Molecular analysis of juvenile hormone action

描述（由申请人提供）：一直存在开发靶标特异性昆虫控制方法的持续需求。保幼激素（JH）和蜕皮甾体（20-羟基蜕皮激素，20 E是最活跃的形式）是调节昆虫发育和生殖的主要激素。由于这些激素不存在于脊椎动物中，它们代表了开发昆虫控制方法的有吸引力的目标。阻碍这一努力的是缺乏对JH作用的分子基础的理解。在目前的资助期间，鉴定了7种蛋白质，它们是参与JH信号转导及其与20 E的串扰的多蛋白质复合物的组分。使用标记的JH进行的配体结合研究表明，这些蛋白质中没有一个以高亲和力与JH结合。虽然这些蛋白质可能参与JH作用及其与20 E的交叉作用，但它们都不是JH受体。寻找一个更好的系统，以研究JH行动没有干扰蜕皮甾类发现JH的生殖赤拟谷盗，一个理想的系统，以研究JH行动没有干扰蜕皮甾类。在这种昆虫中，JH在不受蜕皮激素干扰的情况下调节脂肪体中卵黄蛋白原基因的表达和卵母细胞对卵黄蛋白原的摄取。这两个系统提供了一个独特的机会，研究基因组（脂肪体）和非基因组（卵巢）的作用，在同一昆虫在同一时间。因此，更新申请提出研究JH在调节脂肪体中的卵黄蛋白原基因表达和卵母细胞中的卵黄蛋白原摄取中的作用机制，其主要目标是鉴定JH信号转导的关键受体、转录因子和蛋白质修饰剂。全基因组测序的完成、基因芯片的可用性、系统性RNA干扰（RNAi）的功能以及对JH及其类似物的良好反应使T. castaneum是研究JH作用的理想模式系统。在第一个具体目标中，将采用微阵列、RNAi、qRT-PCR、电泳迁移率变动分析和配体结合分析来鉴定和表征参与JH调控卵黄蛋白原基因表达的受体、转录因子和蛋白修饰物。在第二个具体目标中，将使用一组体内、细胞培养和体外测定来确定在T.卵母细胞对卵母细胞的卵黄蛋白原的吸收受JH的激活。JH类似物被用于蚊子控制，但一些蚊子已经对这些类似物产生了抗药性。这些研究的结果将有助于理解JH类似物的作用模式，从而促进其明智的使用。所鉴定的基因将有助于开发筛选测定法以鉴定新的昆虫病媒控制剂。所鉴定的受体也可以通过开发用于各种应用的基因开关而用于医学，例如基因治疗、组织工程和治疗性蛋白质生产。

DESCRIPTION (provided by applicant): There has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E is the most active form) are the major hormones that regulate development and reproduction in insects. Since these hormones are not present in vertebrates, they represent attractive targets for the development of insect control methods. Hindering this effort is the lack of understanding of the molecular basis of JH action. During the current grant period, seven proteins that are components of multi-protein complexes involved in JH signal transduction and its cross-talk with 20E were identified. Ligand binding studies performed using labeled JH showed that none of these proteins bind to JH with high affinity. While these proteins are likely involved in JH action and its cross-talk with 20E, none of them are JH receptors. Search for a better system to study JH action without interference from ecdysteroids discovered JH regulation of reproduction in Tribolium castaneum, an ideal system to study JH action without interference from ecdysteroids. In this insect, JH without the interference from ecdysteroids regulates vitellogenin gene expression in the fat body and vitellogenin uptake by the oocytes. These two systems provide a unique opportunity to study both genomic (fat body) and non-genomic (ovary) actions of JH in the same insect at the same time. Therefore, the renewal application proposes to study mechanism of JH action in regulation of vitellogenin gene expression in the fat body and vitellogenin uptake into the oocytes with a major goal of identifying receptors, transcription factors and protein modifiers critical for JH signal transduction. Completion of whole genome sequencing, availability of microarrays, functioning of systemic RNA interference (RNAi) and excellent response to JH and its analogs make T. castaneum an excellent model system for studies on JH action. In the first specific aim, microarray, RNAi, qRT-PCR, electrophoretic mobility shift assays and ligand-binding assays will be employed to identify and characterize receptors, transcription factors and protein modifiers involved in JH regulation of vitellogenin gene expression. In the second specific aim, a panel of in vivo, cell culture and in vitro assays will be used to determine whether any of the 51 G- protein-coupled receptors indentified in T. castaneum as those required for egg production and embryogenesis are activated by JH to regulate vitellogenin uptake into the oocytes. JH analogs are being used for mosquito control, but some mosquitoes are already developing resistance to these analogs. The results from these studies will aid in understanding the mode of action of JH analogs, thus facilitating their judicious use. The genes identified will be useful to develop screening assays to identify new insect disease vector control agents. The receptors identified can also be used in medicine by developing gene switches for various applications such as gene therapy, tissue engineering and therapeutic protein production.