利用发根农杆菌体系检测西瓜CRISPR/Cas9系统的靶位点  

Detection of target sites of CRISPR/Cas9 system in watermelon by Agrobacterium rhizogenes system

作　　者：张月乔 葛洁 田树娟 袁黎 ZHANG Yueqiao;GE Jie;TIAN Shujuan;YUAN Li(State Key Laboratory of Crop Stress Biology for Arid Areas/College of Horticulture,Northwest A&F University,Yangling 712100,Shaanxi,China)

机构地区：[1]旱区作物逆境生物学国家重点实验室·西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《中国瓜菜》2020年第4期7-11,共5页China Cucurbits And Vegetables

基　　金：高等学校博士学科点专项科研基金(Z109021848);植物单倍体育种机制与体系研究(Z111021801)。

摘　　要：西瓜遗传转化技术周期长,过程复杂,CRISPR/Cas9基因编辑系统又存在着一定的脱靶效应,为了保障所选择的靶位点的可行性,本研究选择西瓜ClSPO11-1基因(ID:Cla003301)为靶标,利用CRISPR/Cas9基因编辑系统构建双靶点的基因敲除载体,利用发根农杆菌Ar. Qual菌株侵染西瓜子叶外植体,通过简单的组培过程,使西瓜外植体长出不定根,在不定根中检测到了在靶位点1处发生了不同程度的碱基缺失,经过与野生型氨基酸序列比对分析后发现均造成了翻译提前终止和氨基酸的突变,成功实现了对西瓜不定根的基因编辑,该方法周期短,操作简便,实现了快速鉴定在西瓜中选择的靶位点的靶向效率,为研究西瓜基因功能和遗传改良提供了保障。Watermelon genetic transformation technology is time consuming and process complicated. The CRISPR/Cas9 gene editing system has a certain off-target effect. In order to ensure the feasibility of the selected target site, the watermelon ClSPO11-1 gene(ID:Cla003301)was selected in this study as target, using the CRISPR/Cas9 gene editing system to construct a double-target gene knockout vector, using Agrobacterium rhizogenes Ar. Qual strain to infect watermelon cotyledon explants, and to make watermelon explants growing through a simple tissue culture adventitious roots. In the adventitious roots, different degrees of base deletions at target site 1 were detected. After comparison and analysis with amino acid sequences of wild-type, both premature translation termination and amino acid mutations was found. This method has a short cycle and simple operation. It achieves the rapid target identification efficiency of selected target sites in watermelon, and provides a guarantee for the study of watermelon gene function and genetic improvement.

关 键 词：西瓜 CRISPR/Cas9系统 发根农杆菌 基因敲除 

分 类 号：S651[农业科学—果树学]