角鲨烯合成酶特异性mU6-siRNA真核表达载体的构建及鉴定

Construction of Eukaryotic Expression Vector of siRNA Specific to Squalene Synthetase Gene

利用RNA干扰技术靶向构建角鲨烯合成酶基因ERG9特异性真核表达载体。将针对粟酒裂殖酵母(Schizosaccharomyces pombe Linder)ERG9不同部位所设计的3对siRNA序列通过重组技术克隆到质粒mU6 pro中构建真核表达重组体mU6 ERG9 siRNA1、2、3,转化DH5α菌株扩增,提取质粒通过限制性酶切和测序分析对重组表达载体进行鉴定,分析结果表明3个表达载体的设计基因插入正确,成功构建了ERG9特异性真核表达载体mU6 ERG9 siRNA,重组体的成功构建为在裂殖酵母细胞中靶向RNA干扰角鲨烯的合成从而提高辅酶Q10合成途径的代谢通量打下基础。

To construct eukaryotic vector expressing siRNA （small interference RNA）of ERG9.Designing three different siRNA targeting the coding sequence of the ERG9,the mU6 ERG9 siRNA was constructed by inserting the designed siRNA to the eukaryotic expression vector mU6 pro.The constructed eukaryotic vector expressing siRNA of ERG9 was transformed into DH5 α strain,Finally the recombinant plasmid identified by testriction enzyme was used for sequence analysis.It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing siRNA of ERG9 was correct.The results of study lay the foundation for further studying on enhancing the metabolic flux of the CoQ10 pathway by the inhibitive effect on synthesis of squalene.