利用CRISPR-Cas9技术制备水稻LOC＿Os01g07170突变体  

Preparation of LOC＿Os01g07170 Mutant in Rice Using CRISPR-Cas9

作　　者：宋明 付瑞锋 曹怡然 张大兵[1] Song Ming;Fu Ruifeng;Cao Yiran;Zhang Dabing(School of Life Sciences and Biotechnology,Shanghai Jiaotong University,Shanghai,200240)

机构地区：[1]上海交通大学生命科学技术学院,上海200240

出　　处：《分子植物育种》2019年第3期827-833,共7页Molecular Plant Breeding

基　　金：科技部国家重点基础研究发展计划(2013CB126902)资助

摘　　要：在真核生物中,HORMA结构域具高度保守性,含HORMA结构的蛋白在细胞减数分裂中具有重要作用。LOC_Os01g07170蛋白具有HORMA结构域,系统进化树分析表明该蛋白与拟南芥ASY1相似度较高,且该基因在生殖器官大量表达,推测LOC_Os01g07170基因可能参与细胞的减数分裂。本研究利用CRISPR-Cas9技术对LOC_Os01g07170基因进行敲除,在该基因上设计了两个靶位点,构建LOC_Os01g07170基因敲除载体,采用农杆菌介导的方法转化水稻粳稻品种9522,获得T0代转基因苗。结果显示突变靶点1处共获得7棵转基因苗,鉴定得到3株纯合突变体,突变类型为在第3个外显子的第1 137号碱基缺失1个C碱基;突变靶点2处共获得11株转基因苗,有4株纯合突变体,突变类型为在第4个外显子的第1 501号碱基处插入1个C碱基。分析氨基酸序列,发现这2种突变株系都存在移码突变而使氨基酸翻译提前终止。本研究成功利用CRISPR-Cas9基因编辑技术获得了水稻LOC_Os01g07170基因的两个突变类型的株系,为进一步研究LOC_Os01g07170基因功能提供了遗传材料。The HORMA domain is highly conservative in eukaryotes.Proteins containing HORMA structure play an important role in cell meiosis.LOC_Os01g07170 protein contains HORMA domain,and phylogenetic tree analysis indicates that LOC_OsO1g07170 is similar to Arabidopsis ASY1,and LOQOsO1g07170 is highly expressed in reproductive organs,suggesting that LOCOsO1g07170 may participate in cell meiosis.In this study, the LOC_OsO1g07170 gene was knocked out by CRISPR-Cas9 technique.Two target loci were designed to construct the knockout vector of LOQOsO1g07170 gene.Agrobacteriurn-mediated transformation of rice variety 9522 was carried out to obtain transgenic seedlings of T0 generation.The results showed that 7 transgenic seedlings were obtained at the mutation target 1,and 3 homozygous mutants were identified.The mutation type was one C deletion at 1137th base on the 3rd exon.A total of 11 transgenic seedlings were obtained at 2 mutation targets, including 4 homozygous mutants with one C base inserted into the 1 501th base of the 4th exon.The amino acid sequence analysis found that there were frameshift mutations in 2 mutant lines,leading to early termination of amino acid translation.In this study,two mutant lines of LOC_OsOlg07170 gene in rice were successfully obtained by CRISPR-Cas9 gene editing technique,which could provide genetic material for further study of LOQOsO1g- 07170 gene function.

关 键 词：水稻 HORMA 减数分裂 CRISPR-Cas9 

分 类 号：S511[农业科学—作物学]