| Lignocellulosic biomass is the most abundant renewable resource on the planet and has broad application prospects in the production of bulk bio-based products.The effective utilization of biomass resource can not only solve the increasingly serious energy crisis faced by human society,but also turn waste into treasure to solve the environmental pollution problems caused by burning straw.China is a large agricultural country,increasing the share of biomass energy in the new energy field is not only in line with the strategic requirements of sustainable development,but also an urgent need for ecological civilization construction.Trichoderma reesei is a model fungus that degrades biomass and also is a major industrial production strain of cellulase.In recent decades,the synergistic mechanism of the cellulase system of T.reesei in the process of degrading cellulose has been preliminarily analyzed through the study of the physicochemical properties of cellulase and the hydrolysis efficiency of cellulose has been improved using greatly optimized composition of the T.reesei enzyme system.Further increasing the production capacity of T.reesei cellulase to reduce the cost is a prerequisite for promoting the industrial production of biofuel ethanol and other bulk bio-based chemicals.In recent years,studies have shown that the expression of T.reesei cellulase genes are regulated by light signal and extracellular nutrition signal.It is key to analyze mechanism of these signal pathways and integrate them effectively to improve the cellulase production capacity of T.reesei.Although a variety of transcriptional regulators involved in the expression of the T.reesei cellulase genes have been identified,the precise mechanism regulating cellulase gene expression by these factors is not well understood.Therefore,systematically elaboration of the molecular network mechanism of T.reesei cellulase expression will provide new ideas of the effective strain engineering strategy to achieve the above objectives.In this study,we identified the transcriptional regulation complex Cyc8-Tupl?also known as Ssn6-Tupl?in T.reesei and investigated its function in cellulase gene expression.The main research results are as follows:1 The copper-controlled RNAi system for reversible silencing of target genes was established and the physiological functions of several genes in T.reesei were characterized using it.Gene knockout mediated by homologous recombination is a common method of genetic manipulation in T.reesei for characterizing function of target genes,but in practice,inefficiency or unsuccessful knockout of some genes that are essential for the basic growth or sporulation are present.Therefore,our laboratory developed a genetic operating system named promoter replacement system that relied on the controllable promoter tcu1.In that system,the addition of copper ions in the media will repress the expression of target gene to achieve gene knockdown and the target gene will be overexpressed when the copper ions are absent of the media.In order to avoid the possible adverse effects resulted from the overexpression of target gene in genetic manipulation,we further developed a copper-controlled RNA interference?RNAi?system.In this system,konck down of target genes with the help of endogenous RNAi mechanism is achieved when the copper ions are absent in the media and the RNAi process will be closed by adding copper ions into the media to simulate gene replenishment.Both systems rely on the tight control of copper ions for the expression of the tcul promoter.In order to validate the effectiveness of the copper-controlled RNAi system,we successfully achieved knockdown of the constitutively expressed gene pyr4,the cellulose-induced genes cel7a and xyrl and the gene fabl which is difficult to knock out by homologous recombination.More importantly,the relevant phenotype of RNAi strains that integrate this system had been significantly restored after the addition of copper ions,which will eliminate the negative effects of random integration of hairpin RNA?hpRNA?expression cassette into the genome.This fully demonstrated the utility and feasibility of the copper-controlled RNAi system for characterizing the function of target genes in T.reesei.2 Cyc8-Tupl complex and transcriptional activator Xyrl mediate the inducible expression of T.reesei cellulase genes by an interdependent manner.Our co-immunoprecipitation?CoIP?assays showed that T.reesei Cyc8 and Tupl is a stable complex in vivo.The copper-responsive promoter replacement system and RNAi system were used to knock down tupl and cyc8,respectively.The results indicated that the konckdown of cyc8 or tup1 seriously affected the inducible expression of the T.reesei cellulase genes,which is contrary with those researches in which Cyc8-Tupl complex is mostly involved in transcriptional repression,suggesting that T.reesei Cyc8-Tupl complex may play an important activation function in the expression of cellulase genes.In addition,the complex also plays a crucial role in the conidiation.Chromatin immunoprecipitation?ChIP?assays showed that Cyc8-Tupl complex can be recruited to the cellulase gene promoter to directly regulate their transcription and the recruitment of this complex on the promoter is depend on the transcriptional activator Xyr1?xylanase regulator 1?.Moreover,knockdown of cyc8 or tup1 also leaded to the inhibition of nucleosome depolymerization on the promoter region of the cellulase genes under cellulsoe-induced condition,which may repress the assembly of preinitiation complex?PIC?.Overexpression of Xyrl did not restore the cellulase expression in cyc8 or tupl knockdown strain and the binding of Xyrl on the promotor of cellulase genes is also dependent on the presence of Cyc8-Tup1.While simultaneous overexpression of Xyrl and Tupl greatly increased the cellulase production,which further indicated that there is a synergistic regulation mechanism between Xyr1 and Tupl.In conclusion,the Cyc8-Tupl complex and the transcription factor Xyr1 mediate the expression of T.reesei cellulase genes by an interdependent manner.3 Tupl is a global regulator and knockdown of tupl in T.reesei does not relieve carbon catabolite repression.We performed a comprehensive analysis of the regulatory targets of Tupl in the T.reesei genome?10239 genes?using the RNA-Seq.Overall,Tup1 repressed 2207?21.6%of the genes in genome?genes in T.reesei and activated 1641?16%?genes under glucose condition.Under cellulose-induced condition,Tupl repressed 1988?19.4%?genes in T.reesei and activated 1961?19.2%?genes.The above results indicated that Tupl is a global regulator.Surprisingly,the knockdown of tupl in T.reesei did not result in drastically increase of the expression of most?hemi?cellulase genes under glucose condition,which is inconsistent to the CCR of specific gene?such as invertase encoding gene?mediated by Tup1 in saccharomyces cerevisiae.This result indicated that Tupl is not involved in CCR in T.reesei or other regulatory mechanisms other than Tupl are involved in the CCR of T.reesei.Compared with glucose condition,1017 genes were up-regulated?Induction-up?and 1993 genes were down-regulated?Induction-down?in T.reesei genome under cellulose-induced condition,while knockdown of tupl resulted in decreasing expression of 408 genes?40%?in the gene set of Induction-up and increasing expression of 461?23%?genes in the gene set of Induction-down under cellulose-induced condition.About 50%of genes that contains most?hemi?cellulase genes invoved in biomass degradation regulated by Tupl in inducible gene sets?including Induction-up and Induction-down?also were regulated by Xyrl under cellulose-induced condition,which indicted that the regulatory pattern between Tupl and the transcriptional activator Xyrl has some overlaps.4 A S.cerevisiae Cti6-like protein Clpl was identified in T.reesei and it was be proved that Clpl is involved in the regulation of cellulase gene expression and conidiation.The S.cerevisiae Cti6?Cyc8-Tupl interacting protein 6?contains a PHD?plant homeodomain?domain and directly interacts with Cyc8.We identified a Cti6 like protein Tr 27020 in T.reesei by bioinformatic analysis and named it Clpl?Cti6 like protein 1?because it shares low sequence identity with Cti6.Clpl contains two conserved domains:PHD and UIM?ubiquitin interacting motif?,but S.cerevisiae Cti6 only contains the PHD domain.After knockout of clp1,the conidiation and cellulase expression of T.reesei were severely impaired.Overexpression of Xyrl in the ?clp1 strain largely rescued the expression of cellulase to higher level than the control strain,which indicated that overexpression of Xyrl bypassed Clpl to activate the expression of cellulase genes.Cell localization analysis showed that Clpl is a nuclear protein and mutation of PHD domain or deletion of UIM domain did not affect the nuclear localization of Clpl,but mutation of PHD domain leaded to abnormal localization of Clp1 in nucleus.Unlike the UIM deleted strain,the expression of cellulase and conidiation in the PHD mutant strain was significantly reduced,which suggested that the PHD domain of Clpl plays a crucial role in the cellulase gene expression and conidiation.ChIP assays indicated that Clpl was recruited to cellulase gene promoter to regulate their transcription and its recruitment on cellulase gene promoters is dependent on cellulose-induced condition.5 The transcription factor Rxel that modulated the expression of xyrl was identified and it was be proved that Rxel is involved in the cellulase gene expression and conidiation of T.reesei.The Cyc8-Tupl is a highly conserved complex for transcriptional regulation in eukaryotes.Studies have shown that this complex is involved in a variety of cellular physiology processes.Therefore,we speculated that the T.reesei Cyc8-Tupl complex also may participate in the expression of cellulase genes through other pathways besides direct regulation based on its recruitment on the promoters.We identified a C2H2 type transcription factor Tr108357 whose expression is much higher under cellulose-induced condition than glucose condition in T.reesei by RNA-Seq data.Knockdown of tupl resulted in significantly decrease of its expression level under cellulose-induced condition.In addition,it was found that this transcription factor had obvious bindding activity on the xyrl promoter by yeast one-hybrid assay in our preliminary work of laboratory,so it was named Rxel?Regulator 1 of xyrl expression?.In order to characterize the physiological function of Rxel in T.reesei,we tried to knockout the rxel gene in T.reesei but without success.So the copper-controlled RNAi system was used for conditionally knockdown of rxel.The results showed that knockdown of rxel significantly reduced the expression of xyrl and cellulase genes and absolutely abolished conidiation of T.reesei.Constitutive expression of Xyrl in the rxel knockdown strain restored induced expression of the cellulase genes to comparable or even exceed level of control strain,but it can not restore the defect of conidiation resulted from knockdown of rxe1,indicating that Rxe1 may controll the expression of cellulase genes by modulating the expression of xyr1.In conclusion,we identified a novel regulatory factor Rxe1 who not only regulates the expression of xyr1 and cellulase genes but also controls conidiation of T.reesei. |
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