Developmental Analysis of a Putative Juvenile Hormone Receptor

保幼激素（JH）是昆虫发育和繁殖的关键激素。 虽然已经进行了许多JH的整体生物体研究，但对这种激素的分子作用知之甚少。 利用遗传学方法，本PI一直在研究JH如何在果蝇的分子水平上发挥作用。 以前的工作导致分离的突变体，耐甲氧普烯（Met），它具有高抗性JH或甲氧普烯，JH杀虫类似物。 Met果蝇被证明具有细胞内JH结合蛋白，可能是JH受体，对JH III具有降低的亲和力。 有充分的证据表明，这种亲和力的变化是耐药性的原因。 Met抗性表型和JH结合数据均表明Met+基因可能编码受体。 含有Met+基因的DNA区域已被克隆，两个转录本已被鉴定，cDNA分子已被分离并80%测序。 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。 表达Met+ cDNA以产生融合和非融合Met+蛋白。Met+融合蛋白将用于产生Met+蛋白特异性的多克隆抗体。 该抗体将用于鉴定生化制剂中的Met+蛋白以及确定其细胞内定位。 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，推测为转录因子。 在杆状病毒表达系统中表达后，将为两个Met+全长cDNA中的每一个产生真实的Met+非融合蛋白。 将评价这些蛋白质中的每一种结合JH III和JH双环氧化物的能力。 Met+编码JH结合蛋白的假设将在这些实验中直接测试。 最后，在设计用于连接JH在果蝇中作用的未来研究的工作中，在FRT-FLP筛选后将分离第二和第三染色体上的新的甲氧普仑抗性突变。 筛选将被设计为鉴定基因产物可能独立作用或可能与Met+蛋白物理相互作用以赋予抗性的基因座。 这项工作将在以下几个方面具有重要意义：（1）将评估Met+基因产物的细胞内定位和JH结合，从而更好地理解JH作用：（2）可能鉴定出JH作用的新位点;和（3）由于Met突变体对JH杀虫类似物具有抗性，将获得对这些化合物的这种抗性机制的更好理解。 这些知识可能适用于JH杀虫类似物控制下的害虫抗性管理。

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.