无脊椎动物新型受体Parathyroid hormone receptor like (PTHRL) 的鉴定及其对赤拟谷盗表皮发育的调控

Parathyroid hormone receptor (PTHR) mainly regulates kidney and bone development in vertebrates. However, insects do not have these organs, but replaced by epidermis and fat body with similar functions. Meanwhile, the PTHR signal system has not been discovered in any model insects, such as Drosophila melanogaster, Bombyx mori and Anopheles gambiae. Whether this signal system is lost in invertebrates had been a mystery. Until recently, we have originally mined out the putative PTHR-like (PTHRL) receptors from Tribolium castaneum and several other insects. Knocking down the pthrls of T. castaneum resulted that most beetles were arrested before eclosion, and even though some beetles could eclosion into adults, they were with abnormal folded elytra and rougher epidermis than the control. The result indicated that the functions of pthrls have significantly differentiated between insects and vertebrates. And it is not clear whether the receptor and ligands of PTHRL have co-evolutioned in invertebrates, how these receptors participate in insect physiology, and what the mechanism of these signal transduction systems is. Thus we are planning to analyze the adaptive evolution of PTHR in the metazoa, and identify their potential endogenous ligands in T. castaneum by receptor-ligand assay. We will explore the regulation mechanism of PTHRLs in the epidermal development in T. castaneum, detect the interacting proteins for PTHRLs by co-immunoprecipitation, and illustrate the PTHRLs signal transduction pathway on the epidermal development as well as the downstream signal transduction network of PTHRLs by proteomics methods. And finally, we are going to further elucidate the function and the regulation mechanism of PTHRLs in the epidermal development in T. castaneum, and even to discover the novel bio-insecticides which may target for PT

甲状旁腺激素受体（PTHR）主要调节脊椎动物骨骼、肾脏等组织的发育，但昆虫中只有起类似功能的表皮和脂肪体。而且模式昆虫果蝇、家蚕、冈比亚按蚊等均无该信号系统，它是否在无脊椎动物中遗失曾是一个迷，最近我们才首次在赤拟谷盗等昆虫中发现了其类似受体（PTHRL）的存在，敲减该受体导致大部分赤拟谷盗不能顺利羽化，羽化的成虫翅膀粗糙且无法正常折叠。暗示该受体在昆虫与脊椎动物之间具有显著的功能差异性、而且昆虫中关于PTHRL受体与配体的进化关系、如何参与昆虫相关生理功能及信号传导机制尚不清楚。因此我们拟分析PTHR在后生动物中的进化适应性，利用受体配体检测技术鉴定其在赤拟谷盗中的潜在内源性配体，探讨其对赤拟谷盗表皮发育的影响机制，利用免疫共沉淀等实验鉴定其互作蛋白并通过蛋白质组学解析其下游信号传导网络，以阐明PTHRL的生理功能及其对表皮发育的调控机理，为开发特异性杀虫剂靶标提供基础。

神经肽类激素及其受体在昆虫中参与变态发育、求偶生殖、营养与能量代谢等各项生理活动。由弥散神经内分泌系统分泌的甲状旁腺激素 PTH 家族成员及其受体PTHRs在脊椎动物中主要参与调节体内钙和磷的代谢，并涉及骨骼、肾脏等组织的发育。无脊椎动物的PTHRLs被认为是脊椎动物 PTHRs 的同源对应物，继我们实验室在赤拟谷盗、意大利蜜蜂和体虱等昆虫中发现该受体之后，也在其它昆虫如褐飞虱中相继发现。然而，其配体却一直未见报道，通过本项目我们研究分析了脊椎动物、无脊椎动物的 PTHR/PTHRL的系统进化关系，揭示了它们之间独立发生复制与丢失事件，且无脊椎动物PTHRLs数目更多样化，并成功鉴定出昆虫PTHRLs的内源配体iPTH并对其进行了功能解析，利用多组学联合分析了该信号系统影响昆虫表皮形态建成的内在分子机制及信号调控网络，对部分下游基因也进行了功能验证，利用CodeML程序对无脊椎动物PTHRLs进行了适应性进化分析，筛选出21个多位于胞外近膜端及跨膜结构域的正选择位点。这些研究让我们对PTHRs/PTHRLs在后生动物中的进化模式有了更清晰的认识，解决了PTHRLs 在昆虫等无脊椎动物中仍是孤儿受体的现状，同时对该系统影响蜕皮羽化、表皮形态建成的关键基因进行了深度挖掘，已较为全面地阐明了该信号系统在昆虫生长发育过程中的功能及调控机理，可望对以赤拟谷盗、褐飞虱为例的多种害虫的防治提供理论依据，亦可为人类甲状旁腺激素信号系统失衡所造成的多种疾病提供参考。