Studies On The Functional Genome Of Nosema Bombycis

Microsporidia are obligate intracellular, unicellular eukaryotic parasites, which generally exsit in the natural world. They can infect almost all vertebrates and invertebrates, including fish, rabbits and so on. Nosema bombycis is a parasite of Bombyx mori, which can cause destructive pebrine disease and lead serious losses for their vertical transmission. N. bombycis is set to be the only pathogen for quarantine in sericulture. So, preventing the pebrine is an important and difficult task for sericulture.The spore wall(SWP) is thought to firstly and directly interact with the host cell. So, they may play a key role in the process of microsporidia infection. Because of this, SWPs become a focus of research in microsporidian pathogenesis. Recently,13 SWPs were reported in microsporidia, and researches suggest that some SWPs can participate in microsporidia infecting process, but the infection mechanism is still unknown. The whole genome sequence project of N. bombycis has been finished, which provides the basis for studying on the SWPs of N. bombycis.In previous study, we developed two proteomic-based approaches, which were LC-MS/MS analysis and MALDI-TOF-MS analysis, to obtain 14 hypothetical spore wall proteins (HSWPs) from N. bombycis. In this study, we chose one of these genes, named as hswp14. According to the analysis of the N. bombycis protein sequences, one sequence is similar with HSWP14, defined as HSWP15. In our work, we have identified the hswpl4 and hswpl5 of N. bombycis with bioinformatics and RT-PCR analysis. Then we prokaryotically expressed the two hypothetical spore wall proteins and analyzed their localization in N. bombycis. Further functional analysis was carried out on the interaction between N. bombycis and the host B. mori. The main results are as follows:1. Sequences and transcription analysis of N. bombycis hswpl4 and hswp15We analyzed the spore wall proteins (SWPs) by proteomic-based approaches, LC-MS/MS and MALDI-TOF-MS. Fourteen hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. We chose the hswp 14 as tartget gene for research. Based on the analysis of the N. bombycis protein sequences, we found one sequence was similar with HSWP 14, named as HSWP 15. Preliminary sequences analysis with on-line prediction methods, HSWP 14 and HSWP 15 have N-glycosylation and phosphorylation sites, but lack O-glycosylation site and the heparin-binding motifs (HBMs).To detect the transcription level of hswpl4 and hswp15, silkworm were infected by N. bombycis spores at 4th instar, then the infected midguts were collected from the 1st day to the 10th day post-infection (p.i.). RT-PCR results indicated that hswp14 and hswp15 were both transcribed after N. bombycis infection. These results suggested that hswp14 and hswp15 may play roles during the process of infection and proliferation.2. Gene cloning and prokaryotic expression of N. bombycis hswpl4 and hswp15The hswp 14 and hswp 15 genes were cloned into the pCold I vector respectively. The fusion proteins of HSWP 14 and HSWP 15 were expressed in E. coli Rosetta strain and purified. Polyclonal antibodies were raised against HSWP14 and HSWP15 with mouse. Anti-HSWP 14 and anti-HSWP15 have immune reaction with the total proteins of N. bombycis by Western blotting, suggesting hswpl4 and hswp15 genes expressed in mature spores.3. Localization and function analysis of SWP14 and SWP15 from N. bombycisIn order to determine the cellular localization of HSWP 14 and HSWP 15, the mouse polyclonal antibody anti-HSWP14 and anti-HSWP15 were used for IFA. The fluorescence signals were detected not only in the spore wall of mature spores, but also on the spore coat. These results indicated that HSWP 14 and HSWP 15 were definitely spore wall proteins of N. bombycis.Finally, the spore adherence assays were conducted to expore the role of SWP14, SWP15 in infection process. After incubating the anti-serum of SWP14 and SWP15, with spores of N. bombycis respectively, we detected the spore adherence rate of BmE-SWUl, a cell line from Bombyx mori. Results indicated that SWP15 antibody could reduce N. bombycis spores adhering to the host cells significantly, while SWP14 serum was not effective in preventing spore adhering to the host cells.