利用CRISPR/Cas9系统创制紧密连锁的M320与M330基因双突变体  

Obtain of Double Mutant of Closely Linked Genes M320 and M330 by CRISPR/Cas9 System

作　　者：庞奥运 金璐[1] 陈吉杨 刘春林[2] 阮颖[1] Pang Aoyun;Jin Lu;Chen Jiyang;Liu Chunlin;Ruan Ying(College of Bioscienee and Biotechnology, Hu＇nan Agricultural University, Changsha, 410128;Agricultural College, Hu＇nan Agricultural University, Changsha, 410128)

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128 [2]湖南农业大学农学院,长沙410128

出　　处：《分子植物育种》2018年第13期4301-4307,共7页Molecular Plant Breeding

基　　金：国家自然科学基金面上项目(31771838);湖南省教育厅重点项目(17A101)共同资助

摘　　要：为了研究含有多MATH结构域基因的功能,本研究利用CRISPR/Cas9基因编辑系统对拟南芥中两个紧密连锁的功能冗余基因M320(AT2G25320)和M330(AT2G25330)进行共敲除,获得了之前通过杂交无法获得的多MATH结构域基因双突变体材料。通过构建针对M320和M330基因的CRISPR/Cas9共敲除质粒,借助农杆菌介导的浸花法成功转化野生型拟南芥Col-0。最终测序验证显示多MATH结构域基因被成功敲除。M320、M330以及这两个基因同时被敲除的成功率分别是5.13%、17.95%和2.56%;通过测序分析发现突变体共产生了3种可造成移码突变的杂合突变类型:分别为在靶位点插入一个A或T以及缺失一个G。本研究为我们解析多MATH结构域基因的功能提供了材料基础,同时为紧密连锁基因的功能研究提供了创制突变体的有效方法。In order to study the function of genes containing multiple MATH domains, CRISPR/Cas9 genome editing system was used to co-knockout two tightly-linked functional redundancy genes M320（AT2 G25320） and M330（AT2 G25330） in Arabidopsis thaliana in this study. Multiple MATH domain gene double mutant materials which were previously unavailable by hybridization were obtained. Wild-type Arabidopsis Col-0 was successfully transformed by method of Agrobacterium-mediated floral dip by constructing a CRISPRCas9 knockout plasmid targeting M320 and M330 genes. Final sequencing showed that the genes of the multiple MATH domains were successfully knockout. The success rates for M320, M330 and the two genes at the same time were 5.13%, 17.95%and 2.56%, respectively. Sequencing analysis indicated that 3 kinds of heterozygous mutations that could cause the mutagenesis of the mutants were produced： the frameshift mutations were resulted by insertion of a base A or T at the target site, or deletion of a base G. This study laid the material foundation for us to analyze the function of multiple MATH domain genes, and provided an effective method for the creation of mutants for the study of the function of close linkage genes as well.

关 键 词：拟南芥 紧密连锁 多MATH结构域基因 CRISPR/Cas9 定向敲除 

分 类 号：Q943.2[生物学—植物学]