Molecular Analysis of Juvenile Hormone Action

描述(申请人提供)：一些昆虫与我们争夺食物并传播人畜疾病，但其他昆虫是有益的。因此，一直有对开发针对目标的昆虫控制方法的需求。保幼激素(JH)和蜕皮激素是调节昆虫蜕皮、变态和繁殖的主要激素。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。这项建议的主要目标是确定在JH作用中发挥关键作用的基因。利用JH-和20-羟基蜕皮激素(20E)反应性JH酯酶(JHE)基因启动子区域的JH反应元件(JHRE)和蜕皮激素受体(ECR)缺陷的黑腹果蝇细胞系(L57)，建立了一个可用于研究JH作用的分子基础的可靠体系。在L57细胞中，JHRE控制的报告基因被JH诱导，而JH的诱导被20E抑制。从L57细胞中分离的核蛋白与JHRE结合，在ECR存在的情况下，20E可以阻止这种结合。在L57细胞中，ECR和DSP不直接与JHRE结合，也不直接参与JHRE调控的报告基因的JH诱导。鉴定与JHRE结合的蛋白质对于理解JH的作用至关重要。该提案的两个具体目标是：(1)确定在JH作用中起关键作用的基因；(2)确定已识别的基因在JH作用中的作用。JH诱导JHRE调控报告基因的相关基因将通过RNAi和酵母菌鉴定。通过研究表达的蛋白与JHRE和标记的JH的结合、它们的mRNA和蛋白质在黑腹果蝇发育过程中的表达以及对缺乏已鉴定基因的黑腹果蝇突变体的分析，将对已鉴定的基因进行鉴定。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。通过开发用于基因治疗、药物发现和治疗性蛋白质生产等各种应用的基因开关，这些受体还可以用于医学。

DESCRIPTION (provided by applicant): Some insects compete with us for food and transmit human and livestock diseases, but other insects are beneficial. Therefore, there has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids are the major hormones that regulate molting, metamorphosis 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. The main objective of this proposal is to identify genes that play critical roles in JH action. JH response elements (JHRE) identified in the promoter region of JH- and 20-hydroxyecdysone (20E)-responsive JH esterase (jhe) gene and ecdysone receptor (EcR) deficient Drosophila melanogaster cell line (L57) were used to develop a robust system that can be used for studying the molecular basis of JH action. In L57 cells, a reporter gene placed under the control of JHRE is induced by JH, and the JH induction was suppressed by 20E. The nuclear proteins isolated from L57 cells bind to JHRE and 20E in the presence of EcR can prevent this binding. In L57 cells, EcR and DSP do not bind directly to JHRE and are not directly involved in JH induction of a reporter gene regulated by JHRE. Identification of proteins that bind to JHRE is critical for understanding JH action. The two specific aims of the proposal are: (1) to identify genes that play key roles in JH action; and (2) to determine the role of identified genes in JH action. The genes that are involved in JH induction of JHRE regulated reporter gene will be identified using RNAi and yeast assays. The identified gene will be characterized by studying the binding of expressed proteins to JHRE and labeled JH, expression of their mRNA and proteins during D. melanogaster development, and analysis of D. melanogaster mutants deficient in identified genes. 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 can also be used in medicine by developing gene switches for various applications such as gene therapy, drug discovery and therapeutic protein production.