MOLECULAR BASIS OF ECDYSTEROID ACTION IN THE MOSQUITO

该建议的目的是表征调控层次介导的20-羟基蜕皮激素（20 E）的行动，在成年蚊子。在这个项目的前五年，我们实现了一个基本的了解的分子基础上的20 E行动在蚊子卵黄发生。 现在，我们可以问这个蜕皮激素调节的层次结构是如何适应血餐的控制的。 我们证明了蚊子蜕皮激素受体复合物的DNA结合特性，其序列识别和确定的伊蚊和按蚊基因中的自然反应元件。 我们发现了蚊子蜕皮激素受体（EcR）识别蜕皮激素和20 E的独特性质。 我们计划阐明负责识别配体和异源二聚化的EcR蛋白的决定因素。 我们将克隆两个转录单位的ultraspicacle（usp）基因，编码USP-A和USP-B亚型，并阐明如何20 E发挥相反的作用，对同一基因的不同部分。 我们的特点是蚊子E75，一个关键的早期基因介导的20 E行动。 三种E75亚型的表达与卵黄发生基因的激活和维持相关。我们计划阐明通过E75的长期持续表达与卵黄发生基因的刺激实现的分子机制。 脂肪体和卵巢成为主管响应20 E激活作为羽化后的发展的结果。 核受体FTZ-F1在能力获得中起重要作用。 我们将研究这个因素在20 E激活早期和靶基因的作用，以及保幼激素在获得卵黄发生20 E反应能力中的作用。 通过阻断控制卵黄发生基因激活的调控层次，确保了卵黄发生起始需要吸血。 在停滞状态下，超气门（USP）与阻遏物HR 38结合。 我们将寻找维持这种异二聚体结合的特定信号。 我们还确定了肾上腺氧还蛋白还原酶基因，编码一个关键的类固醇激素生成酶在卵黄发生卵巢中表达，并将开始剖析其活动是通过蜕皮神经肽刺激控制的机制。完成这项拟议的研究将导致更深入地了解蜕皮甾类激素在蚊子卵成熟调节中的作用，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.