INTERFERENCE WITH STEROID RESPONSE GENES IN LEPIDOPTERA

University Of Washington, Seattle WA

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Abstract

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.