Studies On The Functional Genome Of Nosema Bombycis

Microsporidia are a group of unicellular and obligated intracellular parasite. Microsporidia can virtually infect all animals, from invertebrates to vertebrates, and even including humans. Nosema bombycis is the pathogen of silk perbrine disease. Due to the vertical transmission, the infection and pathogenesis of N.bombycis can lead to devastating disasters on sericulture. So it has been the statutory quarantine in sericultrue countries.Lectins are closely related to important physiological processes, such as cell adhesion, bacterial infections, natural immune defenses, the engulfing and ingestion of bacteria, thus they play an extremely important role in life activity. We identified a broad existence of multicopied in microsporidia, Encephalitozoon cuniculi, Antonospora locustae, Enterocytozoon bieneusi, Nosema antheraea and Nosema ceranae, by bioinformatics analysis. However, the role of Ricin B-lectin super family in the microsporidia remains unclear.Here, we surveyed the sequence characterization, the second structure and subcellular localization of lectin NbRBL3, using of bioinformatics. Then, the gene of Nbrb13was cloned and expressed in vitro. The recombinant protein was purified, and the polyclonal antibodies against NbRBL3were producted. Subsequently indirect immunefluorescence assay was used to analysis the subcellular location of NbRBL3. Meanwhile, we employed immunoblotting analysis with spore coat protein, germination liquid protein, and the total proteins of BmE cell intected N. bombycis, to verify the secretion of NbRBL3. Furthermore, we gained the efficient expreesion and production of biologically active NbRBL3in BmE cell through the vector of Bombix mori Nuclear Polyhedrosis Viru (BmNPV). We want to investigate the effect on normal cell if the NbRBL3was over-expression. The main results are as follows:1. Sequence characteristics and transcription analysis of NbRBL3in Nosema bombycisNbRBL3is composed of204amino acids, its molecular weight is22.81kDa and isoelectric point is7.86. NbRBL3has a singnal in the N-terminal (1-14aa) and a ricin B-lectin domain (56-172). We find one N-glycosylatio site, sixteen phosphorylation sites, and four C-terminal microbody targeting signals. Subcellular localization prediction found that NbRBL3has none membrane localization signal, or GPI-membrane anchor points, but it has a tendency of exocytosis, which suggesting NbRBL3is a secreted protein. Secondary structure prediction result showed that NbRBL3protein has one a-helix,13β-sheets, and the rest are random coil. The structure is similar to the typical ricin B chains.Furthermore, we extracted the total RNA of midguts from silkworms infected by N. bombycis. RT-PCR showed that the transcript of Nbrb13was expressed from the first day to the tenth day.2. Prokaryotic expressing, polyclonal antibody preparing and Western bloting of NbRBL3We designed the specific primers of Nbrb13and amplified Nbrb13from genomic DNA of N.bombycis. Then the expression vector pET30-Nbrb13was constructed, so we transfered the vector into Rosetta. The recombinant protein were purified and used to produce polyclonal antibodies in mouses and rabbits. The total protein of N.bombycis were extracted and performed for western blotting analysis with anti-NbRBL3. We found the specific band of25kDa, which indicated the presence of NbRBL3protein in the mature N. bombycis, but it is larger than the predicted theoretical molecular weight of NbRBL3. May be NbRBL3was post-translationally modified.3. Subcellular localization of NbRBL3in Nosema bombycisIn order to detect the expression and location of NbRBL3in mature Nosema bombycis, indirect immunofluorencence assays was peformed. The results are as follows:Dispersive fluorescence was observed inside the sprores treated with2%SDS, while no singnal was examinated in the sterile water-treated spores. But the singnal was stronger when the spores were permeabilizated by1%Triton, while the weak signal was determined if the N. bombycis were treated with0.1M K2CO3in28℃for6h. Furthermore no signal was observed in the sprore coat.We obtained spore coats, using of glass beads vibration with Percoll density gradient centrifugation. Spore coats protein and germination liquid protein were extracted and determined with anti-NbRBL3, respectively. The result showed that anti-NbRBL3can hybride a specific band with germination liquid protein, while no specific band with sproe coats protein. In summary, NbRBL3located in the sporoplasm and maybe secreted outside following the spore germination.4. Detection of NbRBL3in BmE cell and haematocyte of silkworm infected by Nosema bombycisBased on proven location of NbRBL3in N. bombycis, we speculated this protein may be a secreted protein. Here indirect immuofluorescence was used to detect the expression of NbRBL3in BmE cell and haematocyte infected by N. bombycis, with the serum from mice and rabbit against NbRBL3. The confocal results showed that the fluorescent signal not only distributed inside the spores, but also in the surrounding of the infected cells. This result further implied that NbRBL3may be secreted outside the spore. Western blotting showed that the NbRBL3was detected in the supernatant protein of BmE cell infected N. bombycis, which also showed the NbRBL3, as a secreted protein, may played a role in the process of infecting and parasitizeing in the host cell.5. An Elementary Study on the function of NbRBL3The gene of Nbrb13was cloned and inserted into pFastBacHTB donor plasmid. Then the purified plasmid was transformed into DH10Bac-host cell, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-Nvrb13. The baculovirus expression vector was successfully constructed, with which would product the over expression of soluble and biologically active recombinant NbRBL3in the BmE cell. We could investigate the over expression of NbRBL3leading to cell agglutination. But the special fuction of NbRBL3required further research.