ITRAQ-based Comparative Proteomic Analysis Of Conidia And Mycelium From The Filamentous Fungus Metarhizium Robertsii

The filamentous fungus Metarhizium robertsii mainly have two different and typical developmental stages--conidia and mycelium.As the asexual reproduction unit,conidia plays an important role in its propagation and infect insects.Mycelium is nutritional intake unit of its vegetative growth stage.Comparative proteomics is a way to identify proteins,draw the mass spectra of the different proteins,explore the nature of biological phenomena by analyze the protein samples from different stages,different tissues,different states and the samples which in different external conditions.Technical means of comparative proteomics mainly include the traditional 2-DE(Two-dimensional gel electrophoresis)technology two-dimensional gel electrophoresis and the emerging high-throughput proteomics approach.However,the high-throughput quantitative proteomic study of Metarhizium has not been reported.Therefore,in order to further improve the effect of biological control of Metarhizium and explore the important and key metabolic and regulatory pathways in the M.robertsii conidia,comparative proteomics of M.robertsii conidia(10d)and mycelium(1d,as control)were analyzed by using i TRAQ high-throughput quantitative proteomic technology in the present study.The main results are as follows:1.The results of the identification and quantitation analysis show that: a total of 2129 proteins were identified,approximately account for 20% of total protein(2129/10581).Protein-species with > 1.5-fold difference in abundance and t-test p-value < 0.05(FDR <1%)was regarded as differentially expressed proteins(DEPs)in our data.Therefore,there are 239(184 up-regulated and 55 down-regulated)were differentially expressed between conidia and mycelia were detected compared with the sample of mycelium.These differentially expressed protein-species(DEPs)mainly included enzymes involved primary metabolic pathways and general(oxidative)stress responsive proteins,such as minor allergen Alt a 7(EFZ03492),heat shock protein 70-like protein(EFZ01368),subtilisin-like protease PR1H(EFY94277),and major proteins participating in biosynthetic and energy metabolism.2.Cluster of Orthologous Group(COG)analysis of all the identified proteins show that these proteins are mainly concentrated in 7 functional categories,such as energy production and conversion,amino acid transport and metabolism,carbohydrate transportand metabolism.These results indicate that the proteins in the conidia and mycelia are mainly involved in the rapid growth and the basic biosynthetic metabolism.3.Gene Ontology(GO)enrichment analysis of the 239 DEPs showed that main molecular functional categories represented were catalytic activity,binding,electron carrier activity,structural molecule activity and enzyme regulator activity.According to the biological process categories,the protein-species were mainly classified into metabolic process,cellular process,single-organism process,biological regulation and response to stimulus.In addition,GO analysis predicted the 239 DEPs in various cellular components,such as cell part,organelle,macromolecular complex and membrane.Overall,these results manifested that the regulation of the gene expression and metabolic pathways was remarkably dynamic between conidia and mycelia,and indicated that multiple biological processes are involved in the physiological,biochemical and morphological differences between conidia and mycelium of M.robertsii.4.KEGG pathway enrichment analysis of the 239 DEPs showed that about 60%(4/7)of significantly enriched pathway were involved in the related process of degradation,that is,styrene degradation(ko00643),ethylbenzene degradation(ko00642),geraniol degradation(ko00281),valine,leucine and isoleucine degradation(ko00280).The other three significantly enriched pathways are microbial metabolism in diverse environments(ko01120),cyanoamino acid metabolism(ko00460)and ubiquitin mediated proteolysis(ko04120).Specially,the pathway of styrene degradation,ethylbenzene degradation,geraniol degradation are involved in the secondary metabolism.These secondary metabolites are involved in inhibiting the germination of conidia,that is,the proteins which enriched in the secondary metabolism can eliminate the inhibitory effect,and plays an important role in the process of maintaining the basic energy metabolism.Moreover,there may be more carbon and nitrogen sources,which respond to the future basal metabolism of germination,provided by the above four degradation pathway combined with cyanoamino acid metabolism.In addition,most of the stress resistance related protein-species significantly enriched in the ubiquitin mediated proteolysis pathway,the results shown that conidia mainly through the degradation of various forms of damaged proteins to resist environmental stress,not the complex of damaged proteins.In conclusion,according to the analysis of protein classification and expression abundance,our result further explained the differences of the metabolic level of conidia and mycelia,and the phenotypic of filamentous fungus in different stage.These results deepened our understanding of conidia proteome in M.robertsii and also provide a basis forfurther exploration of improving efficiency of the fungi as biocontrol agents.