PIWI蛋白介导的piRNAs在鸡精子发生中的功能研究

The piRNAs binding to PIWI protein involved in the epigenetic and post-translational regulation is a cruisal factor for maintaining germ stem cells, gametogenesis and embryonic early development. Compared to mammals, the development of bird embryos is visible in vitro and easy to manual operation, and the gametes are accessible. In this study, we will investigate the expressions of Piwi gene at the different embryonic stem cells and spermatogenesis in chicken by Q-PCR and Western blotting. Meanwhile, using the model of azoospermia and PIWI mutant by TALEN and shRNA vectors,we will fish the piRNAs binding to PIWI protein by RNA Binding Protein Immunoprecipitation, RIP technology, and then purify the specific piRNAs for the analysis of Hiseq2000. With the analysis of bioinformatics,we gain the origin and regulation function of piRNAs within signaling pathway before and during the meiosis stages in spermatogenesis. To verify further the biological function and regulation mechanism of piRNAs and PIWI protein,we detect the change of proliferation and differentiation of germ stem cells and sperm maturation in the experimental groups treated by piRNAs inhibitor and mimics through Nepa high eifficy transfection system,respectively.In summary, we illustrate the biological function of piRNAs and PIWI protein during spermatogenesis in poultry.

PIWI蛋白介导的piRNAs在表观遗传和转录初水平的调控是生殖干细胞维持、配子形成以及胚胎早期发育的关键因素。与哺乳动物相比，鸡胚胎发育体外可见且易于人工操作，配子获得简单。本研究利用Q-PCR和免疫印记技术检测鸡胚生殖干细胞和精子发生过程中PIWI蛋白的表达水平；同时分别构建鸡无精症和PIWI突变体细胞模型（通过TALEN和shRNA干扰技术），利用RNA-蛋白质免疫沉淀技术"钓取"与PIWI蛋白特异性结合的piRNAs，纯化后用于Hiseq2000分析。经生物信息学分析与注释，获得减数分裂前期和减数分裂过程中piRNAs的起源发生、调控功能及相关信号通路。最后，通过Nepa高效转染系统在细胞和个体水平进行候选piRNAs抑制和mimic分析,检测生殖干细胞的增殖、分化状态以及精子成熟过程中的变化，最终揭示piRNAs及PIWI蛋白在鸡精子发生中的功能与调控机制。

PIWI蛋白介导的piRNAs在表观遗传和转录初水平的调控是哺乳动物生殖干细胞维持、配子形成以及胚胎早期发育的关键因素，但在家禽中尚是空白。本研究首次克隆了家禽特有的Piwi基因-Piwi like 1 (Piwil1)序列及其结构特征，证实了鸡piRNAs能特异性结合PIWI蛋白，主要来源于基因及转录因子结合位点。鸡Piwil1基因在不同生精细胞的表达量受到NF-Y和CpG岛甲基化程度共同调控。Piwil1基因具有抑制转座子CR1-F活性的功能，体内、体外抑制(ShRNA)与敲除（CRISPR-Cas9）Piwil1基因后早期胚胎、生殖细胞和配子形成发生障碍。通过二代测序筛选到影响禽类精子发生的减数分裂前期和后期的一批特异性piRNAs，证实了piRNA-19128通过靶向KIT基因调节Melanogenesis信号通路从而参与精子减数分裂的调控机制，最终阐明了鸡piRNAs及PIWI蛋白在早期胚胎发育、精子发生中的功能与作用机理。