参于精子发生过程中的功能性长链非编码RNA的鉴定与分子机制研究（果蝇）

Thousands of long noncoding RNAs (lncRNAs; > 200 nucleotides) have been identifiedby genome-wide transcriptome analyses. Recent studies show that lncRNAs are often predominantly transcribed in testis in both vertebrates and Drosophila, suggesting that these RNAs play similar functional roles in spermatogenesis across a broad range of animal taxa. However, it remains unclear whether lncRNAs are truly.involved in male reproduction in vivo. Thus we attempted to survey the functional roles of lncRNAs in spermatogenesis using our optimized CRISPR system. In this study, first we plan to identify hundreds of testis-specific Drosophila lncRNAs through bioinformatics prediction, published gene expression data analysis and RNA-seq. To comprehensively analyze lncRNA functions in vivo, we plan to inactivate these lncRNA genes by genomic deletion using an optimized three-component CRISPR/Cas9 system. Through quantitative fertility assay, testis.morphology examination, testis squash and DAPI staining et al., we would check phenotypes of lncRNA knockout mutants and select lncRNAs with critical function in spermatogenesis. Then we would explore the mechanism of several lncRNAs with severe phenotypes by which it may act using various methods such as RNA-seq, ChIRP-seq and RNA pull-down. These studies will provide important genetic evidence to the in vivo function and the underlying mechanism of lncRNA in Drosophila spermatogenesis.

高通量测序技术发展使得大量长链编码RNA（lncRNA）被鉴定出来，同时研究表明睾丸为lncRNA特异高表达的两组织之一，指向lncRNA极可能参与精子发生过程的调控。为揭示非编码RNA介导的信息传递方式和分子机制，本项目以模式生物果蝇为代表，系统的鉴定睾丸组织特异性表达的，可能参与精子发生的lncRNA基因。通过CRISPR/phiC31技术大规模建立lncRNA敲除突变品系，并结合原位功能补偿实验系统鉴定lncRNA的生物学功能。通过对lncRNA敲除果蝇突变株进行缺陷表型分析，鉴定出在减数分裂后精子细胞核压缩、形态发生以及精子延伸中具有重要功能的lncRNA分子。针对若干具重要表型的功能性lncRNA基因（包括1株雄性不育突变体lncRNA：CR44456），结合生化和分子生物学手段研究lncRNA的分子调控机理。

睾丸是有性生殖进化发育中一个高度复杂的器官，睾丸组织中精子发生过程（spermatogenesis）是一个复杂、严密而又保守的细胞分裂与分化过程。精子发生过程存在DNA水平和组蛋白等表观修饰，有些小RNA，如piRNA在精子基因组稳定性方面也起着重要作用。然而，精子发生过程中的分子机制还是所知甚少，为目前生命科学研究相对真空领域。尤其是目前在睾丸组织测序鉴定到的大量长链非编码RNA（lncRNA）在精子发生过程中是否发挥功能尚不知晓。因而，有必要通过动物模型用遗传学的方法研究这些长链非编码RNA在动物体内的生物学功能及相应的分子调控机制。.取得的结果与数据：(1)鉴定了果蝇睾丸组织中不同发育阶段特异性表达的lncRNA; (2)构建了 lncRNA基因敲除突变体果蝇；（3）发现大量lncRNA基因敲除会导致雄性果蝇育性下降；（4）生化鉴定了雄性不育lncRNA-CR44456基因的相互作用的蛋白质底物分子。.科学意义：通晓精子发生的形成过程（如各部件如何装配成成熟健康的精子，精子发生中包括基因水平和表观遗传水平等不同层次的分子调控机制，以及精子携带的遗传信息对早期胚胎发育的影响），对探索精子发生的发育学过程，精子传递哪些遗传信息到子代与这些信息对子代健康的贡献等基础生物学问题至关重要。