Construction and Transformation of Arabidopsis CKX3 Gene Editing Vector Based on CRISPR/Cas9

Cytokinin（CK） is a key hormone involved and played important role in plant growth and development.Cytokinin oxidase/dehydrogenase（CKX） is an irreversibly degradation of cytokinin into adenine/adenosine flavinase,as a key enzyme in the degrading branches in cytokinin signaling,plays an important role in maintaining the balance of cytokinin in plants,and effectively controlls the level of endogenous cytokinin in plants.In this study,Arabidopsis CKX3 was selected as the target gene,and an efficient Arabidopsis CKX3 gene editing vector was constructed and transformed into Arabidopsis through the CRISPR/Cas9 vector containing the Arabidopsis seed coat recombinant promoter with fluorescent screening marker gene m Cherry.Inverted fluorescence microscope was used to screen T0 generation seeds with red fluorescence and planted to obtain T1 generation plants.The genome of T1 generation plants was extracted for PCR identification and sequencing analysis to identify the phenotype and hormone determination of homozygous mutant offspring.The results showed that the editing vector pHEE401E-m Cherry-CKX3 was successfully constructed.The target gene CKX3was successfully edited in the progeny of Arabidopsis transformed.The homozygous mutant plants of CKX3showed obvious phenotypes,and the hormone content reached a significant level.The results laid the foundation for analyzing of CKX gene function.