利用发根农杆菌体系检测大豆CRISPR/Cas9系统的靶点  

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

作　　者：熊艳萍 许崇晶 单金明 赵琳[1] XIONG Yan-ping;XU Chong-jing;SHAN Jin-ming;ZHAO Lin(Institute of Soybean Research,Key Laboratory of Soybean Biology in Chinese of Ministry Education,Key Laboratory of Soybean Biology and Breeding(Genetics)of Ministry of Agriculture and Rural Affairs,School of Agriculture,North-east Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学大豆研究所,大豆生物学教育部重点实验室,农业农村部东北大豆生物学与遗传育种重点实验室,黑龙江哈尔滨150030

出　　处：《中国油料作物学报》2021年第1期161-170,共10页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金面上项目(31771820、32072086);高产养分高效利用转基因大豆新品种培育(2016ZX08004-005);黑龙江省自然科学基金重点项目(ZD2020C002)。

摘　　要：作为一种基因编辑的新技术,CRISPR/Cas系统已被广泛应用于各种生物的基因工程中。但CRISPR/Cas9基因编辑系统还存在着一定的脱靶现象,确定基因编辑靶点选择的可行性可提高基因编辑效率。本研究选用大豆GmCO基因为靶标,使用CRISPR/Cas9基因编辑系统来构建靶点的基因敲除载体,并且利用发根农杆菌K599菌株对大豆子叶进行侵染,再通过植物组织培养的方法促使大豆外植体长出不定根,通过不定根检测发现在靶点2处出现了碱基的缺失,说明成功实现了对大豆目标基因的编辑。此方法操作简单,周期短,实现了对大豆中选择靶点的可行性的快速鉴定,为研究大豆的GmCO基因功能和遗传改良方面提供了新的技术支持。As a new gene-editing technology,CRISPR/Cas system has been widely used in the genome engineering of various organisms.Soybean genetic transformation technology is time-consuming and complicated.The CRISPR/Cas9 gene-editing system has a certain possibility of off-target.To ensure the feasibility of selected target,the soybean gene GmCO was selected in this study as a target,using the CRISPR/Cas9 gene-editing system to construct a target gene knockout vector.And then plant tissue culture was used to promote soybean explants to grow adventitious roots by cotyledon explant infection mediated by Agrobacterium rhizogenes K599 strain.The detection of adventitious roots showed that there was a four-base deletion at target 2,indicating that the gene-editing of soybean adventitious roots was successfully realized.Due to its simple operation and short cycle,this method realizes the feasibility of rapid identification of the selected target in soybean and provides a potential way to study soybean gene GmCO function and genetic improvement.

关 键 词：大豆 CRISPR/Cas9系统 发根农杆菌 基因敲除 

分 类 号：S565.1[农业科学—作物学]