水牛YY1基因克隆及其shRNA片段的筛选

Cloning of Buffalo YYl Gene and Screening Its shRNA Fragments

本试验旨在克隆水牛阴阳因子1(YY1)基因,并筛选获得可有效抑制水牛YY1基因表达的shRNA干扰片段。以水牛成纤维细胞cDNA为模板,采用RT-PCR方法,扩增水牛YY1基因CDS序列,将其连接插入到pMD18-T载体中,进行序列测序并分析。采用Xho I和EcoR I双酶切,将水牛YY1基因编码区定向克隆至pEGFP-N1载体中,构建其真核表达载体pYY1-EGFP-N1。设计2条靶向水牛YY1基因的shRNA序列并构建其慢病毒表达载体,命名为pSicoR-GFP-YY1 shRNA1/2。以pSicoR-GFP和pSieoR-GFP-1864分别为空白对照和阴性对照质粒,采用脂质体转染方法,将YY1真核表达载体分别与2条shRNA慢病毒表达载体共转染293T细胞,48h后观察绿色荧光蛋白EGFP表达情况;72h收集共转染细胞样品,采用qRT-PCR和western-blot方法分析YY1基因的表达水平。结果表明,克隆得到1248 bp的水牛YY1基因CDS序列,其与牛YY1基因的同源性达99%。构建的水牛YY1基因真核表达载体pYY1-EGFP-N1能在水牛胎儿成纤维细胞(BFF)中瞬时表达。酶切分析及序列测定结果表明,所构建的shRNA慢病毒表达载体pSieoR-GFP-YY1 shRNA1/2为阳性克隆。质粒共转染293T细胞48h后,可观察到绿色荧光蛋白EGFP的表达。qRT-PCR结果显示,设计合成的2条shRNA对水牛YY1基因的表达均有抑制效果,其中YY1 shRNA2的抑制效果(93.64%)显著高于YY1 shRNA1(50.77%)(P<0.05),Western-blot分析结果显示,2条shRNA对YY1蛋白表达均有抑制效果。以上结果说明克隆得到水牛YY1基因编码区序列,并获得2条有效抑制其表达的shRNA片段,为进一步阐明YY1基因在水牛早期胚胎发育过程中的作用奠定了基础。

The aim of present study was to clone the CDS sequences of buffalo YY1 gene,and screen its effective shRNA fragments.The cDNA of buffalo fetal fibroblast(BFF) was used as the PCR template,the CDS full-length sequence of buffalo YY1 gene was amplified by using touch down RT-PCR methods.The purified CDS fragment was inserted into pMD18-T vector,and the positive plasmid was identified and sequenced.Fusion expression vector of buffalo YY1 gene was constructed by inserting the YY1 CDS fragment into pEGFP-Nl vector,named as pYY1-EGFPN1.The pYY1-EGFP-N1 plasmid was transfected into BFF cells by Lipofectamine~? LTX reagent.Two shRNA fragments targeting buffalo YY1 gene were designed and synthesized,their lentiviral expression vectors were constructed,named as pSicoR-GFP-YY1 shRNA1/2 respectively.YY1recombinant plasmid(pYY1-EGFP-N1) and shRNA lentiviral expression plasmid(pSicoR-GFPYY1 shRNA1/2) were co-transfected into 293T cells by using Lipo-LTX reagent at the ratio of1:1 or 1:6,pSicoR-GFP and pSicoR-GFP-1864 plasmid were used as blank and negative control respectively.At 72 h after transfection,the cells were harvested,and the total RNA was extracted.The expression of YY1 gene in each transfected cells was detected by qRT-PCR and Westernblot methods.The results showed that the CDS sequence of cloned buffalo YY1 was 1 248 bp,the nucleotide homology of YY1 gene between bovine and buffalo was 99%.The constructed pYY1-EGFP-N1 plasmid could transiently express in BFF cells.The results of qRT-PCR showed that the two designed shRNAs could inhibit the expression of buffalo YY1 mRNA.the shRNA2 fragment had significantly higher inhibition effect than shRNAl(93.64%v 50.77%)(P<0.05).The results of Western-blot analysis confirmed that the two shRNAs had inhibition effect on gene expression of buffalo YY1.The above results indicated that the CDS sequence of buffalo YY1 was obtained,two effective shRNA fragments targeting buffalo YY1 gene were selected,which laid foundation for further study the role of YY1 gene in buffalo embryo development.