东方粘虫 U6启动子的克隆及功能验证
Cloning and Functional Verification of U6 Promoter from Mythimna separata (Walker)

为了持续获得大量短片段干扰RNA（short interference RNA, siRNA），通过染色体步移技术克隆得到东方粘虫 U6 snRNA 基因5′-端侧翼启动子序列1 426 bp。经生物信息学分析，该序列含SPH元件、八聚体序列（OCT）、远端序列元件（DSE）、近端序列元件（PSE）及TATA box等RNA聚合酶Ⅲ（RNA polymerase Ⅲ，RNA Pol Ⅲ） U6启动子的特征元件。通过构建RNA干扰（RNA interference, RNAi）载体的方式，对增强型绿色荧光蛋白EGFP基因进行RNAi，证明克隆得到的东方粘虫 U6启动子可成功驱动shEGFP表达，具有 U6启动子功能。为后续构建以东方粘虫V-ATP酶H亚基为靶基因的沉默载体奠定基础。
In order to obtain constantly short interference RNA(siRNA) , the 1 426 bp 5′- flanking promoter sequence of U6 snRNA gene of Mythimna separata(Walker) was cloned by the method of Genome Walking, as well as its function was verified in this study. Based on bioinformatics analysis, the results showed that the promoter fragment contained polymerase Ⅲ core promoter elements SPH, OCT, DSE, PSE and TATA box. The observation of expression level enhanced green fluorescent protein EGFP after being transfected with RNA interference (RNA interference, RNAi) , the vectors by fluorescence microscope showed that the U6 promoter fragment drived the expression of shEGFP successfully. This study had established a foundation for further construction of RNAi vectors which can silence V-ATPase subunit H.