人甘丙肽2型受体基因启动子的克隆与初步功能分析

Cloning and Preliminary Functional Analysis of Human Galanin Receptor 2 Promoter

人甘丙肽2型受体(GalR2)作为G蛋白偶联受体,其功能已被广泛研究,但GalR2基因的转录调控机制尚不明确。我们利用聚合酶链式反应(polymerase chain reaction,PCR)方法扩增人GalR2基因上游1121 bp的片段,再通过PCR方法将其缺失成3段长度不等的片段,然后分别将其克隆到荧光素酶报告基因载体pGL3-Basic中,并将相应的质粒命名为pGL3-B-1121、pGL3-B-922、pGL3-B-521和pGL3-B-200;通过荧光素酶活性分析检测不同长度的GalR2基因启动子在人神经母细胞瘤细胞(SK-N-SH)中的活性,发现GalR2基因上游1121 bp区段具有明显的转录活性,缺失-1121到-922 bp区域引起启动子活性的明显增高,暗示该区域存在负调控元件;利用TESS在线软件分析GalR2基因上游序列,发现该基因启动子含有Sp1、AP-1、AP-2、NF-1、YY1和ERα等多种顺式作用元件。因此,本实验初步确定了人GalR2基因启动子的转录调节区,为进一步研究该基因转录调控机制奠定了基础。

As a G protein-coupled receptor,the role of galanin receptor 2（GalR2） has been studied extensively,but its transcriptional regulation mechanism is still unknown.In this study,5＇-upstream promoter sequence（spanned from-1121 to-1 bp） of human GalR2 gene and its three deletion segments were isolated and cloned into the pGL3-Basic vector.Corresponding plasmids were named as pGL3-B-1121,pGL3-B-922,pGL3-B-521 and pGL3-B-200,respectively.Those four reporter plasmids were transfected into SK-N-SH cells,and luciferase activities were measured.The results showed that deletion of the region between-1121 to-922 bp resulted in a significantly increased activity compared with pGL3-B-1121,indicating the presence of negative elements in this region.Moreover,it was also found that the GalR2 promoter had many putative transcription factor binding sites by using online software TESS,such as Sp1,AP-1,AP-2,NF-1,YY1 and ERα,suggested that those elements might be involved in transcriptional regulation.In conclusion,this study provides important clues for elucidating the transcriptional regulation mechanisms of the GalR2 gene.