利用CRISPR-Cas9基因编辑技术获得AtLCR67基因敲除突变体

Obtaining the Knock-out Mutant of AtLCR67 Gene in Arabidopsis thaliana by CRISPR-Cas9 Gene Editing Technique

拟南芥AtLCR67基因属于低分子量富含半胱氨酸(low-molecular-weight cysteine-rich,LCR)基因家族。在正常生长条件下,该基因在发育的种子中特异表达。为了研究该基因在发育或代谢过程中的可能作用,在没有合适T-DNA插入突变缺乏的情况下,我们利用CRISPR-Cas9定向敲除技术来创制该基因沉默的拟南芥突变体材料。通过构建AtLCR67基因的CRISPR-Cas9敲除质粒,借助农杆菌介导的浸花法转化野生型拟南芥Col-0,最终获得12株T0代转基因植株。对T0转基因植株的AtLCR67基因进行测序分析发现,在设计的靶位点处存在短片段缺失12个碱基和增加一个T碱基两种突变类型,进一步做RT-PCR检测分析发现突变体中AtLCR67基因完全不表达,说明成功获得拟南芥lcr67突变体。突变体材料的获得为进一步研究AtLCR67基因在发育或代谢方面的的功能奠定了良好基础。

AtLCR67 gene is a member of low-molecular-weight cysteine-rich（LCR） gene family in Arabidopsis thliana. Under normal growth conditions, the gene is specifically expressed in the developing seed. In order to study the possible role of the gene in the process of development and metabolism in seed, CRISPR-Cas9 editing technique was used to create the gene knockout mutant because of no suitable T-DNA insertion mutant is available. After constructing CRISPR-Cas9 gene editing plasmid of AtLCR67 gene, 12 transgenic plants（T0） were obt-ained by the transformation method of Agrobacterium-mediated floral dip. According to the results of sequencing of AtLCR67 gene in T0 plants, there were two kinds of mutants, one with deletion of 12 bases and the other with the T base insertion. Further RT-PCR analysis showed that the AtLCR67 gene expression was not detected in lcr67 mutants,which indicated that the Arabidopsis thaliana lcr67 mutant was successfully obtained. The acquisition of mutant materials laid a good foundation for further study on the function of AtLCR67 gene in development or metabolism.