INTERFERENCE WITH STEROID RESPONSE GENES IN LEPIDOPTERA

干扰鳞翅目类固醇反应基因

• 批准号: 6490991
• 负责人: LYNN M RIDDIFORD
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490991
• 关键词: Lepidoptera; Manduca; antisense nucleic acid; complementary DNA; developmental genetics; ecdysone; gene expression; genetic promoter element; hormone receptor; hormone regulation /control mechanism; juvenile hormones; messenger RNA; metamorphosis; molecular cloning; molting; mutant; nucleic acid sequence; oligonucleotides; protein isoforms; skin; steroids; tissue /cell culture; transcription factor

The long-term goal of the parent grant is to understand how ecdysteroids and juvenile hormone (JH) act at the cellular and molecular level to coordinate insect molting and metamorphosis using the polymorphic epidermis of the tobacco hornworm, Manduca sexta, as a model system. We have characterized some of the major members of the ecdysteroid-induced transcription factor cascade that is activated during the molt including a switch in isoforms of the ecdysone receptor (EcR) and its partner USP (a RXR homolog), and how they are regulated by 20-hydroxyecdysone and JH. Our main specific aim in this FIRCA proposal is to determine whether the new technique of double stranded RNA interference will work in Lepidoptera to provide us with a means of studying the effects of loss-of-function of these various factors to ascertain their specific roles in molting and metamorphosis. We will also determine the role of the natural antisense RNA encoding a putative cuticle gene found in the 3' untranslated region of the ecdysone receptor gene. If the RNAi technique does not work, we will begin initial explorations of the use of transgenic Lepidoptera mediated by the piggyBac vector for ectopic expression of candidate genes or dominant negative forms at critical times in development. These studies should provide insight into the key roles of the switching of nuclear receptor isoforms during a well-defined developmental process and into the importance of the action of JH in regulating both the types and quantity of transcription factors induced by ecdysteroids for the prevention of metamorphosis. Since the effects of JH on metamorphosis in Lepidoptera is typical of most insects, the information gained also can provide a basis for the rational design of new insect growth regulators to control insect vectors of human disease and avoid possible side effects of these regulators or their environmental degradation products interfering with the endocrine and developmental roles of the RXR in vertebrates.

父母补助金的长期目标是了解蜕皮激素和保幼激素（JH）如何在细胞和分子水平上起作用，以烟草天蛾（Manduca sexta）的多形表皮为模型系统，协调昆虫蜕皮和变态。 我们已经确定了蜕皮激素诱导的转录因子级联反应的一些主要成员的特征，这些转录因子级联反应在蜕皮期间被激活，包括蜕皮激素受体（EcR）及其伴侣USP（RXR同系物）的亚型中的开关，以及它们如何受到20-羟基蜕皮激素和JH的调控。 在FIRCA的建议中，我们的主要具体目标是确定双链RNA干扰的新技术是否会在鳞翅目昆虫中起作用，为我们提供一种研究这些不同因素功能丧失的影响的方法，以确定它们在蜕皮和变态中的特定作用。 我们还将确定天然反义RNA的作用，该反义RNA编码在蜕皮激素受体基因的3'非翻译区发现的假定角质层基因。 如果RNAi技术不起作用，我们将开始初步探索利用piggyBac载体介导的转基因鳞翅目昆虫在发育的关键时期异位表达候选基因或显性阴性形式。 这些研究应提供深入了解核受体亚型的开关在一个明确的发展过程中的关键作用，并进入行动的JH在调节蜕皮激素诱导的转录因子的类型和数量，以防止变态的重要性。 由于JH对鳞翅目昆虫变态的影响在大多数昆虫中是典型的，所获得的信息也可以为合理设计新的昆虫生长调节剂提供基础，以控制人类疾病的昆虫媒介，并避免这些调节剂或其环境降解产物干扰RXR在脊椎动物中的内分泌和发育作用的可能副作用。