适用于大豆的CRISPR/Cas9体系的构建  

Construction of CRISPR/Cas9 System for Soybean

作　　者：吴楠 姜龙 于晓明 宁夕琳 杨祥波 元明浩 Wu Nan;Jiang Long;Yu Xiaoming;Ning Xilin;Yang Xiangbo;Yuan Minghao(College of Agriculture,Jilin Agricultural Science and Technology University,Jilin,132101)

机构地区：[1]吉林农业科技学院农学院,吉林132101

出　　处：《分子植物育种》2024年第19期6359-6367,共9页Molecular Plant Breeding

基　　金：吉林省教育厅“十三五”科学技术项目(JJKH20200391KJ)资助。

摘　　要：为了验证不同靶点的突变效率以及鉴定突变类型,本试验在基因GmFAD2-1(g3)、GmFAD2-2b(g6)的第一个外显子和第二个外显子处分别设计两对靶点,通过体外活性检测,目的是验证各个靶点的活性,并分别构建不同靶点的基因编辑载体,利用发根农杆菌转化法进一步验证其突变的效率及类型。结果表明:设计在基因第一个外显子区域的靶点,活性明显高于其他位置处设计的靶点;设计的靶点中GC的含量分别为52.6%、36.8%、31.5%、21.1%,说明GC含量越高,靶点的活性越强;通过发根农杆菌介导法快速验证不同靶点的突变效率,以U6为启动子的g3-Target1突变效率为92%,突变类型主要是碱基的插入、缺失、替换;g3-Target2突变效率为36%,突变类型主要是碱基的插入、缺失。以U6为启动子的g6-Target1突变效率为84%,突变类型主要是碱基的插入、缺失、替换;g6-Target2突变效率为8%,突变类型主要是突变类型主要是碱基的插入、缺失。In order to verify the mutation efficiency and identify the mutation type of different targets,two pairs of targets were designed in the first and second exons of GmFAD2-1(g3)and GmFAD-2b(g6)genes respectively,and their activity was verified by in vitro activity test.At the same time,CRISPR/Cas9 vectors with different targets were constructed,and the mutation efficiency and type were further verified by Agrobacterium rhizogenes transformation method.The results showed that the activity of the target designed at the first exon of the gene was significantly higher than that designed at other locations.The content of GC in the designed target was 52.6%,36.8%,31.5%and 21.1%respectively,indicating that the higher the content of GC,the stronger the activity of the target.The efficiency of g3-Target1 with U6 as promoter was 92%,and the mutation types were mainly base insertion,deletion and substitution;the mutation efficiency of g3-Target2 was 36%,and the main mutation types were insertion and deletion.The mutation efficiency of g6-Target1 with U6 as promoter was 84%,and the main mutation types were insertion,deletion and substitution;the mutation efficiency of g6-Target2 was 8%,and the main mutation types were insertion and deletion of base.

关 键 词：大豆 CRISPR/Cas9 突变效率 发根农杆菌 

分 类 号：S565.1[农业科学—作物学]