旨在对绵羊miR-150基因的启动子进行鉴定以及探索生殖激素对miR-150启动子转录调控的影响。本研究利用生物学软件预测雌性绵羊(小尾寒羊)卵巢miR-150启动子区域及繁殖相关转录因子结合位点,构建重组质粒,并利用双荧光素酶报告基因检测miR-150的启动子活性。通过在无血清培养基内添加FSH、LH和E2,进行miR-150启动子重组质粒转染并检测miR-150启动子活性。结果,分别利用Promoter 2.0和PROMO软件预测出miR-150启动子区域和转录起始位点,并获得了与繁殖相关转录因子结合位点。所预测的绵羊miR-150启动子片段序列长度为582 bp,且成功构建miR-150启动子质粒,并确定绵羊颗粒细胞的最佳转染效率(脂质体∶质粒DNA=1∶1)和质粒最佳转染比例(50∶1),从而验证了所预测的区域具有启动子活性。此外,FSH、LH和E2处理组与对照组(无激素组)相比,miR-150启动子的活性均显著下调(P<0.05)。结果表明,生殖激素可以通过影响绵羊miR-150启动子活性来调控miR-150基因的表达,为miR-150调控卵泡发育的机制研究提供了新思路。
The aim of this study was to identify the promoter of miR-150 gene and explore the effect of reproductive hormone on its transcription regulation in sheep. The miR-150 promoter region of ovary in female sheep(Small-tail Han sheep) and the reproduction-related transcription factor binding site were predicted by biological softwares, then recombinant plasmids were constructed and the dual luciferase reporter gene was used to detect the activity of miR-150 promoter. Moreover, the miR-150 promoter recombinant plasmid was transfected and miR-150 promoter activity was detected by adding FSH, LH and E2 in serum-free medium. The miR-150 promoter region and transcription initiation site were respectively predicted using Promoter 2.0 and PROMO, and transcription factor binding sites associated with reproduction were obtained. The predicted length of the sheep miR-150 promoter fragment was 582 bp, and the miR-150 promoter plasmid was successfully constructed. The optimal transfection efficiency(liposomes∶plasmid DNA=1∶1) of the sheep granulosa cells and the optimal transfection proportion(50∶1) of the plasmid were determined. Thus, the predicted region with significant promoter activity was verified. In addition, the activity of the miR-150 promoter was significantly down-regulated in the FSH, LH and E2 treatment groups compared with the control group(hormone-free group)(P<0.05). The results indicated that reproductive hormone could regulate the expression of miR-150 by affecting the miR-150 promoter activity, which provided a new idea for studying the mechanism of miR-150 regulating ovine follicular development.
Chinese Journal of Animal and Veterinary Sciences