CRISPR/Cas9技术编辑新疆甜瓜全缘叶基因

CRISPR/Cas9 Mediated Editing the Whole Leaf Gene of Muskmelon in Xinjiang

【目的】以新疆甜瓜地方品种老汉瓜为研究材料,对其全缘叶基因进行基因编辑,构建新疆甜瓜CRISPR/Cas9敲除载体,分析甜瓜耐受潮霉素最大浓度,为研究新疆甜瓜裂叶基因的功能奠定基础。【方法】在课题组前期确定甜瓜全缘叶基因PLL的基础上,结合甜瓜基因组数据,设计PLL基因外显子的特异靶位点,构建甜瓜全缘叶基因PLL的CRISPR/Cas9敲除载体,将构建好的载体转化到大肠杆菌中,并测序验证,然后对甜瓜耐受潮霉素浓度进行分析。【结果】针对全缘叶基因PLL外显子选择一个长约20 bp的靶位点,根据靶位点设计sgRNA框,并成功将其构建到CRISPR/Cas9敲除载体上,然后将载体成功转化到大肠杆菌中。分析出甜瓜可耐受潮霉素的最大浓度为10 mg/L。【结论】成功获得甜瓜全缘叶基因PLL的敲除载体,并转化到大肠杆菌中。分析出甜瓜耐受潮霉素的最大浓度,为进一步利用CRISPR/Cas9基因编辑技术研究新疆甜瓜裂叶基因pll的功能奠定基础。

【Objective】A local variety called Xinjiang old man melon was used as the research material.The entire leaf gene of Xinjiang melon was edited and the knockout vector of CRISPR/Cas9 of Xinjiang melon entire leaf gene constructed.After that,the the maximum tolerance to hygromycin in the muskmelons was analyzed in the hope of laying a foundation for the study of the function of muskmelon cleavage gene in Xinjiang.【Method】Based on the previous study of entire leaf gene PLL in the melon and melon genomic data,the specific target site of exon of PLL gene was designed,and the CRISPR/Cas9 knockout vector of melon entire leaf gene PLL was constructed Then,the vector was transformed into Escherichia coli and sequenced and the tolerance of melon to hygromycin analyzed.【Result】A 20 bp target site was selected for the exon of the whole leaf gene PLL,and the sgRNA frame was designed according to the target site,and attached it to the CRISPR/Cas9 knockout vector,then the vector was successfully transformed into Escherichia coli.Melons could tolerate the maximum concentration of hygromycin at 10 mg/L.【Conclusion】The knockout vector of melon entire leaf gene PLL was successfully constructed and transformed into Escherichia coli,which analyzed the maximum concentration of cantaloupe tolerance to hygromycin.This has laid a foundation for further study on the function of PLL gene in muskmelon leaves of Xinjiang by using CRISPR/Cas9 gene editing technique.