| Lignocellulose,as one of the main components of the plant cell wall,is the most abundant renewable carbon on earth.Lignocellulose degraded by cellulase can be used in food industry,paper industry,animal husbandry,bio-ethanol research and so on.Many microorganisms,such as Trichoderma,Penicillium and other mesophilic fungi,can produce cellulase,.Compared with medium temperature fungi,thermophilic fungi are able to withstand high temperature environment,which is not susceptible to be polluted by medium temperature fungi.Most of the cellulases produced by the thermophilic fungi have good thermal stability and can degrade the cellulose quickly,which brought them broad application prospects.Myceliophthora thermophile ATCC42464 is a filamentous fungi.The cellulases it produced have good thermal stability and can degrade many kinds of cellulose.Thus,Myceliophthora thermophile ATCC42464 is a potential medium-high temperature enzymes library.In the present study,we selected two potential regulatory factors,MHR1 and MHR2 from M.thermophile ATCC42464 by RNA-Sequencing(RNA-Seq).The roles of MHR1 and MHR2 on gene regulation of cellulase were investigated by using RNA interference and homologous overexpression technology,respectively.The main contents of this study are as follows:(1)The siRNA sequence of mhr1 gene was designed.The silencing expression cassette of mhr1 gene was constructed by using p UC19-M plasmid.The recombinant plasmid was transformed into M.thermophile ATCC42464 with the aid of plasmid p AN7.1 through co-transformation.The transformants were screened by using PDA plates with hygromycin B and genomic DNA PCR analysis.Five transformants were obtained successfully.According to the results of RT-qPCR,the transformant Mt R5 was the best one,which had the highest interference rate of mhr1 gene.Mt R5 and the wild-type strain(WT)were both cultured under inducing and non-inducing mediums,then their extracellular protein concentration,cellulase activities and expression of main cellulase genes were measured.When cultured in non-inducing medium for 144 h,the extracellular protein concentration,the filter paper activity(FPA),the endoglucanase activity(EGase activity)and the xylanase activity in Mt R5 were 1.94,1.52,1.47 and 1.20-fold higher,respectively,than those in WT.The results of qPCR showed that the gene expressions of cbh1,egl3 and xyr1 in Mt R5 were 6-to 10-fold higher than those in WT.When cultured in inducing medium for 72 h,the extracellular protein concentration and the cellulase activities in Mt R5 were higher than those in WT.The results of qPCR showed that the gene expressions of cbh2,egl3 and xyr1 in Mt R5 are 28-to 56-fold higher when compared to the wild type.The results indicated that silencing mhr1 gene can improve gene expressions of some cellulase and increase the cellulase activities.Therefore,the regulatory factor MHR1 is acting as a repressor for the gene expressions of cellulase in M.thermophile.(2)In this study,we obtained the full gene sequence of MHR2 from NCBI database and constructed the overexpression vector of mhr2 gene.By protoplast transformation,screening using PDA plate with hygromycin B,PCR identification,and sequencing comparison,five transformants were obtained successfully.According to the results of RT-qPCR,the transformant Mt O24 had the highest expression of mhr2 gene.When cultured in inducing medium for 72 h,the extracellular protein concentration and the cellulase activities in Mt O24 and WT were both up to peak.The extracellular protein concentration,the filter paper activity,the endoglucanase activity in Mt O24 were 1.58-,1.30-,and 1.24-fold higher,respectively,than those in WT.The results of qPCR showed that the gene expressions of four main cellulases in Mt O24 were several folds higher than those in WT,which was consistent with the increase of cellulase activities.Our studies showed that MHR2 is a regulatory factor on gene regulation of cellulase in M.thermophile,and worked as an activator on cellulase gene expression. |
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