欢迎访问《中国农学通报》,

中国农学通报 ›› 2018, Vol. 34 ›› Issue (8): 27-36.doi: 10.11924/j.issn.1000-6850.casb17110102

• 生物技术科学 • 上一篇    下一篇

CRISPR/Cas9技术在获取双突变体材料中的应用

吕晶森,谭成方,刘春林,阮 颖   

  1. 湖南农业大学生物科学技术学院,湖南农业大学生物科学技术学院,湖南农业大学农学院,湖南农业大学生物科学技术学院
  • 收稿日期:2017-11-23 修回日期:2018-02-26 接受日期:2018-01-24 出版日期:2018-03-21 发布日期:2018-03-21
  • 通讯作者: 阮 颖
  • 基金资助:
    国家自然基金面上项目“油菜SDG8 中新发现VHE基序的功能研究”(31771838)。

Application of CRISPR/Cas9 Technology in Obtaining Double Mutant Materials of Arabidopsis thaliana

  • Received:2017-11-23 Revised:2018-02-26 Accepted:2018-01-24 Online:2018-03-21 Published:2018-03-21

摘要: 为了研究含双MATH结构域基因sb3 与sb6 的功能,需要创制这2 个紧密连锁基因的双突变体。利用CRISPR/Cas9 基因编辑技术对拟南芥Col-0 中的sb3、sb6 基因进行特异性定点双敲除。通过PCR和Singer 测序验证,共获得10 株T0代转基因植株,其中5 株在目标片段上没有发生编辑;另5 株在sb3 基因上的目标序列都发生了编辑,在sb6 基因的目标序列上只有2 株发生了编辑。至此成功获得了sb3 与sb6 基因双敲除的突变体,且获得该突变的类型为核苷酸缺失突变体。这个双突变体的获得为进一步研究双MATH结构域基因在拟南芥中的功能奠定了良好的基础

Abstract: To study the gene function of sb3 and sb6 containing two MATH domains, we need to obtain the double mutants of these two closely linked genes. CRISPR/Cas9 gene editing technology was used to create special double knockout mutants of sb3 and sb6 genes in Arabidopsis thaliana Col-0., 10 T0 transgenic plants were obtained by PCR and Singer Sequencing. Among them, 5 transgenic plants had no editing in target sequence. In the other five transgenic plants, the target sequence of sb3 gene was edited, but the target sequence of sb6 gene was edited only in 2 transgenic plants. So far, the double-knockout mutants of sb3 and sb6 genes are successfully obtained, belonging to nucleotide deletion mutants. The two double mutants lay a good foundation for further research on the function of dual MATH-domain genes in Arabidopsis thaliana.