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

作　　者：李杰[1] 杨予涛[1] 马克乾[1] 闫竹青[1] 李晓晓[1] 赵春礼[1] 徐志卿[1,2] 

机构地区：[1]首都医科大学神经生物学系北京神经科学研究所北京神经再生修复研究重点实验室,北京100069 [2]Department of Neuroscience,Karolinska Institutet

出　　处：《生物物理学报》2011年第5期417-423,共7页Acta Biophysica Sinica

基　　金：国家自然科学基金项目(30870815);北京市自然科学基金重点项目(09G0013);人事部留学人员科技活动择优资助项目;Swedish Research Council(3106);北京市人才强教深化高层次人才计划(PHR20100510)~~

摘　　要：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.

关 键 词：GalR2基因 启动子 转录调节 

分 类 号：Q291[生物学—细胞生物学]