小鼠精胺氧化酶基因启动子结构和功能研究

Structional and Functional Studies for the Promoter of Mouse Spermine Oxidase Gene

目的:克隆和鉴定小鼠精胺氧化酶(mSMO)启动子DNA序列。方法:用Trizol试剂法从小鼠成纤维细胞中提取总RNA,应用5’-RACE法获得mSMO的转录起始位点;巢式PCR方法克隆含有mSMO启动子的DNA序列,并由此构建5’端系列截短的mSMO启动子荧光素酶报告质粒;将报告质粒瞬时转染Cos7细胞后,用荧光素酶分析法测定启动子活性。结果:确定mSMO基因至少有6个转录起始位点(+1,+4,+27,+31,+51,和+63),且均定位于外显子1中。克隆获得mSMO基因转录起始位点上游大约2.1kb的DNA片断,以此片段为基础构建了11个5’端系列截短的启动子报告质粒。报告基因分析证实,该上游DNA片段具有启动子活性,截短至-615和-176bp时,获得2个启动子活性峰值,截短至-373bp时启动子活性最低。结论:mSMO基因含有多个转录起始位点,其上游-176～+124bp为mSMO核心启动子区,-615～-176bp区为重要的转录调控区。

Objective：To clone and identify the promoter in the mouse SMO gene（mSMO）.Method：The total RNA was extracted from NIH 3T3 cells by Trizol reagent.RNA ligase-mediated 5＇-RACE was performed to obtain transcriptional start point of mouse SMO gene.Nest PCR was used to clone DNA fragment that contained the promoter of mSMO gene using genome DNA derived from NIH 3T3 cells as the template.Starting from this DNA fragment,11 reporter plasmids that contained 5＇ serial truncated promoter were constructed.After the plasmids were transiently transfected into Cos7 cells,the luciferase activity were essayed to determine promoter activity.Result：6 transcriptional start points（＋1,＋4,＋27,＋31,＋51,and ＋63bp） were identified in the mSMO gene and all of them located in exon 1.A ~2.1 kb DNA fragment upstream the mSMO gene was obtained.Starting from this fragment,11 reporter plasmids with serial truncated promoter in the 5＇ end were constructed.The reporter gene assay proved the promoter activity for this upstream DNA fragment.When truncated to the positions at-615 and-176bp,two peak values of promoter activity were observed and lowest promoter activity was obtained when the fragment was truncated to the position of-373bp.Conclusion：mSMO gene contains multiple transcriptional start points.The core promoter locates within the area-176 to ＋124bp and the area between-615 and-176 is the important transcriptional regulation region.