刺参夏眠相关神经肽发掘及其功能验证

The emerging role of neuropeptides is a current hot topic in research exploring the regulation of multiple forms of animal dormancy Very little is known about the neuropeptides involved with aestivation in sea cucumbers or their regulation. The present project begins with the localization and structural analysis of the sea cucumber nervous system, followed by identification of neuropeptides that may regulate reversible transitions leading to and from aestivation. With the newly-completed sea cucumber genome as a reference, the neuropeptidome will be profiled in the nervous system of the sea cucumber at different stages of aestivation utilizing bioinformatics and HPLC-QTOF-MS-based technology. Gene structures of the precursors of identified key neuropeptides will be analyzed, and the distribution of key neuropeptides and their precursors will be studied using immunofluorescence and in situ hybridization, respectively. To validate the functional role of key neuropeptides over the whole period of aestivation, sea cucumbers will be bathed in or injected with synthetic neuropeptides dissolved in sea water. These pharmaceutical tests will finally identify the “triggers” that induce sea cucumbers to enter into or arouse from aestivation, and the “stabilizers” that maintain long-term aestivation in sea cucumbers. The results will provide a crucial new perspective on the regulation of stress physiology in marine invertebrate and new biotechnological directions to pursue for aquaculture of this commercially-important animal.

神经肽对动物休眠的诱导和调节机制是动物休眠调控机理研究热点之一，关于刺参夏眠相关神经肽的发掘及其调控机理的研究几近空白。本项目拟从刺参神经系统的整体定位和结构特征观察入手，以神经组织中夏眠相关神经肽的发掘为突破点，结合刺参基因组参考文库采用HPLC-QTOF-MS联用技术构建刺参神经肽文库，查明神经肽在夏眠全过程中的表达特征；基于发掘的关键神经肽，利用生物信息学方法分析其前体蛋白的基因结构特征，分别采用免疫荧光和原位杂交手段定位分析神经组织中关键神经肽及其前体蛋白的分布特征；活体浸泡或注射化学合成的神经肽，应用药理学和生态学实验技术验证关键神经肽在夏眠全过程中的作用，以期锁定诱导刺参夏眠和解除夏眠的“触发器”，以及维持夏眠状态的“稳定剂”，为刺参夏眠的调控提供重要“物质”基础。

神经肽对动物休眠的诱导和调节机制是动物休眠调控机理研究热点之一，而关于刺参夏眠相关神经肽的发掘及其调控机理的研究几近空白。本项目以刺参夏眠相关神经肽的发掘为突破点，利用HE染色，米利根染色以及免疫组化对刺参神经组织分布进行组织学定位观察；构建了刺参神经环转录组文库，结合生物信息学分析方法及HPLC-QTOF-MS 联用多肽组学技术深度发掘刺参神经系统中的神经肽及其前体，首次在棘皮动物中进行连锁群水平的神经肽前体基因定位分析，并从中筛选出关键的候选神经肽；以刺参基因组文库为参考，克隆鉴定并分析了候选关键神经肽前体AjTRHP，AjPPLNP2，AjCTP1& AjCTP2，AjLQP，Ajholotocin precursor，AjSSP，AjMS21P的基因结构特征，利用原位杂交，免疫组化技术和荧光定量PCR技术分别对关键神经肽和神经肽前体进行定位定量分析，为这些神经肽在夏眠过程中作用机制的发掘奠定基础；利用无标记蛋白定量技术测定部分神经肽在夏眠过程中的定量表达，结果表明，神经肽AjPPLH2在夏眠初期有显著的高表达，体外药理学实验结合生态学实验进一步验证了其在诱导刺参进入夏眠中起到的关键作用。本研究以神经肽的发掘为契机，从激素水平上深入开展，系统揭示刺参夏眠的调控机理，为神经肽与水产动物特殊生理机能发生机制内在联系的研究开拓一个新的突破点。