猪紧密连接蛋白1基因克隆及其shRNA片段的筛选

Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments

试验旨在筛选有效抑制猪紧密连接蛋白1(zonula occludens-1,ZO-1)基因表达的shRNA干扰片段,为后期利用RNA干扰技术深入了解ZO-1基因在猪卵母细胞成熟过程中的功能奠定基础。本研究首先克隆了猪ZO-1基因,并构建了其真核表达载体,而后筛选了有效抑制ZO-1基因表达的shRNA干扰片段。结果表明,猪ZO-1基因编码区长度为3 036bp,多重序列比对结果显示,猪ZO-1基因核苷酸序列与牛、羊、马和人相应序列的同源性分别为88%、88%、87%和85%。构建的pDsRed-N1-ZO-1真核蛋白融合表达载体经脂质体法转染HEK293T细胞后,可观察到清晰的RFP红色荧光蛋白表达。将靶质粒与shRNA干扰质粒共转染HEK293T细胞48h后,与对照组相比,可观察到红色荧光表达得到抑制。实时荧光定量PCR结果显示,设计合成的2条shRNA对猪ZO-1基因表达均有抑制效果,其中ZO-1shRNA201的抑制效果显著高于ZO-1shRNA1276(77.8%和67.0%,P<0.05)。

The aim of this study was to screen effective shRNA fragments targeting pig zonula occludens-1（ZO-1）gene,which would lay foundation to further explore its function in pig oocytes in vitro maturation（IVM）by RNAi technology.Pig ZO-1gene was cloned and its eukaryotic expression vector was constructed,its effective shRNA fragments were screened.The results showed that the CDS length of cloned pig ZO-1gene was 3 036 bp.The results of sequence multialigned showed that the sequence of pig ZO-1gene shared 88%,88%,87% and 85% homology with Taurus,Ovis aries,Equns caballus and Homo sapiens,respectively.Clear RFP red fluorescent was observed in transfected cells with the pDsRed-N1-ZO-1eukaryotic expression plasmid transfected into HEK293 Tcells by X-tremeGENE HP DNA transfection reagent.Two shRNA fragments targeting pig ZO-1gene were designed and synthesized,weaker RFP red fluorescent was observed in co-transfected cells with the target and interfered shRNA plasmid groups.The Real-time PCR results showed that the two designed shRNAs could effectively inhibit the expression of pig ZO-1mRNA,shRNA201 fragment had significantly higher inhibition effect than that of shRNA1276fragment（77.8% and 67.0%,P〈0.05）.