Function Analysis Of G Protein α Subunits Genes In Tremella Fuciformis And Application Of The Yeast-Like Cell Expression System

Tremella fuciformis,an edible mushroom as well as a typical dimorphic fungus having the yeast-hypha transition triggered by environmental cues.It has long been believed that dimorphism is necessary for pathogenicity in pathogenetic dimorphic fungi,but the mechanism that regulates this switch has remained a mystery.The heterotrimeric guanine nucleotide-binding proteins(G proteins),universal signaling proteins in eukaryotes,carry different signals from the receptors to various effectors,then regulate a variety of biological processes,including fungal dimorphism.In this paper,three G protein α subunit gene were cloned and characterized their contributions to the T.fuciformis dimorphism.The yeast-like cell was used as a cell factory to produced hydrophobin from Pleurotus ostreatus.The main research contents are as follows:(1)The mycelium and yeast-like cell of T.fuciformis were adopted to construct transcriptome sequencing library.13.16 G data and 16190 Unigenes were generated from de novo splicing.Among the NR database annotated Unigenes,55.75% and 13.35% of them had similar sequence to T.mesenterica and Cryptococcus neoformans,respectively.7203 Unigenes were annotated in the COG database,which could be divided into 25 terms.3921 Unigenes were annotated in the GO database,and could be divided into 46 branches.9991 Unigenes were annotated in the KEGG database.A total of 1105 SSR loci were found from all Unigenes.The tri-nucleotide has the highest repeats of 546,while the repetition of mono-nucleotide was the last.From the KEGG analysis,differential expressed genes was enriched in pathways including amino sugar and nucleotide sugar metabolism,tyrosine metabolism and alanine,aspartate and glutamate metabolism.All results provided abundant data resource for further investigating on molecular mechanism and functional genes of T.fuciformis dimorphism.(2)The open reading frame(ORF)encoding three novel G protein α subunits(Gα),Tr Gpa1,Tr Gpa2 and Tr Gpa3,were cloned from the dimorphic fungus,T.fuciformis.The c DNA of Tr Gpa1 has 1059 nucleotiedes,and encodes a protein of 352 amino acid residues with a molecular weight of 40.18 k Da.The c DNA of Tr Gpa2 has 1071 nucleotiedes,and encodes a protein of 356 amino acid residues with a molecular weight of 40.76 k Da.The c DNA of Tr Gpa3 has 1245 nucleotiedes,and encodes a protein of 414 amino acid residues with a molecular weight of 45.87 k Da.The DNA of Tr Gpa1,Tr Gpa2 and Tr Gpa3 have 1437 bp,1339 bp,1489 bp nucleotiedes,respectively.The deduced protein sequence shares high homology with Gα from other species.The phylogenetic tree revealed Tr Gpa1,Tr Gpa2 and Tr Gpa3 belong to the group I,group III and group II of Gα superfamily,respectively.(3)The roles of Tr Gpa1,Tr Gpa2 and Tr Gpa3 in the T.fuciformis dimorphism were analyzed by gene overexpression and knockdown via Agrobaterium tumefaciens mediated transformation.Stable integration of the target gene into the genome of T.fuciformis was confirmed by PCR and Southern blot hybridization.Quantitative real-time PCR(q RT-PCR)analysis showed that the Tr Gpa1,Tr Gpa2 and Tr Gpa3 in the corresponding overexpression transformants was highly expressed,while in the corresponding knockdown transformants got low expression.Additionally,each transformant was compared with Y32 about the morphological change under different environmental factors,including p H values,cultural time,inoculum size,and quorum-sensing molecules(farnesol and tyrosol).Comparing with Y32,although the Tr Gpa1,Tr Gpa2 and Tr Gpa3 overexpression and knockdown transformants exhibited the same morphological transition,the Tr Gpa1 and Tr Gpa2 overexpression transformant always had higher ratios of pseudohyphae,while the Tr Gpa1 and Tr Gpa2 knockdown transformant always had less proportions of pseudohyphae.The pseudohyphae ratios of Tr Gpa3 overexpression transformant and Tr Gpa3 knockdown transformant showed no differences with those of Y32.Therefore,it was speculated that the Tr Gpa1 and Tr Gpa2 involve in the dimorphim of T.fuciformis and plays a positive role in the promotion of pseudohyphal growth.Additionally,the Tr Gpa3 may not involves in the dimorphim of T.fuciformis.(4)The hygromycin resistance gene expression vectors were constructed based on the p CAMBIA1300.The hygromycin resistance gene was driven by gpd promoters with different length(846 bp,321 bp)and α-tubulin promoters with different length(693 bp,386 bp).According to the q RT-PCR analysis,the gpd promoter drove hygromycin resistance gene more efficiently than the α-tubulin promoters,and the 846 bp gpd promoter fragment exhibited the highest hygromycin resistance gene expression efficiency.(5)Hydrophobins,secreted by filamentous fungi,have the amphiphilic ability and hydrophobicity.They are promising for many industrial applications,especially for the food industry because of their superior stable ability of foams or emulsion.The yeast-like cells of T.fuciformis have great potential to be a microbial cell factory for the food industry.It is edible and easy to culture by fermentation,and it possesses the complete human-like post-translational modification system.The hydrophobin Po.Hyd expression vector p GEH-GH was constructed and transformed into T.fuciformis cells via A.tumefaciens.The heterologous expressed Po.Hyd was extracted and estimated its emulsifying activity.The results showed that the heterologous expressed Po.Hyd exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate.