青鳉DMY基因缺失造成性反转中性别差异miRNAs调控的机制研究

Sex determination is one of the core scientific issues in animal reproduction and development. Sex reversal is one special type of sex determination, which there is no change in genetics yet big change in gonad phenotype. It is a common phenomenon in fish, however, the mechanism is unclear. DMY gene is the first identified male sex determination gene in fish. In our previous study, XYDMY- mutants were generated using TALENs, which developed into females after sex reversal. Sex determination regulatory network and transcriptional regulation of DMY gene were investigated in Medaka. Interestingly, the transcription factor, DMY, was not only directly binding to the regulatory elements to promote targeted gene transcription, but also producing sex-biased miRNAs to participate in mRNA post-transcriptional regulation, which may be indirectly roles of DMY gene during sex determination and sex reversal in Medaka. Therefore, using Medaka as a model and basing on XYDMY- mutants, we intends to explore the generation and regulation mechanism of sex-biased miRNAs in XYDMY- mutants, combined with high-throughput sequencing and analysis of GRO-seq, RNA-seq and miRNA-seq. It is expected to elucidate the novel mechanism of miRNAs in sex reversal, to provide new ideas for further understanding post-transcriptional regulation in animal reproduction and to provide theoretical basis on animal sex determination and gender-controlled breeding.

性别决定是动物生殖发育的核心科学问题之一。性反转是遗传信息不变而性腺变化的特殊性别决定。鱼类广泛存在性反转现象，机制尚不清楚。青鳉DMY基因是第一个被鉴定的鱼类雄性性别决定基因。前期研究，申请人敲除DMY后获得了遗传雄性生理雌性的性反转青鳉，研究了DMY的下游靶基因和转录机制；有趣地发现转录因子DMY除了直接结合调控元件起始靶基因转录，还能通过生成性别差异miRNAs(Sex-biased miRNAs)参与转录后调控。因此，申请人拟以青鳉为模型，通过动物、细胞和分子水平的实验研究，辅以small RNA-seq、RNA-seq和GRO-seq等高通量测序及分析，探索DMY缺失产生sex-biased miRNAs调控性反转的新机制。有望阐释青鳉性反转的miRNAs调控新机制，为深入理解动物生殖发育中转录后调控提供新思路，为动物性别决定及性别控制育种相关研究提供理论依据。

性别决定是动物生殖发育的核心科学问题之一。性反转是遗传信息不变而性腺变化的特殊性别决定。鱼类广泛存在性反转现象，机制尚不清楚。青鳉DMY基因是鱼类中第一个被鉴定的雄性性别决定基因。申请人在前期敲DMY基因获得遗传雄性生理雌性的性反转青鳉的基础上，系统建立了青鳉性腺不同发育阶段（10天、30天和120天）的small RNA-seq、RNA-seq等文库，完成了高通量测序和相关分析，为青鳉性反转研究积累了重要组学数据，青鳉性腺的相关组学数据作为重要信息支撑了首个动物性反转数据库ASER的建立（Genomics Proteomics & Bioinformatics，2021，封面文章）；还参与联合开发了全基因组范围内原位捕获细胞核内与染色质相互作用RNA的新技术GRID-seq（Nature Protocols，2019），有望用于原创性地发现新RNA和研究RNA新功能。系统注释和分析了青鳉雌雄性腺（精巢、卵巢）中miRNA情况，筛选与鉴定了DMY缺失型青鳉中性别（性腺）差异表达miRNAs，发现了ola-miR-7等性腺差异表达的miRNA，并探索了其在青鳉性反转中的功能及其机制，以（共）通讯作者或第一作者发表研究论文4篇。有望阐释青鳉性反转的miRNAs调控新机制，为深入理解动物生殖发育中转录后调控提供新思路，为动物性别决定及性别控制育种相关研究提供理论依据。项目执行期内，负责人罗大极获得了国家自然科学基金优秀青年基金（2019）资助，中国科学院百人计划（2020），湖北省杰出青年基金（2020）等人才计划资助，还获得了湖北五一劳动奖章（2019）等荣誉。培养了1名博士研究生，4名硕士研究生。