Cloning, Expression And Functional Analysis From Helicoverpa Armigera SCP-2

H.armigera is a generalist herbivore with extremely high insecticides resistance. Furthermore, H. armigera has developed resistance to many insecticides including Bt. There is an urgent need for novel insecticides with a new mode of action.SCP-2protein interested us when exploring novel insecticides. In insects, cholesterol is needed for cellular membranes and ecdysteroid biosynthesis. However, insects lack key enzymes in the cholesterol synthesis pathway and therefore depend on dietary and/or symbiotic microbes to provide cholesterol for their physiological needs. This physiological constraint implies that cholesterol absorption/transport is particularly important for insects. As discovered mosquito SCP-2inhibitors that block cholesterol uptake in mosquito and Manduca larvae; Some mosquito SCP-2inhibitors are lethal to susceptible insects of Diptera, Lepidoptera and Coleopteran but have low mammalian toxicities; Resistant insect cell strains exhibited sensitivity to SCP-2inhibitors compared with susceptible cell strain. Therefore insect SCP-2offers a unique target for the development of insecticides.H. armigera has been used as an model system for our study of insecticides. The main result of this study are as follows:(1) HaSCP-x/2gene was identified firstly. HaSCP-x/2gene spans a total of1974bps and encodes an open reading frame (ORF) of536amino acids which included two domains.Homology search indicated that the N-terminal domain was highly homologous to2/3-oxoacyl-CoA thiolase, while the C-terminal domain was highly homologous to SCP-2protein in other species.(2)Exploited high quantity expression model of HaSCP-2protein in vitro. Identified PET22b and pGEX-KG vector could express high quantity of HaSCP-2protein. Provided sufficient protein for HaSCP-2three dimensional structure analysis and high throughput screening of compounds to identify chemical inhibitors of H. armigera.(3) The transcription profiles of HaSCP-x/2were determined by qRT-PCR. The levels of transcripts containing the HaSCP-2domain in the midgut was significantly higher than other examined tissues. Midgut is a major site for chlestrol absorption indicating that HaSCP-2may be involved in absorption and transfer of sterol in midgut.(4)HaSCP-x/2knockdown leading cholesterol of fatbody decreased57%and eggs produced per females decreased33%. There must be links among SCP-x/2gene,cholesterol accumulation and development in lepidopteran insects.(5) AeSCPIs are used as SCP-2inhibitors in biological assays of H.armigera larvae. AeSCPIs are lethal to younger larvae and reduce fecundity of elderly larvae.Our study identified SCP-x/2gene from H. armigera larvae, investigated it’s function by temporal and spatial expression profiles and screened chemical SCP-2inhibitors which intensively affected normal development in H. armigera larvae. We provided theoretical foundation of novel Lepidoptera target for the development of insecticides.