| Lentinula edodes(Berk.)Pegler is an important white-rot mushroom,which mainly degrades lignocellulose by lignocellulases secreted into the extracellular in hydrolase and oxidase mechanism.Because the lignocellulase activity is consistent with the expression of the corresponding genes,it is of great significance to increase the expression level of the extracellular lignocellulose-degradation associated genes of the fungus to promote the effective utilization of the substrate.The expression of the fungal lignocellulase genes are regulated by various transcription factors,but only a few white-rot-fungi associated transcription factors have been reported.Previous studies suggested that an endogenous transcription factor LELCRP1(Lentinula edodes Lignocellulase Genes Regulation Protein 1)with HMG-box domain may be involved in the expression of lignocellulase genes in Lentinula edodes heterocaryotic strain W1.In this study,p CAMBIA1300-g,in which the hygromycin-resistance gene was promoted by galactosylglyceraldehyde triphosphate dehydrogenase gene promoter of L.edodes(legpdh),was used as the vector backbone.The legpdh promoter and the lelcrp1 antisense strand fragment were linked by the double-joint method,and then connected to the linearized vector through homologous recombination to construct lelcrp1 RNAi vector,which was transformed into the mycelia of L.edodes heterotypic strain W1 by Agrobacterium tumefaciens-mediated transformation(ATMT).Four silencing transformants were screened and the expression level of lelcrp1 were reduced to 12.6%,16.5%,15.3% and 16.2% of the original strain,respectively.Analysis of expression profiles of 27 lignocellulase genes in four RNAi transformants revealed that,9 cellulase genes,1 hemicellulase gene,2 auxiliary enzymes AA9 genes and 1 manganese peroxide gene were down-regulated significantly,compared to the original strain.Southern blot and TAIL-PCR analysis proved that the 13 down-regulated lignocellulase genes above were related to the transcriptional regulation of lelcrp1.Detection of mycelial extracellular enzymatic activity in cellulose + lignosulfonate(SLS)medium found that the activity of filter paper cellulase(FPA),cellobiohydrolase(p NPCase)and manganese peroxidase(Mn P)in RNAi transformants were significantly decreased compared to W1,indicating that LELCRP1 protein mainly affected the activity of cellulase and lignin degrading enzymes of L.edodes.The mycelial growth under different carbon source substrates showed that the mycelial growth rate of RNAitransformants in a slightly single carbon source was significantly slower than that of the original strain,but no difference in the complex matrix.The above test results showed that,LELCRP1,an endogenous transcription factor with HMG-box domain in L.edodes,can affect the expression and enzymatic activities of some lignocellulase genes,as well as the utilization of some carbon source substrates.This paper lays a preliminary foundation for further study of the molecular mechanisms and regulatory networks of lignocellulase genes,and provides new strategies to improve the transformation and utilization efficiency of the lignocellulosic substrates of L.edodes,which has important scientific significance. |
