罗氏沼虾性腺发育相关microRNA的鉴定及其调控机制的研究

During gonadal differentiation and development (D&D) in vertebrate, many reports suggested that the microRNA (miRNA) plays an essential role in the regulation of target genes through the mechanism of post-transcription. However, the correspondent information is rarely characterized in shrimps and crabs. Although gonadal D&D affect yield and quality of Macrobrachium rosenbergii larva directly, study of miRNA regulation in this field has not been carried out until now. In this project, we will mainly focus on the differently expressed miRNA in the gonad to explore its expression profile and regulation function during gonadal D&D in M. rosenbergii. Using next generation high-throughput sequencing technique, we will analyze the newly expressed and unique miRNA and mRNA after gonadal differentiation and different stages of gonadal development, respectively; And prediction and preliminary verification of function of miRNA and its target will be performed by bioinformatics, qPCR, in situ hybridization and RNAi; Then compare the expression patterns of miRNA at different stages of gonadal development; Lastly, in vitro and in vivo injections by miRNA agomir and miRNA antagomir will be performed to research on regulation of miRNA to its target gene, to facilitate a gene regulation pathway of gonadal D&D constructed by miRNA as an important node in M. rosenbergii.

脊椎动物性腺分化发育过程中microRNA(miRNA)对其靶基因的转录后调控研究较为丰富，但在虾蟹类中报道较少。罗氏沼虾性腺分化发育直接影响其苗种产量和质量，而miRNA对罗氏沼虾性腺分化发育调控的研究仍未开展。本课题以罗氏沼虾性腺差异表达的miRNA为主要研究对象，探究miRNA在罗氏沼虾性腺分化发育过程中的表达规律及调控功能。通过高通量测序，比对分析性腺分化后新表达的及性腺不同发育期特异表达的miRNA和mRNA；并利用生物信息学、定量PCR、原位杂交和RNA干扰等方法预测和初步验证miRNA与其靶基因的功能；然后比较性腺不同发育时期miRNA的表达规律；最后分别通过体外和体内注射miRNA agomir和miRNA antagomir研究miRNA对靶基因的调控机制，以期在罗氏沼虾中建立以miRNA为重要节点的性腺分化发育的调控通路。

罗氏沼虾（Macrobrachium rosenbergii）是我国重要的淡水养殖经济虾类之一。性腺分化和性腺发育是罗氏沼虾养殖关注的焦点，人工调控性腺发育更能为养殖业带来更高的经济效益。因此，探讨性腺分化和发育相关基因调控的发育机制，优化罗氏沼虾的育种方法和途径，是非常必要的。本课题获得了新的罗氏沼虾基因组DNA分子标记，该标记可以有效地区分不同群体的雌、雄罗氏沼虾个体遗传性别，为后续遗传操作鉴定个体性别提供了基础工具；目前，小RNA对罗氏沼虾性腺分化发育调控的研究仍未开展。本课题以罗氏沼虾性腺差异表达的miRNA为主要研究对象，主要探究了miR-184对罗氏沼虾促雄腺类胰岛素样因子（IAG）基因的调控机制，证明了miRNA通过靶向调控目标基因参与罗氏沼虾性别分化、性腺发育过程。在罗氏沼虾幼体发育时期利用miR-184敲降IAG基因后，众多涉及性别、生长和代谢相关基因的表达水平发生显著变化，暗示了miRNA可能参与了罗氏沼虾多个生理生化过程。同时，本课题首次从罗氏沼虾中分离到一个卵巢特异表达的fem-1基因。在siNRA的研究过程中，还发现了两个负调控fem-1的siRNA靶点，并在体外验证了其有效性，为后续研究siRNA介导的罗氏沼虾卵巢发育提供了新的思路。