Studies On Functional Genome Of Nosema Bombycis

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parastites. Nosema bombycis as one kind of microsporidia is the pathogen of perbrine disease, however, the mechanism of infection and pathogenesis remains unclear. In the life cycle, these parasites were mainly dependent on the nutrients and energy providing by the host. In recent years, many researches focus on the secretory proteinase, which can be used as an important virulence factor in pathogenic fungi. The subtilisin-like protease (SLP) as members of the family of serine protease is widely existed in bacteria, fungi, protists and other organisms. It reported that subtilisin-like protease is related to the pathogenicity, degradation and cell autophagy process. With the completion of genome sequence project of Nosema bombycis, it was found that subtilisin-like protease (SLP) also exist in this species. So we speculate that subtilisin-like protease in N. bombycis may participate in the process of invasion. Based on this, we identified two classes of subtilisin-like serine protease (Nbslpl, Nbslp2-1and Nbslp2-2) in the N. bombycis genome database. This thesis mainly surveyed the sequence features of Nbslp2and analyzed its transcription, the recombinant rNbSLP2was expressed and polyclonal antibody against rNbSLP2was prepared. Furthermore, the indirect immuno-fluorescence assay were employed to probe the subcellular localization of NbSLP2. Meanwhile, co-immunoprecipitation technology was used to screen interacted proteins of NbSLP2. The main results are summarized as follows: 1. Sequence characteristics and transcriptional analysis of Nbslp2in Nosema bombycisNbSLP2is composed of557amino acids, with the predicted molecular weight63.84kDa and pI7.29, respectively. NbSLP2has Peptidase_S8domain and a transmembrane domain in the C terminal of494～516amino acids. Four predicted glycosylation sites existing in NbSLP2suggested NbSLP2maybe a glycoprotein. In addition, we extracted the total RNA of midgut from silkworm which infected N. bombycis at1～10d to analyze transcription activity. The results showed that the transcript of Nbslp2was detected from p.i.72h, however, the quantity of this gene’s transcript had a little reduction with the extension of the infected time, suggesting that Nbslp2may play an important role in N. bombycis proliferation early phase.2. Prokaryotic expression, antibody preparation and Western blot analysis of the NbSLP2Full-length gene and sequence removed the membrane structure domain(Nbslp2and Nbslp2△TM) were amplified using specific primers, respectively. The fragment of Nbslp2△TM was inserted into the expression vector pET30a for prokaryotic expression. After purification. rNbSLP2△TM was used to inject mice to prepare polyclonal antibody. The titer was more than1:25600which determined by Enzyme-Linked Immuno Sorbent Assay (ELISA).The total proteins of N. bombycis were extracted by glass beads. Western blotting was performed with anti-NbSLP2△TM antibody. The result showed anti-NbSLP2△TM can hybride a specific band, which is consistent with the molecular weight of NbSLP2. To verify whether NbSLP2is a membrane protein or not, we used asynchronous extraction method to extract membrane proteins of N. bombycis. And the immunoblotting display a stronger hybridization signal compared with the band in total proteins, implying NbSLP2is most likely a membrane protein.3. Localization analysis of NbSLP2in N. bombycisIndirect immunofluorescence technique was applied to analyze the localization of NbSLP2in N. bombycis mature spore and the spore coat. The subcellular localization features are as follows:(1) Using anti-NbSLP2ATM to detect NbSLP2proteins, the results showed that the signals evenly distributed in the cytoplasm of the mature spores. To observe the difference localization of NbSLPl and NbSLP2, both two kinds of polyclonal antibodies against those proteases were used as the primary antibody. The fluorescence signals were spread in the whole spore. Merging two kinds of fluorescence, we found the distribution signals were overlapped in two ends of spores, which may suggest that they play a collaborative role together in spores.(2) In spore coats, NbSLPl and NbSLP2share similar localization that mainly existing in one end of the spore coat. Further, except the coincidence signal distribution in spore coat, the plasma membrane still has green fluorencence which represent the NbSLP2. In conclusion, NbSLP2is a membrane protein and could be secreted to the anterior end of spores, possibly participating polar tube extrusion process.4. Detection of NbSLP2in BmE cell infected by N. bombycisHere we used indirect immunofluorescence technology to detect the NbSLP2expression in the BmE cells infected by N. bombycis spores. The results showed that in the infected cells, fluorescent signal distributed in spore inside and around the infected cells, which may indicate NbSLP2is not only a membrane protein, but also can secrete into the host cell. Double immunofluorescence assay showed NbSLP2share part of same localization with NbSLPl in the infected cells, and this result suggest that the process of invasion to host cell for proliferation may need both those proteases. Western blotting results revealed NbSLP2existed in the supernatant proteins of the infected BmE cells, further confirming NbSLP2could secret to the host cell.5. Functional analysis of NbSLP2in N. bombycisTo identify the protein NbSLP2interacted, the co-immunoprecipitation technique combined with mass spectrometry were adopted. After co-immunoprecipitation with total proteins of N. bombycis, the specific band was cut from the gel and subject to linear ion trap quadrupole mass. Two candidate target proteins,3-phosphoric acid glycerol dehydrogenase and septin were detected. Considering the coverage, size and molecular weight of the peptides, the septin is thought to be reliable. It reported that septins are involved in the cytokinesis in most eukaryotes and mammals. In yeast, Septin locate in budding neck. To detect the localization of Nbseptin, the expression of Nbseptin-GFP fusion protein in yeast cells were carried out and Nbseptin-GFP could be sorted to budding neck of yeast, suggesting Nbseptin can locate to the budding neck of yeast, In addition, we found that Nbseptin was recruited along with the budding yeast. However, the interaction mechanism between Nbseptin and NbSLP2is still need to explore in next step.