不同种属H1启动子驱动miR-1在PK15细胞中的表达情况

Expression of mi R-1 controlled by H1 promoters from different species in PK15 cells

【目的】筛选出能高效表达外源性成熟miR-1的真核表达载体,为揭示miR-1参与猪肌肉发育及肉质性状形成的分子机制奠定基础。【方法】分别克隆巴马小型猪miR-1前体序列、H1启动子序列及人类H1启动子序列,并构建不同种属H1启动子驱动其表达的真核表达载体,利用脂质体法转染PK15细胞后,采用荧光定量RT-PCR(qRT-PCR)检测不同种属H1启动子表达载体驱动miR-1表达效率。【结果】经酶切鉴定和测序分析证实,成功构建了3个miR-1过表达载体(pEGFP-miR-1、pEGFP-hH1-miR-1和pEGFP-pH1-miR-1),以其转染PK15细胞24和48 h后,荧光显微镜观察发现绝大部分细胞都发出绿色荧光,且含有H1启动子细胞的荧光强于无H1启动子细胞。转染pEGFP-pH1-miR-1细胞、转染pEGFP-hH1-miR-1细胞、转染pEGFP-miR-1细胞的miR-1相对表达量分别为105.02、99.00和79.65,其miR-1表达水平均极显著高于转染pEGFP-C 1细胞和空白对照组细胞(P<0.01)。【结论】重组质粒pEGFP-miR-1、pEGFP-hH1-miR-1和pEGFP-pH1-miR-1均能在PK15细胞中高效表达miR-1,即猪源与人源的H1启动子均能提高外源性miR-1表达,可用于后续的miR-1功能研究。

[Objective]In order to lay the foundation for further research on molecular mechanisms of miR-1 involved in porcine muscle development and meat quality traits,the eukaryotic expression vectors that could express efficiently exogenous mature miR-1 were screened out.[Method]The sequences of porcine pre-miR-1,H1 promoter and human H1 promoter were cloned,respectively.And H1 promoter-controlled miR-1 expression vectors of different species were constructed.Then the recombinant expression vectors were transfected into PK15 cells by liposome-mediated transfection method,and the expression efficiency of miR-1 was analyzed by fluorescent quantitative qRT-PCR.[Result]The enzyme digestion and sequencing results demonstrated that three recombinant vectors（pEGFP-miR-1,pEGFP-hH1-miR-1 and pEGFP-pH1-miR-1） were constructed successfully,and transfected into PK15 cells.After 24 and 48 hours,under the fluorescent microscope,the green fluorescence could be observed in the most of PK15 cells,and the fluorescence intensity of PK15 cell with H1 promoter was stronger than that without H1 promoter.The expression levels of miR-1 in transfected cells containing pEGFP-pH1-miR-1,pEGFP-hH1-miR-1 and pEGFP-miR-1 were 105.02,99.00 and 79.65,respectively,and which were extremely significantly higher than expression levels of miR-1 in transfected cell containing pEGFP-C1 and control cell（P0.01）.[Conclusion]The recombinant plasmids（pEGFP-miR-1,pEGFP-hH1-miR-1 and pEGFP-pH1-miR-1） can express efficiently exogenous mature miR-1.Therefore,the porcine-derived and human-derived H1 promoters are capable of improving efficiently expression level of miR-1,and can be used to research function of miR-1 in the future.