水牛OCT4基因5′调控序列的克隆与功能分析

Cloning and function analysis of OCT4gene 5′regulatory region in buffalo

为探讨水牛OCT4基因的表达调控模式,本研究扩增克隆了水牛OCT4基因5′调控序列片段,并设计了-2 571bp、-2 389bp、-2 338bp和-2 191bp 4个不同长度的调控序列片段,分别构建了EGFP表达报告载体。通过生产转基因猪早期胚胎和转染水牛胎儿成纤维细胞分析了不同长度调控序列片段的转录活性。结果发现,在猪4.5d胚胎中各调控序列均能成功启动下游EGFP表达,但转染水牛胎儿成纤维细胞48h后均未观察到荧光。QRT-PCR分析显示,-2 571bp片段在4.5d猪胚胎细胞中的转录活性极显著高于其他调控序列(P<0.01),-2 389bp与-2 338bp之间差异不显著(P>0.05),二者均极显著高于-2 191bp(P<0.01)。上述结果表明水牛源OCT4基因5′调控序列在猪早期胚胎中具有转录活性,且翻译起始位点上游2 191bp片段足以介导OCT4在多能性细胞中的特异性表达;-2 389^-2 338bp片段上游的CR4亦参与了猪4.5d胚胎中OCT4基因的转录调控。

To investigate the expression regulation mechanism of OCT4 gene in buffalo, the 5r regu-latory region of OCT4 gene was cloned and analyzed. And four different lengths regulatory se-quences （-2 571 bp,-2 389 bp,-2 338 bp and -2 191 bp） were selected to construct their EG FP reporter vectors, respectively. The transcriptional activity of each fragment was analyzed by producting transgenic embryos and transfecting into buffalo fetal fibroblasts. The results showed that the green fluorescent proteins could be observed in all groups at 4.5 d embryos. After tran- fecting the four reporter plasmids into buffalo fetal fibroblast for 48 h, no fluorescence was ob-served in all four groups. QRT-PCR analysis showed that the activity of -2 571 bp fragment was extremely significantly higher than other regulatory sequences （P〈0.01）. There was no signifi-cant difference between -2 389 bp and -2 338 bp （P〉0.05） ,and the both were extremely signif-icantly higher than -2 191 bp （P〈0.01）. These suggest that 5＇ regulatory sequences of buffalo OCT4 also has activity in pig early embryo cells,and the 2 191 bp upstream of start codon is suffi-cient to regulate pluripotential cell-specific expression of OCT4. The CR4 plays a role in transcrip-tional regulation of OCT4 at 4.5 d embryos of pig through -2 389- -2 338 bp fragment.