Molecular Analysis of Juvenile Hormone Action

保幼激素作用的分子分析

• 批准号: 7340781
• 负责人: SUBBA R PALLI
• 金额: $ 19.34万
• 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/7340781
• 关键词: Adult; Aedes; Agonist; Alkaline Phosphatase; Base Pairing; Binding; Binding Proteins; Biological; Biological Assay; Biological Metamorphosis; Cell Line; Cells; Code; Collection; Complementary DNA; Complex; Culicidae; D Cells; Development; Direct Repeats; Disease; Disease Vectors; Dose; Double-Stranded RNA; Drosophila melanogaster; Ecdysone; Ecdysteroids; Ecdysterone; Elements; Esterase Gene; Event; Food; Gene Expression; Genes; Hormone Receptor; Hormones; Human; Insect Control; Insecta; Intestines; Juvenile Hormones; Label; Lead; Length; Life Cycle Stages; Livestock; Luc Gene; Mediating; Medicine; Messenger RNA; Methods; Molecular; Molecular Analysis; Molting; Mosquito Control; Nuclear Protein; Nuclear Proteins; Personal Satisfaction; Phosphorylation; Play; Production; Promoter Regions; Protein Binding; Protein Dephosphorylation; Protein Kinase C; Proteins; RNA Interference; RNA chemical synthesis; Reporter; Reporter Genes; Reproduction; Research; Research Personnel; Resistance development; Response Elements; Role; Screening procedure; Signal Transduction; Switch Genes; System; Techniques; Time; Tissues; Vertebrates; Work; Yeasts; analog; base; drug discovery; ecdysone receptor; gene therapy; genome sequencing; hormone analog; hormone response element; insect disease; juvenile hormone esterase; mRNA Differential Displays; mutant; prevent; programs; receptor; response; therapeutic protein; vector control

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（申请人提供）：有些昆虫与我们争夺食物，传播人类和牲畜疾病，但其他昆虫是有益的。因此，对靶标特异性的昆虫控制方法的开发一直存在持续的需求。保幼激素和蜕皮激素是调节昆虫蜕皮、变态和生殖的主要激素。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。这项建议的主要目标是确定基因在JH行动中发挥关键作用。在JH-和20-羟基蜕皮激素（20 E）-应答性JH酯酶（jhe）基因启动子区鉴定的JH应答元件（JHRE）和蜕皮激素受体（EcR）缺陷的果蝇细胞系（L57）被用于开发可用于研究JH作用的分子基础的稳健系统。在L57细胞中，置于JHRE控制下的报告基因被JH诱导，并且JH诱导被20 E抑制。从L57细胞分离的核蛋白与JHRE结合，并且在EcR存在下20 E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JH诱导受JHRE调控的报告基因。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标是：（1）鉴定在JH作用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和酵母测定来鉴定参与JH诱导JHRE调节的报告基因的基因。将通过研究表达蛋白与JHRE和标记JH的结合，以及它们在D. melanogaster的发育，并对D.黑腹菌突变体缺乏已鉴定的基因。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。这些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，药物发现和治疗性蛋白质生产。