CRISPR-Cas9系统敲除亚麻FAD2基因表达载体的构建  

Construction of Expression Vector for Knocking out FAD2 Gene in Flax by CRISPR/Cas9 System

作　　者：张喻 江海霞[1] 闫文亮[1] 郭栋良[1] 杨亮杰[1] 叶佳丽 王玥[1] 谢丽琼[1] Zhang Yu;Jiang Haixia;Yan Wenliang;Guo Dongliang;Yang Liangjie;Ye Jiali;Wang Yue;Xie Liqiong(College of Life Science and Technology, Xinjiang University, Urumqi, 830046)

机构地区：[1]新疆大学生命科学与技术学院,乌鲁木齐830046

出　　处：《分子植物育种》2019年第7期2185-2192,共8页Molecular Plant Breeding

基　　金：高校科研计划项目(XJEDU2017M002);国家自然科学基金(31160056;C020408);新疆维吾尔自治区高技术项目(201517108)共同资助

摘　　要：CRISPR/Cas9系统是一种新型基因编辑工具,准确编辑目标基因,广泛应用于动植物的基因编辑中。本实验构建了油用亚麻品种‘陇亚10号’FAD2基因的CRISPR/Cas9基因编辑载体,以CRISPR/Cas9基因编辑技术为切入点,根据Gene Bank中公布的FAD2基因序列(DQ222824.1)在FAD2基因外显子部分运用sg RNA在线设计软件CRISPR-P 2.0对亚麻FAD2基因做脱靶分析,在外显子区域选取2个片段作为靶序列进行敲除。以潮霉素作为筛选标记,采用Golden Gate cloning法构建针对‘陇亚10号’FAD2基因的CRISPR/Cas9基因敲除载体,命名为p YLCRISPR/Cas9-FAD2。将pYLCRISPR/Cas9-FAD2载体导入农杆菌并检测其稳定性,保存菌种以便后期利用农杆菌介导法转化‘陇亚10号’,为深入研究亚麻FAD2基因的功能提供参考。CRISPR/Cas9 system is a new gene editing tool, which can edit target genes accurately. It is widely used in animal and plant gene editing. In this research, a gene editing vector of CRISPR/Cas9 for the oil flax cultivar ’LongYa No.10’ was constructed. Taking CRISPR/Cas9 gene editing technology as a breakthrough point,according to the FAD2 gene sequence(DQ222824.1) published in Gene Bank, the FAD2 gene was missed in the exon of FAD2 gene by using sg RNA online design software CRISPR-P 2.0. Two fragments in the exon region were selected as the target sequence for deletion. With hygromycin as the screening marker, the gene knockout vector of CRISPR/Cas9 targeting the FAD2 gene of ’Long Ya No.10’ was constructed by the method of Golden Gate cloning, which was named p YLCRISPR/cas9-FAD2. Afterwards, pYLCRISPR/cas9-FAD2 was introduced into Agrobacterium, the its stability was tested. The positive strain was conserved for later transformation of’LongYa No.10’ by Agrobacterium-mediated method, which could provide a reference for further study on the function of flax FAD2 gene.

关 键 词：亚麻 FAD2 CRISPR/Cas9 基因编辑 

分 类 号：S565.9[农业科学—作物学] Q943.2[生物学—植物学]