赤拟谷盗蛛毒素受体Latrophilin的鉴定及其生理功能与进化适应性分析

Latrophilin (LPH) not only involved in the occurrence of schizophrenia, attention deficit/hyperactivity disorder as well as other diseases among humans and mammals, and could transmit exogenetic signal of latrotoxins (LTXs) in vertebrates as well as alter the sensitivity to some neurological disorder drug. However, up to date, it is unclear about the function of LPH in invertebrates, the mechanism of diversification of its multiple functions between invertebrates and vertebrates, co-evolution of the receptor and ligands, and their mechanism of signal transduction. Thus, we are planning to identify the insect specific endogenous and exogenetic ligands or the interactive proteins for LPH in Tribolium castaneum through the deorphanization method of GPCRs and CoIP methods, respectively, and dissect out whether the insect LPH could transduce the signal of latrotoxins which show specific lethal to insects, and how to generate the specific adaptivity. Furthermore, we will demonstrate the function of LPH on the growth, development of Tribolium castaneum, and analysis how it regulates the neurotransmitters (Acetylcoline and Dopamine) and involves in the drugs (Neurological disorder drugs and Insecticides) sensitivities. Moreover, we will illustrate the downstream signal transduction network in the transcriptional level and the protein level. Thus, it could illustrate the function and the regulation mechanism of LPH in Tribolium castaneum, and reveal the species-specific evolutionary mechanisms between LPH and LTXs. Furthermore, it could play fundamental roles in revealing the interaction between LPH and insecticides, even to discover the novel bio-insecticides which may target for LPH in the furture.

蛛毒素受体Latrophilin (LPH) 不仅涉及人类与哺乳动物多动症、精神分裂症及成瘾等精神疾病的发生，还涉及到对外源蛛毒素的信号传递及多种药物的敏感性。但迄今对无脊椎动物LPH的功能及与脊椎动物LPH之间功能多样性形成、受体与配体的进化关系和信号传导机制研究不详。因此我们拟利用受体配体检测技术鉴定赤拟谷盗LPH的内、外源配体或通过免疫共沉淀鉴定其互作蛋白，并分析其能否介导特异性毒杀昆虫的蛛毒素信号而产生适应性。进而分析LPH对赤拟谷盗生长、发育的作用，及其对神经递质（乙酰胆碱、多巴胺）调控和药物（神经类药物与杀虫剂）效应之间的影响，并在转录水平和蛋白水平解析其下游信号传导网络。从而阐明赤拟谷盗LPH功能和调节机制，揭示物种特异性LPH受体与蛛毒素LTXs的进化机制，并为探讨LPH与杀虫剂的作用关系甚至发现新的生物杀虫剂靶标奠定基础。

蛛毒素受体Latrophilin (LPH) 不仅涉及人类与哺乳动物多动症、精神分裂症及成瘾等精神疾病的发生，还涉及到对外源蛛毒素的信号传递及多种药物的敏感性。进化分析发现数种昆虫LPH均发生了适应性选择，其中赤拟谷盗LPH有两个正选择氨基酸位点被发现，显示昆虫LPH的突变与其功能及自身的环境适应性有一定相关性。因此我们检测了赤拟谷盗LPH的内、外源可能配体，进而分析发现Tclph具有三种不同拼接本，它们在赤拟谷盗生长、发育与生殖功能中具有重要作用，其中以转录本Tclphb效应最显著。这些功能与cAMP/PKA，PLC/IP3和IS信号途径最相关的 foxo，plc和 pka 基因的表达密切相关，也影响Vg合成，进一步降低了dslph昆虫的生殖能力。.而杀虫剂克百威和敌敌畏等可以诱导 Tclph 的高表达，而晚期幼虫中的 Tclph 敲减也增加了赤拟谷盗幼虫对这些杀虫剂的敏感性。研究发现Tclph可以正调控Tcchat和负调控Tcace-1的转录，进而促进ChAT和抑制AChE的酶活，最终增加ACh含量而参与杀虫剂的药物敏感性。其药物敏感性上调也可以通过上调Tcest4基因的表达来补偿Tclph和Tcest6的低表达而实现。.表达谱分析鉴定出274个差异表达基因，我们也对其中的杀虫剂转运相关基因以及杀虫剂代谢相关基因等部分差异基因进行了qRT-PCR与RNAi功能验证，这些研究揭示Tclph参与了0- Ⅱ相的细胞解毒过程，初步阐明了赤拟谷盗LPH生长、发育与代谢解毒功能和调节机制，揭示物种特异性LPH受体的进化机制，这些结果为Tclph在赤拟谷盗中调控药物敏感性和生殖的分子机制提供了新的线索，为开发潜在的新的杀虫剂作用靶标，有害昆虫的免疫与防治研究提供新的理论依据及策略。