Journal of Shenzhen University Science and Engineering (深圳大学学报理工版) 2017/34:2 PP.122-131
Filamentous fungus Trichoderma reesei QM9414 were treated with different concentrations of chemical reagent 5-Aza-2'-deoxycytidine in order to explore epigenetic regulatory mechanism of DNA methylation on cellulase expression. Then the cellulase activities with filter paper and carboxymethyl cellulose-Na enzymatic activities were examined. Real-time fluorescent quantitative PCR (RT-qPCR) was used to analyze the expressions of cellulase gene cbh1, egl1,activator gene xyr1,and metabolic repressor gene cre1 and ace1 in the treated T.reesei QM9414 strains. Methylation status of upstream of gene cbh1, egl1, xyr1, cre1 and ace1 was deteced by methylation specific PCR (MS-PCR) analysis. The results illustrate that the 0.1 mmol/L treated stains manifest the highest enzymatic activities including filter paper and CMC-Na enzymatic activities, 30% and 53% higher than those of the starting strain respectively. RT-qPCR reveals that the expression levels of the gene cbh1, egl1, xyr1 are also higher than those of starting strain, while the expressions of gene cre1 and ace1 are not significantly changed. Further analysis by MS-PCR indicates that the non-methylated statuses of upstream of gene cbh1, egl1, xyr1 are higher than those of the starting strain, as well as the gene cre1 and ace1 are not obviously vary. Furthermore, DNA methyltransferase by the Western blot analysis and DNA methylation quantification show that the expression levels of methyltransferase treated with 5-Aza-2'-deoxycytidine are lower than that of the starting strain, and the methylation levels of genomic DNA also decrease. In short, the cellulase activities of T.reesei QM9414 significantly increase after treatment with 5'-Aza. The expression of cbh1, egl1 and xyr1 genes may be related to DNA methylation by DNA methyltransferase level, and finally regulate cellulase genes expression.
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