CRISPR/Cas9技术在获取拟南芥双突变体材料中的应用

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

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。

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 T_0 transgenic plants were obtained by PCR and Singer Sequencing. Among them, 5 transgenic plants had no editing in targetsequence. In the other five transgenic plants, the target sequence of sb3 gene was edited, but the targetsequence 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.