Study On Apoptosis Of BmN-SWU1 Cell Of Bombyx Mori Induced By Ultraviolet Light

So far, It have been proved that cytc is released from mitochondria to cytoplasm in cell apoptosis induced by Ultraviolet light or baculovirus by the research group of Huangzhong Nomorl University.This result suggests that mitochondria play key role in apoptosis in insect cells like many mammalian cells.As one of the mode insects in biology, heredity and developmental biology, the research on apoptosis of Bombyx mori is still at the primary stage. Yet it is unclear that whether there is the same pathway in Bombyx mori or not .In this works, apoptosis induced by UVC were studied in BmN-SWU1 cells.The main results are as follows:1. The clone of Bmcytc and the bioinformation alaysiswe cloned the gene Cytochrome c in silkworm (Bombyx mori) and analyzed its sequence. The results shows that the length of its cDNA and ORF are 570bp and 324bp (ORF: 84-407) .The deduced protein is 108aa and deduced molecular weight is 11.7KD.It has a typical domain of Cytochrome c . And results obtained by using Clustal W show that the deduced protein shares 75 % identity with human, 83% with rat,75% with Drosophila(fly-d) and 88% with Drosophila (fly-p). The critical amino acids for Cytochrome c:Apaf-l interaction is conservative.Shares 75% identity with human, 83% with rat,75% with Drosophila(fly-d) and 88% with Drosophila (fiy-p). And the results of genome alaysis show that the gene contains 4 exons and they all conformed the GT-AG rule. Also the critical amino acids for Cytochrome c:Apaf-l interaction is conservative in Bombyx mori.2. Apotosis induced by UVC in BmN-SWU1 cellsMicroscopic examination of BmN-SWU1 cells dealled with UV revealed progressive cell blebbing starting at 12h p.i. and culminating in total cell destruction at 24h p.i..The fragmentation of induced cell nuclei was showed with Hoechest33258. gel electrophoresis analysis of DNA extracted from dealled cell showed typical DNA ladder. All these supported that UVC treated BmN-SWU1 cells undergo apoptosis.3. The change of mitochondrion in apoptosis of BmN-SWU1 cells induced by UVCUusing the flow cytometry assay, effort of UVC on mitochondrial membrane potential was invstigated by Rh-123 staining of cells. The result showed that the Rh-123 fluorescentintensity in BmN-SWU1 cells was reduced after treatment for 12h, and fluorescence peak shift left. The effects of UVC on apoptosis-related protein cytc were investigated by western blotting. Cytc was released from mitochondria to cytosolic after induction for 12h. And this result is idential with many mammalian cells?...