牙鲆piRNA及其介导的生殖细胞转座子沉默和基因表达调控机制的研究

Germ cell is an ideal tool for fish genetic resources preservation. Investigation on the development mechanism of fish germ cells will help to open up a new way to fish germplasm preservation and cell engineering breeding. piRNAs (Piwi-interacting RNAs) are a class of germline-specific small non-coding RNAs that form complex with Piwi proteins. The Piwi/piRNA pathway is a germline-specific RNA silencing mechanism which has been implicated to prevent the activity of selfish genomic elements (such as retrotransposon) and regulate various protein coding genes. Piwi/piRNA pathway plays critical roles in embryonic development, sex determination, gametogenesis to maintain the stability and integrity of genome in gamete. Recently we found that in Japanese flounder (Paralichthys olivaceus), the expression levels of several Piwi/piRNA pathway genes were specifically high in testis of adult individuals which indicated that Piwi/piRNA was related to spermatogenesis of Japanese flounder. Based on this interesting result, our project plans to 1) characterize the expression pattern of piRNAs in the gonads of Japanese flounder during different developmental stages, 2) screen the piRNA targeting transposons and coding genes, construct their co-expression network and 3) further explore its regulation mechanism of transposon silencing and gene expression in vitro, providing the key candidate Piwi/piRNA pathway for Japanese flounder gametogenesis. All these works will help to understand the function and regulation mechanism of piRNAs during gametogenesis, pave the way for the preservation of excellent fish breeds and endangered fish germplasms, and lay a foundation for cell engineering breeding in fish.

生殖细胞是鱼类遗传资源保存的理想细胞类型，对其发育分化机制的研究，可以为鱼类种质保存和细胞工程育种开辟新的途径。piRNA是生殖细胞特异性表达的新型非编码小RNA，其与Piwi蛋白结合，通过沉默转座子和调控编码基因等方式在生殖细胞发育分化及性别决定中发挥重要作用。我们前期研究发现，piRNA通路的多个基因在牙鲆精巢中特异高表达，预示其在牙鲆生殖发育过程中的重要作用，但关于牙鲆piRNA的调控机制还不明确。本项目将在此基础上采用转录组测序及分子生物学等手段开展牙鲆不同发育时期雌、雄性腺piRNA的表达鉴定，搜寻piRNA靶定的转座子和编码基因，构建其共表达网络，在细胞水平上对关键piRNA进行人工调控，解析其与靶转座子和靶基因的调控模式及作用路径，阐明其在牙鲆生殖发育过程中诱导转座子和基因沉默的分子机制，为牙鲆生殖发育等研究提供重要参考依据，为种质资源的保存和利用、育种工作的开展奠定基础。

Piwi/piRNA通路通过参与生殖细胞自我更新和减数分裂过程，在精子发生中沉默转座子和相关基因。本研究首先结合牙鲆10个成体组织和6个发育时期的mRNA和small RNA转录组数据，筛选并验证了牙鲆Piwi/piRNA通路的时空表达及分子进化模式，确定了牙鲆Piwi/piRNA通路的生殖特异性表达，特别是精巢高表达，并在胚胎发育早期高表达的模式；项目开展了牙鲆转座子家族的鉴定及其潜在的miRNA和piRNA靶向调控关系构建，发现牙鲆精巢中的piRNA多成簇分布，且更多的靶向DNA转座子，可能与硬骨鱼中DNA转座子的多样性和丰度相关，我们以牙鲆Tc1转座子为例验证了其与Piwi基因的互补表达模式；我们同时鉴定了牙鲆雌雄差异表达piRNA靶向最多的pim基因家族，发现pim家族的扩张可能作为piRNA簇的前体转录本参与次级加工，并参与其它pim基因的靶向调控。本项目为阐明Piwi/piRNA在牙鲆生殖发育过程中诱导转座子和基因沉默的分子机制，为牙鲆生殖发育等研究提供重要参考依据，为种质资源的保存和利用、育种工作的开展奠定基础。