Molecular Analysis of Juvenile Hormone Action

描述（由申请人提供）：有些昆虫与我们竞争食物并传播人类和牲畜疾病，但其他昆虫是有益的。因此，开发针对特定目标的昆虫防治方法一直是一个持续的需求。幼体激素（JH）和蜕皮激素（ecdysteroids）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，它们代表了昆虫控制方法发展的有吸引力的目标。阻碍这一努力的是缺乏对JH作用的分子基础的理解。本提案的主要目的是确定在JH作用中起关键作用的基因。在JH-和20-羟基蜕皮激素（20E）响应的JH酯酶（jhe）基因启动子区发现JH应答元件（JHRE），并利用蜕皮激素受体（EcR）缺陷的果蝇黑ogaster细胞系（L57）建立了一个强大的系统，可用于研究JH作用的分子基础。在L57细胞中，JHRE控制下的一个报告基因被JH诱导，而JH的诱导被20E抑制。从L57细胞中分离的核蛋白在EcR存在的情况下与JHRE和20E结合，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结合的蛋白对于理解JH的作用至关重要。该提案的两个具体目的是：(1)确定在JH作用中起关键作用的基因；(2)确定鉴定的基因在JH作用中的作用。参与JH诱导JHRE调节报告基因的基因将通过RNAi和酵母测定来鉴定。鉴定的基因将通过研究表达蛋白与JHRE和标记JH的结合、D. melanogaster发育过程中它们的mRNA和蛋白的表达，以及分析D. melanogaster缺乏鉴定基因的突变体来进行鉴定。JH类似物被用于控制蚊子，但一些蚊子已经对这些类似物产生了耐药性。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。所鉴定的基因将有助于开发筛选方法，以鉴定新的昆虫病媒控制剂。这些受体也可以用于医学，通过开发基因开关，用于各种应用，如基因治疗、药物发现和治疗性蛋白质生产。

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.