策勒黑羊与和田羊卵巢组织差异表达繁殖相关miRNA的筛选及鉴定

The fecundity and seasonal estrous traits of Qira black sheep and Hetian sheep have obvious differences. miRNA is a non-coding small RNA, and play an important role in the growth development and function of the ovary, ootid and other reproduction-related tissues. In this study, the ovarian tissues represent oestrus and anestrus stages from the Qira black sheep and Hetian sheep are used as experimental materials，presenting a global survey of the sheep ovarian tissue miRNAs with Solexa depth sequencing, and combine with bioinformatics analysis, screening the candidate miRNAs that related with sheep reproductive traits; based on this results, the significant differences expressed miRNAs are used for target miRNA to detect the expression levels of miRNA and its target genes in ovarian tissues in different experimental groups with Real-time PCR; further construct miRNA over expression lentiviral vector and inhibition expression lentiviral vector, transfecting the ovarian Granulosa cells and Mesangial cells, using Real-time PCR and western blot analysis to detect its target gene, as well as reproduction related genes (such as FSHR of LHR, E2, INH and P4)in mRNA and protein expression levels; and combine with the LNA-miRNA in situ hybridization results, to determine the important miRNAs of sheep reproductive traits, and to explore the molecular mechanism of miRNA regulation of reproductive traits in sheep. Aiming to provide a theoretical basis for further applied them to the reproductive traits with artificial regulation, and speed up the progress of sheep genetic breeding.

策勒黑羊、和田羊在产羔和发情性状上存在明显的差异，miRNA是一类非编码小RNA，在卵巢、卵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。本研究以发情期和休情期策勒黑羊、和田羊为对象，运用miRNA solexa测序技术，检测卵巢组织miRNAs差异表达情况，结合生物信息学分析，筛选与绵羊繁殖性状相关的miRNAs;在此基础上，运用实时定量PCR方法检测miRNAs及靶基因差异表达情况；进一步构建miRNA过表达和抑制表达慢病毒载体，转染体外培养的卵巢颗粒细胞和膜细胞，基于实时定量PCR和Western blot技术，在mRNA和蛋白水平上检测miRNA对其靶基因及FSHR、LHR、E2、INH和P4等绵羊繁殖相关基因的影响；结合LNA-miRNA原位杂交结果，确定绵羊繁殖性状相关的重要miRNAs及调控机理, 为进一步应用于人工调控繁殖性状、加快绵羊遗传育种进展提供理论依据。

miRNA是一类非编码小RNA，在卵巢、卵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。筛选鉴定繁殖力差异显著两绵羊品种间差异表达的miRNA，对了解miRNA在绵羊繁殖性状中的作用具有重要意义。本研究以繁殖性状具有明显差异的策勒黑羊和和田羊发情期、乏情期卵巢组织为实验材料，构建卵巢组织miRNA库并进行高通量测序，4个小RNA文库共检测到672个已知miRNA和147个新miRNA。miRNA Sloexa测序结果表明，和田羊与策勒黑羊发情期、乏情期，策勒黑羊发情期、乏情期以及和田羊乏情期、发情期卵巢组织中分别筛选到差异表达miRNAs 125、28、83、29个；4个随机选择的差异表达miRNAs实时定量PCR检测结果与 Sloexa测序结果一致；利用双肾荧光素酶基因表达系统，对miR-375靶基因BMPR1B、TGFB2，miR-125b靶基因STAT3，miR-503-5p靶基因CDC25A进行了验证，结果表明miR-125b可通过STAT3上相应的3’UTR位点调控报告基因的表达，认为STAT3基因为miR-125b的靶基因，结合位点在329-351和1152-1175；miR-125b细胞水平功能检测表明miR-125b mimics可促进FSHR基因的表达、明显下调其靶基因STAT3的表达；miR-125b mimics促进颗粒细胞增殖；DGE测序结果表明，和田羊与策勒黑羊发情期、乏情期，策勒黑羊发情期、乏情期以及和田羊乏情期、发情期卵巢组织中分别筛选到差异表达基因1252个、357、651和537个，8个随机选择的差异表达基因实时定量PCR检测结果与 DGE测序结果一致。本研究鉴定了在策勒黑羊、和田羊卵巢组织差异表达的miRNA和基因，并对部分miRNAs靶基因的调控及细胞水平功能进行了研究，为深入揭示miRNA影响绵羊泡发育和绵羊繁殖控制的分子机理奠定基础。