Transcriptome And Quantitative Proteomic Analysis Of Non-germinated And Germinated Spores Of Nosema Bombycis And Functional Analysis Of NbRBL Protein

Microsporidia are obligate intracellular parasitic fungi with over 1500 species in approximately 187 genera.Owing to the wide host range and special biological evolution taxonomic status and infection mechanism,microsporidia are attracted a lot of attention.Nosema bombycis.the first reported microsporidium.can transmit a highly mortal disease referred to as pebrine.which causes huge economic losses in the silk-producing industry in most nations around the world.N.bombycis utilizes spores germination for infecting Bombyx mori.The germination process.which is the inevitable process of infecting the host cell by microsporidian.is regarded as a key developmental turning point from dormant state to reproduction state.At present,there are few studies on the germination process of N.bomycis.The changes of spores in the molecular biology level during the germination period and the role on the subsequent invasion are limited known.To investigate the basis of the germination process,we performed a series of experiments and obtained research results as follow.1.we sequenced and analyzed the transcriptome of Nosema bombycis non-germinated spores(NGS)and germinated spores(GS).More than 60 million transcript high-quality reads were generated from two groups using RNA-Seq technology.After assembly,sets of 2756 and 2690 unigenes were identified and subsequently annotated based on known proteins.After differentially expressed genes(DEGs)analysis,66 genes were identified(P-value<0.05)between two groups.of which 57 were down-regulated and nine were upregulated.A protein phosphatase-associated gene was first identified as significantly up-regulated by RNA-Seq and immunoblotting analysis and it was indicated that dephosphorylation potentially contributed to germination.We also focused our attention on the DEGs that were involved in glycometabolism,spore wall proteins and ricin B lectin of Nosema bombycis.Gene ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses revealed biological functions and processes and the most enriched pathway were pentose phosphate pathway and amino sugar and nucleotide sugar metabolism pathways.The dataset generated in this study provides a basic characterization of the transcriptome changes in Nosema bombycis during the germination process.The analysis of transcriptome data and identified certain functional genes as robust candidates related to germination could help to make a deep-understanding for spores germination and invasion.2,To gain an in-depth understanding of the germination process,a label-free quantitative proteomic investigation was performed on N.bombycis non-germinated spores(NGS)and germinated spores(GS)with five biological replicates in each group.A total of 1136 proteins were identified,127 proteins being significantly changed(P-value<0.05)between the NGS and GS groups with 60 proteins being up-regulated and 67 proteins being down-regulated after the germination process.The quantitative proteomic analysis implied that SWP4 might play roles in germination and invasion.Additionally,bifunctional polynucleotide phosphatase/kinase and flap endonucleases 1 might help N.bombycis spores to resist host immunity and contribute to the proliferative phase.Combined with classifications and pathways analyses,the significant changes were observed in glycolysis and the pentose phosphate pathway after germination,with certain proteins related to transcription and translation showing significant differential regulation.This finding implies that changes in energy metabolism and protein synthesis in spore invasion and proliferation began during the germination process.This work provides the first in-depth proteomic analysis of the N.bombycis germination process and could be regarded as a crucial theoretical foundation for the overall understanding of microsporidia germination and invasion.Findings from this study pave the way for further quantitative studies at metabolomic levels.3,According to the results of transcriptome analysis,NbRBL gene was significantly down-regulated after spore germination process.We combined the related research results,and performed some experiments to study the protein function.The protein isoelectric point of NbRBL was 6.00 and the molecular weight of NbRBL was 57.7kDa.The signal peptide of NbRBL was from 1 to 16 in the protein sequence.NbRBL was predicted as secretory pathway.Twenty-one O-glycosylation sites and four N-glycosylation sites were predicted.The relative espression level of NbRBL gene was low at the first thirty hours after infecting to BmN cells.The relative espression level of NbRBL gene was high from 42 hours to 96 hours after infecting to BmN cells and reached the highest point at the 60 hours after infecting to BmN cells.Gene cloning,prokaryotic expression and antibody preparation experiments for NbRBL were performed.The specific bands in N.bombycis total proteins and secreted proteins after spore germination for NbRBL were detected using Western Blotting.With the help of indirect immunoinfluscent assay and confocal laser scanning microscope,the bright green fluorescence signal of NbRBL displayed a dispersed distribution in the spore.NbRBL could enhance the adhesion effect of spore to the BmN cells.The proliferative activity of BmN cells which were incubated with anti-NbRBL were higher than the control group at different time point after N.bombycis infection,the treatment groups were supported less damage from spore invasion.According to these results,it was implied that NbRBL protein could enhance the adhesion effect of spore to the BmN cells and make contribution to the spore invasion process,finally increase the infection efficiency.In summary,To gain an in-depth understanding of the germination process,transcriptome analysis and label-free quantitative proteomic investigation was successfully performed on N.bombycis non-germinated spores(NGS)and germinated spores(GS).Combining with the analysis of bioinformatics databases and Western Blotting、qRT-PCR results,dephosphorylation and SWP4 should play an important role in the process of spore germination;Glycolysis pathway and the pentose phosphate pathway were activated and made preparations for the spore invasion and proliferation.According to the results of sequence analysis,espression pattern at different development stage of spores,Western Blotting,CLSM location analysis,effects of NbRBL on the spores infecting BmN cells,NbRBL protein could enhance the adhesion effect and increase the infection efficiency.