绵羊CYP19基因卵巢启动子的克隆及其表达活性研究

Cloning and Expression Assay of Sheep CYPl9 Gene Ovarian Promoter

本研究旨在克隆绵羊CYP19基因卵巢启动子序列片段,构建真核表达载体,并在细胞水平检测其组织特异性表达情况。参考已知序列设计特异性引物,PCR扩增绵羊CYP19基因卵巢启动子1.1和0.5kb两个片段,并与已公布的序列进行同源性比较;将测序正确的两个片段分别定向克隆到去除CMV的pEGFP-N2载体骨架中,构建真核表达载体pCYP19-1.1-EGFP-N2和pCYP19-0.5-EGFP-N2,重组质粒在脂质体LipofectamineTMLTX+PLUS介导下,分别转染绵羊的颗粒细胞和胎儿成纤维细胞,并于转染后24、48和72h观察EGFP表达情况。结果表明,扩增获得片段与已公布序列高度同源;应用转录因子结合位点预测软件对所得序列分析表明,该扩增片段含有类似于TATA-box核心启动顺式元件,并含有多个潜在转录因子的结合位点。转染24h后,发现pCYP19-1.1-EGFP-N2在颗粒细胞可观测到绿色荧光表达,48h荧光细胞数量有所增加;转染72h时荧光细胞数最多;在胎儿成纤维细胞也有少量EGFP基因表达。pCYP19-0.5-EGFP-N2在颗粒细胞和成纤维细胞中均未检测到荧光。结果表明,扩增所得的绵羊CYP19基因卵巢启动子1.1kb片段可引导外源基因在颗粒细胞中的表达,可用于与繁殖相关的基因功能及转基因动物研究,但并非卵巢特异性启动子。

The research was conducted to clone the sheep CYP19 gene ovarian promoter fragments and detect the tissue-specific expression in cells level.According to the known sequence the specific prmiers were designed,1.1and 0.5kb of sheep CYP19 gene ovarian promoter fragment were amplified by PCR,then the sequences were analyzed by software with published sequences.After eukaryotic expression vector pCYP19-1.1-EGFP-N2 and pCYP19-0.5-EGFP-N2 were constructed by cloning promoter fragments into the pEGFP-N2 vector without CMV,then the recombinant plasmids were transfected into sheep granular cells and fetal fibroblast cells by liposome Lipofectamine TMLTX+PLUS.The EGFP fluorescence expression was observed under the microscope after transfection for 24,48 and 72h.The sequenced results showed that sheep CYP19 gene promoter fragments which were cloned were 1.1and 0.5kb length and had highly homologous with the published sequences.The sequence analyzing with transcription factor binding sites prediction software indicated that the promoter fragment contained a core promoter cis-element smilar withTATA box and transcription factor binding sites.24 hafter transferring with pCYP19-1.1-EGFPN2,the EGFP-expressing positive granulosa cells could be observed,48 hafter transferring,the EGFP-expressing positive granulosa was increased,72 hafter transfection the EGFP-expressing positive granulosa increased to the most.But after transfection the EGFP which was expressed in sheep fetal fibroblast cells was little.The results also showed that no EGFP-expressing positive granulosa cells and fetal fibroblast cells were observed at 24,48 and 72hafter transfection with pCYP19-0.5-EGFP-N2.The sheep CYP19 promoter 1.1kb can drive foreign gene expressing in sheep granulosa cells and it can be used for the functional studies of fecundity-related genes and transgenic animal research,but it is not ovarian specific expression promoter.