利用CRISPR/Cas9技术获得水稻恢复系Bsr-d1基因突变体  

Construction of the Bsr-d1 Gene Mutants in Rice Restorer Line by CRISPR/Cas9 Technology

作　　者：卿冬进 邓国富[2] 戴高兴[3] 高利军[1] 黄娟[1] 高菊 潘英华[3] 周维永[3] 梁海福[3] Qing Dongjin;Deng Guofu;Dai Gaoxing;Gao Lijun;Huang Juan;Gao Ju;Pan Yinghua;Zhou Weiyong;Liang Haifu(Guangxi Crop Genetic Improvement and Biotechnology Laboratory,Guangxi Academy of Agricultural Sciences,Nanning,530007;Guangxi Academy of Agricultural Sciences,Nanning,530007;Rice Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007)

机构地区：[1]广西农业科学院,广西作物遗传改良生物技术重点开放实验室,南宁530007 [2]广西农业科学院,南宁530007 [3]广西农业科学院水稻研究所,南宁530007

出　　处：《分子植物育种》2020年第16期5343-5350,共8页Molecular Plant Breeding

基　　金：广西科技基地和人才专项(桂科AD18281069);广西农业科学院科技发展基金(桂农科2018JZ34);广西自然科学基金项目(2017GXNSFAA198266);广西农业科学院基本业务专项(桂农科2018YT24)共同资助。

摘　　要：为研究水稻抗稻瘟病基因Bsr-d1在杂交水稻恢复系中的功能,我们利用CRISPR/Cas9定点基因编辑方法,设计2个敲除靶标位点,构建了Bsr-d1基因编辑载体,并通过农杆菌介导的方法转化水稻恢复系品种‘GH998’,成功获得了转基因植株,并在T1代筛选到4个无转基因成分的纯合突变株系。纯合突变株系有4种突变类型,包括第199号碱基处缺失17个碱基、第212号碱基处缺失4个碱基、第216号碱基处插入1个碱基、第216号碱基处插入2个碱基突变类型。通过蛋白序列分析,发现纯合突变株系都存在移码现象导致氨基酸变化并提前终止翻译。本研究成功利用CRISPR/Cas9基因编辑方法敲除水稻恢复系‘GH998’中Bsr-d1基因,获得无转基因成分的纯合突变株系,为进一步研究Bsr-d1基因在杂交水稻上的功能提供了理论依据和品种资源。To study the function of rice blast resistance gene Bsr-d1 in hybrid rice restorer line,we designed two knock-out targets and constructed the Bsr-d1 gene editing vector using the CRISPR/Cas9 system,and transformed it into the rice restorer line'GH998'by Agrobacterium mediated transformation approach.We successfully obtained the transgenic plants,and screened out four homozygous mutants without transgenic components in T 1 generation.There are four mutation types in homozygous mutant lines,including 17 bases deletion at 199th base,4 bases deletion at 212th base,1 base insertion at 216th base and 2 base insertion at base 216th.Protein sequence alignment analysis showed that bases deletion and insertion of the four mutation types caused the amino acids change and premature stop.In this study,the Bsr-d1 gene in the rice restorer line'GH998'was successfully knocked out by CRISPR/Cas9 gene editing method,and homozygous mutant lines without transgenic components were obtained.It provided theoretical foundation and variety resources for further study the function of Bsr-d1 gene in hybrid rice.

关 键 词：水稻 稻瘟病 Bsr-d1 基因编辑技术 CRISPR/Cas9 

分 类 号：S511[农业科学—作物学]