| Microsporidia is a class of eukaryotic organisms that can be parasitic on vertebrates and invertebrates. As a common pathogen of fish, shellfish and economic insects,microsporidia has brought huge loss to agricultural production. Nosema bombycis is the first microsporidia found by human. The pebrine disease caused by Nosema bombycis is an important disease. It usually brings serious influence to sericulture production, because it is able to pass through the egg to the offspring and the diffusion rate is so fast. Up to now,although many studies have been made on the transmission rule and control techniques of Nosema bombycis, the infection mechanism has not been fully understood.When the microsporidia infect the host cells, spores must germinate firstly and then inject the infective sporoplasm into the host cell cytoplasm through the polar filament. Now,it is commonly believed that spores' germination and infection comprising a total of 5events, respectively:(1) spores' adhesion to host cells;(2) spores are activated;(3) spores' internal osmotic pressure increased;(4) the polar filament ejected;(5) sporoplasm were injected into the cytoplasm of the host cell though the polar filament. Germination is the prerequisite for the infection, it is important to study the mechanism of germination in order to prevent and control the disease. It is suggested that microsporidia may exist water channel protein(aquaporin), which can specifically transport the water from the out of spore to the inside of spore, causing the increase of osmotic pressure and making the spores germinate. This paper aims to explore whether there is an aquaporin in Nosema bombycis, and whether it is involved in the process of germination and infection.In the experiment, we found a gene named as NbAQP in Nosema bombycis by retrieving the microsporidiaDB and MIPModDB. Then we cloned and analyzed NbAQP gene of Nosema bombycis. Sequence analysis showed that NbAQP gene contains an ORF with 750 bp encoding a polypeptide of 249 amino acids, the protein has a molecular weight about 26.66 kD, the isoelectric point is 5.12, no signal peptide was found. The homology was more than 50% when it compared with 5 aquaporins of other microsporidia. NbAQP sequence contains six membrane domains and tertiary structure prediction analysis showed that it has six long helices and two short helices, each of two short helix extending a chain of amino acids, amino acid chain returns surround the starting point. Semi-quantitative RT-PCR was used to study the transcription of NbAQP gene after Nosema.bombycis infected the silkworms. The results showed that within seven days after the infection, thetranscription of NbAQP could be detected. Sequence analysis and transcriptional analysis showed that the NbAQP protein of Nosema bombycis has the conservative structure as the aquaporin. And it was able to be transcriped normally in the Nosema bombycis. There is an aquaporin in Nosema bombycis.Then we expressed the NbAQP protein of Nosema bombycis using eukaryotic expression system of Saccharomyces cerevisiae. NbAQP gene was cloned into eukaryotic expression vector which named pYES2-NTC, and the recombinant expression plasmid was constructed and transformed into yeast cells. Subsequently, protein expression was induced in the INVSc1 strains, using beta galactose. After that, the His-NbAQP recombinant protein bands of 31 kD were detected by Western-blotting. This shows that the NbAQP protein of Nosema bombycis can be expressed in the eukaryotic expression system,which provides the basis for the study of the orientation and function of the protein.Polyclonal antibody against the protein was prepared, and the location of NbAQP protein in the Nosema bombycis was analyzed by immunoelectron microscopy. The results showed that, the colloidal gold particles were loosely distributed in the spore wall and the membrane of the spore, while there was no distribution of colloidal gold in the same position when it was observed by electron microscope in the control group. This revealed that the NbAQP protein was mainly localized in the spore wall and the plasma membrane of the spores. And this has important significance to its function.We did antibody blocking experiments, in order to study whether the NbAQP protein is involved in the germination process of Nosema bombycis, it could or not affect the infection to the host. We detected the change of the spore germination rate, Results showed that the germination rate of the treated group which was incubated with polyclonal antibody for an hour was 16.58%, when it compared with the germination rate of the control group, which is 23.05%, the difference was significant(P<0.05). After incubation with specific antibodies, the inhibition rate of spore germination was about 28%. This shows that when NbAQP proteins have been blocked by its antibody, the spore germination rate can be reduced. It is proved that the NbAQP protein is indeed involved in the process of spore germination, and this result has important significance for the study on the germination and infection mechanism of Nosema bombycis. |
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