Functional Study Of Fv-gs6 Gene Encoding β-1,6-glucan Synthase From Flammulina Velutipes

β-1,6-glucan is an important component of fungal cell wall,which plays an important role in the formation of fungal cell wall structure and helps to stimulate cell activity and trigger inflammatory responses.The encoding gene of β-1,6-glucan synthase is the key gene for synthesis ofβ-1,6-glucan synthase.In order to reveal the function of β-1,6-glucan synthase encoding gene,following methodology was adopted;Real-Time Quantitative PCR(RT-qPCR)analysis of fv-gs6 in different tissues at various growth stages of Flammulina velutipes.The expression level of fv-gs6 under mechanical stress and H2O2 stress were also analyzed;Construction of overexpression(fv-gs6-OE)vector and agrobacterium-mediated transformation of F.velutipes strain FvOl into the dikaryon mycelium.Later,fv-gs6 expression analysis,mycelial growth speed measurement,cell wall sensitivity analysis and phenotype of fruit body were studied in fv-gs6 mutants.The results of the purposed study are as follows:(1)the expression level of fv-gs6 in mycelium,stipe elongation,pileus elongation,mature stipe,and pileus,were 0.10,0.72,87.87,0.314,and 56.56,0.07 times of primordial stage in Fv01 strain.It was also found that expression level of fv-gs6 increased 3.44,3.79,1.88,1.96 and 3.59 times in different incubation intervals under mechanical stress.Under different H2O2 concentrations(0 mmol/L,1.25 mmol/L,2.5 mmol/L,5 mmol/L,and 10 mmol/L),the fv-gs6 expression and growth of Fv01 gradually decreased with the increase of H2O2 concentration.(2)The fv-gs6-OE mutants were obtained by transformation and were named as fv-gs6-OE5,fv-gs6-OE10,fv-gs6-OE12,fv-gs6-OE14,and fv-gs6-OE16.RT-qPCR results showed that expression level of fv-gs6 in transformants(fv-gs6-OE5,fv-gs6-OE10,fv-gs6-OE12,Jv-gs6-OE14,fv-gs6-OE16)were significantly higher than wild type strain Fv01.(3)Observations on Cell morphology(cell wall thickness and cell width at the clamp connection of the F.velutipes transformants):the results showed that cell wall thickness at the clamp connection septum was significantly increased in different mutants(fv-gs6-OE5,fv-gs6-OE10,fv-gs6-OE12,fv-gs6-OE14,fv-gs6-OE16)as compared to Fv0l.Cell width were noted as 0.974,1.192,1.156,1.132,1.083,1.167 um in Fv01 fv-gs6-OE5,fv-gs6-OE10,fv-gs6-OE12,fv-gs6-OE14,and fv-gs6-OE16 respectively.The fv-gs6 overexpression transformant cells were different in shape and wider at the clamp connection than in wild type strain Fv01.(4)Mycelial growth analysis showed that the growth speed of some overexpression strains were faster than Fv01 while growing on PDA medium,but some were later.It indicated that fv-gs6 may have no influence on the mycelium cultivated on PDA.The results also showed that growth speed of the overexpression strains were relatively faster than Fv01 on PDA containing exogenous H2O2.However,growth speed of the overexpression strains were slightly fluctuates as compared to Fv01 on PDA containing exogenous SDS.Overall,strains showed high growth on simple PDA as compared to amended with exogenous H2O2 or SDS.It also indicated that H2O2 and SDS have inhibitory effects on mycelial growth.While mycelial growth inhibition was significantly higher in Fv01 than fv-gs6 overexpression strains.These results indicated that the fv-gs6 overexpression mutantant were less sensitive for H2O2 and SDS than Fv01.Under exogenous stress conditions(H2O2 and SDS),the mycelial growth inhibition rate of overexpression transformants,was lower than Fv01.It indicated that fv-gs6 may be involved in stress regulation mechanism.(5)Hyphal recovery conditions under mechanical damage:Results exhibited that overexpressed transformants accumulated more mycelium as a result of mechanical injury than Fv01,and contained dense mycelium.We speculated that fv-gs6 overexpression transformants may involved in promote mycelia recovery.(6)The fruiting body initiation test showed that the fv-gs6 had little influence on growth speed in the culture flask.After scratching,fv-gs6 overexpression transformants,promoted mycelium on the cultivated material surface to recovery.The primordium formation period for fv-gs6 overexpression transformants and the wild type strains were same.However,stipe length of four transformants were shorter than FvOl,but fv-gs6-OE12 was longer than Fv01.(7)Pearson correlation analysis showed that expression of fv-gs6 was weakly correlated with the mycelial growth speed on PDA but had a strong positive correlation with the growth speed on PDA media amended with H2O2 or SDS.Analysis also suggested a strong positive correlation with the cell wall thickness and cell width at the clamp connection while a weak connection with stipe length.