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）共同调节昆虫的发育和繁殖。虽然在分子水平上的Ec作用的知识现在构成了一个大的身体，JH作用的分子模式仍然知之甚少。在本项目中，作者利用按蚊和果蝇进行了以下研究。作者在查阅文献的基础上，筛选出几种JH受体的候选转录因子，其中，作者从斯氏按蚊（Anopheles Stephensi）中克隆了一个芳香烃受体同源物（Ahr）。当转染到果蝇S2细胞中时，Ahr激活了带有二恶英响应元件（XRE）的模型报告基因的转录，以响应JH。然而，进一步的研究表明，Ahr不直接结合XRE，表明观察到的AH依赖性反式激活Ahr不是一个直接的行动。在寻找S2细胞中JH反应的转录因子过程中，作者克隆了果蝇耐甲氧普仑（Met）基因，并对其产物MET进行了功能分析：MET与JH Ⅲ结合的Kd值为5.3nM，与酵母GAL 4 DNA结合域融合的MET在转染的S2细胞中对具有QAL 4 UAS的报告基因具有JH依赖性调控作用;无论是否存在JH，MET均定位于细胞核。这些结果表明，MET可能作为JH依赖性转录因子，即JH受体发挥作用。

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.