基于CRISPR/Cas9的拟南芥CKX3基因编辑载体构建及转化研究

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

细胞分裂素(cytokinin,CK)是参与植物生长发育过程中的关键激素,在植物生长、发育过程起着非常重要作用。细胞分裂素氧化酶/脱氢酶(cytokinin oxidase/dehydrogenase,CKX)是一种不可逆降解细胞分裂素为腺嘌呤/腺苷的黄素酶,作为细胞分裂素信号中降解分支的关键酶,对于维持植物体内的细胞分裂素平衡有着重要作用,有效的控制了植物内源细胞分裂素的水平。选取拟南芥CKX3为目标基因,通过含有拟南芥种皮重组启动子与荧光筛选标记基因mCherry的CRISPR/Cas9载体,来进行高效的拟南芥CKX3基因编辑载体构建并转化拟南芥。利用倒置荧光显微镜筛选具有红色荧光的T0代种子并种植培养获得T1代植株。提取T1代植株基因组进行PCR鉴定和测序分析,鉴定纯合突变后代的性状表型及激素测定。结果表明,本实验成功构建了编辑载体pHEE401E-mCherry-CKX3,拟南芥转化后代中目标基因成功被高效编辑,CKX3的纯合突变植株表型与野生型差异明显,内源激素含量差异达到显著水平。为研究CKX基因功能奠定了基础。

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 mCherry.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-mCherry-CKX3 was successfully constructed.The target gene CKX3 was successfully edited in the progeny of Arabidopsis transformed.The homozygous mutant plants of CKX3 showed obvious phenotypes,and the hormone content reached a significant level.The results laid the foundation for analyzing of CKX gene function.