MOLECULAR BASIS OF ECDYSTEROID ACTION IN THE MOSQUITO

这项建议旨在描述在成年蚊子中介导20-羟基蜕皮酮(20E)作用的调控体系。在这个项目的头五年里，我们对20E在蚊子卵黄发生中作用的分子基础有了基本的了解。现在，我们可以问，这种蜕皮激素调节的层次结构是如何适应血餐的控制的。我们证明了蚊虫蜕皮激素受体复合体的DNA结合特性与其序列识别有关，并在伊蚊和按蚊基因中鉴定了自然反应元件。我们发现蚊子蜕皮激素受体(ECR)在识别蜕皮激素和20E方面具有独特的特性。我们计划阐明ECR蛋白中负责识别配体和异源二聚体的决定因素。我们将克隆超阿司匹林(USP)基因的两个转录单位，编码USP-A和USP-B亚型，并阐明20E如何在同一基因的不同部分发挥相反的作用。我们鉴定了蚊子E75，这是一个关键的早期基因，介导了20E的作用。三种E75亚型的表达与卵黄生成基因的激活和维持有关。我们计划阐明E75在卵黄生成基因的刺激下实现长期持续表达的分子机制。由于羽化后的发育，脂肪体和卵巢变得有能力对20E的激活做出反应。核受体FTZ-F1在能力获得中起重要作用。我们将研究该因子在20E早期和靶基因激活中的作用，以及保幼激素在获得卵黄素20E反应能力中的作用。控制卵黄生成基因激活的调控体系被阻断，从而确保了启动卵黄发生所需的血液供应。在被捕状态下，超阿司匹林(USP)与一种阻滞剂结合，HR38。我们将寻找维持这种异二聚体联系的特定信号。我们还鉴定了肾上腺素还原酶基因，该基因编码卵黄生成卵巢中表达的一种关键的类固醇合成酶，并将开始剖析其活性受蜕皮性神经肽刺激控制的机制。这项拟议研究的完成将使人们更深入地了解蜕皮类固醇在蚊子卵成熟调控中的作用，并可能导致开发新的蚊子控制方法。

This proposal aims to characterize the regulatory hierarchy mediating 20-hydroxyecdysone (20E) action in the adult mosquito. During the first five years of this project we achieved a fundamental understanding of the molecular basis of 20E action in mosquito vitellogenesis. Now, we can ask how this ecdysteroid- regulated hierarchy is adapted for control by a blood meal. We demonstrated the DNA binding properties of the mosquito ecdysteroid receptor complex with respect to its sequence recognition and identified natural response elements in the Aedes and Anopheles genes. We found unique properties of mosquito Ecdysteroid Receptor (EcR) with respect to recognition of ecdysone and 20E. We plan to elucidate determinants in the EcR protein responsible for recognition of ligands and heterodimerization. We will clone two transcription units of the ultraspiracle (usp) gene, encoding USP-A and USP-B isoforms, and elucidate how 20E exerts an opposite effect on different portions of the same gene. We characterized the mosquito E75, a key early gene mediating 20E action. Expression of three E75 isoforms correlates with activation and maintenance of vitellogenic genes. We plan to elucidate the molecular mechanisms through which the long-term sustained expression of E75 in conjunction with the stimulation of vitellogenic genes is achieved. The fat body and ovary become competent to respond to 20E activation as a result of post-eclosion development. A nuclear receptor FTZ-F1 plays an important role in competence acquisition. We will investigate the role of this factor in 20E activation of early and target genes, and the role of juvenile hormone in the acquisition of vitellogenic 20E response competence. The requirement of blood feeding for initiation of vitellogenesis is assured by the blockage of the regulatory hierarchy controlling vitellogenic gene activation. At the state-of arrest, Ultraspiracle (USP) is bound to a repressor, HR38. We will search for a specific signal that maintains this heterodimeric association. We have also identified the Adrenodoxin Reductase gene, encoding a key steroidogenic enzyme expressed in vitellogenic ovary, and will begin to dissect the mechanisms through which its activity is controlled by ecdysiotropic neuropeptide stimulation. Accomplishment of this proposed research will result in a deeper understanding of the action of ecdysteroids in the regulation of egg maturation in mosquitoes and may lead to the development of novel approaches to mosquito control.