牛胚胎发育相关精子特异miRNAs的鉴定和功能研究

Accumulated evidences have shown that sperm miRNAs might play important roles in embryonic development and nuclear epigenetic reprogramming. The aims of the present objective are to construct miRNA libraries of bovine matured sperm, oocyte and skin fibroblast using the next generation of high-throughput sequencing technique, and screen the developmentally important miRNAs specially expressed in matured sperm. The target genes of key miRNAs are predicted by bioinformatics analysis and dual luciferase reporter system. The effects of key miRNAs on the development, gene expression, and epigenetic reprogramming of early embryo are studied by function-deprivation strategy. The effects of key miRNAs on the efficiency of the somatic cell nuclear transfer are evaluated by function-obtaining strategy. We hope that the project would reveal the biological function of the sperm-specific miRNAs systematically, clarify their regulatory mechanisms on development and (or) epigenetic reprogramming of early embryo, expand the knowledge of regulation of early embryonic development and epigenetic reprogramming, and provide new ideas for improving somatic cell cloning and in vitro induced stem cell technology.

越来越多的证据表明精子受精过程中带入胚胎的miRNAs可能对胚胎的发育和表观重编程修饰起着重要的调控作用。本项目拟利用新一代高通量测序技术构建牛成熟精子、卵母细胞及皮肤成纤维细胞miRNAs文库，筛选精子特异表达miRNAs。通过生物信息学分析和双荧光素酶报告试验揭示关键精子特异miRNAs的预测靶基因及其生物调控作用；利用功能缺失策略研究精子特异关键miRNAs对受精胚胎发育、基因表达及表观重编程的影响；利用功能获得试验研究精子特异关键miRNAs对体细胞克隆胚胎的质量及克隆效率的影响。系统揭示精子特异miRNAs的生物学功能，阐明其对胚胎发育和（或）表观重编程的调控机制，扩展对早期胚胎发育及表观重编程调控的认识，为改进体细胞克隆技术和体外诱导干细胞技术提供新的思路。

通过对精子、卵母细胞和体细胞小RNAs测序及生物信息学分析，筛选出精子高表达且与早期胚胎发育事件相关的miRNAs进行下一步的研究, 预测并验证了它们的靶基因和生物学功能。通过功能缺失和获得实验，重点研究了精子特异miRNA-202、miRNA-301a及miRNA-183对早期胚胎发育的影响，发现这些精子miRNAs对早期胚胎发育过程中多个重要发育事件起关键作用，包括原核形成、卵裂速度、囊胚细胞分化、凋亡、体内发育及出生后动物的健康等，并阐明了其导致这些表型可能存在的调控机制。同时通过对牛体细胞克隆胚进行精子总miRNAs和关键单个miRNAS功能获得实验，明确了精子miRNAs在牛体细胞克隆胚胎发育中的关键作用，为体细胞克隆技术的优化提供了新的思路。