人CREG启动子报告基因质粒的构建及转录活性分析

Construction and transcriptional activity of human CREG promoter reporter gene plasmid

目的构建人E1A激活基因阻遏子基因(hCREG)不同截短片段的启动子增强型绿色荧光蛋白(pEGFP1)报告基因载体,比较各启动子转录活性,从而确定hCREG核心启动子区。方法通过查询美国国立生物技术信息中心(NCBI)数据库hCREG序列并结合启动子特点,获得长度为2003(–1925/+78) bp的启动子片段。利用PCR法及双酶切法截短5个启动子片段并克隆至pEGFP1中,构建pEGFP1_hCREG_2003、pEGFP1_hCREG_945、pEGFP1_hCREG_586、pEGFP1_hCREG_478、pEGFP1_hCREG_358报告基因载体质粒。并将其与内参质粒pGL4.73[hRluc/SV40]瞬时共转染入293T细胞48 h,荧光显微镜下观察pEGFP1hCREG启动子报告基因的绿色荧光表达;采用实时定量PCR检测各启动子mRNA的表达,并确定核心启动子区。采用生物信息学方法预测核心启动子区可能结合的转录因子。结果通过双酶切和基因测序法证实成功构建5个hCREG启动子报告基因载体。荧光显微镜下的观察结果和实时定量PCR结果均显示,pEGFP_1hCREG_586转录活性最高(P<0.05),pEGFP1_hCREG_358转录活性最低(P<0.05),pEGFP1_hCREG_2003、pEGFP1_hCREG_945、pEGFP1_hCREG_478转录活性差异均无统计学意义(P>0.05),提示–867/–509 bp为负性调控区,–400/–281 bp和–508/–401 bp均存在增强子序列,故hCREG基因核心启动子区位于基因5’上游序列–508/–281 bp区域。生物信息学预测关键启动子区–508/–281 bp可能结合的转录因子为Pax5/P53、C/EBPβ、GR-β、GATA-1、GR-α、c-Jun、PRB/PRA、YY1、RXR-α、AP-2、FOXP3、GR、TFIID、STAT4、c-Ets-1。结论成功构建了hCREG基因启动子重组质粒,其核心启动子位于–508/–281 bp区域,此区域可结合多个转录因子。

Objective To construct the promoter enhanced green fluorescent protein(pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1 A-stimulated genes(hCREG),and compare the transcriptional activity of each promoter to determine the hCREG core promoter region.Methods The promoter fragment with length of 2003(–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information(NCBI)database and combining with the characteristics of the promoter.Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003,pEGFP1_hCREG_945,pEGF P1_hCREG_586,pEGFP1_hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid.The 293 T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours.The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope,and the mRNA expression of each promoter was detected by real-time quantitative PCR,and the core promoter region was determined.Bioinformatics was used to predict the transcription factors that might bind to the core promoter region.Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing.The results showed that the transcription activity of pEGFP1_hCREG_586 was the highest(P<0.05),and of pEGFP1_hCREG_358 was the lowest(P<0.05),and no significant difference on the transcription activity among pEGFP1_hCREG_2003,pEGFP1_hCREG_945 and pEGFP1_hCREG_478(P>0.05),implying that –867/–509 bp is a negative regulatory region,and there existed enhancer sequences in –400/–281 bp and –508/–401 bp,so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp.Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53,C/EBPβ,GR-β,GATA-1,GR-α,c-Jun,PRB/PRA,YY1,RXR-α,AP-2,FOXP3,GR,TFIID,STAT4 and c-Ets-1.Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed,the core promoter of which is located in –508/–281 bp,where several transcription factors might be bound.