5-氮杂-2-脱氧胞苷对里氏木霉产纤维素酶的影响
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摘要
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-q PCR)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,0.1mmol/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-q PCR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 chemicalreagent 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-q PCR) 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-q PCR 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.
Filamentous fungus Trichoderma reesei QM9414 were treated with different concentrations of chemicalreagent 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-q PCR) 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-q PCR 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.
引文
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[12]林剑青,赵西西,贺竹梅.基于转录组测序的黄曲霉对5-氮杂胞苷应答机制研究[C]//第四届全国微生物基因组学学术研讨会论文集,济南:[s.n.],2012:1.Lin Jianqing,Zhao Xixi,He Zhumei.Response mechanism of Aspergillus flavus to 5-Azacytidine based on sequencing of transcriptome[C]//Proceedings of the Fourth National Symposium on Microbial Genomics.Jinan:[s.n.],2012:1.(in Chinese)
[13]Yang Kunlong,Zhuang Zhenhong,Zhang Feng,et al.Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor[J].Food Additives and Contaminants:Part A,2015,32(4):554-563.
[14]Yang Kunlong,Liang Linlin,Ran Fanlei,et al.The Dmt A methyltransferase contributes to Aspergillus flavus conidiation,sclerotial production,aflatoxin biosynthesis and virulence[J].Scientific Reports,2016,6:23259.
[15]Ghose T.Measurement of cellulase activities[J].Pure and Applied Chemistry,1987,59(2):257-268.
[16]Jeon J,Choi J,Lee G W,et al.Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus,Magnaporthe oryzae[J].Scientific Reports,2015,5:8567.
[17]Logan P C,Ponnampalam A P,Rahnama F A,et al.The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells[J].Human Reproduction,2010,25(11):2859-2869.
[18]Armstrong R N,Colyer H A,Sharpe D J,et al.5-aza-2'-deoxycytidine,a DNA methylation inhibitor,induces miRNA expression changes in acute myeloid leukaemia cell lines that transcend subtype[J].British Journal of Haematology,2011,153(1):14-15.
[19]Chen Taiping,Li En.Structure and function of eukaryotic DNA methyltransferases[J].Current Topics in Developmental Biology,2004,60:55-89.
[20]Christman J K.5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation:mechanistic studies and their implications for cancer therapy[J].Oncogene,2002,21(35):5483-5495.
[21]龚映雪,台艳,肖文娟,等.酵母多基因表达载体在纤维素生物转化中应用[J].深圳大学学报理工版,2010,27(1):82-87.Gong Yingxue,Tai Yan,Xiao Wenjuan,et al.Construction of saccharomyces cerevisiae integrated expression vector and its application in cellulose bioconvesion[J].Journal of Shenzhen University Science and Engineering,2010,27(1):82-87.(in Chinese)
[2]Evans H H,Evans T E,Littman S.Methylation of parental and progeny DNA strands in physarum-polycephalum[J].Journal of Molecular Biology,1973,74(4):563-572.
[3]Kouzminova E,Selker E U.dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora[J].The Embo Journal,2001,20(15):4309-4323.
[4]Martienssen R A,Colot V.DNA methylation and epigenetic inheritance in plants and filamentous fungi[J].Science,2001,293(5532):1070-1074.
[5]Chachadi V B,Cheng H,Klinkebiel D,et al.5-Aza-2'-deoxycytidine increases sialyl Lewis X on MUC1 by stimulating beta-galactoside:alpha 2,3-sialyltransferase 6gene[J].International Journal of Biochemistry&Cell Biology,2011,43(4):586-593.
[6]李伟,施明亮,毕京鹏,等.DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷的抗胃肠道肿瘤机制研究[J].中国生化药物杂志,2015,35(2):80-83.Li Wei,Shi Mingliang,Bi Jingpeng,et al.Mechanism of DNA methyl transferase inhibitor,5-Aza-2'-deoxycytidine against colorectal and gastric cancer[J].Chinese Journal of Biochemical Pharmaceutics,2015,35(2):80-83.(in Chinese)
[7]辛琪.瑞氏木霉纤维素酶基因转录因子功能及染色质修饰在酶基因转录调控中的作用研究[D].济南:山东大学,2013.Xin Qi.Functional analysis of transcription factors and the involvement of chromatin modification in the transcription regulation of cellulase gen in Trichoderma reesei[D].Jinan:Shandong University,2013.(in Chinese)
[8]张伟.化学表观遗传修饰法研究海葵来源月状旋孢腔菌代谢产物多样性初探[D].青岛:中国海洋大学,2014.Zhang Wei.Preliminary exploration on the secondary matebolites from the sea Anemone-Derived fungus cochliobolus lunatus using chemical epigenetic modification[D].Qingdao:Ocean University of China,2014.(in Chinese)
[9]孙鉴昕.表观遗传修饰剂对三株刺松藻内生真菌次级代谢产物生物活性的影响[D].青岛,青岛科技大学,2016.Sun Jianxin.The effect of epigenetic modification on the biological activity of secondary metabolites of three strains of endophytic fungi from codium fragile[D].Qingdao:Qingdao University of Science&Technology,2016.(in Chinese)
[10]刘云凤.两株中国南海柳珊瑚来源真菌次级代谢产物及其抗菌活性研究[D].青岛:中国海洋大学,2015.Liu Yunfeng.The secondary metabolites and antimicrobial activities of two gorgonian-derived fungi from the South China sea[D].Qingdao:Ocean University of China,2015.(in Chinese)
[11]Vasanthakumari M M,Jadhav S S,Sachin N,et al.Restoration of camptothecine production in attenuated endophytic fungus on re-inoculation into host plant and treatment with DNA methyltransferase inhibitor[J].World Journal of Microbiology&Biotechnology,2015,31(10):1629-1639.
[12]林剑青,赵西西,贺竹梅.基于转录组测序的黄曲霉对5-氮杂胞苷应答机制研究[C]//第四届全国微生物基因组学学术研讨会论文集,济南:[s.n.],2012:1.Lin Jianqing,Zhao Xixi,He Zhumei.Response mechanism of Aspergillus flavus to 5-Azacytidine based on sequencing of transcriptome[C]//Proceedings of the Fourth National Symposium on Microbial Genomics.Jinan:[s.n.],2012:1.(in Chinese)
[13]Yang Kunlong,Zhuang Zhenhong,Zhang Feng,et al.Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor[J].Food Additives and Contaminants:Part A,2015,32(4):554-563.
[14]Yang Kunlong,Liang Linlin,Ran Fanlei,et al.The Dmt A methyltransferase contributes to Aspergillus flavus conidiation,sclerotial production,aflatoxin biosynthesis and virulence[J].Scientific Reports,2016,6:23259.
[15]Ghose T.Measurement of cellulase activities[J].Pure and Applied Chemistry,1987,59(2):257-268.
[16]Jeon J,Choi J,Lee G W,et al.Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus,Magnaporthe oryzae[J].Scientific Reports,2015,5:8567.
[17]Logan P C,Ponnampalam A P,Rahnama F A,et al.The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells[J].Human Reproduction,2010,25(11):2859-2869.
[18]Armstrong R N,Colyer H A,Sharpe D J,et al.5-aza-2'-deoxycytidine,a DNA methylation inhibitor,induces miRNA expression changes in acute myeloid leukaemia cell lines that transcend subtype[J].British Journal of Haematology,2011,153(1):14-15.
[19]Chen Taiping,Li En.Structure and function of eukaryotic DNA methyltransferases[J].Current Topics in Developmental Biology,2004,60:55-89.
[20]Christman J K.5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation:mechanistic studies and their implications for cancer therapy[J].Oncogene,2002,21(35):5483-5495.
[21]龚映雪,台艳,肖文娟,等.酵母多基因表达载体在纤维素生物转化中应用[J].深圳大学学报理工版,2010,27(1):82-87.Gong Yingxue,Tai Yan,Xiao Wenjuan,et al.Construction of saccharomyces cerevisiae integrated expression vector and its application in cellulose bioconvesion[J].Journal of Shenzhen University Science and Engineering,2010,27(1):82-87.(in Chinese)