LncRNA与miRNA参与多胎湖羊卵泡发育精准调控机制研究

Hu sheep which is very famous for its higher prolificacy is one of the sheep breeds carrying the FecB gene in the world. Currently, it is a hot topic that how the long noncoding RNA (lncRNA) and micro RNA (miRNA) regulate the high prolificacy in mutton sheep breeding. However, due to limitations of materials and techniques, most studies are based on entire ovary, where the transcripts from different sizes of follicles were mixed, which restrained the screening of valuable lncRNA and miRNA and negatively affected the study for the high prolificacy mechanism. Therefore, in this study, Hu sheep with different genotypes and reproductive capability are selected and grouped. The healthy follicles are collected by sizes at estrus stage for transcriptome analysis and RNA immunoprecipitation. The differential expressed lncRNA and miRNA during follicular development are then screened to build the regulatory network of lncRNA-miRNA-mRNA. Afterwards, the lncRNA and miRNA responsible for targeting key genes in BMP/Smad pathway are screened while their methylation status are also explored. A series of verification tests will be performed to investigate the regulatory mechanism of lncRNA and miRNA in follicular development of Hu sheep with high prolificacy. Our research will not only provide a theoretical reference for the identification of candidate genes related to prolificacy, but also show a great practical significance for accelerating mutton sheep molecular breeding and accurate marker assisted selection.

湖羊以繁殖力高而著称，是世界上少数几个携带FecB基因的绵羊品种，探索长链非编码RNA（lncRNA）和微小RNA（miRNA）参与调控其多胎机制是当前肉羊繁育研究的热点。但因受材料和技术限制，多数研究均以卵巢为对象，造成不同大小卵泡转录本混合，导致筛选有价值lncRNA和miRNA受限而影响多胎机制研究。为此，本研究以不同基因型高低繁湖羊的、发情期不同大小的健康卵泡为研究对象，采用转录组测序和RNA免疫沉淀等手段寻找卵泡发育过程中差异表达lncRNA和miRNA，构建lncRNA-miRNA-mRNA调控网络，从中筛选靶向BMP/Smad通路关键基因的lncRNA和miRNA，并初步探究其甲基化状态，通过系列验证试验，以揭示lncRNA和miRNA对多胎湖羊卵泡发育的调控机制。这不仅为挖掘与多胎性相关的候选基因提供理论参考，而且对加快肉羊分子育种与精准标记辅助选择具有重要的现实意义。

湖羊以“全年发情、繁殖力高”而著称，是世界上少数几个携带FecB基因的绵羊品种，探究长链非编码RNA（lncRNA）和微小RNA（miRNA）参与调控湖羊多胎机制是当前肉羊繁育研究的热点。本项目分别以发情期不同基因型高低繁湖羊的卵巢与不同大小卵泡、子宫内膜为研究对象，采用RNA-Seq等手段筛选出与雌性动物繁殖相关的差异表达lncRNA和miRNA，通过系列验证试验，揭示lncRNA和miRNA参与湖羊多胎性状形成的分子机制。项目（1）分析了高低繁湖羊外周血生殖激素与卵泡发育差异规律；（2）分别阐明了lncRNA和miRNA在高低繁湖羊卵巢与不同大小卵泡、子宫内膜中的表达规律，构建了雌性动物繁殖相关lncRNA-miRNA-mRNA调控网络；（3）从卵巢水平上揭示了lncRNA FDNCR通过miR-543-3p/DCN/TGF-β通路调控湖羊多胎性的分子机制；（4）从卵泡水平上揭示了lncRNA-412.25通过miR-346/LIF/STAT3通路精准调控湖羊多胎性的分子机制；（5）从子宫内膜水平上解析了lncRNA366.2做为ceRNA通过靶向WNT/β-catenin信号通路，进而参与调控子宫内膜上皮（Endometrial epithelial cells，EECs）细胞增殖、迁移及分泌功能的分子机制；（6）揭示了差异表达miRNA（unconservative_15_2570409）通过靶向PGR基因调控湖羊颗粒细胞（Granulosa cells，GCs）激素分泌的作用机理；（7）分别解析了关键基因CITED4和INHBA参与湖羊GCs功能与状态的调控机制。项目完成了各项研究内容，实现了研究目标，研究结果已正式发表SCI论文8篇（第一标注6篇，第二标注2篇），培养1名青年教师，博士研究生2名，硕士研究生2名。研究结果不仅挖掘与多胎性相关的候选基因提供理论参考，而且对加快肉羊分子育种和精准标记辅助选择具有重要的现实意义。