不同猪种曲细精管转录组分析及公猪性机能发育与生精调控研究

Chinese indigenous boars have some unusual endocrine and physiological attributes, such as earlier sexual maturity, thereby providing a valuable animal model for study of the mechanism of boar sexual development and sperm production. The seminiferous tubules are the sites of sperm production. Its diameter, volume density and other traits relate significantly to sperm production.The existence of differences between breeds for these traits would lead to differences in reproductive maturation and fertility. In addition, transcriptional regulation is a major controller of tissue growth. RNA deep sequencing technology is a revolutionary tool for transcriptomics, which can detect and analyze the overall transcriptional activity of the organization at single-nucleotide resolution. In this project, RNA deep sequencing technology will be used to analyze the transcriptomes of seminiferous tubules of Meishan and Duroc boars in different developmental stages. The gene and cSNP, regulating boar sexual development and sperm production, will be screened. The altered splicing and allele-specific expression will be identified. The promoters of differentially expressed gene will be isolated. The differentially expressed mechanism will be analyzed. The differentially expressed genes, new transcripts and spliced variants will be located to cells. The regulatory network will be analyzed. The results will lay a foundation to reveal the molecular mechanism of earlier sexual maturity of Chinese indigenous boars and differences of sperm production between breeds, and provide new approachs for further development and utilization of superior genetic resources of Chinese indigenous pig breeds.

我国地方品种公猪具有性成熟早等罕见的生理特性，为我们研究公猪性机能发育和生精调节机制提供了珍贵的动物模型。曲细精管是产生精子的地方，其体积密度、直径等性状均与生精能力显著相关，会引起生殖成熟和繁殖力的差异。转录水平的调控是生物体最主要的调控方式。RNA深度测序技术是转录组学研究的革命性工具，能在单核苷酸水平上对组织的全部转录活动进行检测和分析。因此本项目拟利用RNA深度测序技术分析梅山公猪和杜洛克公猪曲细精管发育与成熟阶段的转录组；筛选对公猪性机能发育和生精具有重要调控功能的基因和cSNP；鉴定转录本的可变剪切方式以及等位基因特异表达的调控；分离差异表达基因的启动子，鉴定其差异表达机制；进行差异表达基因、新转录本和可变剪切体的细胞精细定位，解析其调控网络，为揭示我国地方猪种性成熟早和不同猪种生精能力差异的分子机理奠定基础，并为我国地方猪种优异种质资源的进一步开发利用提供新思路。

我国地方品种梅山公猪具有罕见的生理特性，与杜洛克公猪相比，它性成熟早，但睾丸尺寸小、支持细胞的比例低及日产精量少，这为研究公猪性机能发育和生精调节机制提供了珍贵的动物模型。睾丸中曲细精管是产生精子的地方，其体积密度、直径等性状均与生精能力显著相关，会引起生殖成熟和繁殖力的差异。因此，本项目利用苏木精-伊红染色方法(Haematoxylin-eosin staining，HE染色)和RNA 深度测序技术（RNA-seq）分析比较了梅山公猪和杜洛克公猪曲细精管发育与成熟阶段(20日龄，75日龄及270日龄)的组织学结构和转录组图谱。两个品种间的睾丸曲细精管的直径在发育起始阶段均存在着显著的差异。梅山公猪产生精子的年龄早于杜洛克公猪，在75日龄之前就已经开始了，但成年以后精原细胞和支持细胞的数目却比杜洛克的数目少得多。通过RNA-seq，筛选出一批对公猪性机能发育和生精具有重要调控功能的差异表达基因、新转录本和cSNP，富集到6条与雄性性成熟和精子生成相关的信号途径。通过qPCR检测了8个重要的功能性差异基因，它们的表达趋势基本与深度测序结果一致。其中，Neurotrophin 3 （NTF3）和GnRH (type 2) receptor 2 （GnRHR2 ）分别属于影响公猪繁殖力的MAPK信号通路和GnRH信号通路。我们对NTF3和GnRHR2的表达进行了定量和细胞精细定位分析，鉴定了GnRHR2的可变剪切体，分离了NTF3和GnRHR2的启动子，确定了它们的核心启动区域、转录起始位点、启动子活性片段及与之结合的转录因子，并分析了其差异表达机制和调控网络。到目前为止，发表SCI收录论文3篇，会议论文3篇，授权专利1项，取得了阶段性的研究成果，达到了预期目标，为揭示我国地方猪种性成熟早和不同猪种生精能力差异的分子机理奠定了基础。