提高营养水平诱导舍饲哈萨克羊非繁殖季节发情的分子调控机制及microRNA鉴定

The seasonal estrus is a bottleneck which makes it difficult to improve the production efficiency of sheep farms. The current studies on estrus regulation in sheep are mainly focused on the photoperiod regulation. However, it is proved that improving the nutritional levels can induce sheep estrus in so-called non-breeding season. What is the molecular mechanism under the phenomenon? Does it involve the same regulating pathways to photoperiod? To answer these questions, Xinjiang Kazakh sheep will be housed, randomly grouped and given diets with different nutrition levels. The estrus performance and reproductive hormones will be monitored during breeding and non-breeding seasons. The differentially expressed genes and MicroRNAs in hypothalamus, pituitary and ovary between breeding and non-breeding seasons will be scanned using DGE and small RNAsequencing technology. Functions of the differently expressed genes and miRNA will be confirmed by experiments using appreciate cell lines and individual animals. Together with expression information of kiss1/GPR5, the regulatory network which controls non-breeding estrus will be constructed. In summary, this study will identify important genes and miRNAs within the estrus regulatory pathway in sheep non-breeding season. it will help to further understand the molecular mechanism of estrus in sheep.

季节性繁殖是制约绵羊高效养殖的重要瓶颈，目前对于其发情调控的研究主要集中在光周期调控方面。然而在实际生产中，提高营养水平可以诱导部分绵羊在非繁殖季节发情，并可配种产羔，这种调控的分子机制如何？与光周期调控是否一致，目前尚未见有研究。本研究将首先构建不同营养水平的哈萨克羊群体，在繁殖季节和非繁殖季节观察其发情表现及生殖激素变化规律。同时拟利用DGE和small RNA测序技术来筛选乏情季节和发情季节哈萨克羊下丘脑、垂体、卵巢中差异表达的基因和miRNA，对筛选到的差异基因及miRNA在细胞和绵羊个体水平验证其调控作用，结合TSH/Dio系统和Kisspeptin/GnRH系统的表达规律，共同构建绵羊非繁殖季节发情的分子调控机制。本研究将初步鉴定绵羊非繁殖季节发情调控通路中重要基因、miRNA，对进一步了解绵羊发情性状的分子调控机制具有重要的促进意义。

季节性繁殖是制约绵羊高效养殖的重要瓶颈，目前对于其发情调控的研究主要集中在光周期调控方面。然而在实际生产中，提高营养水平可以诱导部分绵羊在非繁殖季节发情，并可配种产羔，这一营养改变绵羊季节性发情性状影响绵羊繁殖性腺轴的作用机制如何，目前尚不清楚。本研究首先通过构建不同营养水平的哈萨克羊群体，在非繁殖季节和繁殖季节观察发情表现、检测生殖内分泌激素变化规律，先后构建分别构建了DGE 9个库、miRNA12个库，利用DGE和small RNA测序技术筛选了非繁殖季节和繁殖季节哈萨克羊下丘脑、垂体、卵巢中差异表达的基因和miRNAs，并对其筛选后的差异表达基因与miRNAs在细胞水平进行了功能作用验证，随后又结合目前已有的发情调控两大通路即TSH/Dio系统和Kisspeptin/GnRH系统的表达规律，进行了绵羊非繁殖季节发情的分子调控机制的联合分析，最终成功筛选出miR-200b并锁定靶标基因GNAQ，总结出从两大营养物质因素即酪氨酸和叶酸出发，不同于目前已有的光周期调控发情的通路的营养诱导绵羊非繁殖季节发情调控网络机制，对探究营养水平诱导绵羊季节性繁殖提高生产效率具有一定的指导作用，并为全面完善非季节性营养调控绵羊发情网络调控机制奠定了基础。

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