血管囊在光周期诱导花鲈季节性繁殖节律中的作用机制研究

The saccus vasculosus (SV) is unique to fish. Although its existence was first described in the 17th century, its physiological function remained a mystery for several centuries. Recently, it was the discovery of functions of SV ranging from photoreception to neuroendocrine response and the verification of SV acting as a sensor of seasonal changes in day length start a new chapter of the research on mechanism of seasonal reproduction rhythm in fish. So far, the study about the function of SV is still in its infancy and it is necessary to conduct wider and deeper studies. The sea bass (Lateolabrax maculatus) is a short-day breeder. Its breeding period can be regulated by changes of the photoperiod. In view of the important roles of SV during the transmission of photoperiod information, and the characteristics of seasonal reproductive rhythms in L. maculatus, the present research project will carry out the transcriptome analysis of SV under different photoperiods to screen photoperiodic response genes, and explore their spatiotemporal expression profiles under mRNA and protein level in vivo. And in vitro, to study the regulating characteristics of photoperiodic response genes responded to photoperiodic change in independently cultured SV, and expression profiles of reproductive endocrine key genes in the hypothalamus or the pituitary induced by the secretion of SV. In addition, the regulation relationships between genes involved in pivotal signaling pathways will be investigated using RNAi technology. According to the above study, we aim to explore the regulatory mechanism of SV in the processes of mediating seasonal reproductive regulation of photoperiod in L. maculatus, so as to provide theory support for the artificial regulation of reproduction period in L. maculatus, and to promote the understanding of the regulatory mechanism for seasonal reproduction in fish.

血管囊是鱼类特有的结构，它的生理功能在几个世纪以来都是未解之谜。最新发现血管囊具有光接收和神经内分泌输出的双重功能，并被证明是鱼类季节性繁殖的传感器，开启了鱼类季节性繁殖节律机制研究的新篇章，然而相关研究仍处于初级阶段，需要开展更广泛、更深入的研究。花鲈属短日照繁殖鱼类，可以通过光周期改变来调控其繁殖期。基于血管囊在光周期信息传递中的重要作用，针对花鲈季节性繁殖的特点，本项目通过（1）分析光周期调控下血管囊的转录组，挖掘光周期应答关键基因，明确其在mRNA及蛋白水平的时空表达特征；（2）在离体水平，掌握光周期调控下血管囊重要应答基因的表达规律，以及血管囊分泌物对下丘脑和垂体中生殖内分泌基因表达的调控规律，并应用RNAi探寻关键光周期效应基因参与的信号通路。综合解析血管囊在光周期诱导的花鲈季节性繁殖中的作用机制，为花鲈繁殖期的人工调控提供理论支持，同时促进对鱼类季节性繁殖节律调控机制的理解。

本研究以花鲈为研究对象，以鱼类光周期传感器——血管囊为核心，开展光周期调控下的花鲈血管囊组织学和转录组学研究，挖掘季节性繁殖光周期效应重点调控基因，研究其时空表达特征；进而通过原代培养，探究光周期对单独血管囊的影响，以及血管囊分泌物对下游生殖内分泌基因表达的影响，并应用RNAi技术研究重点光周期效应基因参与的信号通路。通过活体和离体两种方式，解析血管囊在光周期诱导花鲈季节性繁殖中的作用机制。.（1）利用光学显微镜和透射电镜对5个光周期条件下花鲈血管囊进行组织学观察，证明了不同的光周期对血管囊冠细胞形态及分泌物具有一定的影响。.（2）掌握了血管囊响应光周期的转录组学特征，构建了3个光周期条件下血管囊的差异基因数字表达谱。获得了血管囊、下丘脑、垂体和性腺的转录组数据。筛选获得28个季节性繁殖调控基因。.（3）对Kiss2、GnRH2、Dyn、LHb、TSH、TRH、GP、CYPA2、DOI2、DOI3、Opsin5以及Dyn基因的组织分布，光周期条件下的表达、不同性腺发育时期的表达以及亚细胞定位情况进行了研究，结果表明不同光周期条件通过诱导花鲈血管囊、下丘脑和垂体中生殖调控基因的变化来影响花鲈的生殖内分泌，进而能够影响花鲈的性腺发育水平。.（4）血管囊营养液孵育实验和离体光照实验证明血管囊能够调控垂体和下丘脑中的生殖相关激素，进一步证明血管囊在光周期调控花鲈生殖过程中的重要地位。.（5）Kiss基因与受体结合可与PKA/PKC途径偶联，Dyn基因与受体结合后可与PKA途径偶联.Kiss2通过PKC和PKA途径对Kiss2-GnRH2信号通路中的下游基因的表达进行正向调控；而Dyn通过PKA途径对GnRH信号通路中的下游基因进行负向调控。