Developmental Analysis of a Putative Juvenile Hormone Receptor

假定的保幼激素受体的发育分析

• 批准号: 9419774
• 负责人: Thomas Wilson
• 金额: $ 31.45万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9419774&HistoricalAwards=false
• 关键词: Developmental; Analysis; Putative; Juvenile; Hormone

Juvenile hormone (JH) is critical for insect development and reproduction. Although numerous whole-organismal studies of JH have been carried out, little is understood of the molecular action of this hormone. Using a genetic approach, this PI has been studying how JH acts at the molecular level in Drosophila melanogaster. Previous work led to the isolation of a mutant, Methoprene tolerant (Met), which has high resistance to JH or to methoprene, a JH insecticidal analog. Met flies were shown to have an intracellular JH binding protein, perhaps a JH receptor, possessing a reduced affinity for JH III. There is good evidence that this change in affinity is responsible for the resistance. Both the phenotype of Met resistance and the JH binding data suggest that the Met+ gene may encode the receptor. The DNA region containing the Met+ gene has been cloned, two transcripts have been identified, and cDNA molecules have been isolated and 80% sequenced. The work is now poised for a molecular developmental study of this gene and its product. Met+ cDNAs will be expressed to generate both fusion and non-fusion Met+ protein. Met+ fusion protein will be used to raise a polyclonal antibody specific for Met+ protein. This antibody will be used to identify Met+ protein in biochemical preparations as well as determine its intracellular localization. Particular attention will be given to possible interaction of the antibody with polytene chromosomes to determine if the Met+ protein interacts with chromosomes, presumably as a transcription factor. Authentic Met+ non-fusion protein will be generated for each of the two Met+ full-length cDNAs following expression in a baculoviral expression system. The ability of each of these proteins to bind JH III and JH bisepoxide will be evaluated. The hypothesis that Met+ encodes a JH binding protein will be directly tested in these experiments. Finally, in work designed to bridge to future studies of JH action in Drosophila, novel methoprene-resistant mutations on the second and third chromosomes will be isolated following FRT- FLP screening. The screen will be designed to identify loci whose gene product may act independently or may physically interact with the Met+ protein to confer resistance. This work will have significance in several ways: (1) the intracellular localization and JH binding of the Met+ gene product will be evaluated, leading to a better understanding of JH action; (2) novel loci involved in JH action may be identified; and (3) since Met mutants are resistant to JH insecticidal analogs, a better understanding of this resistance mechanism to these compounds will be gained. This knowledge may be applicable for resistance management of pest insects under control with JH insecticidal analogs.

保幼激素(JH)对昆虫的发育和繁殖至关重要。虽然已经对JH进行了大量的整体研究，但对这种激素的分子作用知之甚少。利用遗传学方法，这位PI一直在研究JH如何在分子水平上作用于黑腹果蝇。以前的工作导致了一个突变体的分离，耐甲氧丁二烯(Met)，它对JH或JH杀虫类似物甲氧丁二胺具有高抗性。MET果蝇细胞内有一种JH结合蛋白，可能是一种JH受体，对JH III的亲和力降低。有很好的证据表明，这种亲和力的变化是产生耐药性的原因。Met抗性的表型和JH结合数据都表明Met+基因可能编码该受体。含有Met+基因的DNA区域已被克隆，两个转录本已被鉴定，并已分离出cDNAs分子并进行了80%的序列测定。这项工作现在正准备对该基因及其产物进行分子发育研究。Met+cDNA将被表达以产生融合和非融合Met+蛋白。Met+融合蛋白将用于制备针对Met+蛋白的多克隆抗体。该抗体将用于鉴定生化制剂中的Met+蛋白并确定其在细胞内的定位。将特别注意抗体与多线染色体的可能相互作用，以确定Met+蛋白是否与染色体相互作用，可能是作为转录因子。在杆状病毒表达系统中表达后，将为两个Met+全长cDNA中的每一个产生真正的Met+非融合蛋白。这些蛋白质中的每一个与JH III和JH双环氧化物结合的能力将被评估。Met+编码JH结合蛋白的假设将在这些实验中直接得到验证。最后，在旨在衔接JH在果蝇中作用的未来研究的工作中，将在FRT-FLP筛选后分离第二和第三染色体上新的甲氧丁二烯耐药突变。该筛查将被设计用来识别其基因产物可能独立起作用或可能与Met+蛋白物理相互作用的基因座，以授予抗药性。这项工作将在以下几个方面具有重要意义：(1)Met+基因产物在细胞内的定位和JH结合，有助于更好地理解JH的作用；(2)可能会发现参与JH作用的新基因座；(3)由于Met突变体对JH杀虫类似物具有抗性，因此将更好地了解这些化合物的抗性机制。这一认识可能适用于JH杀虫类似物控制下的害虫抗性管理。