厚壳贻贝肾上腺素能受体和5-羟色胺受体调控幼虫变态的分子机理研究

Marine Mussels form dense aggregations that dominate temperate rocky shores, and they are typical macrofouling organisms and important aquaculture species in the world. The molecular mechanism underlying larval metamorphosis has been a hot topic of biofouling and ecological research. However, current understanding of G-protein coupled receptors–mussel larval metamorphosis interactions is based primarily on pharmacological bioassays. Thus, we use the marine mussel Mytilus coruscus as a model organism to analyze the mechanism of underlying the adrenergic receptors (ARs) and 5-serotonin receptors (5-HTRs) in mediating larval metamorphosis in this species. Basing on cloning genes of ARs and 5-HTRs in M. coruscus, we analyze their expression and distribution profiles in different developmental stages by qRT-PCR and the whole mount in situ hybridization. The development of adrenaline and serotonin-containing cells in mussel larvae with- and without chemical treatment will be investigated. The comparative proteomic profiles of pre-competent and competent larvae will be checked, and the receptors-ligands interactions will be conducted by the surface plasmon resonance methods by the Biacore X system. Finally, we would develop a method to introduce siRNA into M. coruscus larvae and test the impact of siRNA transfection on the phenotypic changes of larvae. The knowledge obtained in this project will contribute to advancing our understanding of the molecular mechanisms underlying larval metamorphosis of marine invertebrates including M. coruscus, providing novel insights into the identification of molecular targets or the signaling pathways responsible for the larval metamorphosis, and developing novel environmentally and eco-friendly antifouling technology.

贻贝有较强的繁殖力以及对生态环境的很强适应性，广泛分布于世界各地的潮间带,同时贻贝也是代表性大型海洋污损生物。然而，关于贻贝附着的分子机制尚未清楚。因而，本课题选用贻贝作为研究对象，在厚壳贻贝肾上腺素能受体和5羟色胺受体基因序列克隆基础上，通过实时荧光定量PCR和原位杂交技术分析这些基因的时空表达；通过免疫细胞化学技术和荧光染色技术确定暴露于上述神经递质前后的幼虫受体活性细胞的分布变化；探索上述神经递质作用于眼点幼虫前后蛋白质组的变化，运用表面等离子共振技术，分析受体与神经递质的结合作用，通过RNAi技术确定受体基因调控作用机制。本项目的顺利实施，有助于进一步解析贻贝幼虫变态的分子机制，为后续调控该种乃至其他海洋无脊椎动物的附着变态提供新的思路，最终为实现研发生态友好型防污新技术奠定良好基础。

厚壳贻贝是研究受体调控幼虫变态发育的大型附着模式生物。针对调控厚壳贻贝发育的G蛋白偶联肾上腺素能受体和5羟色胺受体的作用机制尚不清楚的问题，本项目从分子生物学和药理学水平开展了G蛋白偶联受体对厚壳贻贝发育的调控机理研究工作。我们发现5-羟色胺类受体激动剂能有效促进厚壳贻贝幼虫变态发育，其受体抑制剂能够有效抑制激动剂对幼虫变态诱导作用，表明5-羟色胺受体可能参与幼虫变态发育过程。利用PCR技术从厚壳贻贝cDNA中获得5-羟色胺2A受体（5-HT2AR）基因以及α2-肾上腺素受体（α2AR）基因全长序列。通过mRNA水平分别检测受体基因在厚壳贻贝雌、雄成体各组织的绝对表达以及幼虫变态前后发育阶段的绝对表达。研究发现5-HT2AR广泛存在于贝雌、雄成体组织、幼虫和稚贝发育阶段，表明5-HT2AR是该种可能是厚壳贻贝基本生理活动的必需受体，且参与调控幼虫的变态发育。在α2AR的研究中，我们同样证明α2AR广泛存在于厚壳贻贝雌、雄成体组织、不同发育阶段幼虫和稚贝体内；特别是，α2AR的mRNA水平在血淋巴组织表达最高，且在幼虫变态发育前后表达差异显著，推测α2AR可能通过免疫信号通路调控厚壳贻贝幼虫变态发育。我们通过RNAi技术对α2-肾上腺素受体基因进行靶向沉默，发现幼虫变态发育受到明显的抑制，证实了α2AR参与调控厚壳贻贝幼虫变态发育。通过比较蛋白质组学分析不同发育阶段幼虫蛋白质、以及肾上腺素等作用于感受态幼虫前后蛋白质组的变化。研究发现这些蛋白主要涉及到肾上腺素信号通路及其上下游相关通路MAPK信号通路、cAMP信号通路等。靶向蛋白质绝对定量实验进一步证实了肾上腺素信号通路在厚壳贻贝变态中的调控作用。我们研究结果对于后续深入开展该种以及其他海洋无脊椎动物变态分子机制和研发新型海洋防污技术奠定基础。