利用CRISPR-Cas9基因编辑技术获得AtLCR67基因敲除突变体

发布时间：2018-06-24 13:21

本文选题：拟南芥 + AtLCR　；参考：《分子植物育种》2017年06期

【摘要】：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。
[Abstract]:Arabidopsis thaliana AtLCR67 gene belongs to low-molecular-weight cysteine-rich LCR gene family. Under normal growth conditions, the gene was specifically expressed in developing seeds. In order to study the possible role of the gene in the development or metabolism of Arabidopsis thaliana, in the absence of suitable T-DNA insertion mutation, CRISPR-Cas9 targeted knockout technique was used to create the silenced Arabidopsis mutants. The CRISPR-Cas9 knockout plasmid of AtLCR67 gene was constructed and transformed into wild-type Arabidopsis thaliana Col-0 by Agrobacterium tumefaciens. Finally, 12 transgenic plants of T0 generation were obtained. Sequencing analysis of AtLCR67 gene in T0 transgenic plants showed that there were two types of mutations at the designed target site: short fragment deletion of 12 bases and addition of one T base. Further RT-PCR analysis revealed that AtLCR67 gene was not expressed at all in the mutant, which indicated that the Arabidopsis lcr67 mutant was successfully obtained. The acquisition of mutants laid a good foundation for further study on the developmental and metabolic functions of AtLCR67 gene.
【作者单位】：湖南农业大学生物科学技术学院;湖南农业大学农学院;
【基金】：国家自然基金面上项目(31571707) 国家重点研发计划项目子课题(2016YFD0100202-8)共同资助
【分类号】：Q943.2

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