| Microsporidia, widely distributed in nature, are extremely ancient and highly specialized obligate intracellular parasites that infect a broad range of animals including vertebrates and invertebrates and are important pathogens for many economic insects such as Bombyx mori and Apis mellifera. The silkworm, Bombyx mori are also infected by Endoreticulatus besides Nosema bombycis. Endoreticulatus.sp Shengzhou is a micro microsporidia found in silkworm eggs, and this microsporidia can also infect so many kinds of other Lepidoptera insects such as Phthonandria atrilineata, Helicoverpa armigera and Prodenia litura besides Bombyx mori. There are different species of microsporidium which can bring infection to the silkworm, Bombyx mori. These microsporidium all have different hosts and internal cell environment and it is reasonable to presume that different species of microsporidium own different infection mechanism. It is a good method to make a precise identification on species of microsporidium by comparative study. In this research, we employed a sample method directly using purified spores suspension with a concentration of109spores/mL instead of genomic DNA as PCR templetes for amplifying complete rRNA genes of Endoreticulatus sp. Shengzhou, and studied its phyletic evolution of the isolate by comparing it with that of other microsporidia. The rRNA gene sequences of LSUrRNA, SSUrRNA, ITS and IGS are1757bp,1254bp,499bp and419bp, respectively. The complete rRNA gene sequences of the isolate is4,43lbp (GenBank number. JN792450) in length with43.35%G+C and the arrangement of the rRNA genes was reversed as LSU-ITS-SSU-IGS-5S. Morphological character and phylogenetic analysis based on the rRNA gene sequences indicated that Endoreticulatus sp. Shengzhou is closely related to Endoreticulatus genus, which provides some theoretical basis for inspection of the pebrine in silkworm eggs production.Though there exist different infection mechanism about different species of microsporidium, infection percentage on the silkworm, Bombyx mori is always effected by germination rate of microsporidia. It is reported that the spore wall proteins of Nosema bombycis play an important role in the process of spores germination and infection. Five kinds of spore wall proteins have been identified which contain two kinds of endosporal proteins (SWP25and SWP30) and three exosporal proteins (SWP5, SWP26, and SWP32) up todate. In the current study, we identified another endosporal protein SWP12with calculated molecular mass of25.56kDa and an isoelectric point (pI) of6.69through gene cloning, SDS-PAGE analysis, protein expression, polyclonal antibody production, indirect immunofluorescence antibody test, and immunoelectron microscopy analysis, the results indicated that this protein is localized to the endospore and the special polyclonal antibody has no obvious inhibition on adherence to host cells. The characterization of this novel spore wall protein from Nosema bombycis may facilitate our further investigation on the relationship between Nosema bombycis and its hosts.Infection of microsporidia on the silkworm, Bombyx mori is not only related to spore wall proteins, but also induce differential genes expression on host cells. At the same time, it is switch on the related immune genes that results in differential genes expression. However, there are few reports on differential expression genes of Bombyx mori after infection by Nosema bombycis. Differential expression genes of BmN cells after infection by N.bombycis were investigated, and ten differential expression genes induced or suppressed by N. bombycis in BmN cells had been identified. Based on blast analysis on the sequences of transcript derived fragments, these differentially expressed genes were divided into five categories such as protein synthesis, signal transduction on cells, transporting ATP synthase and NADPH participate in electron transfer through mitochondria respiratory chain, cell energy metabolism, cellular immunity; and unknown function protein. Identification of differential expression genes in BmN cells is helpful for revealing infect mechanism of Nosema bombycis on BmN cells and also helpful for making an illumination on mechanism of host-pathogen interactions.Akirin gene, one of the differential expression genes, is reported to be a new nuclear factor which was discovered in the innate immune response of Drosophila melanogaster and plays a critical role in the immune deficiency (Imd) pathway. In the present study, Akirin gene of Bombyx mori was identified by screening differential expression genes in BmN cells and was cloned with the method of RT-PCR. Analysis on sequence shows that its cDNA was564bp and encoded a hypothetical proein of195amino acids with a predicted molecular mass of21.77kDa and an isoelectric point (pI) of8.97. The hypothetical protein belongs to alkaline proteins without any signal peptide site. Sikmap software analysis indicated that Akirin gene is located on chromosome22of Bombvx mori, and microarray expression analysis demonstrated that the gene is expressed in a variety of organizations such as ovary, spermary, fat body and midgut. The recombinant protein was obtained by gene cloning and prokaryotic expression, and these assays made a foundation work for the further research on the immunity of Akirin gene in Bombyx mori. |
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