Functional Roles Of A UDP Glucose Pyrophosphorylase Gene In The Polysaccharide Synthesis Of Grifola Frondosa

Grifola frondosa is an edible and medicinal mushroom and rich in polysaccharide,protein,trace elements and other nutrients.It has various functional properties such as anti-tumour,immune enhancement and hypoglycemia.Our previous study has eludicated the structural characteristics and functions of G.frondosa polysaccharides,and deduced their synthesis pathways,which mainly contain UDP glucose pyrophosphorylase?gfugp?to synthesize UDP glucose and glucan synthetase to polymerize UDP glucose to glucan.However,the gene bioinformatics and function of gfugp in the growth and polysaccharide synthesis of G.frondosa has still not been clarified.Therefore,based on the whole genome sequence of G.frondosa published in NCBI database,gfugp was cloned and sequenced,and its bioinformatics analysis was carried out.By constructing the gfugp silencing vectrors,the effect of gfugp on the G.frondosa morphology,yield of mycelia/polysaccharide,composition of polysaccharide and the transcription level of related genes in its synthesis pathway were studied.The outputs of the present study would provide a reference for elucidating the main functions of gfugp in mycelial growth and polysaccharide synthesis during G.frondosa fermentation.?1?A UDP glucose pyrophosphorylase gene gfugp from G.frondosa was cloned,bioinformatically analyzed and reannotated.The total length of gfugp was 2036 bp,which shared over 80%identity to those of Trametes versicolor,Sparassis crispa and Gloeophyllum trabeum.The gene encodes 463 amino acids.GFUGP belongs to the UDP glucose pyrophosphorylase family,which contains a UGPase domain.There are 16 substrate binding sites in amino acid residue 103³11.GFUGP is located in the cell without transmembrane signal peptide and has an octamer model tertiary structure.A phylogenetic tree was constructed using the neighbor-joining method on the basis of amino acids sequences of 89 UDP-glucose pyrophosphorylases blasted in NCBI.Over 90%of UGPs derived from fungi and their amino acids sequences shared 80%to 91%homology to that of GFUGP.Among them,UGPs from basidiomycota and polyporaceae mushrooms such as Wolfiporia cocos,Daedalea quercina,Laetiporus sulphureus and G.frondosa were classified into the same branch,which means that they have similar evolutionary time and high homology.However,its homology with UGP from yeast,plant,animal and human is only 30%⁵0%.?2?The gfugp gene of G.frondosa was silenced to compare the gfugp effect on the mycelial growth of G.frondosa on PDA plates.The conserved region of gfugp gene was selected to construct the silencing plasmid pAN7-gfugp-dual,and transferred into G.frondosa protoplast using PEG-CaCl₂.Eight iGFUGP positive transformants were screened using PDA plates containing 80?g/mL of hygromycin.qRT-PCR results showed that gfugp transcription levels in the positive transformants were significantly down-regulated to 0.23⁰.68 compared with WT strain.WT strain had the average growth rate of 4.93±0.17 mm/d while gfugp silenced strains?iGFUGP1,iGFUGP5 and iGFUGP6?had an significant decrease of mycelial growth rate.Among them,the average growth rate of the strain was the lowest?2.25±0.22 mm/d?,which was only 45.63%of WT.Microscopic observation showed that WT had the dense and multi-layered mycelia while the mycelia of iGFUGP strains were sparse with increased diameters and branches.?3?The effects of gfugp silencing on mycelial and polysaccharide production monosaccharide composition in polysaccharide and the transcription level of genes related to polysaccharide synthesis were investigated.Results showed that,after 7days of fermentation,the diameter and mycelial production of WT were about4300±93?m and 25.5 g/L,respectively,while those of iGFUGP decreased to1044±84?m²082±76?m and 12.2 g/L¹7.3 g/L,respectively.Compared to mycelial and exopolysaccharide production of 0.39 g/g and 6.9 g/L,iGFUGP strains produced an decreased mycelial and exopolysaccharide production of 0.08 g/g⁰.14 g/g and 3.10 g/L⁵.07 g/L,respectively.The iGFUGP mycelial polysaccharides had the similar monosaccharide compositions to that of the WT with an decrease of glucose content by 2%¹3%and an increase of arabinose content by 2%¹3%.Interestingly,glucose content of in iGFUGP exopolysaccharide significantly decreased to 66.79%⁷2.08%,and arabinose content increased to 18.02%and 33.21%,respectively.Over 2.3%of mannose and7.3%of galactose were observed.qRT-PCR results indicated that the expression levels of ugp,uge and gls in iGFUGP strains decreased significantly with a significant increase of expression levels of uxe,which was consistent with the changing trend of monosaccharide composition in mycelial and exopolysaccharides.Therefore,it is speculated that gfugp silencing might reduce UDP glucose supply,further affect the yield and monosaccharide composition of mycelial and exopolysaccharides,and finally interfere with the growth and fermentation performance of G.frondosa.