利用CRISPR/Cas9技术创建OsIPCS家族基因突变体  

Mutants of OsIPCS Family Genes Created by CRISPR/Cas9 Technology

作　　者：彭威[1] 廖鹏飞[1] 阎新[1] 欧阳解秀[1] 李绍波[1] 王鑫 Peng Wei;Liao Pengfei;Yan Xin;Ouyang Jiexiu;Li Shaobo;Wang Xin(Key Laboratory of Molecular Biology and Gene Engineering of Jiangxi Province, College of Life Sciences, Nanchang University, Nanchang, 330031)

机构地区：[1]南昌大学生命科学学院江西省分子生物学与基因工程重点实验室,南昌330031

出　　处：《分子植物育种》2018年第11期3556-3562,共7页Molecular Plant Breeding

基　　金：国家自然科学基金(31560383;31760080)资助

摘　　要：肌醇磷酸神经酰胺合酶(inositol phosphoryl ceramide synthase,IPCS)是植物鞘脂合成途径中的一种关键酶,在植物生长发育和逆境适应中具有重要作用。本研究在OsIPCS基因外显子区域设计带有PAM序列的靶位点,利用PCR扩增获得OsIPCS基因sg RNA表达盒,并成功构建CRISPR/Cas9敲除载体;采用根癌农杆菌介导的遗传转化获得3个OsIPCS基因的T0代CRISPR转基因植株;通过PCR扩增和测序确定突变类型,其中2株不同突变类型的OsIPCS1突变单株,27株不同突变类型的OsIPCS2突变单株,9株不同突变类型的OsIPCS3突变单株,为进一步开展OsIPCS基因功能的研究提供科学依据。Inositol phosphoryl ceramide synthase（IPCS） is an essential enzyme in sphingolipid biosynthesis of plant, which plays an important role in the growth and development and adaptation of adversity of plant. In this study, we designed a target site with PAM sequence in the exons region of OsIPCS genes, The sg RNA expression cassettes of OsIPCS gene were obtained by using PCR amplification, which were then assembled into CRISPR/Cas9 knockout vectors. 3 CRISPR transgenic plants in T0 generation of OsIPCS gene were obtained by Agrobacterium-mediated transformation. PCR amplification and sequencing were applied to determine the mutation types. 2 mutant lines of OsIPCS1, 27 mutant lines of OsIPCS2 and 9 mutant lines of OsIPCS3 were obtained, which would lay a foundation for further research on the function of OsIPCS genes.

关 键 词：水稻 OsIPCS家族基因 CRISPR 载体构建 

分 类 号：Q943.2[生物学—植物学]