水牛SOX2基因5′调控序列的克隆与功能分析

Cloning and Function Analysis of Buffalo SOX2 Gene 5′ Regulatory Region

为探讨水牛SOX2基因的转录调控机制,本试验克隆获得其长2555bp的5′调控序列片段,结合生物信息分析设计了-2263、-1816、-1275、-660和-407bp 5个缺失体,并分别构建其EGFP表达报告载体,通过生产转基因早期胚胎和转染水牛胎儿成纤维细胞分析各缺失体片段的转录活性。结果发现,除-407bp以外的各缺失体在猪4.5d早期胚胎细胞中均能成功启动下游EGFP的表达,且随着片段缩短,其转录活性呈极显著递减趋势(P<0.01);而转染水牛成纤维细胞48h后,除p-407-EGFP以外的各缺失体报告载体转染组均观察到少数细胞发光,转录活性两两之间差异均极显著(P<0.01),转录活性从高到底排布分别为-2263、-660、-1275和-1816bp。p-407-EGFP载体在胚胎水平和细胞水平均未观察到荧光。以上结果表明,-660～-407bp是构成水牛SOX2基因表达不可缺失的部分,-2263～-1816bp中有非多能细胞特异性的增强子元件存在,而-1816～-1275bp和-1275～-660bp均含有多能性细胞特异性的增强子元件。

To explore the transcriptional regulatory mechanism of huffalo SOX2 gene, its 5＇ regulatory region （2555 bp） were cloned, then five deletion mutants --2263, --1816, --1275, 660 and --407 bp were designed and constructed as EGFP reporter vectors respectively. The transcriptional activity of each deletion mutant was analysed by producing transgenic embry os and transfecting into buffalo fetal fibroblast （BFF）. The results showed thai the green fluorescent protein could be observed in all groups in pig embryos （4.5 d） except p 407-E（;FP, and with the gradual reduction of the fragment, the activity of the deletion mutants had a highly significant decreasing trend（P^0.01）. After transfecting into BFF 48 h, a small number of cells was able to see fluorescence in all groups except p 407 EGFP, and the transcriptional activity differences from each other were highly significant（P^0.01）, the --2263 bp fragmenl had the highesl activity, followed by 660 bp, then --1275 bp, and finally --1816 bp. No fluorescence was observed in p 407-EGFP group both embryo and BFF level. The results suggested that the 660-- 407 hp was an integral part of the buffalo SOX2 gene hasic promoter, --2263--1816 bpcontainedthenon-plu ripotential cell specific enhancer element, there were pluripotential cell-specific enhancer elements existed in --18164 --1275 bp and 1275-- 660 bp.