山羊脂肪酸合酶基因(FASN)启动子结构与功能的初步分析

Structural and Functional Analysis of Fatty Acid Synthase Gene Promoter of Goat

本研究旨在对山羊脂肪酸合酶基因(Fatty acid synthase,FASN)启动子进行结构与功能的初步分析,进而对其转录调控机制进行探讨。采用PCR技术从西农萨能羊基因组DNA中克隆FASN基因启动子,通过缺失分析,构建7个包含不同缺失片段的荧光素酶报告基因载体,转染山羊乳腺上皮细胞和MCF-7细胞,利用双荧光素酶系统检测不同片段的启动活性。结果表明,克隆得到FASN基因的启动调控序列2 589bp,生物信息学分析发现,该启动子序列含有典型的启动转录元件TATA-box和E-box,分别位于转录起始位点(+1)上游-41和-74bp处。报告基因分析表明,启动子核心区域定位在-293～-79bp,在线软件预测发现,该区域含有Sp1、NF-Y、USF和SREBP等转录因子结合位点。结果显示,FASN基因启动子前端存在负调控元件,Sp1、NF-Y、USF和SREBP等转录因子可能参与FASN基因的转录调控。

The objective of this study was to analyze the structure and function of fatty acid syn- thase gene（FASN）promoter of goat, to further reveal the transcriptional regulatory mechanism of FASN gene. In this study, the promoter of goat FASN gene was obtained by PCR. Seven promoter fragments in different length were obtained by deletion and cloned into luciferase reporter gene expression vectors. Then, the vectors were transfected into goat mammary epitheli- al cells and MCF-7 cells, their expression activity were determined by using the dual-luciferase reporter assay system. The promoter sequence of 2 589 bp of FASN gene was obtained. The bioinformatics analysis for sequence showed that there were a TATA-box at -41 bp and an E- box at -74 bp of transcription initiation site （q-l）. Luciferase reporter assays demonstrated that the core region of the promoter was from -293 bp to -79 bp conferring basal transcriptional ac- tivity. Meanwhile, some transcriptional factor binding sites including Spl, NF-Y, USF and SREBP were identified in this core region. The results indicated that the transcriptional factorssuch as Spl, NF-Y, USF and SREBP might be involved in the transcriptional regulation of FASN gene.