家蚕神经系统piRNA的系统挖掘及功能解析

Small RNA play important roles during silkworm development. Interestingly, in previous studies, we found four piRNAs could origin from intermidiate-size non-coding RNAs (ncRNA) in the head of silkworm, including snoRNAs and tRNA-like ncRNAs, which was first found that piRNAs could origin from ncRNAs with explicit secondary stuctures. To further elucidate the mechanisms and function of these piRNAs generating from nervouse system, we will construct the library of sRNAs from nervous tissues of silkworm through co-immunoprecipitation of Ago3 and Piwi proteins then by deep sequencing. In order to parse the new generation mechanisms of piRNAs, we will blast all the piRNAs to the known ncRNAs of silkworm to find more piRNAs precursors of ncRNAs. As well, we will find whethter the expression of piRNAs could be changed following the over-expression, RNA interference (RNAi) and mutant of the precosur ncRNAs as well as making mutation of ago3 and piwi genes. Finally, we will explore the function of these piRNAs through over-expression, RNA interfering the expression of piRNAs as well as mutants of ago3 and piwi genes. This work will help us to understand the biogenesis and functions of piRNAs in the nervous system of silkworm.

小分子RNA在家蚕发育过程中具有重要调控作用。项目组在前期研究中发现4个piRNA能够在家蚕头部表达，且其前体为非编码RNA（ncRNA），包括snoRNA和tRNA-like的ncRNA，这是首次发现具有明确二级结构的ncRNA加工产生piRNA。为解析神经系统piRNA的产生机制及功能，本项目拟通过免疫共沉淀从神经组织捕获与Piwi亚家族蛋白（Ago3和Piwi）互作的sRNA，深度测序鉴定家蚕神经系统的piRNA。在此基础上，利用家蚕ncRNA数据库预测产生piRNA的ncRNA，解析ncRNA加工产生piRNA的机制。为解析piRNA功能，拟采取过表达、干涉piRNA表达，构建ago3和piwi的RNAi突变体等，在细胞水平解析piRNA对家蚕神经可塑性及神经发育的调控机理。

小分子RNA在家蚕发育过程中具有重要调控作用。项目组通过提取家蚕神经组织RNA，系统构建了家蚕神经组织sRNA文库，获得家蚕已知的miRNA 684个，新的miRNA 74个。获得已知的piRNA 1个，新的piRNA 5805个。同时对这些sRNA的基因组织形式、基因组定位及可能的靶基因进行了研究。表达谱研究发现，piRNA可能在神经组织、精巢和卵巢中发挥不同的功能。进一步研究发现，pi-4682和miR-305可从基因组同一位点产生，而且二者存在不同的表达模式，体外共转染pi-4682和miR-305及其靶基因small wing发现，过表达pi-4682和miR-305均可使small wing表达量下降。通过在家蚕个体中过表达pi-4682，发现其靶基因small wing表达也下调。表明同一基因位点产生的miRNA和piRNA可能在家蚕发育过程中发挥不同的功能。