Preliminary Study On Molecular Mechanism Underlying The Germination Of Antrodia Camphorata Arthraconidia In Submerged Culture

Antrodia comphorata,an antrodia fungal,has good application prospects for various pharmacological activities.A.comphorata is only grown in the rotten trunk of Cinnamomum camphora in Taiwan,the basswood cultivation and solid-state culture of A.comphorata possess several drawbacks,such as long production period,low yield,and high cost.Submerged fermentation(SMF)was considered as the most important productioin way of A.comphorata and its bioactive metabolites.In the previous studies,the optimal culture conditions for liquid fermentation of A.comphorata were obtained.A.comphorata can produce arthrospores at the end of submerged fermentation,which can be used as inoculum for subsequent fermentation.Arthroconidial inoculation method exhibits several advantages,such as shorter fermentation period and excellent stability.However,few studies on effect factors and molecular mechanism of A.comphorata arthroconidia germination were conducted.In this study,we compared the mRNA expressioin levels of transcriptome during arthroconidia germination process by RNA sequencing and inferred the signaling pathway of arthroconidia germination.Because of host specificity of A.comphorata,we evaluated the effects of metabolites from Camphor tree and A.comphorata on arthroconidia germination and screened small molecule effectors that promoted or inhibited the arthroconidial germination.Proteomics analysis(2DE,iTRAQ)were used to investigate the differential expression proteins of A.comphorata arthroconidia effected by different effectors to validate the function of germination-related genes.The main conclusions of this study are as follows.The differential expression genes of A.comphorata during germination were assessed by RNA-Seq.A total of 54938 transcripts and 32697 Unigenes were obtained.The cluster analysis showed that the expression levels of most of genes were significantly different between dormancy and germination cnonidia.After bioinformatics analysis,79 genes were found to be related to arthroconidia germination,in which 16 genes(rac1,ric8,ganB,sfaD,cyaA,pkaR,pkaA,cre-1,bmh1,ras2,pmr1,gapA,rasA,cdc42,pak A,cmk1)invovled in G?-cAMP-PKA and Ras-MAPK signaling pathways of arthroconidia germination.Antrodia camphorata has host specificity.Here,we first evaluated the effects of 20 compounds from Camphor tree,A.comphorata,and their 34 structural analogues on A.comphorata arthroconidia germination which invubated with 96 well plates.Among the 54 compounds,29 germination promoters and 17 germination inhibitors were obtained.The effects of 22 effectors were investigated with shake flask incubation.The results show that vanillin,vanillic acid and ferulic acid were considered as the major germination promoter,their germination growth rates were 110.18%,66.67%,and 64.13% respectively.While o-vanillin,1-Octene-3-ol and caffeic acid were considered as negative regulators of arthroconidia germination,their germination growth rates were-58.81%,-41.61%,-45.83% respectively.The differential proteomics of A.comphorata arthroconidia between effected by germination promoter and germination inhibitor were analyzed by two-dimensional electrophoresis(2DE)and isobaric tags for relative and absolute quantitation(iTRAQ).44 proteins were successfully identified by 2DE,including 10 germination-related genes(ger O,ubc1,cat-1,snf1,cas2,sfaD,chaperonin,fad5,tyrosine-P,chiA).A total of 3109 proteins were identified by iTRAQ.After bioinformatics analysis,61 proteins related to arthroconidia germination were obtained,in which 17 proteins invovling in signaling pathways of arthroconidia germination.The expression levels of germination-related protein from signaling pathway of arthroconidia germination were investigated.The expression levels of key genes in G?-cAMP-PKA pathway were regulated by vanillin,such as gan B,cyaA,cre-1,pkaA.These genes can improve laccase content and activity,which markedly promotes arthroconidia germination.In the Ras-MAPK pathway,the expression levels of cdc42,which was considered as a key protein to regulated arthroconidia germination,significantly increased in vanillin-treated arthroconidia and decreased in 1-Octene-3-ol-treated arthroconidia.Lastly,the expression levels of 26 germination-related genes were analyzed by RT-qPCR,and we found that the mRNA transcription levels of 8 genes(ger O,fad5,chiA,sfa D,cyaA,cre-1,gapA,cmk1)were consistent with the results of RNA-seq.The results show that these genes are related to A.comphorata arthroconidia germination.