Functions Of Grifola Frondosa Glucan Synthase (GFGLS2) In Mycelium Growth And Exo-glucan Synthesis

Grifola frondosa is an important edible Polyporaceae fungi,rich in polysac-charides,protein,flavonoids and other nutrients.Existing studies have shown that G.frondosa polysaccharide can significantly enhance immunity,prevent tumors,lower blood sugar,and regulate intestinal flora.Our previous study had showed that G.frondosa exo-polysaccharide belonged to glucan,and G.frondosa Glucan synthase(GFGLS)is one of the key enzymes involved in glucan synthesis.On this basis,a putative G.frondosa?-1,3-glucan synthase gene GFGLS(named GFGLS1 in this context)had been cloned and re-annotated.However,following studies indicated that genome of G.frondosa strain 9006-11 released on NCBI contained another putative?-1,3-glucan synthase gene(GFGLS2).So the identity or difference of between the GFGLS1 and GFGLS2 in terms of gene structure and functions in mycelium growth and polysaccharide synthesis should be classified and elucidated systematically.Therefore,based on the previous research of our lab,the nucleotide sequence of GFGLS2 was cloned and sequenced,and structural differences between GFGLS2 and GFGLS1 was compared and analyzed.Using constructed dual promoter RNAi system and PEG-Ca²⁺transformation system,single-silencing and co-silencing strains of GFGLS1 and GFGLS2 were constructed and their roles in mycelial growth rate,morphology,fermentation performance and monosaccharide compositions of G.frondosa were well investigated,respectively.The main conclusions are as follows(1)GFGLS2 showed identical and different bioinformatics with GFGLS1.Based on the complete genome sequence of G.frondosa 9006-11 strain published by NCBI,the nucleotide sequence of GFGLS2 was cloned and sequenced,with a total length of5944 bp,and the length of the coding protein sequence is 5142 bp,encoding 1713 amino acids;Compared with the GFGLS1 re-annotated by our lab,the similarity of the gene sequence encoding amino acids is only 72.56%.The protein domain of GFGLS2p is similar to GFGLS1p,contains two conserved domains of the glucan synthase gene family;the conserved domains of GFGLS1p are located at 261 aa-369 aa and 761 aa-1576 aa;while GFGLS2p are located at 243 aa-352 aa and 692 aa-1510 aa.Both GFGLS2p and GFGLS1p are membrane proteins containing two large transmembrane domains and a hydrophilic cytoplasmic domain,mainly 6 transmembrane helices(TMHs)at the N-terminal and 9 TMHs at the C-terminal.(2)Based on the established G.frondosa binary promoter gene interfering and PEG-Ca²⁺transformation system,single-silencing and co-silencing strains of GFGLS1and GFGLS2 were constructed respectively,and the mycelial growth rate and morphological changes of the silent strains were analyzed.The nucleotide sequence of GFGLS1 and GFGLS2 are completely different,and the highly similar conservative sequence(Ci GFGLS)were selected,and constructed the silence plasmids p AN7-i GFGLS1-dual,p AN7-i GFGLS2-dual,p AN7-Ci GFGLS-dual.The plasmids were transferred into G.frondosa protoplasts by PEG-mediated method,and the target transformants were screened out by selective solid medium(Hygromycin).Compared with the wild strain(WT),the transcription level of the glucan synthase gene in the selected i GFGLS1,i GFGLS2 and Ci GFGLS strains decreased significantly,among which the transcription level of i GFGLS1 decreased by 20%?69%;the transcription level of i GFGLS2 decreased by 25%?70%,and the transcription level of Ci GFGLS decreased by 46%?85%.The growth of WT and the silent strain with significant silencing effect on the PDA plates were observed for 15 days.The average growth rate of WT was 4.93±0.2 mm/d,and the average growth rate of single-silencing strains i GFGLS1,i GFGLS2 and co-silencing strain Ci GFGLS decreased 12.2%?20.3%,23.5%?31.65%and37.7%?44.6%.It can be seen RNAi of GFGLS1 or GFGLS2 slowed down the growth of mycelium,and that GFGLS2 can inhibit the growth of mycelium more than GFGLS1;the growth rate of the co-silencing strain Ci GFGLS-5 is the slowest,with a decrease of44.6%.(3)The changes in the pellet morphology,fermentation performance,polysaccharide production and monosaccharide composition of the silent strains in submerged fermentation were investigated.After 7 days of fermentation,the diameter of WT mycelium pellet is about 4053±93?m,the mass is 25.59 g/L,the mycelium/exopolysaccharide is 0.45 g/L and 5.95 g/L,respectively;The pellets diameter was significantly reduced to 1206±42?2792±76?m,the mass also decreased to 8.68?17.81 g/L,and the amount of mycelium/exo-polysaccharides were significantly reduced to 0.12?0.25 g/L and 1.3?3.2 g/L.Among them,the amount of polysaccharides of Ci GFGLS silent strains decreased most significantly,and mycelium/exo-polysaccharides decreased to 27.28%and 21.76%.The results of monosaccharide composition analysis showed that the proportion of glucose in exopolysaccharides by single-silence and co-silence strains decreased from98%to 61.78%?80.14%,while the proportions of arabinose and mannose increased to 7.8%?16.14%and 3.7%?16.61%,respectively.At the same time,6.87%?14.32%galactose was also detected in the exo-polysaccharides of i GFGLS2 and Ci GFGLS silent strains.The percentage of glucose in mycelial polysaccharides by single-silence and co-silence strains decreased by 18.58%?58.57%,and the ratio of mannose increased.The results of q RT-PCR showed that single silencing or co-silencing of GFGLS1 and GFGLS2 had little effect on the transcription levels of GFGEL4 and GFUGP in the glucan synthesis pathway.