CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

【目的】构建甜瓜e IF4E基因的CRISPR-Cas9植物基因编辑系统的gRNA表达载体。为研究eIF4E基因功能奠定基础。【方法】以新疆主栽甜瓜皇后为材料,在eIF4E基因的第一个外显子区设计gRNA引物,以载体pP1C.4为模板,扩增获得sgRNA克隆框,使用EcoR I、XbaI双酶切pP1C.4载体,利用DNA重组酶构建重组载体p P1C.4-eIF4E。【结果】经菌落PCR检测和测序,gRNA已经成功连接到植物基因敲除载体pP1C.4。【结论】构建了植物基因敲除载体pP1C.4-e IF4E,pP1C.4-e IF4E能对eIF4E基因进行定向编辑。

【Objective】The objective of this study is to construct gRNA expression vector of eIF4E gene in melon by using CRISPR-Cas9 plant gene editing system.【Method】The gRNA primer was designed in the first exon region of eIF4E gene from"Queen"of melon in Xinjiang.The sgRNA cloning box was amplified by using the vector pP1C.4 as a template,the pP1C.4 vector was cut by EcoRI and XbaI enzymes,and the recombinant vector pP1C.4-eIF4E was constructed using DNA recombinase.【Result】The results of colony PCR detection and sequencing showed that gRNA had been successfully linked into plant gene knockout vector pP1C.4.【Conclusion】The plant gene knockout vector pP1C.4-eIF4E is constructed successfully and eIF4E can be directionally edited by pP1C.4-eIF4E,which is of great significance for further study on the function of eIF4E gene.