Establishment Of A Chinese Kale CRISPR/Cas9 Gene Editing System

Chinese kale(Brassica oleracea var.alboglabra)is a Brassica vegetable,and rich in nutrients such as vitamin C,carotenoids,glucosinolates and polyphenols in their bolting stem and tender leaves.ζ-carotene desaturase(ZDS)and phytoene desaturase(PDS)are both important rate-limiting enzymes in the carotenoid biosynthesis pathway.After the ZDS or PDS genes are silenced,the transgenic plants will display a clearly albino phenotype.Therefore,these genes are often used as the reporter target gene to establish the CRISPR/Cas9 and VIGS systems.It is an efficient and accurate method to screen excellent CRISPR target sites using protoplast transient transformation technology.Our previous study found that the ZDS gene has only one member,while the PDS genes has two members in Chinese kale.Therefore,in this study,Chinese kale was used as the material,the BaZDS and BaPDSs were used as the target genes,and then the protoplast transient transformation system,and the stabilize genetic system of single gene and polygene editing of CRISPR/Cas9 gene editing technology were systematically established for the first time in Chinese kale.The results of this study provide theoretical basis and technical support for the study of gene function and breeding of new varieties using the CRISPR/Cas9 gene editing technology on the Chinese kale.The main results are as follows:1.The CRISPR/Cas9 protoplast transient transformation system of Chinese kale was successfully established,which can be used for target site screening.Using CRISPR-P online software,the target site 1 was selected on the first exon of BaZDS gene,and the target site 2 was selected on the second exon to construct pCC-BaZDS-sgRNA1 and pCC-BaZDS-sgRNA2 recombinant plasmid,respectively.The pCC-BaZDS-sgRNA1 vector was transformed by the protoplast transient transformation system,and cultured for 24 h,36 h,and 48 h,respectively.The results showed that the transformation efficiency was the highest at 48 h,and the ratio was 92.6%.Therefore,the optimal culture time was 48 h.Further analysis of the mutations revealed that the type of mutation was mainly due to the deletion of large fragments at the target site,accounting for 86.11%.After transformation of the pCC-BaZDS-sgRNA2 vector,the mutation rate was 54.8% at the target site,and the mutation type was mainly composed of a single base substitution mutation of 2 ~ 5 bp,accounting for 38.71%,followed by the insertion mutation of 1 ~ 2 bp.2.The CRISPR/Cas9 stable genetic system for single gene of Chinese kale was successfully established.Agrobacterium-mediated genetic transformation was used to transform pCC-BaZDS-sgRNA1 vector,and 19 transgenic positive plants were obtained.The positive rate of transgene was 95%,among which 13 strains were mutated,and the mutation efficiency was 68.42%.The type of mutation was mainly based on the insertion of large fragments,accounting for 26.32%,followed by single base substitution mutation,and all mutant plants showed obvious whitening phenotype.3.The CRISPR/Cas9 stable genetic system of the Chinese kale was successfully established,and it has the ability to simultaneously edit multiple genes in Chinese kale.Using CRISPR-P online software,the common target sites of two genes were selected on the fourth exon of BaPDS1 and BaPDS2 genes respectively,and the target sequence was synthesized to construct pCC-BaPDS-sgRNA recombinant plasmid.A total of 68 transgenic positive plants were obtained by Agrobacterium-mediated genetic transformation,and the positive rate of transgene was 90.76%.The mutation analysis showed that the mutation rate was 76.47%,and the ratio of BaPDS1 and BaPDS2 double mutation was about 1/5,and the ratio of single mutation was more than a half.The mutation type was mainly based on the change of short nucleotides,and no more than 10 bp,and the mutant plants produce a distinct whitening phenotype.