绵羊繁殖性状相关lncRNA的鉴定及其调控机制研究

Long non-coding RNAs (Long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nt, widely exist in various organisms and obvious spatio-temporal expression specificity, which have been found to play important roles in regulating gene expression level by epigenetic, transcriptional and post-transcriptional regulation. In this study, the ovarian tissues represent different estrous period (dioestrus, proestrus and estrus) from Qira black sheep and Hetian sheep, which had significant differences in the reproductive traits, are used as experimental materials. RNA-Seq sequencing is used to analyze the differently expressed lncRNA, and combine with bioinformatics analysis, functional enrichment analysis of target genes, lncRNA-mRNA coexpression analysis and lncRNA-miRNA coexpression analysis based on the miRNA expression information obtained in previous study, screening the candidate lncRNA that related with sheep reproductive traits; based on these results, further construct lncRNA interference and over expression vector, transfecting the sheep ovarian granulosa cells, detection the effect of lncRNA on cell proliferation, cell cycle, apoptosis and hormone secreting function, and the effect of lncRNA on the target gene (including pathway related genes) and reproduction related genes in mRNA and protein expression levels; and combine the studies on subcellular localization and interaction proteins screening, to determine the important lncRNA associated with sheep reproductive traits, and to explore the molecular mechanism of lncRNA regulation of reproductive traits in sheep. Aiming to enrich the ncRNA database, and speed up the progress of sheep reproductive traits genetic breeding.

长链非编码RNA(lncRNA)广泛存在于各种生物体中，在表观遗传、转录以及转录后调控等多种层面上调控基因的表达，具有明显的时空表达特异性。本研究运用转录组测序技术，检测繁殖性状具有明显差异的策勒黑羊与和田羊（发情周期不同阶段）卵巢组织lncRNA差异表达情况，结合生物信息学、靶基因的功能富集、lncRNA-mRNA共表达分析及结合项目组前期获得的miRNA信息进行的lncRNA-miRNA联合分析等，筛选与绵羊繁殖性状相关的lncRNA；在此基础上，构建lncRNA干扰/过表达载体，检测lncRNA对颗粒细胞增殖、细胞周期、凋亡、激素分泌功能以及对其靶基因及相关调控网络基因、繁殖相关基因mRNA和蛋白表达的影响；结合lncRNA细胞表达定位及相互作用蛋白的筛选，确定绵羊繁殖性状相关的重要lncRNA及调控机理，为丰富绵羊卵巢组织ncRNA数据、加快绵羊繁殖性状的遗传进展提供理论依据。

长链非编码RNA(lncRNA)广泛存在于各种生物体中，在表观遗传、转录以及转录后调控等多种层面上调控基因的表达。lncRNA在卵巢、生殖细胞等繁殖相关组织的生长发育和功能作用中扮演着重要角色。筛选鉴定发情周期不同阶段、不同繁殖力绵羊品种间差异表达的lncRNA，对了解lncRNA在绵羊繁殖性状中的作用具有重要意义。本研究运用转录组测序技术，以策勒黑羊发情周期卵巢组织为材料，获得Qira-E vs Qira-D、Qira-E vs Qira-P 、Qira-P vs Qira-D、Qira-E vs Qira-M差异表达lncRNA分别为14、24、18和 18个；发情期策勒黑羊、发情期和田羊卵巢组织间获得差异表达lncRNA 36个；同时对发情周期策勒黑羊、发情期和田羊卵巢组织miRNA、发情期和田羊与策勒黑羊卵巢组织circRNA进行了研究；并在上述研究基础上，针对差异表达lncRNA、miRNA、mRNA进行了lncRNA-miRNA-mRNA、circRNA-miRNA-mRNA联合分析；构建lncRNA过表达载体，转染颗粒细胞，初步检测发现lncRNA LOC105611671对颗粒细胞增殖及孕酮分泌有一定的促进作用；pull down技术检测发现LOC105611671相互作用蛋白132个，并进行了相关生物信息学分析。本研究为丰富绵羊卵巢组织ncRNA数据、深入揭示lncRNA影响绵羊泡发育和绵羊繁殖控制的分子机理奠定基础。