| Cellulose is the one of the most abundant and widely distributed carbohydrates on the earth, and it is also the main component of cell wall of plants. About 1.5-1.7x 1011 tons of cellulose was produced by plants through photosynthesis every year.. Production and degradation of cellulose are the important components of the total Earth’s carbon cycle. On the other hand, with the exhaustion of fossil fuels, more and more attentions were paid to the new biomass regenerated energy, and biofuels transforming from cellulose had been a hot field of research for many years. Currently, filamentous fungi have been the main industrial strains for production of cellulases, and Trichoderma reesei is one of the typical representatives. Trichoderma reesei could be capable of secreting three kinds of cellulases, exocellulases, endocellulases and glucosidases, which could account for more than 60% of the extracellualsr proteins. Meanwhile, an important feature for Trichoderma reesei was that the production of cellulases are largely induced by inducing signal, such as Avicel, lactose and sophorose so on, and the production could also be inhibited under glucose. Although that the mechanism of large amout of cellulases for Trichoderma reesei could be efficiently indcued was paid attention in recently years, it is still unclear.Based on the prior research, among the cellulase genes transriptional regulators of Trichoderma reesei, Xyrl is the most key transcription factor to regulate the trancription of cellulase genes. And the transcription of almost all of the cellulase genes except for bgl2 were abolished in the absence of xyrl. So based on the function of Xyrl, we would study the mechanism of celluase genes transcription regulation deeply, and then illuminate the cellulases genes transcripitonal regulatory networks. Meantime the research will also provide theoretical guidance for the rationally genetical modification of industrial strains and impvoing the production of cellulases in industry.This thesis was about the function of Xyr1, and the major studies and results are as follows:1. Analysis of the structures and biological properties of Trichoderma reesei cellulase genes transactivator, Xyr1.According to bioinformatic analysis, Xyrl, a celullulases genes transcription factor in T.reesei, was a zinc binuclear cluster transcription factor like Ga14 in Saccharomyces cerevisiae. The homologues of Xyrl were found widespread in filamentous fungi by phylogenetic analysis. Based on the structure of Ga14, the structure of Xyr1 was divided into DNA binding domain (DBD), Middle Homo logy Region (MHR) and Activating domain (AD). Xyr1 DBD was found that it could bind to cbh1 promoter by yeast one-hybrid and EMSA. Meantime, unlike Ga14AD, Xyr1 AD is not consist of just one acidic region, it contains a acidic-hydrophobic-basic domain. While Xyrl AD was found capable of interacting with MHR domain, and it is supposed that the interaction may be related to the self-regulating of Xyr1.In order to analysis the function of Xyr1 domains in vivo, a series of Xyr1 mutants were constructed in Axyr1 and then activating ability to cellulases genes was measured. Xyr1 DBD alone in Axyr1 could not activated the transcription of cellulase genes and it also could not combine with cbhl promoter by ChIP analysis. While the expression of MHR-AD domain, namely the DBD-deficient mutant, showed that it localized in cytoplasm on glucose and then transferred into vacuole on glucose depletion or inducing conditions. As for the MHR-deficient mutant of Xyr1, no fulorescent signal could be detected, which suggested that MHR domain played an important role in maintaining the stablity of Xyr1. However, the AD-deficient mutant of Xyr1 had a weak cellluase genes activating acitvity, although the fulorescent signal were fainter than Xyr1 ORF. Surprisingly, the absence of Xyr1 AD basic domain led to loss of activating ablility and fulorescent signal. So it suggested that the function of Xyrl AD was more important for structure maintance than its acidic activating ability. So the above results showed that the MHR and AD are important for maintaining the stablility of Xyr1 and opening the chromatin of promoter for recruitment of transcriptional complex.2. The analysis and application of a copper responsive promoter Ptcu1.In order to analyisis of the function of Xyr1, a GFP tagged Xyr1 was built drived by Pgpd. In 1% glucose cultural condition, there was bright fluorescent signal, but in the lactose or Avicel culural conditions, hardly fluorensent signal was detected. And then the gfp-xyr1 showed weaking cellulase genes activating ability compared to the parent strain, QM9414. It is sugested that the driving ability of Pgpd depended on the carbon source and it is weak on Avicel. So in order to study the transcription of cellulase genes, we had to seek for a promoter which had a strong driving ability and worked indpendent of carbon source. We identified a copper transporter encoding gene tcul (Tr52315) in T.reesei by BlastP with homologues of yeast and N.carassa. By qPCR it showed that the transcription of tcu1 was very high on glucose, glycerol or Avicel as sole carbon source and repressed by copper. The transcription of tcul was reduced in accordance with the addition of copper, and the shut-off of copper was very fast. However, only a weak recovery of tcul transcription after copper chelator addition. In the strain of Ptcul-gfp in QM9414, copper could regulated the expression of GFP observed by fluorescence microscope. When cbhl and egl was driven by Ptcu1 in TU6, a large amount of CBH1 and EG1 were secreted into supernatant and the secretion could be repressed by copper. And when the native promoter of a gene was replaced with Ptcul, overexpression and limit-expression of the gene could be analyzed in one strain by copper regulating. So that Ptcul could not only high-efficiently express heterologous proteins in T.reesei, but also it could regulated transcription of a specific gene by copper in vivo indpendent of carbon source.3. Overexpression of Xyr1 led to constitutive production of cellulases and the putative mechanism for the cellulase genes regulation by Crel.The N-terminus of Xyr1 was modified by GFP, and then it showed that Xyr1 were appeared in the nucelus no matter on inducing or non-inducing conditions. And Ptcu1-xyr1 were randomly inserted into the genome of △xyr1 and get the constitutive expression of xyr1, then it showed that the transformant could derepress the carbon catabolite repression on glucose and glycrol, and on glycerol and glucose Ptcu1-xyr1 could product equal amount of cellulases compared the parent strain QM9414 on Avicel. The ability of cellulases production releasing from CCR of Ptcu1-xyr1 on non-inducing condition was in accord with the amount of the expression of Xyr1. Although the production of cellulases of Ptcu1-xyr1 was not higher than QM9414 on Avicel, on lactose Ptcu1-xyr1 could product more cellulases than QM9414. And the overexpression of Xyr1 led to be more sensitive to the stress. Moreover, With the analysis of chromatin immunoprecipitation (ChIP), no matter on Avicel or glucose, the efficiency of Xyr1 binding to cbhl promoter of Ptcu1-gfp-xyr1 was higer 20-30 times than that of QM9414 on Avicel. Meantime by Chromatin accessiblity by real-time PCR (CHART-PCR), it suggested that the cbhl promoter of Ptcu-gfp-xyrl was more accessible than that of QM9414 on glucose. Therefore the improvement of Xyr1 binding effiency and then open the structure of chromatin of cellulase genes promoters may lead to the expression of cellualses on glucose.Cre1, an important repressor for cellulases expression, played an important role in carbon catabolite repression in Trichoderma reesei. It is reported that the Crel could bind to cbhl promoter by EMSA and then repress its transcription. Whereas we found that Cre1 could also bind to xyrl promoter by yeast one-hybrid in vitro or ChIP analysis in vivo. By regulating the expression of Ptcu1-cre1 by copper it showed that lack or overexpression of Crel both decreased the production of cellulases. However, the overexpression of Crel in the strain of Ptcul-gfp-xyrl did not influence the production of cellulases no matter on glycerol or Avicel. So it showed that the repression of cellulase genes transcription caused by Crel may be due to regulating transcription of xyr1.4. The function of Xyr1-related proteins in cellulase genes transcription.In the process of cellulase genes transcription, there were other factors interacted with Xyr1, regulating the transcription commonly. In order to seek for the proteins directly interacted with Xyr1, a yeast two-hybrid library screening was done by XYR1AD. And the C-terminal part of Tr30478 was fished by Xyr1AD, meanwhile it also found that it could also interacted with Xyr1MHR. In Trichderma reesei, Tr30478 was found localized in cytoplasm, and identified as a repressor for cellulases production by knockout and overexpression. It is supposed that Tr30478 maybe take part in the inner-interaction of Xyr1 AD and MHR.Accompanied by the cellulase inducing, the transcription of xyrl could also be induced. In order to identify the trancriptional factors which regulated the transcription of xyrl, yeast one-hybrid library screening was done to fish the protein interacted with xyrl promter. And 3 proteins was fished from the cellulose-inducing library, two transcription factors and one kinase Sch9, which maybe participate in inducing of Xyrl. |
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