| Microsporidia are unicellular,obligate intracellular,eukaryotic parasites,which have a broad host range.Pebrine disease is one of the important diseases in silkworm-mulberry industry,bringing serious losses to sericulture production.In recent years,the interaction between host and pathogen has become a hot spot.Silkworm as the host,its research on the response mechanism of microsporidia infection can help us to understand the strategy of coping with pathogen invasion.N.bombycis as the pathogen of silkworm,its infection mechanism has been widely concerne and research,germination process as an important part of infection,its research is of great significance.The increase in osmotic pressure in microsporidia is a prerequisite for spore germination.There are reports that aquaporins and trehalase may be the key proteins that promote osmotic pressure in microsporidia.Therefore,in this paper,the proteomics of the response of the BmN cells to microsporidian infection and the function of trehalase in N.bombycis was studied.The main results are as follows: 1.Proteomic Analysis of BmN Cells infected by N.bombycis based on iTRAQBy using N.bombycis in vitro infection-proliferation system,infected and uninfected BmN cells by N.bombycis for research materials,the protein was quantitatively labeled and quantified by iTRAQ in the infected group(6 h,12 h,24 h,48 h,96 h and 8 d)and the control group.The differentially expressed proteins were identified by GO and KEGG enrichment for functional analysis.A total of 33 513 polypeptide sequences and 4 872 protein sequences were retrieved from the original mass spectrometry data of the silkworm proteome database(http://www.uniprot.org/proteomes/UP000005204),of which 4 077 proteins that were reliable,and the total number of differential proteins in the infected and control groups was 1 597.In the early stages of infection,a large number of proteins showed different expression,indicating that BmN cells in the N.bombycis infection early infection has an efficient response process.In the mid-infection,the number of differentially expressed proteins was the largest,and the number of down-regulated proteins reached a peak,possibly due to the increased response of Bm N cells due to proliferative division of microsporidia.At the late stage of infection,the number of differentially expressed proteins began to decrease,possibly due to microsporidia entering the spore formation and BmN cells died in large numbers,and weakened their response.This suggests that BmN cells respond to the infection of N.bombycis to make protein expression regulation have a certain relevance with N.bombycis infection process.GO and KEGG enrichment analysis were used to explore the biological function and the biological pathways of differentially expressed proteins.The results of GO analysis showed that a large number of differentially expressed proteins were involved in biosynthesis,translation,protein modification and transport process,indicating that the infection of N.bombycis had a great influence on the physiology and development of the host,and the host need to synthesize large amounts of amino acids to cope with pathogen invasion.The results of the KEGG analysis showed that the infection of N.bombycis activated a large number of channel associated with innate immunity,such as: Endocytosis,ubiquitin-proteasome,Glutathione metabolism,Lysosome,Peroxisome,Phagosome,Oxidative phosphorylation,JAK-STAT signaling pathway,Regulation of autophagy and so on.Indicating that the host response to microsporidian infection is a complex regulatory process,and the host uses its own immune response mechanism to actively and comprehensively react against the invasion of microsporidia.2.Sequence analysis and preliminary function study of trehalase from N.bombycisThe N.bombycis used as the research material,based on microsporidian database,the bioinformatics analysis and preliminary function of trehalose in N.bombycis were studied by means of molecular biology techniques and methods.The results showed that four trehalase gene copies were found in the microsporidian genome database.By comparing the four amino acid sequences and other microsporidia homologous amino acid sequences,it was found that N.bombycis trehalase and other microsporidia trehalase amino acid sequence similarity is low.Select one of the more conservative sequences(NbTre1)for further analysis,NbTre1 did not have a signal peptide structure,and the subcellular localization was predicted to be located in the cytoplasm,eleven of the 14 phosphorylation sites were serine and threonine sites,suggesting that NbTre1 may be modified by post-translational phosphorylation and dephosphorylation to change their vitality.At the same time,Nb Tre1 phosphorylation and dephosphorylation reactions may also be involved in a certain degree of cell signaling and regulation of trehalose degradation.Through the construction of an expression vector pET28a-NbTre1 and transformed into E.coli Rosetta(DE3)competent cells to obtain NbTre1 protein expression strain and expression of NbTre1 fusion protein was induced with IPTG,the protein molecular weight size is consistent with the expected results.After purification by affinity chromatography,the fusion protein was used to immunize New Zealand white rabbits for preparing polyclonal antibody,then used ELISA to determine antibody titer was 1?25600,western blot showed that the obtained antibodies could be used to detect NbTre1 protein.The expression level of NbTre1 gene in different stages of silkworm infection was detected by qPCR.The expression of NbTre1 gene was detected in all time,and the expression of NbTre1 gene was higher in the early stage of infection,which indicated that the gene could be involved to the germination process of N.bombycis,the trehalase may be the key protein of microsporidia germination.The location of NbTre1 in microspores was analyzed by IFA using the prepared polyclonal antibody,the results showed that the NbTre1 protein is distributed within spores,but the specific location information remains to be further studied.The results of this study provide a good understanding of the response and interaction of silkworm to N.bombycis,and provide a preliminary understanding of the sequence and transcriptional characteristics and localization of NbTre1,which lays a foundation for further study of its function. |
PDF Full Text Request