Expression Profile And Metabolome Analysis On The Pre-, Intra- And Post Spore-discharging Stages Of Pandora Neoaphidis And Its Electroporation Study

Most of entomophthoralean fungi can infect host by actively discharging conidium from the cadaver,and cause a devastating epizootic in population.This unique ability is vital for infection and colonization of entomophthoralean fungi,which makes the fungi survive through hitting and sticking to the "hard-met" target insect.Pandora neoaphidis,one of representative obligated aphid pathogenic fungi in entomophthoralean fungi,however,since its molecular biology is remain poorly understood,the potential genes of interest about fungal pathogens and biocontrol application are severely limited.Here,P.neoaphidis hyphae of three different stages including BD(before discharge),DD(during discharging)and AD(after discharge)were analyzed by using RNA-seq and metabolome analysis.The differentially expressed genes of hyphae of different stages,were summarized to the potential genes related to spore discharge.Meanwhile,an electroporation system was developed preliminarily for the genetic transformation of P.neoaphidis.The results were as followed.1.RNA-seq for pre-,intra-and post spore-discharging stages of P.neoaphidis Using RNA-seq,three different stages hyphae of P.neoaphidis including BD,DD and AD were analyzed to screen the differentially expressed genes of each comparison group using the existing transcriptome information as reference.372 differentially expressed genes were totally obtained in DD vs AD,in which 113 genes were up-regulated and 259 genes were down-regulated.1509 differentially expressed genes were totally obtained in AD vs BD,in which 1325 genes were up-regulated and 184 genes were down-regulated.478 differentially expressed genes was totally obtained in DD vs BD,in which 383 genes were up-regulated and 95 genes were down-regulated.Based on GO functional enrichment analysis and KEGG pathway enrichment analysis,the function of differentially expressed genes in BD stage were mainly to maintain hyphae normal growth and development and metabolic regulation;those in DD stage were mainly to participate in carbohydrates,lipids,and amino acid metabolism;those in AD stage mainly to participate in cellular processes,metabolism processes and protein binding.2.Screening the spore discharge-related genes of P.neoaphidisBased on GO functional enrichment analysis and KEGG pathway enrichment analysis,and the spore actively discharge-related research existed,13 spore discharge-related genes were screened mainly to the perspective of the ATP binding,ion channels,and transport-related metabolic pathways.those genes were annotated in following pathways:TR26574|c0_g3_i1 and TR20190|c0_g2_i1 for Ca2+ transport related pathways,TR26574|c0_g3_i1 and TR20190|c0_g2_i1 for Na+/H+ transporters genes,TR26996|c1_gl_i1 for sodium:dicarboxylate symporter,TR12238|c0_g1_i1,TR33495|c0_g1_i1,TR33495|c0_g2_i1,TR15871|c0_gl_i1,and TR8321|c0_g1_i1 for the pathway related to amino acid transporter,AAT family,TR2884|c0_g1_i1 and TR11581|c0_g2_i1 for the pathway related to solute carrier family 32,and TR25948|c0_g1_i1 for sugar/H+ symporter.Because of poor information on the spore discharge,these 13 genes might be associated withspore discharge according to the existing data.3.Metabolome analysis for pre-,intra-and post spore-discharging stages of P.neoaphidisMetabolites of three different stages hyphae BD,DD,and AD were analyzed by LC-MS detection technology,respectively.Data identified by the quality control were measured using univariate and multivariate analysis,and the different ions of each comparison group were screened on the basis of following parameters including VIP ? 1,fold change ? 1.2 or fold change ?0.8333,and q-value<0.05.In DD vs BD,1252 and 878 different ions were screened in the cationic mode and anionic mode,respectively.In AD vs DD,1101 and 970 different ions were screened in the cationic mode and anionic mode,respectively.In AD vs BD,1374 and 1212 different ions were screened in the cationic mode and anionic mode,respectively.Additionally,based on VIP>3,different metabolites were screened for the further studies.In DD vs BD,126,92 metabolites were screened in the cationic mode and anionic mode,respectively.And 79 of 218 metabolites were identified,and 23 metabolites participate in specific metabolic pathways.In AD vs DD,186,90 metabolites were screened in the cationic mode and anionic mode,respectively.And 114 of 276 metabolites were identified,35 metabolites participate in specific metabolic pathways.In AD vs BD,178,86 metabolites were screened in the cationic mode and anionic mode,respectively.And 119 of 264 metabolites were identified,31 metabolites participate in specific metabolic pathways.4.Preliminary study on genetic transformation system for P.neoaphidisBased on plasmids pAN52-1N,pET-29b,pABeG and pUMa932,a new plasmid pACeG including enhanced green fluorescent protein gene(egfp)and carboxin resistance gene(cbx)were constructed.Subsequently,plasmid pACeG were transformed into germinated spores of P.eoaphidis by electroporation following 6-7 kV/cm field intensity.At last transformation was realized as GFP instantaneous expression.In the study,the differentially expressed genes and the small moleculer metabolites were obtained for pre-,intra-and post spore-discharging stages of P.neoaphidis using RNA-seq and metabolome technology.Meanwhile,the main biological function,metabolic pathway and participated signal transduction were also summarized.Those provide theoretical help for further function studies on the spore discharge-related genes.Furthermore,an electroporation genetic transformation system was developed for the further pathogenesis study of P.neoaphidis at the molecular level,and might provide helpful promotion for comprehensive pests management and the field application of entomophthoralean fungi,especially for P.neoaphidis.