Molecular Analysis of Juvenile Hormone Action

保幼激素作用的分子分析

• 批准号: 6867842
• 负责人: SUBBA R PALLI
• 金额: $ 20.4万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867842
• 关键词: Drosophilidae; RNA interference; biological signal transduction; ecdysone; gene induction /repression; genetic regulation; genetic regulatory element; hormone receptor; hormone regulation /control mechanism; juvenile hormones; messenger RNA; metamorphosis; protein binding; protein localization

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.

描述（由申请人提供）：一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，人们对靶标特异性的昆虫控制方法的发展一直存在。青少年激素（JH）和雌激素是调节昆虫中摩擦，变形和繁殖的主要激素。由于这些激素不存在于脊椎动物中，因此它们代表了开发昆虫控制方法的有吸引力的靶标。阻碍了这一努力是对JH作用的分子基础缺乏理解。该提案的主要目的是确定在JH行动中起关键作用的基因。 JH响应元件（JHRE）在JH-和20-羟基甲虫的启动子区域（20E） - 反应性JH酯酶（JHE）基因和ecdysone受体（ECR）缺乏Drosophila melanogaster细胞系（L57）用于开发一个可用于研究JH HORECS基础的强大系统。在L57细胞中，JH诱导了放置在JHRE控制下的报告基因，JH诱导被20E抑制。从L57细胞中分离出的核蛋白在存在ECR的情况下与JHRE结合，可以防止这种结合。在L57细胞中，ECR和DSP不直接与JHRE结合，也不直接参与JH诱导由JHRE调节的报告基因。鉴定与JHRE结合的蛋白质对于理解JH作用至关重要。该提案的两个具体目的是：（1）确定在JH行动中起关键作用的基因； （2）确定已鉴定基因在JH作用中的作用。将使用RNAi和酵母测定法鉴定出参与JHRE调节报告基因的JH诱导基因的基因。鉴定的基因将以研究表达蛋白与JHRE的结合并标记为JH，在D. melanogaster发育过程中表达其mRNA和蛋白质的表达，以及对鉴定基因缺乏的D. melanogaster突变体的分析。 JH类似物被用于蚊子控制，但是一些蚊子已经在对这些类似物产生抗性。这些研究的结果将有助于理解JH类似物的作用方式，从而促进其明智的使用。确定的基因将对开发筛选测定方法很有用，以鉴定新的昆虫疾病载体控制剂。该受体也可以通过为各种应用（例如基因疗法，药物发现和治疗性蛋白质的产生）开发基因开关来用于医学中。