棉铃虫神经肽促咽侧体素GPCR的鉴定及新型配体筛选

Neuropeptides and their G-protein coupled receptors (GPCRs) play key roles in regulation of insect growth, development, reproduction, nervous transduction, metabolism, behavior and other aspect of life, which can be used as a target model to develop novel agents for pest control. Insect allatotropin can stimulates JH synthesis in the corpora allata, which can regulate insect development. In the present research, the full length of cDNA of the gene enconding allatotropin G-protein coupled receptor in Helicoverpa armigera (Har-ATR) will be cloned and sequenced. Based on the cell model, the binding activity of ATs to ATR will be detected, and the best ligand to Har-ATR will be decided. The second message of signal pathway of AT to ATR will be illuminated in Helicoverpa armigera. The mimetic peptides or chemicals will be designed and synthesized based on the ligands to Har-ATR, which will be used to detect the ability of agonist or antagonist to Har-ATR. If showing high ability of agonist or antagonist, the mimetics will be selected as the candidate artificial ligands. The effects of candidate artificial ligands to insect will be clarified using injection of insect or in vitro assay. And then, the ligands those showed high effects will be selected as novel agents for pest control. The results of the present research provide the basic knowleges for developing novel pesticide to control the growth and development of Helicoverpa armigera. The platform of G-protein coupled receptor in the present research also can be used to screen the agonist or antagonist to other insect neuropeptide receptors, which is useful to the screening of green pesticides with a wide range and controlling the harmfulness of pests.

昆虫神经肽及其G蛋白偶联受体(GPCR) 在调控其生长、发育、生殖、神经传导、代谢以及行为等方面起重要的作用，是绿色农药设计重要的靶基因。昆虫的促咽侧体素(AT)具有促进JH合成等重要功能。本研究拟克隆棉铃虫神经肽促咽侧体素(Har-AT)的G蛋白偶联受体(Har-ATR)基因的全长序列，基于细胞模型明确其相应配体，明确棉铃虫AT与ATR作用的第2信使；设计和合成不同模拟AT结构的小分子化合物，分析其对棉铃虫ATR的激活活性或拮抗作用，筛选出棉铃虫AT的GPCR的高效结合的新型配体；采取直接注射或体外培育的方法，进一步调查高效结合的新型配体的生理作用，筛选出具有生理功能的人工新型配体。本研究为进一步把这些新型配体开发成控制棉铃虫生长发育的绿色农药建立基础；建立的G蛋白偶联受体研究平台，可以用来筛选昆虫其他神经肽受体的激活剂或拮抗剂，为高通量筛选绿色农药、控制害虫的危害建立基础。

昆虫神经肽促咽侧体素(allatotropin，AT)主要调控保幼激素的合成，进而调控昆虫的生长、发育及生殖。对棉铃虫的allatotropin的GPCR进行克隆和鉴定，为进一步把这些新型配体开发成控制棉铃虫生长发育的绿色农药建立基础。.克隆获得棉铃虫allatotropin(Har-AT)受体基因(Har-ATR)全长cDNA序列， ORF为1341bp，编码466个氨基酸。Har-ATR包含G蛋白偶联受体典型的7次跨膜结构域。Har-AT在胸腺中表达最高，其次是脑，在中肠与脂肪体中不表达。而Har-ATR在脑中表达量最高，在各组织均有表达。在脑组织中，Har-AT在蛹8天表达量最高，其次为蛹10天；Har-ATR在5龄幼虫第2天表达量最高，其次为蛹10天，与Har-AT发育表达模式稍有差别。.4种AT与HATR均引起细胞内Ca2+水平的增加，呈现特异性的响应关系。Har-AT、Har-ATL-I、Har-ATL-II、Har-ATL-III的EC50值分别为6.9364×10-5 mM、2.8744×10-7mM、1.3061×10-6 mM和2.38×10-3 mM。上述结果证明我们克隆的Har-ATR是Har-AT的受体，Ca2+是Har-ATR的信号通路中的第2信使；与Har-ATR结合活性最高的为Har-ATL-I的，其次是Har-AT和Har-ATL-II，Har-ATL-III最低。进一步检测配体与Har-ATR的结合激活，是否会导致细胞内cAMP水平的改变。配体和Har-ATR结合后cAMP响应不明显，因此cAMP并非Har-AT激活Har-ATR的主要第2信使通路，Ca2+是Har-ATR的信号通路中主要的第2信使。.合成3个配体的C端的5个氨基酸模拟肽（命名为AT、ATL1和ATL2），ATL1和HAR-ATR结合后引起的Ca2+的响应较弱，ATL1与Har-ATR结合的EC50值为2.16 ×10-3mM，与Har-ATLIII的结合活性相当，AT和ATL2与Har-ATR的结合更弱。因此，具有高结合活性的模拟肽设计和筛选工作还需要进一步开展。.注射后不同肽对棉铃虫幼虫的JH的合成均有促进作用，Har-ATL-I和Har-ATL-II效果最好，提高倍数达到1.8-1.92倍；其次为Har-AT，比正常对照达到1.5倍；Har-ATLIII与其