Kisspeptin调控绵羊GnRH基因表达的作用机制解析

项目名称： Kisspeptin调控绵羊GnRH基因表达的作用机制解析

项目编号： No.31501929

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 畜牧学与草地科学

项目作者： 李转见

作者单位： 河南农业大学

项目金额： 21万元

中文摘要： 促性腺激素释放激素（GnRH)对调控HPG轴下游其他元件的活动和维持动物正常的繁殖周期具有重要的作用。最近研究表明，几乎所有脊椎动物GnRH的合成和分泌都受kisspeptins及其受体GPR54调控。但有关绵羊GnRH的合成和分泌调控的分子机理依然不清。本项目以当地小尾寒羊为研究对象，首先，克隆绵羊下丘脑GnRH基因的启动子区，利用生物信息学分析启动子特征及相关的转录因子；其次，构建系列缺失的GnRH启动子与荧光素酶融合载体，转染下丘脑原代细胞并kisspeptins处理，利用荧光报告系统筛选绵羊GnRH启动子受kisspeptins控制的关键元件；接着，利用FAIRE技术鉴定kisspeptin处理时被激活的GnRH启动子元件；最后，利用ChIP技术确定与关键元件相互作用的转录调控因子；RNAi验证确定的转录调控因子。综上结果，阐明绵羊kisspeptin调控GnRH基因表达分子机制。

中文关键词： 羊；数量性状；繁殖性状；表达调控；基因功能

英文摘要： The gonadotropin-releasing hormone (GnRH) operates as the first and most important output signal for the hypothalamic regulation of the downstream elements of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining normal reproductive cycle. Recent development in reproductive regulation has revealed that kisspeptins, a family of neuropeptides encoded by the kiss1 gene and their receptor GPR54, are pivotal upstream regulators of GnRH neurons. Though regulatory process of GnRH expression via kisspeptin has been explored using experimental animals such as mouse, the detailed mechanism is unclear until now. In the proposed research project, firstly we will clone the promoter region of sheep GnRH gene, and look into the potential transcript factors binding loci in the region, and possible transcript factors which interact with the loci; then we will determine the key cis-acting GnRH promoter elements by sequential deletions of the sheep GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene; thirdly, we will localize the kisspeptin-response element (KsRE) by using formaldehyde assisted isolation of regulatory elements (FAIRE) technique; finally we will identify factors that interact with KsRE of GnRH promoter to regulate sheep GnRH expression by using chromatin immunoprecipitation (ChIP), and confirm their functions by RNAi assay. Take all the information together, we will be able to expound molecular mechanism of GnRH expression regulated by kisspeptin in sheep.

英文关键词： sheep;Quantitative traits;Reproductive traits;Expression regulation;Gene function