Cloning and activity determination of fatty acid synthase (FAS) gene promoter of xinong saanen dairy goat | 西农萨能奶山羊脂肪酸合酶基因启动子的克隆及活性测定

صينى. 【目的】克隆测定西农萨能奶山羊脂肪酸合酶基因（Fatty Acid Synthase,FAS）启动子的全长序列,进行活性区域分析,为奶山羊FAS基因功能和表达调控机理研究提供依据。【方法】根据牛和人脂肪酸合酶基因启动子的同源序列以及西农萨能奶山羊脂肪酸合酶基因5′UTR区域,分别设计上、下游引物,以西农萨能奶山羊全血DNA为模板克隆启动子序列。依据生物信息学分析结果重设引物,将FAS启动子基因分段克隆并连接到荧光素酶表达载体PGL-3,与Psv-β-半乳糖苷酶对照载体共转染293、MCF-7细胞,进行荧光素酶活性检测和β-半乳糖苷酶的活性检测。【结果】FAS基因启动子序列全长2 640 bp,与牛、人FAS启动子序列同源性90％以上,包括数个潜在SP1、Ets、LSF等转录因子结合位点和CCAAT框、GC框。-1 040―-340 bp处可能包含启动子活性中心,通过启动子缺失片段试验将活性中心范围缩小至-721―-540 bp之间。【结论】通过克隆FAS基因启动子区域,分析表明启动子前端存在负调控元件,找出了启动子最小活性中心。

إنجليزي. 【Objective】 The fatty acid synthase (FAS) gene promoter of Xinong Saanen dairy goat was cloned and sequenced to analyze the active region, thus providing an evidence for function determination and expression regulation of FAS gene. 【Method】 According to the homologous sequences of bovine and homo, and to 5‘UTR of Xinong Saanen dairy goat FAS gene, upstream and downstream primers were designed, respectively. Using genome DNA as the template, the FAS gene promoter was cloned. Primers were redesigned according to the results of bioinformatics analysis, the FAS gene promoter was cloned by segmenting and linking up with the PGL-3 expression vector, co-transfecting 293 and MCF-7 cells with the β-galactosidase control vector, and luciferase and β-galactosidase expression were detected. 【Result】 There are 2 640 bp in FAS gene promoter totally, the homology with bovine and homo is over 90%. The promoter contains several potential transcription factor bind sites such as SP1, Ets, LSF bind sites, several CCAAT boxes and a GC box. The core promoter sequences potentially locate in -1 040―-340 bp, and reduced the range to -721―-540 bp by promoter deletion experiment. 【Conclusion】 The full length of Xinong Saanen Dairy Goat FAS gene promoter was cloned, the potential negative control element was analyzed, and the core promoter sequences were found.