Developmental Analysis of a Putative Juvenile Hormone Receptor

幼虫激素（JH）是昆虫发育和繁殖的重要激素。尽管对JH进行了大量的全身研究，但对这种激素的分子作用知之甚少。利用遗传方法，该PI一直在研究JH在果蝇的分子水平上如何起作用。先前的工作导致分离出一种突变体，耐甲氧二烯（Methoprene resistant, Met），它对JH或JH杀虫剂类似物甲氧二烯（Methoprene）有很高的抗性。研究表明Met蝇具有细胞内JH结合蛋白，可能是JH受体，对JH III具有较低的亲和力。有充分的证据表明，这种亲和力的变化是导致耐药性的原因。Met抗性的表型和JH结合数据都表明Met+基因可能编码受体。已经克隆了含有Met+基因的DNA区域，鉴定了两个转录本，并分离了cDNA分子并进行了80%的测序。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。Met+ cdna将表达生成融合和非融合Met+蛋白。利用Met+融合蛋白制备针对Met+蛋白的多克隆抗体。该抗体将用于生化制剂中Met+蛋白的鉴定以及确定其在细胞内的定位。将特别关注抗体与多染色体的可能相互作用，以确定Met+蛋白是否作为转录因子与染色体相互作用。在杆状病毒表达系统中表达后，两个Met+全长cdna中的每一个都将产生真实的Met+非融合蛋白。这些蛋白结合JH III和JH二环氧化物的能力将被评估。Met+编码JH结合蛋白的假设将在这些实验中得到直接验证。最后，为了进一步研究JH在果蝇中的作用，我们将在FRT- FLP筛选后分离出第二和第三染色体上的新型甲氧丁烯抗性突变。筛选将用于识别基因产物可能独立起作用或可能与Met+蛋白物理相互作用以赋予抗性的位点。这项工作将有几个方面的意义：(1)将评估Met+基因产物的细胞内定位和JH结合，从而更好地了解JH的作用；(2)可能发现与JH作用有关的新位点；(3)由于Met突变体对JH杀虫类似物具有抗性，因此可以更好地了解其对这些化合物的抗性机制。这些知识可能适用于用JH杀虫类似物控制的害虫的抗性管理。

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.