鸡SIRT1基因的表达调控及其对肝脏脂类代谢的功能研究

项目名称： 鸡SIRT1基因的表达调控及其对肝脏脂类代谢的功能研究

项目编号： No.31472091

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 畜牧学与草地科学

项目作者： 顾志良

作者单位： 常熟理工学院

项目金额： 88万元

中文摘要： SIRT1是一种NAD+-依赖性蛋白去乙酰化酶，哺乳动物中SIRT1基因对脂类代谢具有调节作用。鸡脂类合成主要在肝脏中完成，而SIRT1基因在鸡脂类代谢中的功能尚未报道。本研究将采用RACE方法，获得鸡SIRT1基因全长cDNA，结合生物信息学方法、报告基因分析手段、ChIP和EMSA等技术，弄清鸡SIRT1基因启动子结构和该基因的转录调控机制；通过生物信息学的方法，筛选出能与SIRT1基因配对结合的miRNA，并用双荧光素酶报告基因进行细胞学验证以研究其转录后调控的机制；同时利用转染siRNA的方法，敲低鸡肝细胞中SIRT1的表达，结合RNA-Seq技术检测肝细胞中差异表达基因；在肝细胞中过表达SIRT1，通过荧光定量PCR和Western Blotting方法检测SIRT1信号通路中基因表达情况，综合分析SIRT1基因在鸡脂类代谢中的作用，从另一角度探讨鸡脂类代谢和脂肪沉积的调控机理。

中文关键词： 鸡；SIRT1基因；表达调控；脂类代谢；microRNA

英文摘要： Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that regulates lipid metabolism in mammalian. De novo fatty acid synthesis in chickens takes place mainly in the liver, whereas the role of SIRT1 gene in lipid metabolism in chickens has not been reported. In this study, the RACE method will be used to obtain full length cDNA sequence of SIRT1 gene, and the bioinformatics, reporter gene assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) will be combined to understand the structure of SIRT1 promoter and regulatory mechanism of SIRT1 gene in chickens. Through bioinformatics, miRNAs were screened which can directly bind to SIRT1 3'UTR. Then, the mechanism of post-transcription regulation of SIRT1 gene was verified by co-transfection and dual-luciferase reporter assays. Using siRNA to knockdown the expression level of SIRT1 in chicken hepatocytes, and differentially expressed genes in the chicken hepatocytes were detected by RNA-Seq technology in the meantime. Furthermore, detection of the expression of genes in signaling pathways was performed in chicken hepatocytes after over-expressing SIRT1 by fluorescence quantitative PCR and Western Blotting. Accordingly, the role of SIRT1 gene in chicken lipid metabolism will be concluded comprehensively. The regulatory mechanism of lipid metabolism and fat deposition of chicken will be explored from another angle in this study.

英文关键词： Chicken;SIRT1 gene;Regulation of gene expression;Lipid metabolism;microRNA