Molecular analysis of juvenile hormone action

描述(由申请人提供)：一直有对开发针对目标的昆虫控制方法的持续需求。保幼激素(JH)和蜕皮激素(20-羟基蜕皮酮，20E是最活跃的形式)是调节昆虫发育和繁殖的主要激素。由于这些激素不存在于脊椎动物中，它们是昆虫控制方法发展的有吸引力的目标。阻碍这一努力的是对JH作用的分子基础缺乏了解。在目前的资助期间，鉴定了7个蛋白质，它们是参与JH信号转导及其与20E的串扰的多蛋白质复合体的组成部分。用标记的JH进行的配基结合研究表明，这些蛋白都不能高亲和力地与JH结合。虽然这些蛋白可能参与JH的作用及其与20E的串扰，但它们都不是JH受体。寻找在不受蜕皮激素干扰的情况下研究JH作用的更好的系统，发现了JH对赤霉菌生殖的调节，这是研究JH作用而不受蜕皮激素干扰的理想系统。在这种昆虫中，JH在没有蜕皮类固醇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。这两个系统为同时研究JH在同一昆虫中的基因组(脂肪体)和非基因组(卵巢)作用提供了独特的机会。因此，更新申请提出研究JH在调节脂肪体内卵黄蛋白原基因表达和卵母细胞吸收卵黄蛋白原中的作用机制，主要目标是寻找JH信号转导关键的受体、转录因子和蛋白修饰物。全基因组测序的完成、微阵列的可获得性、系统RNA干扰(RNAi)的功能以及对JH及其类似物的良好响应使卡氏毛滴虫成为研究JH作用的良好模式系统。在第一个特定目标中，将使用微阵列、RNAi、qRT-PCR、凝胶迁移率改变分析和配基结合分析来鉴定和鉴定参与JH调节卵黄蛋白原基因表达的受体、转录因子和蛋白修饰物。在第二个特定目标中，将通过一组体内、细胞培养和体外测试来确定在卡氏锥虫中鉴定的51个G蛋白偶联受体中是否有任何一个被JH激活以调节卵黄蛋白原的摄取。JH类似物正被用于控制蚊子，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，从而促进它们的明智使用。确定的基因将有助于开发筛选试验，以确定新的昆虫疾病媒介控制剂。已识别的受体还可以通过开发用于基因治疗、组织工程和治疗性蛋白质生产等各种应用的基因开关而用于医学。

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.