基于CRISPR/Cas9技术对水稻细胞质分裂关键基因OsMAP65-3s的编辑  

Editing of OsMAP65-3s,a Key Gene for Cytoplasmic Division in Rice,Based on CRISPR/Cas9 Technology

作　　者：林小丽 姜志树 贺榕 胡嘉敏 朱昌兰[1] 周大虎 贺浩华[1] 徐杰[1] LIN Xiaoli;JIANG Zhishu;HE Rong;HU Jiamin;ZHU Changlan;ZHOU Dahu;HE Haohua;XU Jie(College of Agronomy,Jiangxi Agricultural University/Key Laboratory of Crop Physiology,Ecology and Genetic Breeding,Ministry of Education/Research Center of Super Rice Engineering and Technology,Jiangxi Province/Collaboration Center for Double-season Rice Modernization Production,Nanchang,Jiangxi 330045)

机构地区：[1]江西农业大学农学院/作物生理生态与遗传育种教育部重点实验室/江西省超级稻工程技术研究中心/双季稻现代化生产协同中心,江西南昌330045

出　　处：《核农学报》2022年第4期716-727,共12页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金资助项目(31960403);江西省自然科学基金-杰出青年基金项目(20212ACB215003)。

摘　　要：MAP65-3是植物胞质分裂的重要调节因子。为探究水稻OsMAP65-3基因的功能,本研究采用CRISPR/Cas9技术对水稻中OsMAP65-3基因(OsMAP65-3.1和OsMAP65-3.2)进行编辑。针对每个基因各设计了2个不同的靶位点,并将OsMAP65-3.1与OsMAP65-3.2的靶位点放在同一个载体上,构建了CSMAP65-3A和CSMAP65-3B 2个双基因编辑载体。通过农杆菌介导的遗传转化分别获得17和21个植株,PCR鉴定阳性植株分别为16和20株。对T_(0)代植株靶位点附近序列进行测序分析,结果表明OsMAP65-3s基因均被成功编辑,OsMAP65-3.12个位点编辑效率为17.50%和9.38%,OsMAP65-3.2编辑效率为15.62%和45.00%;T_(0)代已获得osmap65-3.2、osmap65-3.2/OsMAP65-3.1(+/-)和OsMAP65-3.2(+/-)/OsMAP65-3.1(+/-)材料。本研究为进一步创制OsMAP65-3s突变体,开展基因功能研究和水稻胞质分裂分子机制解析奠定了理论基础。MAP65-3 is a key regulation factor in plant cytokinesis,to explore the function of OsMAP65-3 gene in rice,by CRISPR/Cas9 system,both OsMAP65-3 genes,OsMAP65-3.1 and OsMAP65-3.2,were edited in this study.Two different target sites were selected for each gene,and the target sites of both genes were placed on the same vector.Then two double-gene-editing vectors,CSMAP65-3 A and CSMAP65-3 B were obtained.Seventeen CSMAP65-3 A transformed plants and 20 CSMAP65-3 B plants were generated by agrobacterium-mediated genetic transformation,and 16 and 20 positive plants were identified by PCR,respectively.Sequencing analysis of the OsMAP65-3s in the T_(0) generation plants showed that OsMAP65-3s were edited.Two sites in OsMAP65-3.1 were edited by 17.5%and 9.38%,respectively;while two sites in OsMAP65-3.2 by 15.62%and 45%;osmap65-3.2,osmap65-3.2/OsMAP65-3.1(+/-)and OsMAP65-3.2(+/-)/OsMAP65-3.1(+/-)materials were obtained in T_(0) generation.This study lays a solid foundation for further creation of OsMAP65-3s mutants,gene function research and elucidation of the molecular mechanism of cytoplasmic division in rice.

关 键 词：水稻 胞质分裂 OsMAP65-3 CRISPR/Cas9 基因编辑 

分 类 号：S511[农业科学—作物学]