Isolation And Purification Of A Regulatoral Factor For Diapause Hormone, Cloning And Expression Of The N-ethylmaleimide-sensitive Factor

Recently, man have made great progress in the reseaches of neuropeptides and focused their interest on the regulation of the known neuropeptides and the exploration of the new functional neuropeptides.On the one hand, we tried our best to find new functional neuropeptides. Primary reseaches showed that a factor in brain is necessary for the regulation of diapause hormone (DH) in the cotton bollworm, Helicoverpa armigera (Har). Using conventional column chromatography and HPLC, we finally isolated and purified the factor from the extracts of the pupal brain. MS-TOF analysis indicated that the molecular weight of this purified factor was about 1.1 kDa. In vivo injection of the purified factor demonstrated it could break diapause efficiently as DH, and in vitro experiment showed that the brain factor could effectively stimulate subesophageal ganglion (SG) to produce or release DH.On the other hand, we selected the N-ethylmaleimide-sensitive fusion protein (NSF) to begin our work in the regulation of neuropeptides. NSF is an essential component for the neurotransmitter or neurohormone release apparatus present in all eukaryotic cells. Here, a new NSF orthologue was characterized from H. armigera. Northern blot exhibited a high expression in larval brain. In situ hybridization further localized the Har-NSF mRNA in the molecular layers and the granule cell layers of the brain, SG, and other ganglia. Southern analysis indicated that a single copy of the gene is present in a haploid genome. Using antibodies labeled with fluoresceins, we directly proved that NSF is co-localized with prothoracicotropic hormone (PTTH) and DH. both of which regulate insect development. These findings suggest that Har-NSF may be involved in regulating insect neurohormone release. The developmental profiles of Har-NSF show that both mRNA and protein in pupal brain-SG complexes are significantly higher in nondiapause-destined individuals than in diapause-destined individuals. The expression patterns are consistent with those of PTTH and DH. These data suggest a central function of Har-NSF in developmental process through regulating neurohormone release.