SREBP1转录因子在奶牛乳腺MAC-T细胞中对SCD基因启动子的作用研究

项目名称： SREBP1转录因子在奶牛乳腺MAC-T细胞中对SCD基因启动子的作用研究

项目编号： No.31201869

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 畜牧学与草地科学、兽医学、水产学

项目作者： 韩立强

作者单位： 河南农业大学

项目金额： 22万元

中文摘要： 固醇调节元件结合蛋白1（SREBP1）与硬脂酰辅酶A脱氢酶（SCD）在奶牛乳腺细胞的脂类代谢基因调控中发挥重要作用，序列比对发现在SCD启动子上具有与SREBP1转录因子的结合位点，但SREBP1对于SCD的作用机制还需要进一步证明。本项目首先构建SCD基因的启动子载体与SREBP1蛋白真核表达载体，然后二者共转染奶牛乳腺MAC-T细胞，通过荧光素酶报告基因研究SREBP1蛋白的过表达对于SCD启动子转录活性的调控作用，凝胶迁移率实验和抗体超迁移实验确证SREBP1与SCD启动子位点的特异结合。然后在共转染的基础上，细胞培养液加入共轭亚油酸（CLA）进行处理，westen-blotting检测膜结合的前体SREBP1蛋白和核内成熟SREBP1蛋白的含量变化，探讨在不饱和脂肪酸处理下，SREBP1调控SCD基因启动子的作用机制。

中文关键词： 固醇调节元件结合蛋白1；脂酰辅酶A去饱和酶；乳腺上皮细胞；转录；表达

英文摘要： SREBP-1（sterol regulatory binding protein-1）and SCD (Stearoyl-CoA desaturase) may plays a crucial role in the regulation of gene expression involved in the milk fat synthesis of bovine mammary gland.Sequence comparison of the bovine promoters indicated that there are possible SREBP1 binding site，however,the machanism of this regulation need further clear and definite. In this study, we constracted the SCD promoter fragments and cloned into a luciferase reporter vector .we constracted the SREBP fragments and cloned into eukaryotic expression vector.The SREBP expression vector and SCD promoter vector were transiently co-transfected into MAC-T cell. The effect of SREBP-1 overexpression on the transcriptional activity of the SCD promoter was analysed through the firefly luciferase activities. The binding site was further confirmed by gel shift assay and super gel shift assay.Upon co-transfection of the two vectors, MAC-T celle was treated with conjugated linoleic acid （CLA）. The abundance of nuclear and membrane SREBP1 was analysed by western-blotting.The aim of the test was to explore the mechanism of SREBP1 regulating SCD promoter transcription during the CLA treatment。

英文关键词： SREBP1；SCD；mammary epithelial cell；transcription；expression