Studies On The Functional Genome Of Nosema Bombycis

Microsporidia are intracellular eukaryotic parasites, can infect nearly all invertebrates to vertebrates, including some important economic animals, such as shrimp, silkworm, fish, and human beings. Microsporidia can infect the economic silkworm and cause pebrine disease, resulting in big economic losses every year in sericulture countries. When scientists found that the microsporidia can infect human, more and more people start to pay attention to these small parasites. With several microsporidia genomes having been published, the research of microsporidia entered the stage of functional genomics era.Nosema bombycis (N. bombycis) is a typical species of Nosema genus and is the first microspridia identified by scientist. By genome sequencing, we predicted more than 4400 genes in N. bombycis genome. Comparative genomics analyses indiated that the serine protease inhibitor (serpin) only exists in microsporidia of Nosme genus. In the N. bombycis genome, there are 19 serpin genes,15 serpins among of them have more than 200 amino acids.Serpins are a class of proteins with serpin domain, play important roles in living beings, serving as protease inhibitor or functioning in energy transportion, protein storage processes. To explore the function and localization of microsporidia serpin, we choose NbSPN13 as the target to analysis its’transcription, expression, subcellular localization in Saccharomyces cerevisiae and spore of N. bombycis. The results are as following:1. The sequence characteristics and transcriptional analysis of NbSPN13.We retrieved the complete sequence of NbSPN13 from genome database of N. bombycis and found that it has six N-glycosylation sites and five crystine residues, without O-glycosylation site and GPI modificative site. These resultes suggests that the NbSPN13 may be N-glycosylation modified and contain disulfide bonds. The result of multiple sequence alignment of NbSPN4, NbSPN13, NbSPN18 and NbSPN19 showed a high identical signal peptide sequence. By the comparison of signal peptide of NbSPN13 with some organelles’ leading peptides, we found these signal peptides have similarity of 35-40% on their amino acid sequences. Subsequently, the subcellular localization prediction result of the NbSPN13 demonstrates that it may be leaded to endoplasmic membrane or mitochondria. In order to get the transcription activity analysis of NbSPN13, We collected the silkworm midguts infected by N. bombycis from 1d to 10d post infection, the results of transcription of NbSPN13 illustrates that this gene was transcribed from 1d to 10d post infected by N. bombycis.2. Prokaryotic expression and antibody preparation of NbSPN13We cloned the△NbSPN13 (without codons of signal peptide) sequence into pGEX vector, and then transformed this recombinant vector into Rosetta srtain of E. coli. With the inducing reagent IPTG treatment, the GST-△NbSPN13 was expressed in inclusion body. We purified the proteins by splicing the gel, and give mice immunization with the mixture of GST-△NbSPN13 and adjuvant. After the fourth immunization, we get the serum of mice with the method of eyeball blood collection. In agarose diffusion test of antibody, we found the serum and antigen can produce precipitation line at 32 times dilution of antibody. The result shows that our serum against NbSPN13 can meet experiment’s requirment subsequently. In the Western blotting of the antibody incubated with total protein of mature spores, we found there are two bands with signal, and the molecular weight of these two protein bands were bigger than the expected size, so we speculated that the NbSPN13 may undergo protein modification as predicted by the sequence analysis.3. Localization analysis of the NbSPN13 protein in yeast and N. bombycis As the model of unicellular eukaryotes, Saccharomyces cerevisiase is a useful tool to explore the heterologous protein localization. Here we also selected Saccharomyces cerevisiae to check the sub-cellular localization of NbSPN13 protein. The construction of the pUG35-NbSPNl3 and pUG35-ANbSPNl3 vectors can fuse protein of NbSPN13 and ANbSPNl3 with GFP (NbSPNl3-GFP and ANbSPNl3-GFP) in yeast. After transformed the pUG35-NbSPNl3 and pUG35-△NbSPNl3 into Saccharomyces cerevisiae by electric shock, the fusion protein of NbSPN13-GFP and ANbSPNl3-GFP were expressed and we observed the green light signal and judged the localization of the fusion protein is in mitochondria. It was reported that serpins of human being can be lead to mitochondria and was involved in apoptosis of cancer cells. At the same time, the indirect immunofluorescence assay by using the antibody of NbSPN13 and mature spores of N. bombycis. The result illustrates that the NbSPNl3 localized at peripheral part of N. bombycis. The localization of NbSPNl3 made a good basis for further work on the function of NbSPN13.