基于转录组测序的绵羊常年发情候选基因、miRNA和lncRNA的功能分析

Seasonal estrus is a main bottleneck restricting the efficient production in sheep industry. If the ovine seasonal estrus would be changed using genetic methods, the annual parities of ewes will increase and the stable production of mutton will be maintained throughout the year. However, the molecular mechanism and major genes for ovine year-round estrus are still unclear. In this study, the Small Tail Han (STH) sheep (year-round estrous breed) and Tan sheep (seasonal estrous breed) are selected as test breeds. The hormone levels between Tan sheep in anestrus and two breeds in estrous seasons will be compared. By transcriptome sequencing technology (RNA-seq), the differentially expressed genes, miRNAs and lncRNAs in pineal body and reproductive tissues will be screened between Tan sheep in anestrus and STH sheep in the same season, and between Tan sheep in anestrus and in estrous season. The differentially expressed genes, which are categorized into reproduction or circadian rhythms clusters by GO enrichment analysis and also targets of miRNAs selected above, and their corresponding miRNAs will be focused on and their biological functions will be further analyzed at cellular level by over-expression and knockout of genes. The functions of new lncRNAs will be predicted by combining the results of genes and miRNAs. The gene-miRNA-lncRNA co-expression regulatory network related to ovine year-round or seasonal estrus will be constructed. This research will provide important clues for revealing the molecular mechanism of ovine year-round estrus, and offer a theoretical basis and some key genes for breeding of year-round estrous sheep by molecular techniques.

季节性发情是限制绵羊高效生产的主要瓶颈，若能从遗传上改变绵羊季节性发情的特性，则可增加母羊年产胎次，维持羔羊肉的全年稳定生产。然而绵羊常年发情性状的分子机制和主效基因尚未明确。本研究将以小尾寒羊（常年发情）和滩羊（季节性发情）为实验材料，分析滩羊乏情期与2种绵羊发情季节激素水平的差异；利用RNA-seq技术筛选乏情期与发情季节滩羊间、乏情期滩羊与同季节小尾寒羊间松果体和繁殖组织中差异表达的基因、miRNA和lncRNA，选择基因功能聚类为繁殖或昼夜节律相关类别且基因和差异miRNA间存在靶关系的组合，利用过表达和敲除的方法在细胞水平揭示它们的生物学功能；结合基因和miRNA数据预测新lncRNA的功能；构建绵羊常年发情和季节性发情相关基因-miRNA-lncRNA的共表达调控网络。本研究可为揭示绵羊常年发情分子机制提供重要线索，为通过分子手段选育常年发情绵羊品种提供依据和基因来源。

季节性发情是限制绵羊高效生产的主要瓶颈，然而绵羊常年发情性状的分子机制和主效基因尚未明确。本研究以小尾寒羊（常年发情）、苏尼特羊和滩羊（季节性发情）为实验材料，利用人工自动控光绵羊发情性状模型和高通量测序手段，筛选获得与绵羊常年发情性状相关分子标记。主要结果如下：.（1）探明了不同发情性状绵羊在不同季节下内分泌变化规律。常年发情小尾寒羊与季节性发情滩羊四个季节血液中E2浓度变化趋势相似，两个品种间差异不显著（P>0.05）。两个品种四个季节血液中P4、FSH和LH浓度变化趋势相似，但不同季节下两个品种间浓度差异显著（P<0.05）。结果表明E2和P4可能与绵羊的发情有关，高水平的FSH和LH对开启或维持绵羊的发情具有重要作用。.（2）成功建立了人工自动控光绵羊OVX+E2模型。光照改变使OVX+E2模型绵羊体内生殖激素分泌发生了显著变化，且该变化与本试验中正常绵羊3种激素变化趋势一致，暗示OVX+E2处理并不影响绵羊外周血血清中FSH、LH、PRL等生殖激素的分泌。.（3）高通量测序筛选获得了绵羊发情季节性性状相关的候选基因、miRNA及lncRNA，构建了lncRNA和mRNA共表达互作调控网络。在人工模拟绵羊休情和发情条件下，差异表达的miRNA主要富集在核苷酸切除修复、细胞因子-细胞因子受体互作以及JAK-STAT信号转导通路。差异表达的mRNA和lncRNA经过功能富集均筛选到了胰岛素释放、类固醇激素合成、甲状腺激素通路、催产素等信号通路。并发现了38个差异表达lncRNA，它们共调控8个靶基因。.（4）筛选验证获得了绵羊常年发情性状的分子标记。Per1、Per2和DIO2基因在绵羊繁殖组织中均表达，在季节性发情绵羊品种中的表达量与常年发情小尾寒羊中表达量呈现显著差异。发现Per1、Per2和DIO2基因中共3个位点基因型频率和等位基因频率在季节性发情和常年发情的绵羊品种中达到显著差异（P≤0.05），与绵羊季节性发情性状显著相关。绵羊BAD基因可通过胰岛素信号通路来调节细胞凋亡进而影响动物所处的发情状态。TSHR基因的S505R突变与小尾寒羊的常年发情性状相关，此突变位点通过使T3浓度降低延迟了突变型中LH水平的下降时间，从而推迟了突变型出现休情状态的时间。. 本项目研究结果为常年发情绵羊新品种选育提供了有效的分子标记。