深圳大学学报（理工版）2017，Vol.34Issue(2)：122-131,10.DOI:10.3724/SP.J.1249.2017.02122
5-氮杂-2-脱氧胞苷对里氏木霉产纤维素酶的影响
Effect of 5-Aza-2′-deoxycytidine on the expression of cellulases in Trichoderma reesei
摘要
Abstract
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.关键词
丝状真菌/里氏木霉/5-氮杂-2-脱氧胞苷/DNA甲基化/DNA甲基化转移酶/甲基化特异性PCRKey words
filamentous fungus/Trichoderma reesei (T.reesei)/ 5-Aza-2′-deoxycytidine (5′-Aza)/DNA methylation/DNA methyltransferase/methylation specific PCR (MS-PCR)分类
生物科学引用本文复制引用
周娇娇,佘炜怡,王浩入,谢宁,田生礼..5-氮杂-2-脱氧胞苷对里氏木霉产纤维素酶的影响[J].深圳大学学报（理工版）,2017,34(2):122-131,10.基金项目
National Natural Science Fundation of China (31601014) （31601014）
Shenzhen Science and Technology Research Foundation(JCYJ20150525092940997)国家自然科学基金资助项目(31601014) （JCYJ20150525092940997）
深圳市科技计划资助项目(JCYJ20150525092940997) （JCYJ20150525092940997）
