| Currently, the fossil fuels are at the danger of being used up. Every country of the world is looking for the new substitutable energy, in order to ensure the energy supply and the society development. Cellulose is one of the most abundant renewable energy sources in the nature. Therefore, effective use of cellulose will be an excellent way to alleviate the energy crisis. Most of cellulose in the nature could be degraded by microorganisms, among which filamentous fungi play an important role. The lignocelluloses-degrading enzymes have been widely used in many areas such as energy, textile, pulp and paper, dyeing and food. Since the low production yield and specific activity of the secreted cellulase in filamentous fungi, researchers have been paying much attentions to improve the production and enzyme activity of cellulase, especially focus on the selection and breeding of strains. Methods such as traditional physical or chemical mutation, cell fusion and other genetic operations are the universal strategies. The cellulase expression level of filamentous fungi is related to the environmental carbon source. Due to the little knowledge about the regulation mechanism of cellulase expression, the induction of cellulase is difficult to control exactly and the production level is not stable. To understand the synthesis and regulation of cellulase expression is not only important for theoretical studies, but also helpful to renovate the strain directly on the genetic level.Current research is mainly around the cellulase synthesis regulation of Trichoderma and Aspergillus. Although many achievements have been obtained on varied levels about the Trichoderma and Aspergillus, there is few research regarding the transcriptome analysis of cellulase synthesis regulation in other filamentous fungi, such as Penicillium. We studied the transcription profiling of Penicillium decumbens 114-2 by directly comparing the gene expression profiling obtained from the glucose medium and wheat bran-microcrystalline cellulose medium, respectively, which would provide some information about the regulation mechanism of cellulase synthesis. Moreover, we established the transformation system in the P. decumbens, and it would be helpful to directly renovate the strain by genetic engineering technology. The main results of the thesis are as follows:1 Analysis of the transcription profiling of gene expression of Penicillium decumbens using digital gene expression tag profiling (DGE) technology.The differences on gene expression level were reported by directly comparing the gene expression profiling produced in the glucose medium and in the wheat bran-microcrystalline cellulose medium by Digital Gene Expression tag profiling (DGE) technology. Two libraries were built from P. decumbens 114-2 cultured with glucose and wheat bran-microcrystalline cellulose, which were named G library and W library, respectively. The G library had 3,161,822 total tags including 230,285 distinct tags, and the W had 3,137,364 sequenced total tags with 215,955 distinct tags. 114,548 differentially expressed distinct tags were detected between the two libraries, including 55,050 up- and 59,498 down-regulated distinct tags in the comparison of W library with G library. We use FDR (false discovery rate)< 0.001 and the absolute value of log2Ratio≥1 as the threshold to judge the significance of gene expression difference, there were 4,051 tags up-regulated and 6,595 tags down-regulated significantly under induction condition.The sequencing tags were mapped to the P. decumbens 114-2 genome and the result showed that 51.05% and 55.15% tags of library W and G mapped to reference genes from P. decumbens 114-2. The other no mapping tags were mapped to genome sequence, result showed that 37.83% and 39.18% tags were mapped to the genome. Finally there were about 90% tags could be mapped to the P. decumbens genome.Using FDR<0.001 and the absolute value of log2Ratio≥1 as significant different expressed standard, there was 2,396 different expressed genes between the two library. Under induced condition,924 genes were up-regulated and 1,472 genes were down-regulated. The cellulase and other glycoside hydrolases genes were up-regulated significantly, indicating the co-regulation of these cellulose degradation genes. And the expression level of some genes encoding transport proteins and membrane-bound proteins were up-regulated too. The expression levels of genes encoding ion transport proteins were down-regulated under the induced condition. The result of Gene Ontology functional enrichment analysis for the different expressed genes showed that extracellular carbohydrate hydrolases were the most significant differentially expressed genes.2 Confirmation of the changed-folds of cellulase genes expression under induced and repressed conditions from the DGE result using real-time quantitative PCR analysis.The expression of 5 genes coding cellulolytic enzymes (cel5A, cel7A, cel7B, cel45A and swollenin) was up-regulated significantly under wheat bran-microcrystalline cellulose culture condition when detected using DGE. The result of RT-qPCR showed that the 5 genes, swo, cel7A, cel7B, cel5A and cel45A, were all up-regulated about 325-fold,149-fold,109-fold,58-fold and 58-fold under induction condition, respectively. cel3A was down-regulated about 0.57-fold under the same condition. The other 3 regulator genes were all down-regulated slightly under induction condition. The results of RT-qPCR were consistent with the result of DGE, indicating that DGE was a believable method to analyse the regulation of cellulase expression in fungi.Moreover, we investigated theβ-glucosidase activity of P. decumbens under the two conditions. Extracellularβ-glucosidase activity was much higher, and the band of 120 kDaβ-glucosidase was much stronger under induction condition than that under repression condition. On the other hand, the intracellularβ-glucosidase activity under repression condition was higher than that under the induction condition. These results suggested that cel3A might be regulated via different mechanisms from other cellulases. The cellular location of this protein may be regulated under the two conditions.3 The construction of genetic transformation system for P. decumbens and the expression of cel5A from T. reesei in P. decumbens M12. A P. decumbens pyrG gene deficient strain Ml2 was obtained by spontaneous mutation. Subsequently, the gene encoding endonucleases II (Cel5A) from Trichoderma reesei was transformed into the mutant strain Ml2 using protoplast-mediated transformation technology. Native gel electrophoresis and measurement of endoglucanase activity and filter paper activity suggested that cel5A from T. reesei was successfully expressed in P. decumbens.4 The construction of deletion vector of vacuole serine protease gene to study its function in P. decumbens.The gene encoding vacuole serine protease (VSP) was found to be significantly down-regulated when cultured with inducing cellulose according to DGE result. The fragment of vsp gene in P. decumbens was amplified, and then the flanking sequence was obtained by using TAIL-PCR and SEFA-PCR. The deletion vector of vsp was constructed using fusion PCR and was transformed into P. decumbens. Transformants were selected and identified using PCR. Unfortunately, no vsp deletion has been detected in the transformants so far. |
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