大豆多基因编辑表达载体的构建及应用  

Construction and application of soybean CRISPR/Cas9 multiplex editing vector

作　　者：陈向前 姜奇彦[1] 孙现军[1] 牛风娟 张慧媛 胡正[1] 张辉[1] CHEN Xiang-Qian;JIANG Qi-Yan;SUN Xian-Jun;NIU Feng-Juan;ZHANG Hui-Yuan;HU Zheng;ZHANG Hui(Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院作物科学研究所,北京100081

出　　处：《作物学报》2022年第11期2706-2714,共9页Acta Agronomica Sinica

基　　金：海南省重点研发计划项目(ZDYF2022XDNY135);中国农业科学院科技创新工程(CAAS-ZDRW202201);国家重点研发计划项目(2021YFD1201603-2);国家自然科学基金项目(31601302)资助。

摘　　要：大豆基因家族往往存在多个功能相似的基因,开发多基因编辑载体对多基因或基因家族进行编辑,对遗传转化效率低的大豆的基因编辑及基因功能研究具有重要的应用价值。本研究利用大豆特有的不同U6启动子驱动表达sgRNA,利用载体上同尾酶将不同大豆U6启动子表达的sgRNA进行串联,成功构建了可以同时对大豆多个基因进行编辑的CRISPR/Cas9多基因编辑表达载体pCambia3301-Cas9-GmU6n-gDNAn。并利用该载体,在大豆发状根中成功实现了大豆GRF(Growth-Regulating Factor)基因家族不同成员同时被编辑的目的。该载体的创建有效提高了大豆基因编辑效率,为大豆基因编辑及基因功能研究提供重要的工具。Most members of one gene family have similar function in soybean.Construction of soybean multiplex editing vector to edit multiple genes or gene families have important application value in soybean gene editing and gene functions,especially for the soybean with low transformation efficiency.Here,we reported a CRISPR/Cas9 multiplex editing vector,pCambia3301-Cas9-GmU6n-gDNAn,that enabled the editing of multiple genes in soybean.In this vector,different sgRNAs were driven by different soybean U6 promoters,and multiple sgRNA expression cassettes were assembled into pCambia3301-Cas9 vector by isocaudamers.The result showed that this system could simultaneously produce multiple mutations in soybean hairy roots by targeting multiple GRF genes via single transformation events.This vector can improve the efficiency of gene editing in soybean,and provide a simple toolbox for studying functions of multiple genes and gene families in soybean for basic research and genetic improvement.

关 键 词：大豆 U6启动子 CRISPR/Cas9多基因编辑载体 发状根 

分 类 号：S565.1[农业科学—作物学]