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

项目名称： 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

项目编号： No.31260536

项目类型： 地区科学基金项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 曾献存

作者单位： 石河子大学

项目金额： 50万元

中文摘要： 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。

中文关键词： miRNA；卵巢；差异表达；绵羊；

英文摘要： 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, an

英文关键词： miRNA；ovary；differential expression；sheep；