CRISPR/Cas9介导的多基因编辑系统在大豆中的应用  

Application of CRISPR/Cas9-mediated multiplex genome editing system in soybean genome editing

作　　者：关贝贝 王燕娟 陈海峰[2] 陈水莲[2] 沙爱华[1] 曹东 GUAN Bei-bei;WANG Yan-juan;CHEN Hai-feng;CHEN Shui-lian;SHA Ai-hua;CAO Dong(College of Agriculture,Yangtze University,Jingzhou 434025,China;Key Laboratory of Biology and Genetic Im-provement of Oil Crops,Ministry of Agriculture and Rural Affairs,Oil Crops Research Institute,Chinese Academy of Agricultural Sciences,Wuhan 430062,China)

机构地区：[1]长江大学农学院,湖北荆州434025 [2]中国农业科学院油料作物研究所,湖北武汉430062

出　　处：《中国油料作物学报》2021年第1期149-160,共12页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金面上项目(32072097);中央级科研院所基本科研业务费专项(1610172018008)。

摘　　要：自CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)基因组编辑技术发现以来,迅速在作物中得到广泛应用。但是,CRISPR/Cas9多基因编辑系统在大豆中的研究尚待开发。本文利用CRISPR/Cas9介导的多基因编辑系统,分别构建了两个载体,一个载体含6个靶点,编辑7个大豆基因(4个Glycine max ASYMMETRIC LEAVES1(GmAS1)同源基因和3个GmAS2同源基因),另一个载体含8个靶点,编辑11个G.max AGAMOUS家族同源基因(4个GmAG同源基因,2个G.max SEEDSTICK(GmSTK)同源基因和5个G.max SHATTERPROOF1(GmSHP1/2)同源基因)。大豆遗传转化后,经表型鉴定和靶点检测发现,CRISPR/Cas9介导的多基因编辑系统在大豆中成功实现了多基因编辑。当3个GmAS1同源基因和3个GmAS2同源基因同时突变时,导致大豆叶片向远轴面弯曲、皱缩且叶柄变短的表型。当2个GmSHP1同源基因和2个GmSTK同源基因同时突变时,导致豆荚停止发育的不育表型。To apply CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)based genome editing system on soybean functional genomics and breeding,CRISPR/Cas9-mediated targeted mutagenesis were obtained for more than 5 genes in soybean.One CRISPR/Cas9 plasmid conferred 6 sgRNA expression cassettes targeting 4 Glycine max ASYMMETRIC LEAVES1(GmAS1)genes and 3 GmAS2 genes,and another plasmid conferred 8 sgRNA expression cassettes targeting 6 G.max AGAMOUS1(GmAG)genes and 5 G.max SHATTERPROOF1(GmSHP1/2)genes.From the double mutagenesis of 3 GmAS1 genes and 3 GmAS2 genes,a phenotype of upward curling leaves and shorten leaf petiole length was discovered.From the double mutagenesis of 2 GmSHP1/2 genes and 2 GmSTK genes,sterility phenotype was found.

关 键 词：大豆 CRISPR/Cas9 GmAS1/2 GmSHP1/2 GmSTK 多基因编辑 

分 类 号：S565.1[农业科学—作物学]