小麦淀粉分支酶CRISPR/Cas9基因敲除系统gRNA表达载体构建  

Expression Vector Construction of Starch Branching Enzyme gRNA With CRISPR/Cas9 Gene Knockout System of Triticum aestivum L.

作　　者：李苗[1] 陈晓军[1] 马斯霜 白海波[1] 郑国保[1] 朱金霞[1] 张源沛[1] Li Miao;Chen Xiaojun;Ma Sishuang;Bai Haibo;Zheng Guobao;Zhu Jinxia;Zhang Yuanpei(Agricultural Bio-Technology Centre,Ningxia Academy of Agricultural and Forestry Sciences,Yinchuan,750002)

机构地区：[1]宁夏农林科学院农业生物技术研究中心

出　　处：《分子植物育种》2019年第20期6668-6672,共5页Molecular Plant Breeding

基　　金：宁夏回族自治区农业育种专项(2013NYYZ02);宁夏回族自治区重点研发计划项目(对外科技合作)共同资助

摘　　要：本研究以小麦淀粉分支酶(starch branching enzyme, SBEIIa)为研究对象,选取SBEIIa基因外显子5’端的第一及第二个外显子区域的4个SBEIIa-gRNA参试靶点进行靶位点活性检测,确定最佳基因敲除靶点。采用试剂盒法,以选定基因敲除靶点序列设计gRNA引物,通过oligo二聚体(Oligoduplex)合成、oligo二聚体插入载体、转化及鉴定等步骤,构建具有表达小麦淀粉分支酶gRNA (guide RNA)的CRISPR/Cas9基因敲除系统,获得小麦淀粉分支酶基因敲除载体。根据载体中插入序列,进行测序分析;测序结果能够确认gRNA已经完整准确地连入载体。研究构建的小麦淀粉分支酶基因敲除载体能对小麦SBEIIa基因表达量进行负向调控,对后续小麦淀粉分支酶基因及向其它相关基因编辑调控研究提供科学依据。Starch branching enzyme(SBEIIa) is the research object of this study. Four SBEIIa-gRNA reference targets site in the first and second exon regions at the 5’ end of the SBEIIa gene were selected and target site activity assay was performed. Identify the best gene knockout target site. Using the kit method, designing gRNA primers with selected gene knockout target sequences, construction of CRISPR/Cas9 gene knockout system with expression of Starch branching enzyme(SBEIIa) gRNA(guide RNA) by series of steps such as oligoduplex synthesis, oligo dimer insertion, transformation and identification, obtain wheat starch branching enzyme gene knockout vector.Sequencing analysis was performed based on the inserted sequence in the vector, the sequencing result confirmed that the gRNA was completely and accurately ligated into the vector. The constructed wheat starch branching enzyme knockout vector can negatively regulate the expression of wheat SBEIIa gene. This project is of great significance for the study of wheat starch branching enzyme gene and editing and regulation of other genes.

关 键 词：小麦(Triticum AESTIVUM L.) CRISPR/Cas9系统 淀粉分支酶基因 gRNA 载体构建 

分 类 号：Q78[生物学—分子生物学]