利用CRISPR/Cas9技术创建OsGPRP家族基因突变体  

Mutants for OsGPRPs Constructed Based on CRISPR/Cas9 Technology

作　　者：王鑫 刘丹阳 欧阳解秀[1,2] 彭辉 李绍波[1] 阎新[1] Wang Xin;Liu Danyang;Ouyang Jiexiu;Peng Hui;Li Shaobo;Yan Xin(School of Life Sciences,Nanchang University,Nanchang,330031;Medical Experimental Teaching Division,Nanchang University,Nanchang,330031)

机构地区：[1]南昌大学生命科学学院,南昌330031 [2]南昌大学医学实验教学中心,南昌330031

出　　处：《分子植物育种》2018年第15期4943-4949,共7页Molecular Plant Breeding

基　　金：国家自然科学基金(31560298;31460279)资助

摘　　要：本研究利用CRISPR/Cas9技术对水稻OsGPRP家族基因进行敲除,在3个水稻OsGPRP基因的第一个外显子PAM序列区域各设计一个靶位点,并将3个靶位点的sg RNA进行串连,成功构建了3个OsGPRP基因同时敲除的CRISPR/Cas9载体;采用根癌农杆菌介导的遗传转化获得了60株T0代CRISPR转基因植株;通过PCR扩增和测序分析,我们获得了2种不同类型的OsGPRP3单基因突变材料;6种不同类型的OsGPRP2/OsGPRP3两基因突变植株;1种类型的OsGPRP1/OsGPRP3两基因纯合突变植株和4种不同类型的OsGPRP1/OsGPRP2/OsGPRP3三基因突变材料,为进一步研究OsGPRP家族基因的生物学功能及其相互关系提供遗传材料。In this study, OsGPRPs family genes in rice were knocked out based on CRISPR/Cas9 technology. We designed a target site in PAM sequence region in the first exon of 3 OsGPRPs genes in rice correspondingly and serialized the sgRNA on 3 target sites. Later we successfully constructed CRISPR/Cas9 vectors that knocked out with 3 OsGPRPs genes. 60 T0 CRISPR transgenic plants were obtained through Agrobacterium-mediated genetic transformation. Target PCR amplification and sequencing were applied to these plants. We obtained two single gene mutant plants of OsGPRP3 in different types, 6 mut plants of OsGPRP2/OsGPRP3 genes in different types, homozygous mutant plant of OsGPRP1/OsGPRP3 genes in one type and 4 mutant plants of OsGPRP1/OsGPRP2/ OsGPRP3 genes in different types. This work would provide the genetic material for further researches on the biological function and interrelationships of OsGPRPs family genes.

关 键 词：水稻 OsGPRP家族基因 CRISPR/Cas9 载体构建 

分 类 号：Q943.2[生物学—植物学]