrasound-assisted treatment.The genetic transformation system is established,the genetic transformation system is as follows:Agrobacterium state: GV3101,bacterial liquid OD600=0.5,infecting medium(DM)with 20 mg·L-1 AS,Infection process 15 min and without sonication,co-culture medium(MC)with 20 mg·L-1 AS(dark treatment for 48 h).Resistant callus and resistant bud differentiation are induced in explant culture medium(MS).Resistant seedlings are identified by PCR detection and GUS staining.It is confirmed that the DNA had been introduced into the Chinese cabbage.The positive strains are analyzed by q PCR for the expression level of Bra A1000785 gene in Chinese cabbage leaves.Finally,the system is verified by repeated tests to obtain an average efficiency of 10.83%.(2)This study further established the Chinese cabbage CRISPR/Cas9 gene editing system based on the genetic transformation system of Chinese cabbage.Chinese cabbage gene Bra Sca000221 is used as the target gene.The gene editing efficiency of the T0 generation resistant plants is 72%,which were all heterozygous mutants.T0 generation strains obtained 28 T1 generation mutants by self-crossing,of which homozygous edited plants is accounted for 35.7%,and heterozygous edited plants is accounted for 64.3%.The above results indicate that the single-target editing of the Chinese cabbage genome can be carried out by the CRISPR/Cas9 system through the Chinese cabbage genetic transformation system.