| Brown rot fungi,as one of the wood-rot fungi,can secrete large amounts of cellulases and hemicellulases.Even though with a little of lignin modification,its degradation role can rapidly depolymerize cellulose and hemicellulose,which plays an important role in the natural biological cycle.In our study,three kinds of brown rot fungi were collected from Maoershan experimental forest farm of Northeast Forestry University,including Fomitopsis pinicola,Piptoporus betulinus and Pholiota adiposa.The three kinds of brown rot fungi were studied with white birch sawdust induced treatment,no sawdust induced as control.Then,we detected the growth rate,biomass,cellulase and hemicellulase activities of those fungi and analyzed the genetic diversity of cellulase and hemicellulase degradation related enzyme gene expression by target region amplified polymorphism?TRAP?markers.The three kinds of brown rot fungi were cultured at 23? and 28? with PDA culture medium,and measured the diameter of colony everyday.The result showed that the average growth rate of Fomitopsis pinicola and Piptoporus betulinus was 0.91cm/d at 23?,and 1.04cm/d at 28?,respectively;while in Pholiota adiposa,the growth rate was 0.30cm/d and 0.33cm/d.Therefore,those fungi were suitable for incubation at 28?.Interestingly,the growth rate of Pholiota adiposa was not greatly influenced with temperature changes.With the condition of 28?,we used two different cultivate methods?static and shaking cultivation?to incubate fungi,and measured dry weight of mycelium every 48 hours,to determine the biomass changes in different conditions.The results showed thatthe biomass in Piptoporus betulinus reached the maximum value(7.45g·L-1)on the 6th day with static condition,while the highest value in Fomitopsis pinicola and Pholiota adiposa maximum was 4.47 g·L-1?6.71 g.L-1 on the 10 th day,respectively;under the shaking cultivation,the highest value in Piptoporus betulinus reached 4.17g·L-1 on the 8th day,while on the 12 th day and 14-day,Fomitopsis pinicola and Pholiota adiposa reached the highest value with 2.83g·L-1 respectively.This result suggested that those brown-rot fungi were more suitable for cultivate in static condition.The enzyme activity change of three brown rot fungi was determined by DNS method within 12 days.Our results showed that the highest value of cellulase activities were in b-endoglucanase,and the lowest was found in b-exoglucanase;in hemicellulase the ?-glucosidase activity was the higest,while xylanase and mannose harboed the similar values.The results of variance analysis demonstrated that the expression level of the six kinds of enzymes was significantly difference in different treatment and culture days.The ?-glucosidase in Fomitopsis pinicola and Pholiota adiposa,was significant and non-significant differences in culture days,respectively.In addition,compared with the control group and sawdust-inducted group,the induced group enzyme activities in Fomitopsis pinicola and Pholiota adiposa were higher than the control,suggesting that the sawdust could promote the enzyme sectetion.However,the enzyme activities in Piptoporus betulinus revesaled the reverse phenonmenon.The genetic diversity of these three brown rot fungi was studied by TRAP molecular marker to cellulose and hemicellulose degradation related enzyme genes.With the optimized TRAP-PCR system,out of 27 pairs of primers with good polymorphism clear and reproducible bands were selected from 96 pairs.The polymorphisms of the related enzyme genes in three brown rot fungi were detected by electrophoresis,and 676 bands were amplified,among 496 polymorphic bands and the polymorphic ratio was 73.37%.The number of bands yielded by the cellulose degradation related enzyme gene was 431,and the number of polymorphic bands was 311 with the percentage of polymorphism was 72.16%;the number of bands amplified by hemicellulose degradation related enzyme gene was 245 with 185 polymorphic bands,and the percentage of polymorphism was 75.51%.The results showed that the expression of the enzyme gene in three brown rot fungi had great genetic diversity and strong genetic variation. |
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