周娇娇,佘炜怡,王浩入,等.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(No.2(111-220)):122-131.[doi:10.3724/SP.J.1249.2017.02122]
Zhou Jiaojiao,She Weiyi,Wang Haoru,et al.Effect of 5-Aza-2'-deoxycytidine on the expression of cellulases in Trichoderma reesei[J].Journal of Shenzhen University Science and Engineering,2017,34(No.2(111-220)):122-131.[doi:10.3724/SP.J.1249.2017.02122]
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5-氮杂-2-脱氧胞苷对里氏木霉产纤维素酶的影响
周娇娇,佘炜怡,王浩入,谢 宁,田生礼

深圳大学生命与海洋科学学院,深圳市微生物基因工程重点实验室,广东深圳 518060

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PCR

Effect of 5-Aza-2'-deoxycytidine on the expression of cellulases in Trichoderma reesei
Zhou Jiaojiao, She Weiyi, Wang Haoru, Xie Ning, and Tian Shengli

Zhou Jiaojiao, She Weiyi, Wang Haoru, Xie Ning, and Tian ShengliCollege of Life Science and Marine Science, Shenzhen University, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen 518060, Guangdong Province, P.R.China

filamentous fungus; Trichoderma reesei(T.reesei); 5-Aza-2'-deoxycytidine(5'-Aza); DNA methylation; DNA methyltransferase; methylation specific PCR(MS-PCR)

DOI: 10.3724/SP.J.1249.2017.02122

备注

为探究脱氧核糖核酸(deoxyribonucleic acid, DNA)甲基化修饰对丝状真菌里氏木霉(Trichoderma reesei,T.reesei)产纤维素酶基因表达的表观遗传调控机制,利用不同浓度(0～1.0 mmol/L)的化学修饰剂5-氮杂-2-脱氧胞苷(5-Aza-2'-deoxycytidine, 5'-Aza)培养T.reesei QM9414.通过测定滤纸酶活性(filter paper enzymatic activities,FPA)和羧甲基纤维素钠酶活性(carboxymethyl cellulose-Na enzymatic activities, CMCA)确定纤维素酶活性的变化; 利用实时荧光定量PCR(real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测0.1 mmol/L 5'-Aza培养的T.reesei QM9414中纤维素酶基因cbh1、 egl1、 酶激活因子xyr1基因以及分解代谢阻遏因子cre1与ace1基因的表达水平; 通过甲基化特异性PCR(methylation specific PCR,MS-PCR)分析cre1、 ace1、 cbh1、 egl1和xyr1基因上游序列的甲基化状态; 利用Western blot和DNA甲基化定量测定分析了DNA甲基转移酶和DNA的甲基化水平.结果显示,0.1 mmol/L 5'-Aza处理后的T.reesei QM9414菌株的FPA和CMCA最高,比出发菌株T.reesei QM9414分别高30%和53%; RT-qPCR结果显示,处理后的T.reesei QM9414菌株中纤维素酶基因cbh1、 egl1与酶激活因子xyr1基因的相对表达量均高于出发菌株,而分解代谢阻遏因子cre1和ace1基因的相对表达量与出发菌株无明显差异; MS-PCR结果显示, cbh1、 egl1和xyr1基因的非甲基化状态高于出发菌株,而分解代谢阻遏因子cre1与ace1基因的非甲基化状态也无明显差异; Western blot和DNA甲基化定量测定结果分别显示,5'-Aza处理后菌株的DNA甲基转移酶表达比出发菌株明显降低,全基因组DNA甲基化程度也有下降.研究结果表明,5'-Aza处理T.reesei QM9414菌株后,纤维素酶活性明显增加,纤维素酶基因cbh1、 egl1和激活因子xyr1基因表达水平的增加可能是通过DNA甲基转移酶影响DNA甲基化水平,最终调控基因的表达.

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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