组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响
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摘要
以蛇足石杉Huperziaserrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因(histone methyltransferases,HMT)CgClr4和组蛋白去乙酰化酶基因(histonedeacetylase,HDAC)CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR(RT-qPCR)检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzineA(HupA)产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。
A histone methyltransferases(HMT) gene CgClr4 and two histone deacetylase(HDAC) gene, CgClr3 and CgSir2, were knocked out and then complemented in the endophytic fungus Colletotrichum gloeosporioides strain Cg01 in Huperzia serrata by the PEG-mediated genetic transformation system. The expression of the corresponding genes in the complemented strains were detected by RT-qPCR and huperzine A(HupA) production in the mutant strains were analyzed by high performance liquid chromatography(HPLC).It was found that the HupA production of the 3 gene knockout mutant strains CgClr4, CgClr3 and CgSir2 were 255μg/L, 270μg/L and 244μg/L respectively, decreased by 21.3%, 16.6% and 24.7% as compared with wild type(WT). There is no significant difference between the gene expression of the corresponding gene in the complemented strains CgClr4/CgClr4, CgClr3/CgClr3 and CgSir2/CgSir2 and that in the wild type strain, with the HupA production to be 351.9μg/L, 334.7μg/L and 331μg/L, respectively. The HupA production of complemented strains was restored to that of the wild type strain. The results showed that these three genes had the function of regulating the biosynthesis of HupA, which provided a theoretical basis and a new insight for studying the synthetic mechanism of HupA in endophytic fungi of Huperzia serrata.
A histone methyltransferases(HMT) gene CgClr4 and two histone deacetylase(HDAC) gene, CgClr3 and CgSir2, were knocked out and then complemented in the endophytic fungus Colletotrichum gloeosporioides strain Cg01 in Huperzia serrata by the PEG-mediated genetic transformation system. The expression of the corresponding genes in the complemented strains were detected by RT-qPCR and huperzine A(HupA) production in the mutant strains were analyzed by high performance liquid chromatography(HPLC).It was found that the HupA production of the 3 gene knockout mutant strains CgClr4, CgClr3 and CgSir2 were 255μg/L, 270μg/L and 244μg/L respectively, decreased by 21.3%, 16.6% and 24.7% as compared with wild type(WT). There is no significant difference between the gene expression of the corresponding gene in the complemented strains CgClr4/CgClr4, CgClr3/CgClr3 and CgSir2/CgSir2 and that in the wild type strain, with the HupA production to be 351.9μg/L, 334.7μg/L and 331μg/L, respectively. The HupA production of complemented strains was restored to that of the wild type strain. The results showed that these three genes had the function of regulating the biosynthesis of HupA, which provided a theoretical basis and a new insight for studying the synthetic mechanism of HupA in endophytic fungi of Huperzia serrata.
引文
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Bok JW,Chiang YM,Szewczyk E,Reyes DY,Davidson AD,Sanchez JF,Lo HC,Watanabe K,Strauss J,Oakley BR,Wang CC,Keller NP,2009.Chromatin-level regulation of biosynthetic gene clusters.Nature Chemical Biology,5(7):462-464
Christoph Z,Agnieszka G,Michael S,Martin W,Joseph S,Kathrin R,2013.Small chemical chromatin effectors alter secondary metabolite production in Aspergillus clavatus.Toxins,5(10):1723-1741
Gross H,2007.Strategies to unravel the function of orphan biosynthesis pathways:recent examples and future prospects.Applied Microbiology and Biotechnology,75(2):267-277
Hedden P,Phillips AL,Rojas MC,Carrera E,Tudzynski B,2001.Gibberellin biosynthesis in plants and fungi:a case of convergent evolution?Journal of Plant Growth Regulation,20(4):319-331
Hiragami K,Festenstein R,2005.Heterochromatin protein 1:a pervasive controlling influence.Cellular and Molecular Life Sciences,62(23):2711-2726
Ju Z,Wang J,Pan SL,2009.Isolation and preliminary identification of the endophytic fungi which produce hupzine A from four species in Hupziaceae and determination of huperzine A by HPLC.Fudan University Journal of Medical Sciences,36(4):445-449(in Chinese)
Liang L,Li J,Cheng L,Ling J,Luo Z,Bai M,Xie B,2014.A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.Microbiological Research,169(11):835-843
Lee I,Oh JH,Shwab EK,Dagenais TR,Andes D,Keller NP,2009.HdaA,a class 2 histone deacetylase of Aspergillus fumigatus,affects germination and secondary metabolite production.Fungal Genetics and Biology,46(10):782-790
Nakayama J,Rice JC,Strahl BD,Allis CD,Grewal SI,2001.Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science,292(5514):110-113
Ro DK,Paradise EM,Ouellet M,Fisher KJ,Newman KL,Ndungu JM,Ho KA,Eachus RA,Ham TS,Kirby J,Chang MC,Withers ST,Shiba Y,Sarpong R,Keasling JD,2006.Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,440(7086):940
Scherlach K,Hertweck C,2009.Triggering cryptic natural product biosynthesis in microorganisms.Organic&Biomolecular Chemistry,7(9):1753-1760
Shen BM,Xiao Jl,Dai LY,Huang YH,Mao ZH,Lin RM,Yao YR,Xie BY,2015.Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.Microbiological Research,170:18-26
Shilatifard A,2006.Chromatin modifications by methylation and ubiquitination:implications in the regulation of gene expression.Annual Review of Biochemistry,75:243-269
Stewart MD,Li J,Wong J,2005.Relationship between histone H3 lysine 9 methylation,transcription repression,and heterochromatin protein 1 recruitment.Molecular and Cellular Biology,25(7):2525-2538
Stierle A,Strobel G,Stierle D,1993.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew.Science,260(5105):214
Studt L,Schmidt FJ,Jahn L,Sieber CM,Connolly LR,Niehaus EM,Freitag M,Humpf HU,Tudzynski B,2013.Two histone deacetylases,FfHda1 and FfHda2,are important for Fusarium fujikuroi secondary metabolism and virulence.Applied and Environmental Microbiology,79(24):7719-7734
Vakoc CR,Mandat SA,Olenchock BK,2005.Histone H3 lysine9 methylation and HP1γare associated with transcription elongtion through mammalian chromatin.Molecular Cell,19(3):381-391
Wang Y,Yan RM,Zeng QG,Zhang ZB,Wang D,Zhu D,2011.Producing huperzine A by an endophytic fungus from Huperzia serrata.Mycosystema,30(2):255-262(in Chinese)
Wang YE,Yue DX,Tang XC,1986.Anti-cholinesterase activity of huperzine A.Zhongguo Yao Li Xue Bao,7(2):110-113
Yuan JQ,Zhou XL,Wang S,Yan ZG,Feng SX,Jiang JM,Zhu DY,Ma XJ,2012.Simultaneous determination of huperzine Aand huperzine B in different parts of Huperzia serrata from different habitats by HPLC.Chinese Journal of Pharmaceutical Analysis,32(9):1541-1544,1549(in Chinese)
Zheng R,Zhang ZB,Yan RM,Wang Y,Xiao YW,Zhu D,2018.Isolation of secondary metabolites with acetylcholinesterase inhibitory activity of endophytic fungus Pezicula sp.JR14 from Huperzia serrata.Mycosystema,37(1):102-109(in Chinese)
鞠錾,王峻,潘胜利,2009.4种石杉科植物中产生石杉碱甲的内生真菌的分离及初步鉴定和HPLC测定石杉碱甲的含量.复旦学报,36(4):445-449
汪涯,颜日明,曾庆桂,张志斌,汪娣,朱笃,2011.一株产石杉碱甲的蛇足石杉内生真菌的分离和鉴定.菌物学报,30(2):255-262
袁经权,周小雷,王硕,闫志刚,冯世鑫,蒋娇梅,朱大元,马小军,2012.HPLC测定不同产地与不同部位千层塔中石杉碱甲和石杉碱乙含量.药物分析杂志,32(9):1541-1544
郑瑞,张志斌,颜日明,汪涯,肖依文,朱笃,2018.蛇足石杉内生真菌无柄盘菌Pezicula sp.JR14中抑制乙酰胆碱酯酶的次级代谢产物分离.菌物学报,37(1):102-109
Bannister AJ,Zegerman P,Partridge JF,Miska EA,Thomas JO,Allshire RC,Kouzarides T,2001.Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.Nature,410(6824):120-124
Bok JW,Chiang YM,Szewczyk E,Reyes DY,Davidson AD,Sanchez JF,Lo HC,Watanabe K,Strauss J,Oakley BR,Wang CC,Keller NP,2009.Chromatin-level regulation of biosynthetic gene clusters.Nature Chemical Biology,5(7):462-464
Christoph Z,Agnieszka G,Michael S,Martin W,Joseph S,Kathrin R,2013.Small chemical chromatin effectors alter secondary metabolite production in Aspergillus clavatus.Toxins,5(10):1723-1741
Gross H,2007.Strategies to unravel the function of orphan biosynthesis pathways:recent examples and future prospects.Applied Microbiology and Biotechnology,75(2):267-277
Hedden P,Phillips AL,Rojas MC,Carrera E,Tudzynski B,2001.Gibberellin biosynthesis in plants and fungi:a case of convergent evolution?Journal of Plant Growth Regulation,20(4):319-331
Hiragami K,Festenstein R,2005.Heterochromatin protein 1:a pervasive controlling influence.Cellular and Molecular Life Sciences,62(23):2711-2726
Ju Z,Wang J,Pan SL,2009.Isolation and preliminary identification of the endophytic fungi which produce hupzine A from four species in Hupziaceae and determination of huperzine A by HPLC.Fudan University Journal of Medical Sciences,36(4):445-449(in Chinese)
Liang L,Li J,Cheng L,Ling J,Luo Z,Bai M,Xie B,2014.A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.Microbiological Research,169(11):835-843
Lee I,Oh JH,Shwab EK,Dagenais TR,Andes D,Keller NP,2009.HdaA,a class 2 histone deacetylase of Aspergillus fumigatus,affects germination and secondary metabolite production.Fungal Genetics and Biology,46(10):782-790
Nakayama J,Rice JC,Strahl BD,Allis CD,Grewal SI,2001.Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science,292(5514):110-113
Ro DK,Paradise EM,Ouellet M,Fisher KJ,Newman KL,Ndungu JM,Ho KA,Eachus RA,Ham TS,Kirby J,Chang MC,Withers ST,Shiba Y,Sarpong R,Keasling JD,2006.Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,440(7086):940
Scherlach K,Hertweck C,2009.Triggering cryptic natural product biosynthesis in microorganisms.Organic&Biomolecular Chemistry,7(9):1753-1760
Shen BM,Xiao Jl,Dai LY,Huang YH,Mao ZH,Lin RM,Yao YR,Xie BY,2015.Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.Microbiological Research,170:18-26
Shilatifard A,2006.Chromatin modifications by methylation and ubiquitination:implications in the regulation of gene expression.Annual Review of Biochemistry,75:243-269
Stewart MD,Li J,Wong J,2005.Relationship between histone H3 lysine 9 methylation,transcription repression,and heterochromatin protein 1 recruitment.Molecular and Cellular Biology,25(7):2525-2538
Stierle A,Strobel G,Stierle D,1993.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew.Science,260(5105):214
Studt L,Schmidt FJ,Jahn L,Sieber CM,Connolly LR,Niehaus EM,Freitag M,Humpf HU,Tudzynski B,2013.Two histone deacetylases,FfHda1 and FfHda2,are important for Fusarium fujikuroi secondary metabolism and virulence.Applied and Environmental Microbiology,79(24):7719-7734
Vakoc CR,Mandat SA,Olenchock BK,2005.Histone H3 lysine9 methylation and HP1γare associated with transcription elongtion through mammalian chromatin.Molecular Cell,19(3):381-391
Wang Y,Yan RM,Zeng QG,Zhang ZB,Wang D,Zhu D,2011.Producing huperzine A by an endophytic fungus from Huperzia serrata.Mycosystema,30(2):255-262(in Chinese)
Wang YE,Yue DX,Tang XC,1986.Anti-cholinesterase activity of huperzine A.Zhongguo Yao Li Xue Bao,7(2):110-113
Yuan JQ,Zhou XL,Wang S,Yan ZG,Feng SX,Jiang JM,Zhu DY,Ma XJ,2012.Simultaneous determination of huperzine Aand huperzine B in different parts of Huperzia serrata from different habitats by HPLC.Chinese Journal of Pharmaceutical Analysis,32(9):1541-1544,1549(in Chinese)
Zheng R,Zhang ZB,Yan RM,Wang Y,Xiao YW,Zhu D,2018.Isolation of secondary metabolites with acetylcholinesterase inhibitory activity of endophytic fungus Pezicula sp.JR14 from Huperzia serrata.Mycosystema,37(1):102-109(in Chinese)
鞠錾,王峻,潘胜利,2009.4种石杉科植物中产生石杉碱甲的内生真菌的分离及初步鉴定和HPLC测定石杉碱甲的含量.复旦学报,36(4):445-449
汪涯,颜日明,曾庆桂,张志斌,汪娣,朱笃,2011.一株产石杉碱甲的蛇足石杉内生真菌的分离和鉴定.菌物学报,30(2):255-262
袁经权,周小雷,王硕,闫志刚,冯世鑫,蒋娇梅,朱大元,马小军,2012.HPLC测定不同产地与不同部位千层塔中石杉碱甲和石杉碱乙含量.药物分析杂志,32(9):1541-1544
郑瑞,张志斌,颜日明,汪涯,肖依文,朱笃,2018.蛇足石杉内生真菌无柄盘菌Pezicula sp.JR14中抑制乙酰胆碱酯酶的次级代谢产物分离.菌物学报,37(1):102-109
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