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

利用保幼激素受体对蜕皮激素信号 II 的干扰进行鉴定

基本信息

批准号：
15580040
负责人：
MIURA Ken
金额：
$ 2.43万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

幼虫激素（JHs）与另一种激素——表皮甾体激素（Ec）共同调控昆虫发育和繁殖的方方面面。虽然在分子水平上对Ec作用的认识已经相当丰富，但对JH作用的分子模式仍然知之甚少。在本项目中，作者使用按蚊和果蝇进行了以下研究。作者在查阅有关JH对Ec信号干扰的文献的基础上，筛选出了JH受体的几个候选转录因子，并从按蚊（Anopheles Stephensi）中克隆了一个芳烃受体同源物（Ahr）。当转染到果蝇S2细胞时，Ahr激活了一个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不直接与XRE结合，这表明观察到的jh依赖性的Ahr转激活不是直接作用。在寻找S2细胞中与JH反应有关的转录因子过程中，作者克隆了果蝇耐甲氧丁烯（Met）基因，并对其产物Met进行了功能分析：Met与JH III结合，Kd为5.3 nM；在转染的S2细胞中，与酵母GAL4 DNA结合域融合的MET对具有QAL4 UAS的报告基因进行jh依赖性调控；无论是否存在JH， MET的定位都是核的。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。