转录因子SOX2 (sex determining region Y-box2)在早期胚胎发育第一次谱系分化以及内细胞团多能性维持等方面具有重要的作用。但是目前有关SOX2基因启动子的系统研究较少,尤其是在猪(Sus scrofa)中尚无相关报道。为系统分析猪SOX2基因启动子在早期胚胎中的活性,本研究通过优化显微注射体系中绿色荧光蛋白表达载体种类和注射时间,构建了适合猪SOX2基因启动子活性分析的参照体系和显微注射体系;对猪SOX2基因翻译起始位点上游5000 bp区域进行转录因子结合位点的预测,发现该区域存在4个转录因子结合位点簇;针对上述区域设计并构建相应的缺失型SOX2基因启动子报告载体,利用建立的显微注射体系将其导入胚胎,通过mCherry荧光强度以及qRT-PCR定量分析SOX2基因启动子中不同转录因子结合位点簇对启动子活性的影响。结果表明,与全长SOX2基因启动子相比,SOX2基因翻译起始位点上游2254~2442 bp区域缺失后,猪4-细胞和8-细胞胚胎中SOX2基因启动子活性下降至17.8%,该缺失区域中仅包含两个NF-AT(nuclear factor of activated T cells)转录因子结合位点。因此,本研究结果推测猪SOX2基因启动子中NF-AT转录因子结合位点是影响猪早期胚胎SOX2基因启动子活性的关键位点。本研究为揭示猪早期胚胎发育中SOX2基因表达调控机制提供了数据支持。

SOX2 (sex determining region Y-box2) is one of the critical pluripotent factors that play a crucial role in the first lineage differentiation and maintenance of pluripotency in inner cell mass during early embryonic development. However, there are few researches about the regulation of the SOX2 promoter, especially in Sus scrofa. To analyzed the activity of SOX2 promoter in early porcine embryos, we determined the control system and established the microinjection system for assessing SOX2 promoter activity by analyzing the embryonic development and the expression of enhanced green fluorescence protein (EGFP) after micro-injected different EGFP plasmids at different times after activation of the oocytes. Then, we analyzed the structure of 5000 bp upstream of the SOX2 translation initiation site and found there were four transcription factor binding site clusters. Next, we designed and constructed promoter-containing plasmids to analyze the function of each cluster. To detect the activity of different promoters, we assessed the mCherry expression in protein levels and mRNA levels by analyzing the mCherry fluorescence intensity and qRT-PCR after injecting plasmids into embryos. These results showed that the activity of the shorted promoter, with the region from 2254 bp to 2442 bp upstream of translation initiation site deleted, decreased to 17.8% at 4-cell and 8-cell stages compared with the full-length promoter. This region included two NF-AT transcription factor binding sites, which indicated that the NF-AT binding site is a key region to regulate the activity of the SOX2 promoter. The results provide important data for determination the mechanism of porcine SOX2 regulation.