徐淮山羊 Sox2 基因启动子的克隆及其活性的初步分析

Cloning and Preliminary Activity Analysis of Xuhuai Goat Sox2 Promoter

本研究旨在确定徐淮山羊Sox2基因启动子区域,找出该基因启动子的核心调控区,初步探讨Sox2基因的表达调控机制。根据GenBank已公布的绵羊Sox2基因的启动子序列,设计特异性PCR引物扩增Sox2基因的一系列启动子缺失片段,经酶切、测序及生物信息学分析,构建包含Sox2基因5′侧翼区一系列启动子缺失片段的pGL3-Sox2双荧光素酶表达载体,转染COS-7和GC1细胞,并进行5-aza-2′-deoxycytidine诱导,检测不同片段的启动子活性。结果表明:徐淮山羊Sox2基因5′侧翼区-1249—+49 bp区域的启动子活性最强(COS-7细胞),-1792—+49 bp区域活性最强(GC1细胞),-224—+49 bp区域为Sox2基因启动子基本活性区域。进一步研究发现,-484―-109 bp区域存在正调控元件,-755—-484 bp区域存在负调控元件。本实验通过构建包含Sox2基因启动子不同片段的重组报告基因载体并比较其转录活性,确定了Sox2基因启动子的核心区域。另外,5-aza-2′-deoxycytidine可以显著增强Sox2启动子的活性。为进一步研究Sox2基因的表达调控机制奠定了基础。

The aim of this study was to determined and found the promoter core regulatory regions of Xuhuai goat＇s Sox2 gene, and investigate Sox2 gene regulation mechanism. According to the sequence of Ovis aries Sox2 gene published in GenBank, the primers were designed and the deletion fragments of Ovis aries Sox2 gene promoter 5＇ flanking regions were amplified, and then were recombined into PGL3-Basic plasmid by digestion ,sequencing and bioinformatics analysis. And then were transfected into COS-7 and GC1 cells, and the activity of dual-luciferase reporter gene was detected. The studies showed that the region contained-1 249--＋49/-1 792--＋49 of Sox2 promoter had the maximal promoter activities, respectively transfected in CSO-7 cell and GC1 cell. sequence from -224--＋49 had the basal promoter activities, Further studies showed that there were positive （the region of -484--109） and negative （the region of -755---484 bp） regulatory domains, respectively. By constructing the recombinant expression vectors contained different fragments of Sox2 gene promoter and comparing their activity, the core regulatory region of Sox2 gene promoter was identified. In addition, 5-aza-2＇-deoxycytidine significantly enhanced the activity of Sox2 promoter. Lay a foundation for further research on the mechanism of regulation and expression of Sox2 gene.