旨在筛选牛DNA聚合酶β(DNA polymerase β,POLB)基因核心启动子并对调控该基因表达的转录因子进行预测和鉴定.以牛POLB基因CDS区上游1751bp序列为模板,构建5个包含不同截短长度的启动子报告基因载体,利用双荧光素酶报告基因系统鉴定核心启动子,利用AnimalTFDB3.0预测核心启动子结合转录因子,过表达转录因子后分析其对牛POLB基因的转录调控功能.结果:在牛肌肉原代细胞中,构建的5个截短的启动子报告基因载体均具有转录起始活性,pGL3-151(-151～-1 bp)和pGL3-1351(-1 351～-1 bp)的相对荧光素酶活性显著高于pGL3-551(-551～-1 bp)(P＜0.001,P＜0.000 1),表明-151～-1 bp 和-1 351～-551 bp 为牛 POLB 基因核心启动子区;AnimalTFDB 3.0 预测核心启动子区存在转录因子特异性蛋白1(specificity protein 1,SP1)的结合位点;在牛肌肉原代细胞中过表达转录因子SP1,能够显著降低POLB基因的转录活性.本试验结果为进一步研究该基因表达调控机制提供参考.
Identification and analysis of transcriptional regulation of the core promoter for the bovine POLB gene
The aim of this study was to screen the core promoter of the bovine POLB gene and to identify the transcription factors regulating POLB gene expression.In this study,the 1 751 bp upstream sequence of the CDS region of the bovine POLB gene was used as the template.Five promoter reporter gene vectors containing different truncated lengths were constructed to identify the core promoter region by dual lucifer-ase reporter gene activity assay.Candidate transcription factors within the core promoter region were predicted by the AnimalTFDB 3.0 soft-ware.The transcription factor was then overexpressed in the bovine muscle primary cells to analyze its transcriptional regulation of the bovine POLB gene.The results showed that all the five truncated promoter reporter vectors had transcriptional initiation activity.The AnimalTFDB 3.0 predicted that transcription factor SP1 was able to bind the core promoter region of the POLB gene.The relative fluorescence activity of pGL3-151(-151 to-1 bp)was significantly higher than that of pGL3-551(-551 to-1 bp)(P＜0.001).The pGL3-1351(-1 351 to-1 bp)was significantly higher than pGL3-551(-551 to-1 bp)(P＜0.000 1).The present results indicated that the core promoter re-gions were-151 to-1 bp and-1 351 to-551 bp at the upstream of CDS.Overexpression of transcription factor SP1 in bovine muscle prima-ry cells could significantly reduce the activity of the POLB gene(P＜0.000 1).This study identified the core promoter regions of the bovine POLB gene and found that the transcription factor SP1 was able to regulate the transcriptional activity.These findings provided references for further research on the regulatory mechanism of POLB gene expression.
万方数据
维普
