Structural Characteristics And Functional Roles Of A Glucan Synthase Gene In Grifola Frondosa

Grifola frondosa is a medicinal fungus rich in bioactive components including polysaccharides and proteins.Polysaccharides such as glucans obtained from fruiting bodies or mycelia of G.frondosa had proved to have antioxidant,immunomodulatory,antitumor and antiviral effects.Our previous studies had indicated that the submerged exo-polysaccharide of G.frondosa belonged to glucan,which possibly was synthesized from glucose converted to UDP-glucose by UDP-glucose pyrophosphorylase,and then polymerized by glucan synthase.Hence,?-1,3-glucan synthase(GFGLS)is a potentially key enzyme in G.frondosa for the glucan synthesis,However,no studies about the structural and functional analysis of GFGLS had not been reported yet.Recently,the development of gene sequencing technology,especially,the publishied whole genome sequence and annotations of G.frondosa provided a source to molecularly investigate the gene structure and functional roles of GFGLS.However,the lack of an effecient genetic manipulation system limited the analysis of the important functional genes of G.frondosa.Hence,this study would aim to: 1)clone the GFGLS gene sequence and analyze bioinformatics based on the publishied whole genome sequence of G.frondosa,2)establish a gene silencing system for G.frondosa based on reverse genetics,and 3)investigate effect of GFGLS on the fermentation performance of G frondosa.Our findings will provide a reference to know the functional role of a glucan synthase gene GFGLS on mycelial growth and polysaccharide production of G.frondosa.The obtained results are as follows:(1)The glucan synthase gene GFGLS of G.frondosa was cloned by PCR and analyzed with bioinformatic tools.The cloned GFGLS gene of 5927 bp has 11 introns and the 12 exons,sharing 98% of homology to that in G.frondosa 9006-11 strain.The cDNA with the full length of 5346 bp encoded a total of 1781 amino acids,which had a relatively high homology with the glucan synthase of Polyporus brumalis(90.66%)and Trametes versicolor(90.38%).Hydrophilic analysis showed that GFGLSp is a membrane protein having two large membrane domains containing 15 transmembrane structures with 6 transmembrane helices(TMHs)at the N-terminus and 9 TMHs at the C-terminus,which were connected with a large hydrophilic cytoplasmic domain of 581 amino acids between residues 672 and 1253.The large hydrophilic domain was generally regarded as a typical catalytic domain with the substrate UDP-glucose.(2)The dual promoter gene silencing system of G.frondosa was established.The pAN7-gfgls-dual silencing vector was successfully constructed by cloning the GFGLS fragment,the GPD promoter of G.frondosa fragment and the 35 S promoter of Aspergillus nidulans fragment.The conditions for preparing the protoplasts of G.frondosa were optimized with a combination of lysing enzyme from Guangdong Institute of Microbiology and yatalase,thencultivated for 7 days and digested for 3-h.The mycelium of G.frondosa did not grow at a concentration of 80 ?g/mL of hygromycin,which laid a foundation for the selection of transformants.Based on the previous experiments,the silencing vector pAN7-gfgls-dual was successfully transferred into the protoplasts of G.frondosa.The 10 transformants were screened and 9 positive transformants were obtained by PCR amplification for the marker gene HPH.The transformant iGFGLS-3 had the lowest GFGLS expression with 26.1% level of WT strain by q-RTPCR.(3)The effect of GFGLS on the growth and fermentation performance of G.frondosa was investigated.The WT strain showed a radial growth rate of 4.3 mm/d while the GFGLS silenced strain grew at a lower rate of 3.04 mm/d during 7-d cultivation on the PDA Petri dishes,and growth rate decreased by 29.3%.Similar phenomena of iGFGLS-3 strain having extremely few mycelial pellets were also observed in the 250 mL liquid fermentation flasks The morphologies of G.frondosa mutants displayed significantly difference to those of wide type.During 7-d cultivation on PDA Petri dishes,the WT strain appeared the thick and highly branched phenotype,while iGFGLS-3 strain became shorter in length and thinner appearance of hypha.After 7 days of fermentation,the biomass of the iGFGLS-3 mycelia was only 5.02 g/L while the biomass of WT strain mycelia was reached 25.12 g/L.The yield of EPS of 1.23 g/L in the WT strain was approximately four times the yield of EPS of 0.38 g/L in the strain iGFGLS-3.The results of monosaccharide composition analysis showed that the monosaccharide composition and ratio of the transformed strain iGFGLS-3 mycelial polysaccharide and exo-polysaccharides were not significantly different compared with WT.Therefore,it can be speculated that GFGLS has a significant effect on the synthesis of the cell wall and the elongation of the hyphae by down-regulating the glucan synthesis.