徐淮山羊c-Myc基因启动子的克隆及其功能的初步分析

Cloning and Preliminary Function Analysis of Xuhuai Goat c-Myc Promoter

本研究旨在确定徐淮山羊c-Myc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c-Myc基因的表达调控机制。根据UCSC基因组数据库已公布的绵羊c-Myc基因的启动子序列,设计特异性PCR引物扩增c-Myc基因的一系列启动子缺失片段,定向克隆至pEGFP-N1和PGL3-c-Myc,分别转染gFF、COS7及P19细胞,并进行TSA和NFAT1诱导,同时对-402^-249bp区域的转录因子SP1结合位点进行定点突变,最后进行双荧光报告基因活性检测。结果表明,徐淮山羊c-Myc基因5′侧翼区-1 334^+1bp区域的启动子活性最强,-402^+1bp区域为c-Myc基因启动子基本活性区域。进一步研究发现,-1 334^-971bp、-587^-147bp区域存在正调控元件,-1 976^-1 334bp、-971^-587bp区域存在负调控元件。TSA和NFAT1均能增强cMyc启动子的活性,SP1结合位点定点突变后,启动子活性降低。本试验通过构建包含c-Myc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c-Myc基因启动子的核心区域,发现转录因子SP1是c-Myc基因启动子核心区域的调控元件,为进一步研究c-Myc基因的表达调控机制奠定了基础。

The aim of this study was to determine and find the promoter core regulatory regions of Xuhuai goat's c-Myc gene,and to investigate c-Myc gene regulation mechanism.According to the sequence of ovis aries c-Myc gene published in GenBank,the primers were designed and the different deletion fragments of 5′flanking region of c-Myc gene promoter were amplified,and then were cloned into pEGFP-N1and PGL3-Basic,then were transfected into gFF,COS7and P19cells and treated with TSA and NFAT1.SP1transcription factor binding sites in the region of-402--249 bp was mutated,and the activity of dual-luciferase reporter gene was detected.The study showed that the region containing-1 334-+1bp of c-Myc promoter had the maximal promoter activities, and sequence of-402-+1bp had the basal promoter activities,further study showed that there were positive(the region of-1 334--971bp,-587--147bp)and negative(the region of-1 976--1 334bp,-971--587bp)regulatory domains,respectively.TSA and NFAT1significantly enhanced the activity of c-Myc promoter.The activity was declined by the point mutation of SP1binding site.By constructing the recombinant expression vectors containing different frag-ments of c-Myc gene promoter and comparing their activity,the core regulatory region of c-Myc gene promoter was identified.SP1is an important regulatory element of c-Myc promoter core region,which lay a foundation for the further research on the mechanism of regulation and expression of c-Myc gene.