鸡Perilipin1基因启动子的克隆及分析

Promoter Cloning and Analysis of Chicken Perilipin1 Gene

旨在研究鸡脂滴包被蛋白基因(Perilipin1,Plin)启动子的结构及特征。本研究采用PCR方法扩增了鸡Perilipin1基因5′侧翼区约2kb的DNA片段,并对其进行了克隆、测序及生物信息学分析;同时,构建了其全长及系列截短突变的报告基因表达载体,瞬时转染鸡胚成纤维细胞系(DF-1),用双荧光素酶报告基因系统测定了荧光素酶活性,确定了该基因的核心启动子区域。生物信息学分析结果表明,鸡Perilipin1基因的启动子区不存在典型的TATA box结构和CpG岛,但可能存在TFIID、Sp1、AP2、PPAR、RXR、SREBP1、C/EBP、GATA、ER、KLF5等多个转录因子结合位点;报告基因分析结果表明,本研究克隆的鸡Perilipin1基因启动子能够极显著地启动报告基因的表达(P<0.01),并且随着启动子片段由5′端逐渐截短,报告基因活性表现出逐渐增强的趋势,其中-360/-11片段具有最强的报告基因活性。综上表明,鸡Perilipin1基因-360/-11区域的启动子片段具有最强的转录活性,包含该基因的核心启动子序列。

The purpose of this study was to analyze the structure and characteristics of promoter of the chicken Perilipin1 gene.A DNA fragment with 2kb was cloned from the 5′-flanking region of Perilipin1,then sequenced and analyzed by bioinformatics methods.Subsequently,the luciferase reporter gene plasmids containing the full-length region of Perilipin1 gene promoter and its serial truncation mutations were constructed,and transfected into chicken embryonic fibroblast cell line（DF-1）.Through detecting the level of luciferase activity,the core promoter region was determined.The result of bioinformatics analysis revealed that the promoter region of the chicken Perilipin1 gene had no typical TATA box and CpG islands,but many binding sites of transcription factors,such as TFIID,Sp1,AP2,PPAR,RXR,SREBP1,C/EBP,GATA,ER and KLF5.Luciferase reporter assays demonstrated that the chicken Perilipin1 gene promoter could significantly trigger the expression of the reporter gene（P〈0.01）.With the promoter fragment gradually truncated from the 5′end,luciferase activity increased correspondingly,and the-360/-11 promoter fragment showed the strongest activity.Taken together,the promoter region from-360/-11 bp has the highest transcriptional activity,which could be the core regulatory region of the chicken Perilipin1 gene.