人PNPLA3基因启动子转录调控的初步分析

摘要

Objective To construct luciferase reporter plasmids with different promoter segments of human PNPLA3 gene,compare their luciferase activitiy in LO2 cells,and initially investigate the mechanism of transcriptional regulation of PNPLA3 gene.Methods Approximately 1400bp fragment of 5' flanking region of PNPLA3 was bioinformatically analyzed with computer to predict the promoter region.Different promoter segment of PNPLA3 gene were amplified by PCR from genomic DNA of human blood and identified by restriction endonucleases enzyme digestion and sequencing,then they were ligated with luciferase gene in PGL3-Basic vector.These report vectors were transiently transfected into LO2 cells and luciferase assay was performed to analyse the transcription activation of these promoters.We also use online analysis system to predict the potential transcription factor binding sites in the PNPLA3 gene transcriptional regulatory region.Results Five segments of different lengths PNPLA3 gene upstream promoter region of reporter vector were successfully constructed.Although promoters with different length had different activity in LO2 cells,the segments of-1015,-647,-553 and-333 have similar activities,and the activity of the segment of-8 was almost disappeared.Sequence analysis with Mach database showed that a number of putative transcription factors may locate on the333bp to-8bp promoter region.Conclusions According to the different activities of different lengths,it is suggested that the area of-333bp to-8bp might be the main transcriptional regulatory region of PNPLA3 gene.A variety of transcription factor may participate in the transcriptional regulation of PNPLA3 gene by combiningwith the binding sites in the region of-333 to-8.%目的 构建人PNPLA3基因不同长度的上游启动子荧光素酶报告基因裁体,在LO2细胞中比较不同长度启动子片段的活性,为研究PNPLA3基因的转录调控机制提供初步依据.方法 对PNPLA3基因5’侧翼区约1400个碱基进行生物信息学分析,预测其转录调控区域；利用PCR及酶切方法,以人外周血中全基因组DNA为模版扩增不同长度的PNPLA3基因上游启动子区序列,分别构建荧光素酶基因报告载体.瞬时转染LO2细胞,利用双荧光素酶报告基因分析系统检测各启动子的转录活性.利用在线分析软件预测PNPLA3基因主要转录调控区的潜在转录因子结合位点.结果 成功构建5段不同长度的PNPLA3基因上游启动子区的报告载体；在LO2细胞内启动子活性随片段长度变化,表现为当PNPLA3启动子片段从-1015截短到-647,从-647截短至-553,从-553截短至-333时活性变化不大,而从-333截短至-8时活性显著下降(P＜0.05)；Match分析显示-333～-8片段具有多个转录因子结合位点.结论 初步判断-333 ～8区是PNPLA3基因的主要转录调控区.