Research On The Function Of DNA Methylation And Protein Phosphorylation For The Growth And Development Of Metanrhizium Robertsii

Metarhizium robertsii is the species of entomopathogenic fungi which is widely used for biological control,and it is used as a model species of research entomogenous.On the one hand,the epigenetics could affect the growth and development of organism.On another hand,the epigenetics was able to provide new ways to solve the problem.Studies indicated that genomic DNA methylation regulate and control the growth and development of microorganisms,protein phosphorylation and dephosphorylation plays a very important role in the whole process of biological activities.In recent years,well researchs on the epigenetics of filamentous fungi are reported,but relatively few studies focus on the level of genome and proteomics.So this article was attempt to research on DNA methylation and protein phosphorylation of two important stages?conidia and mycelium?of M.robertsii and further analyzed the relationship between the growing development of M.robertsii and genomic methylation,and the relationship between the growing development of M.robertsii and protein phosphorylation.Firstly,the full-length cDNA of two DNA methyltransferases genes?GenBank accession No: MAA₀4944 and MAA₀3836?were cloned from M.robertsii using SMART RACERT-PCR.The analysis of this sequence showed that the full-length cDNA of MRDIM-2 genes was 3911 bp,with3501bP,168 bP and 242 bP of open reading frame?ORF?,5'-untranslated region?5'-UTR?and 3'-untranslated region?3'-UTR?,respectively.The open reading frame?ORF?encoded 1166 amino acids.The mature protein had a molecular mass of 130.257 kDa with a calculated Pl of 7.64.The full-length cDNA of MRRID genes was 2686 bp,with1965bP,483 bP and 238 bP of open reading frame?ORF?,5'-untranslated region?5'-UTR?and 3'-untranslated region?3'-UTR?,respectively.The open reading frame?ORF?encoded 654 amino acids.The mature protein had a molecular mass of73.913 kDa with a calculated Pl of 8.96.The analysis of the homogenous relations between the amino acid sequence of encoded gene for M.robertsii MRDIM-2 and MRRID and the DNA methyltransferase amino acid sequence of other species showed as the following aspects.Firstly,fifteen kinds of protease were divided into five groups and Cordyceps militaris DIM-2 protein which kept close relationship with M.robertsii MRDIM-2.Secondly,there was a close homogeneous relationship between protein of C.militaris DMTA and Neurospora crassa RID.Furthermore,the relative quantitative transcription analysis of mycelium?2d,pure mycelium?and conidia?15d,mature spore?of M.robertsii methyltransferase,the results displayed that there was falling trend for the methylated levelof conidia because of small-scale expression of methyltransferase compared with mycelium through the method of real-time PCR technology.Second,baseding on the results of the real-time PCR analysis,this dissertation performed a comparative genome-wide DNA methylation BS-Seq analysis of the two-day mycelia and fifteen-day conidia of an M.robertsii at single-base resolution,the results showed that approximately 0.39 % of cytosines are methylated in conidia,lower than the DNA methylation level?0.42 %?in mycelia.To understand the function of DNA methylation in the development of M.robertsii,through Gene Ontology?GO?analysis and KEGG analysis of DMR-associated genes,DMR-associated genes were defined as genes with a total of 132 DMRs located in their gene bodies or within 2 kb upstream,and thus,102 were identified.The results showed that the 102 methylated genes belonging to three main GO domains: biological processes?55?;cellular components?9?;and molecular functions?28?.60 predicted pathways were enriched,including amino acid,carbohydrate,fatty acid metabolism and ATP synthase.To investigate the effect of DNA methylation on gene transcription,22 genes of the 108 DMR-associated genes were chosen for gene transcription analysis.Remarkably,moderate methylations,either 2 kb upstream of the start codon?in the promoter?or in both the promoter and the gene bodies,?overlap:promoter + gene body?were associated with higher gene expression.In contrast,genes with either high or low DNA methylation levels trended to be at lower expression levels with no significant difference in gene expression between the two types.However,the level of DNA methylation in the gene bodies alone was not predictive of changes in gene expression.A total of fifty-nine genes with a DMR less than 2kb upstream of their start codon,out of the 102 DMR-associated genes,were analyzed to explore the relationship between DNA methylation and fungal development in M.robertsii.Further analysis showed that genes that are essential for energy synthesis are moderately methylated and highly expressed in the mycelia stage.Examples of such genes include an ATP synthase?MAA₀4235?,and another gene named acetyl-CoA acetyltransferase?MAA₀6480?.Other genes participates in energy metabolism such as MAA₀5759?sucrose transporter?,MAA₀2043?Fructose-1,6-bisphosphatase?,MAA₀1615?glucose oxidase?and inregulating signal transduction associated with G protein-coupled receptors such as MAA₀1167?arrestin?were found from those fifty-nine genes.Thirdly,in order to further analyze the relationship between growth,development and epigenetics of M.robertsii,iTRAQ multiple labeling techniques was selected in this article for studying on Phosphoproteomic of M.robertsii at two different developmental stages.The results showed that totally 1487 Phospho Peptides were found at samples,among them,168 Phospho proteins Peptides have significant differences,including 116 proteins.Comparing with spores,83 Phospho Peptides were up-regulated and 85 were down-regulated.Gene Ontology?GO?and KEGG were select to further analysis the differences among proteins Phospho Peptides,the results showed that the significant differences stands for 6 biological processes?49.08%?,6 cellular components?22.14%?and 4 molecular functions?28.78%?.Pathway annotation of differential proteins both of spores and mycelium was implemented followed by significance test,it is found that differential proteins of spores and mycelium accumulate at six typical pathway?P<0.05?,also known as Ribosome biogenesis in eukaryotes,Glutathione metabolism,RNA transport,Carbapenem biosynthesis,Styrene degradation,and ABC transporters.In addition,the results showed that these proteins expression were up-regulated in mycelia,including ribosomal protein?lQQDAAAQSkPVkEEEDsEMEDQSPSELPPLAR?,centromere/microtubule binding protein CBF5?gEAGsDDDsDaAkGEAGsDDDsD?,WD repeat-containing protein 75?aDGNNtPtPQk?,5-oxoprolinase?lGPGESVIIESPGGGGWGsE?,ribonucleoside-diphosphate reductase?sGQsEDGDDSGER?,and ABC transporters?sVSsIALQk?.These proteins are involved in ribosome biogenesis in eukaryotes,glutathione metabolism and material transport inside the cell.Besides that,it both close related to high metabolic activity of vegetative mycelium and low level of transcription,protein synthesis and metabolic activity of dormant spore phase.In particular,nitrilase?vAssQPsPTRrssTFTDQGFEIALPSVSPtR?expression was found to be up-regula ted in spore.It means productivity of the nitrilase is higher in spore than mycelium.Possible roles both of DNA methylation and phosphorylation in the growing development of M.robertsii have been investigated in this paper.It is found that DNA methylation and phosphorylation are significant different between conidia and mycelium which are two seperated growing stages of M.robertsii,and these expressed proteins with significant difference are also lay out accordingly.Metabolic regulation role of theexpressed proteins in the growing development of M.robertsii is revealed.This finding can help further analyzing the potential function of DNA methylation and protein phosphorylation in filamentous fungi growth and development,and meanwhile provided theoretical basis of filamentous fungi epigenetic.