| Grifola frondosa polysaccharides,especially?-glucan,have been proved to have significant anti-tumor,hypoglycemic and immunomodulatory activities.To obtain?-glucan with high yield and bioactivity,it is necessary to systematically investigate the synthesis pathways and regulation strategies of Grifola frondosa polysaccharides.Previously,our research group had systematically optimized the submerged fermentation process of Grifola frondosa,analyzed the structure and functions of mycelial polysaccharide/exo-glucan,and established the techniques of protoplast preparation based on incomplete enzymatic hydrolysis and PEG-mediated transformation.On this basis,the roles of gfugp and gfgls in mycelial growth and polysaccharide/exo-glucan synthesis of Grifola frondosa were studied.However,the questions including how the branched sugar chain of exo-glucan of Grifola frondosa is formed and which enzymes are involved in glucan branching have not been clearly answered.Therefore,on the basis of the previous work,a gfgel4 gene which may be involved in glucan branch ing was excavated and cloned for the first time,and a comprehensive bioinformatics analysis was carried out.Furthermore,through the previous constructed dual promoter gene silencing and overexpression systems,the gfgel4 silenced and overexpressed strains were constructed,and their growth characters on PDA were observed.To clarify the main roles of gfgel4 gene in the mycelial growth and exo-glucan synthesis of G.frondosa,effects of gfgel4 gene silencing and overexpression on the mycelial pellet formation,fermentation performance and branching structures of exo-glucan were finally investigated.(1)The full-length sequence of gfgel4 gene was obtained by cloning and sequencing.The full-length g DNA sequence of 1945 bp is 1665 bp and contains 5introns and 6 exons.It encodes a total of 554 amino acids and the theoretical molecular weight is 57.127 k Da.Protein domain analysis showed that gfgel4p contained a signal peptide in amino acid residue 1-25 belonging to secretory protein.gfgel4p had two complete domains of GH72 catalytic domain and X8 domain,and no transmembrane domain.Based on the phylogenetic tree and protein structure information,gfgel4p belongs to the GH72~+subfamily,and its similarity with Trametes coccinea and Ganoderma boninense is 74.9%and 71.88%,respectively.(2)The gfgel4 silenced and over-expressed strains were constructed.The silencing plasmid p AN7-gfgel4-dual and over-expression plasmid p AN7-35s-gfgel4and p AN7-gpd-gfgel4 were constructed and transferred into G.frondosa protoplasts using PEG mediated transformation.The silencing strains GFGEL4-i-4,GFGEL4-i-6,over-expressing strains GFGEL4-gpd-2,GFGEL4-gpd-3 and GFGEL4-35s-2,GFGEL4-35s-4 were obtained.After 15-d cultivation on CYM plates,the growth rate of mycelia of the control strain was 4.56±0.12 mm/day,while the growth rates of mycelia of the gfgel4 over-expressed strains GFGEL4-gpd and GFGEL4-35s were increased to 4.87±0.21 mm/day and 4.67±0.14 mm/day,and the number of mycelium bifurcations increased significantly,and the hyphae were stout.The mycelial growth rates in strains GFGEL4-i was decreased to 4.3±0.15 mm/day,The mycelium distribution was sparse and closely adhered to the plate.(3)The effects of gene gfgel4 silence and over-expression on mycelial pellet formation,fermentation performance and exo-glucan branches of Grifola frondosa were investigated.After 7-d fermentation,compared with WT,the silenced transformat GFGEL4-i showed a significant decrease of the diameters of mycelial pellets by 21.8%with the decrease of mycelial biomass and exo-glucan to 98.35%,and 90.06%,respectively.Over-expressing strains GFGEL4-gpd and GFGEL4-35s had an increased fermentation performance with mycelial biomass and exo-glucan to21.07 g/L,and 7.86 g/L,respectively,and diameters of mycelial pellets to 4499±82?m,Among them,GFGEL4-gpd-2 had the highest mycelial biomass concentration of 22.1 g/L.The monosaccharide composition showed that the Glucose percentage in mycelial polysaccharide decreased by 2.07%to 2.58%in gfgel4 over-expressed strains GFGEL4-gpd and GFGEL4-35s compared to that of the WT while its percentage in exo-polysaccharide increased to 88.07%to 91.55%.Furthermore,silence of gfgel4 decreased glucose content in exo-polysaccharide and slightly increased glucose content by 1.44%to 1.93%in mycelial polysaccharide.Compared to WT strains,the silence of gfgel4 increased the percentage of?-1,3-linked glucose residues in exo-glucans to 62.87%,and slightly increased the percentage of terminal glucose and residues at the branching point?3,6-Glcp-1?to 14.17%and 13.15%,respectively,indicating that the branching degree of the silenced strain was Slightly lower.Over-expression of gfgel4decreased the percentage of?-1,3-linked glucose residues in exo-glucans to 50.1%correspondingly,with the increase of branching degree to 0.35.q RT-PCR results showed that the transcription levels of the corresponding genes gfugp,gfgls and gfgel4in GFGEL4-i decreased to 0.78,0.67,0.54.Transcription levels of gfgel4 in GFGEL4-gpd and GFGEL4-35s increased to 1.71,Among them,GFGEL4-gpd-3 had the highest transcription levels of gfgel4 increased to 1.94.Transcription levels of gfugp,gfgls in GFGEL4-gpd and GFGEL4-35s increased to 1.47,1.44.It could be concluded that the silence or overexpression of gfgel4 significantly affected the mycelial growth,polysaccharide synthesis,and branching structures of exo-glucans. |
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