家蚕硫激肽及其受体的鉴定与相关信号通路及生理功能研究

Insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, play critical roles in many physiological processes including food intake, locomotion, metabolism, growth and development, as well as learning and memory. However, the related molecular mechanisms remains largely unknown.The physiological functions of sulfakinins are mediated by its membrane receptors, belonging to G-protein coupled receptors. Activation of the receptor by the neuropeptide initiates a series of events which leads to changes of second messengers (Ca2+, cAMP) within the cell, and then trigger a series of molecular interactions that alter the physiologic state of the cell. So identification of sulfakinin receptors is essential for regarding the regulatory mechanisms underlying sulfakinin mediated signal pathways. In our preliminary studies, the gene coding a putative sulfakinin receptor in Bombyx mori, namely BNGR-A9, was cloned and functionally studied in the HEK293 and CHO cells. The BNGR-A9 expressing cells were activated by synthetic sulfakinin, resulting in increase in CRE-driven luciferase activity and intracellular Ca2+ level as well as receptor internalization. Our preliminary findings will provide a platform to investigate the nature of molecular mechanisms underlying sulfakinin-involved functions, also be helpful for understanding of novel physiological functions of sulfakinin in insects. Based on our existing research results, we look forward to conducting in-depth study of signal transduction mechanisms underlying sulfakinin-involved pathways, to clarify its physiological regulation of Bombyx mori. This project will give us better understanding of regulatory mechanisms and to explore the new physiological functions of sulfakinin peptides and provide theorical foundation for related research in other species.

硫激肽是一类与脊椎动物胃泌激素/肠促胰酶肽同源家族的神经肽，具有较为保守的C末端序列，在动物体内可能参与调控进食、运动、代谢、生长发育、学习记忆等重要生理过程。然而，目前对家蚕硫激肽及其受体及相关调控机制仍不清楚。前期研究中，我们克隆并利用异源表达系统发现，家蚕孤儿受体BNGR-A9 在受到人工合成硫激肽刺激后，CRE报告基因表达明显上升，胞内Ca2+水平亦发生显著变化，且能够诱导受体内吞，因此推断BNGR-A9可能是硫激肽受体。我们拟根据这一新发现利用生物学研究手段系统证实BNGR-A9为硫激肽受体，并在此基础上揭示家蚕硫激肽受体的信号转导机制，并从组织和个体水平探索其生理功能。本课题的开展不仅有助于了解家蚕神经肽及其受体调控机制，为改良家蚕养殖技术、提高经济价值提供理论依据，也可以为害虫的生物防治和人类相关生理活动的调控机制研究提供借鉴。

硫激肽是一类与脊椎动物胃泌激素/肠促胰酶肽进化同源的昆虫神经肽，在昆虫体内参与调控进食、运动、代谢、生长发育、学习记忆等多种重要的生理过程。申请人前期对家蚕硫激肽及其受体进行了系统进化和序列比对分析，发现家蚕孤儿神经肽受体BNGR-A9 （Bombyx mori neuropeptide G protein-coupled receptor A9）与已知的昆虫硫激肽受体具有较高的同源性和序列相似度，并对该受体进行了克隆和功能性检测，结果发现该受体能被人工合成的家蚕硫激肽所激活并引起胞内信号的响应。在此基础上，本项目通过家蚕BmN和HEK293细胞异源表达系统，对该受体进行了功能性鉴定与信号转导机制研究。通过CRE-荧光素酶报告基因系统和Furo2-AM法分别对受体活化引起的cAMP和钙离子水平进行了检测，发现BNGR-A9选择性地结合家蚕硫激肽(Bombyx mori sulfakinin，BmSK)，而对家蚕其他神经肽没有响应，而且磺酸化修饰的硫激肽活性远高于非磺酸化修饰的硫激肽，据此判断BNGR-A9是家蚕磺酸化硫激肽受体，将其命名为BmSKR（Bombyx mori sulfakinin receptor）。通过特异性抑制剂预处理，我们发现家蚕硫激肽介导的受体活化主要结合Gαq蛋白，并通过PLCβ/Ca2+/PKC通路激活MAPK/ERK信号通路。此外，本项目通过荧光定量PCR分析法检测了家蚕硫激肽受体BmSKR及硫激肽的时间与空间表达谱，发现家蚕硫激肽及其受体表达与家蚕进食周期呈相关性。因而，本项目进一步采用dsRNA介导的BmSKR表达下调，对家蚕的进食及代谢水平进行了研究，结果发现BmSKR表达下调后，家蚕的进食与体重均有显著提高，表明家蚕硫激肽可能通过其受体特异性发挥其饱腹因子的作用。同时，研究还发现注射硫激肽后，家蚕血淋巴中的海藻糖浓度明显提高，这种升高能被受体表达下调所抑制。因此推测BmSKR可能参与调控家蚕摄食与糖代谢等生理功能。本研究对家蚕神经肽sulfakinin及其受体的信号转导分子机制以及BmSK/BmSKR系统介导的生理功能进行了较为详细的研究，为进一步深入地、系统地理解家蚕神经肽及其受体介导的信号通路以及对家蚕生理功能的调控提供了实验依据。