利用CRISPR/Cas9系统对林烟草基因编辑的研究

Research of Gene Editing by CRISPR/Cas9 System in Nicotiana sylvestris

摘要: 本研究以野生林烟草(Nicotiana sylvestris)为研究材料，进行基于CRISPR/Cas9系统的基因编辑的研究。本文以林烟草无菌苗叶片为外植体，利用农杆菌介导的叶盘法将真核表达载体pBI121-EGFP转化至林烟草中，获得可发绿色荧光的转基因植株；然后构建含有GEP基因guide序列的CRISPR/Cas9基因敲除载体，并以pBI121-EGFP转基因植株叶片为外植体，进行农杆菌介导的遗传转化，经检测发现已成功将绿色荧光敲除，从而构建了利用CRISPR/Cas9系统对林烟草的基因进行编辑的系统，为烟草中基因功能研究平台的建立提供了技术基础。

Abstract: In this study, Nicotiana sylvestris was used as research material for carrying out the CRISPR/Cas9 gene editing researches. In this paper, the leaves of aseptic seedlings of tobacco (Nicotiana sylvestris) were used as explants. The eukaryotic expression vector pBI121-EGFP was transformed into tobacco by Agrobacterium tumefaciens mediated leaf disc method to obtain transgenic plants with green fluorescence. Then, the CRISPR/Cas9 gene knockout carrier containing the guide sequence of the GEP gene was constructed and the transgenic plant leaves of pBI121-EGFP were used as explants to conduct genetic transformation by Agrobacterium mediated transformation, and the transgene transformation of Agrobacterium mediated by Agrobacterium tumefaciens was carried out. It was found that the green fluorescence was knocked out successfully, and the CRISPR/Cas9 system was constructed to edit the gene of forest tobacco, which provided a technical basis for the establishment of the gene function research platform in tobacco.