Identification of the juvenile hormone receptor using its interference with ecdysone signaling II

保幼激素(JHS)与另一种激素--蜕皮激素(EC)共同调节昆虫发育和繁殖的各个方面。虽然在分子水平上关于EC作用的知识现在已经构成了一个很大的整体，但JH作用的分子模式仍然知之甚少。在本项目中，作者以按蚊和果蝇为研究对象进行了以下研究。在查阅有关JH对EC信号干扰的文献的基础上，作者筛选了JH受体的几个候选转录因子，其中，作者从斯氏按蚊中克隆了一个芳烃受体同源基因(Ahr)。当转染果蝇S2细胞时，AhR激活了携带二恶英反应元件(XRE)的模型报告基因的转录，以响应JHS。然而，进一步的研究表明，AhR并不直接与XRE结合，表明AhR对JH的依赖反式激活不是直接作用。在寻找负责S2细胞JH反应的转录因子的过程中，作者克隆了果蝇耐甲基丁二烯(Met)基因，并对其产物进行了功能分析，MET：MET与JH III结合，Kd为5.3 nM；MET与酵母GAL4 DNA结合域融合，对S2细胞中具有QAL4 UAS的报告基因实施JH依赖性调控；MET的定位是核的，无论有没有JH。这些结果提示MET可能是一种JH依赖的转录因子，即JH受体。

The juvenile hormones(JHs) regulate a wide aspect of insect development and reproduction in combination with another hormone, ecdysteroid(Ec). While the knowledge on Ec action at molecular level now constitutes a large body, the molecular mode of JH action is still poorly understood. In this project the author performed the researches below using Anopheles mosquitoes and Drosophila. Based on some literatures that describe the interferences by JH with Ec signaling, the author selected several candidate transcription factors for JH receptors, Among them, the author cloned an arylhydrocarbon receptor homologue(Ahr) from the mosquito, Anopheles Stephensi. When transfected into Drosophila S2 cells, Ahr activated the transcription from a model reporter gene bearing dioxin response elements(XRE) in response to JHs. Further studies, however, revealed that Ahr does not bind directly to the XRE, suggesting the observed JH-dependent transactivation by Ahr is not a direct action. During the course in search for transcription factors responsible for JH responses in the S2 cells, the author cloned the Drosophila Methoprene-tolerant(Met) gene, and performed functional analysis on its product, MET : MET bound to JH III with a Kd of 5.3 nM ; MET fused to yeast GAL4 DNA binding domain exerted JH-dependent regulation on a reporter gene having QAL4 UAS in the transfected S2 cells ; localization of MET was nuclear irrespective of the presence or absence of JH. These results suggest that MET might function as a JH-dependent transcription factor, namely the JH receptor.