一种有效的嗜热真菌杜邦嗜热菌靶向基因替代系统
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摘要
以嗜热真菌杜邦嗜热菌ThermomycesdupontiiNRRL2155为研究材料,利用同源重组原理和真菌原生质体转化方法,以潮霉素抗性基因替换嗜热真菌目标基因,获得抗潮霉素的靶向基因敲除突变菌株。优化的遗传转化体系为:用15mg/mL裂解酶,在28℃下酶解2g杜邦嗜热菌菌丝5.5h以获得原生质体,经STC缓冲液洗涤重悬后,利用PEG(polyethylene glycol)介导的遗传转化方式,将10μg线性敲除全长片段转化至杜邦嗜热菌原生质体中,通过潮霉素筛选及PCR验证得到基因替换突变菌株,同源重组率达到20%。本研究首次将原生质体转化方法应用在杜邦嗜热菌,并成功建立稳定高效的基因替换体系,为快速构建杜邦嗜热菌的遗传转化体系和研究该嗜热真菌的基因功能提供有效方法。
A strain NRRL 2155 of the thermophilus fungus Thermomyces dupontii served as recipient, and a combination of homologous recombination and fungal protoplast transformation was applied to generate the targeted gene replacement mutant with a hygromycin resistance gene. The optimum condition for targeted gene replacement was as follows: 2 g of fungal mycelia was treated with 15 mg/mL lysing enzyme at 28°C for5.5 h to yield protoplasts. After being washed twice with STC buffer, the protoplasts were treated with 10μg of linear replacement DNA fragment through PEG-mediated genetic transformation method. The putative transformants were screened out with hygromycin B marker and further confirmed with PCR analysis. The efficiency rate of this homologous recombination method was up to 20%. This was for the first time that PEG-mediated protoplast transformation method was applied to T. dupontii, and a stable and efficient gene replacement system was successfully established.
A strain NRRL 2155 of the thermophilus fungus Thermomyces dupontii served as recipient, and a combination of homologous recombination and fungal protoplast transformation was applied to generate the targeted gene replacement mutant with a hygromycin resistance gene. The optimum condition for targeted gene replacement was as follows: 2 g of fungal mycelia was treated with 15 mg/mL lysing enzyme at 28°C for5.5 h to yield protoplasts. After being washed twice with STC buffer, the protoplasts were treated with 10μg of linear replacement DNA fragment through PEG-mediated genetic transformation method. The putative transformants were screened out with hygromycin B marker and further confirmed with PCR analysis. The efficiency rate of this homologous recombination method was up to 20%. This was for the first time that PEG-mediated protoplast transformation method was applied to T. dupontii, and a stable and efficient gene replacement system was successfully established.
引文
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Chadha BS,Kaur R,Saini HS,Singh S,2000.Characterization of hygromycin-resistant transformants of thermophilic fungus Thermomyces lanuginosus.World Journal of Microbiology and Biotechnology,16(3):303-306
Chávez R,Fierro F,García-Rico RO,Vaca I,2015.Filamentous fungi from extreme environments as a promising source of novel bioactive secondary metabolites.Frontiers in Microbiology,6:903
Cooney DG,Emerson R,1964.Thermophilic fungi.An account of their biology,activities,and classification.W.H.Freeman&Company Ltd,San Francisco and London.1-188
Cui X,Wei Y,Xie XL,Chen LN,Zhang SH,2017.Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development,stress resistance and longevity of Thermomyces lanuginosus.Fungal Genetics and Biology,103:42-54
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Della Torre CL,Kadowaki MK,2017.Thermostable xylanase from thermophilic fungi:biochemical properties and industrial applications.African Journal of Microbiology Research,11(2):28-37
de Oliveira TB,Gomes E,Rodrigues A,2015.Thermophilic fungi in the new age of fungal taxonomy.Extremophiles,19(1):31-37
Gangavaram LP,Mchunu N,Ramakrishnan P,2009.Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus.Journal of Microbiological Methods,77(2):159-164
Guo JP,Zhu CY,Zhang CP,Chu YS,Wang YL,Zhang JX,Niu XM,2012.Thermolides,potent nematocidal PKS-NRPShybrid metabolites from thermophilic fungus Talaromyces thermophilus.Journal of the American Chemical Society,134(50):20306-20309
Guo RF,Li DC,Wang R,2005.Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus.Acta Microbiologica Sinica,45(2):270-274
Han H,2012.Establishment a transformation system for Thermomyces lanuginosus and subcellular location and construction gene knockout vector of protein kinase.Master Thesis,Shandong Agricultural University,Jinan.46(in Chinese)
Hou YP,Gao T,Zheng ZT,Chen CJ,Zhou MG,2013.An economic and highly efficient system of protoplast transformation for Fusarium graminearum.Mycosystema,32(5):891-898
Jain S,Durand H,Tiraby G,1992.Development of a transformation system for the thermophilic fungus Talaromyces sp.CL240 based on the use of phleomycin resistance as a dominant selectable marker.Molecular and General Genetics,234(3):489-493
Khan FI,Bisetty K,Singh S,Permaul K,Hassan MI,2015.Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications.Extremophiles,19(6):1055-1066
Kück U,Hoff B,2010.New tools for the genetic manipulation of filamentous fungi.Applied Microbiology and Biotechnology,86(1):51-62
Li CH,Li ZF,Feng ZL,Zhao LH,Shi YQ,Wang LF,Liu YJ,Zhu HQ,2013.Applications of gene knockout by ATMT in filamentous fungus functional genomics.Cotton Science,25(3):262-268(in Chinese)
Li DC,Li AN,Papageorgiou AC,2011.Cellulases from thermophilic fungi:recent insights and biotechnological potential.Enzyme Research,2011(1):308730
Liu Q,Gao RR,Li JG,Lin LC,Zhao JQ,Sun WL,Tian CG,2017.Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering.Biotechnology for Biofuels,10(1):1
Maier FJ,Malz S,L?sch AP,Lacour T,Sch?fer W,2005.Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marke.FEMS Yeast Research,5(6-7):653-662
Niu XM,Chen L,Yue Q,Wang BL,Zhang JX,Zhu CY,Zhang KQ,Bills GF,An ZQ,2014.Characterization of thermolide biosynthetic genes and a new thermolide from sister thermophilic fungi.Organic Letters,16(14):3744-3747
Peng YN,Wang PY,Gong XF,Zhu Y,Yang H,Wang ZY,Zhou JP,2017.Preparation and regeneration of protoplasts from Pythium oligandrum.Mycosystema,36(6):679-690(in Chinese)
Pan YY,Li EW,Che YS,Liu G,2015.Research progresses and trends of secondary metabolites in filamentous fungi.Mycosystema,34(5):890-899(in Chinese)
Pudake RN,Kumari M,Sahu BB,Sultan E,2017.Targeted gene disruption tools for fungal genomics.In:Varma A,Sharma AK(eds.)Modern tools and techniques to understand microbes.Springer,Cham.81-102
Schiestl RH,Zhu JIE,Petes TD,1994.Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.Molecular and Cellular Biology,14(7):4493-4500
Shen HM,Li C,Gao L,Liu TG,Liu B,Chen WQ,2017.Research progress in transformation of fungi mediated by protoplasts.Plant Protection,43(2):25-28(in Chinese)
Tian L,Chen JY,Wang JN,Wang JL,Dai XF,2011.High efficient gene knockout in Verticillium dahliae.Acta Microbiologica Sinica,51(7):906-913(in Chinese)
Wubie AJ,Hu Y,Li W,Huang J,Guo Z,Xu S,Zhou T,2014.Factors analysis in protoplast isolation and regeneration from a chalkbrood fungus,Ascosphaera apis.International Journal of Agriculture&Biology,16(1):89-96
Xu J,Li JG,Lin LC,Liu Q,Sun WL,Huang BQ,Tian CG,2015.Development of genetic tools for Myceliophthora thermophila.BMC Biotechnology,15(1):35
Xu Y,Tu Z,2007.Application and progress of filamentous fungi gene targeting.Journal of Food Science and Biotechnology,26(1):120-126(in Chinese)
Yang F,Gong YF,Liu G,Zhao SM,Wang J,2015.Enhancing cellulase production in thermophilic fungus Myceliophthora thermophila ATCC42464 by RNAinterference of cre1 gene expression.Journal of Microbiology and Biotechnology,25(7):1101-1107
Yang H,2017.The disruption,complementary and function analysis of CAP(adenylate cyclase-associated protein)in Thermomyces lanuginosus.Master Thesis,Jilin University,Changchun.43(in Chinese)
Yao TT,Wang ZX,2006.Preparation,regeneration and transformation of Aspergillus niger protoplasts.Journal of Food Science and Biotechnology,25(4):116-120(in Chinese)
Yu JH,Hamari Z,Han KH,Seo JA,Reyes-Domínguez Y,Scazzocchio C,2004.Double-joint PCR:a PCR-based molecular tool for gene manipulations in filamentous fungi.Fungal Genetics and Biology,41(11):973-981
Zhang LG,Luo SF,Zhou HS,Hu HL,Li PX,2018.Establishment of genetic transformation system of Epicoccum nigrum using PEG-mediated method.Mycosystema,37(2):205-214(in Chinese)
Zhang YP,Luo SC,Huang YP,Tang YP,Yi KX,Xie BY,Chen GY,2014.Isolation and regeneration of Phomopsis asparagi protoplast.Journal of Plant Protection,41(2):182-186(in Chinese)
Zhao CQ,Li DC,2007.A review of taxanomic research on thermophilic fungi.Mycosystema,26(4):601-606(in Chinese)
韩华,2012.疏绵状嗜热丝孢菌遗传转化体系的建立及蛋白激酶定位和敲除载体的构建.山东农业大学硕士论文,济南.46
李彩红,李志芳,冯自力,赵丽红,师勇强,王玲飞,刘义杰,朱荷琴,2013.农杆菌介导的基因敲除技术在丝状真菌基因功能研究中的应用.棉花学报,25(3):262-268
彭轶楠,王沛雅,巩晓芳,祝英,杨晖,王治业,周剑平,2017.生防真菌寡雄腐霉原生质体的制备及再生.菌物学报,36(6):679-690
潘园园,李二伟,车永胜,刘钢,2015.丝状真菌次级代谢产物的研究现状与发展趋势.菌物学报,34(5):890-899
沈慧敏,李超,高利,刘太国,刘博,陈万权,2017.原生质体法介导真菌遗传转化的研究进展.植物保护,43(2):25-28+42
田李,陈捷胤,汪佳妮,王金龙,戴小枫,2011.高效大丽轮枝菌(Verticillium dahliae)基因敲除体系的构建.微生物学报,51(7):906-913
许杨,涂追,2007.丝状真菌基因敲除技术研究进展.食品与生物技术学报,26(1):120-126
杨欢,2017.疏绵状嗜热丝孢菌中CAP的敲除、互补及功能研究.吉林大学硕士论文,长春.43
姚婷婷,王正祥,2006.黑曲霉原生质体的制备、再生及转化条件.食品与生物技术学报,25(4):116-120
张雷刚,罗淑芬,周宏胜,胡花丽,李鹏霞,2018.PEG介导的黑附球菌遗传转化体系的建立.菌物学报,37(2):205-214
张岳平,罗绍春,黄燕萍,汤泳萍,易克贤,谢丙炎,陈光宇,2014.芦笋茎枯病菌原生质体的制备及再生.植物保护学报,41(2):182-186
赵春青,李多川,2007.嗜热真菌的分类研究概况.菌物学报,26(4):601-606
Berka RM,Grigoriev IV,Otillar R,Salamov A,Grimwood J,Reid I,Ishmael N,John T,Darmond C,Moisan MC,Henrissat B,Coutinho PM,Lombard V,Natvig DO,Lindquist E,Schmutz J,Lucas S,Harris P,Powlowski J,Bellemare A,Taylor D,Butler G,de Vries RP,Allijn IE,van den Brink J,Ushinsky S,Storms R,Powell AJ,Paulsen IT,Elbourne LD,Baker SE,Magnuson J,Laboissiere S,Clutterbuck AJ,Martinez D,Wogulis M,de Leon AL,Rey MW,Tsang A,2011.Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris.Nature Biotechnology,29(10):922
Chadha BS,Kaur R,Saini HS,Singh S,2000.Characterization of hygromycin-resistant transformants of thermophilic fungus Thermomyces lanuginosus.World Journal of Microbiology and Biotechnology,16(3):303-306
Chávez R,Fierro F,García-Rico RO,Vaca I,2015.Filamentous fungi from extreme environments as a promising source of novel bioactive secondary metabolites.Frontiers in Microbiology,6:903
Cooney DG,Emerson R,1964.Thermophilic fungi.An account of their biology,activities,and classification.W.H.Freeman&Company Ltd,San Francisco and London.1-188
Cui X,Wei Y,Xie XL,Chen LN,Zhang SH,2017.Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development,stress resistance and longevity of Thermomyces lanuginosus.Fungal Genetics and Biology,103:42-54
Dantas-Barbosa C,Araújo EF,Moraes LM,Vainstein MH,Azevedo MO,1998.Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var.thermoidea to hygromycin B resistance.FEMS Microbiology Letters,169(1):185-190
Della Torre CL,Kadowaki MK,2017.Thermostable xylanase from thermophilic fungi:biochemical properties and industrial applications.African Journal of Microbiology Research,11(2):28-37
de Oliveira TB,Gomes E,Rodrigues A,2015.Thermophilic fungi in the new age of fungal taxonomy.Extremophiles,19(1):31-37
Gangavaram LP,Mchunu N,Ramakrishnan P,2009.Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus.Journal of Microbiological Methods,77(2):159-164
Guo JP,Zhu CY,Zhang CP,Chu YS,Wang YL,Zhang JX,Niu XM,2012.Thermolides,potent nematocidal PKS-NRPShybrid metabolites from thermophilic fungus Talaromyces thermophilus.Journal of the American Chemical Society,134(50):20306-20309
Guo RF,Li DC,Wang R,2005.Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus.Acta Microbiologica Sinica,45(2):270-274
Han H,2012.Establishment a transformation system for Thermomyces lanuginosus and subcellular location and construction gene knockout vector of protein kinase.Master Thesis,Shandong Agricultural University,Jinan.46(in Chinese)
Hou YP,Gao T,Zheng ZT,Chen CJ,Zhou MG,2013.An economic and highly efficient system of protoplast transformation for Fusarium graminearum.Mycosystema,32(5):891-898
Jain S,Durand H,Tiraby G,1992.Development of a transformation system for the thermophilic fungus Talaromyces sp.CL240 based on the use of phleomycin resistance as a dominant selectable marker.Molecular and General Genetics,234(3):489-493
Khan FI,Bisetty K,Singh S,Permaul K,Hassan MI,2015.Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications.Extremophiles,19(6):1055-1066
Kück U,Hoff B,2010.New tools for the genetic manipulation of filamentous fungi.Applied Microbiology and Biotechnology,86(1):51-62
Li CH,Li ZF,Feng ZL,Zhao LH,Shi YQ,Wang LF,Liu YJ,Zhu HQ,2013.Applications of gene knockout by ATMT in filamentous fungus functional genomics.Cotton Science,25(3):262-268(in Chinese)
Li DC,Li AN,Papageorgiou AC,2011.Cellulases from thermophilic fungi:recent insights and biotechnological potential.Enzyme Research,2011(1):308730
Liu Q,Gao RR,Li JG,Lin LC,Zhao JQ,Sun WL,Tian CG,2017.Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering.Biotechnology for Biofuels,10(1):1
Maier FJ,Malz S,L?sch AP,Lacour T,Sch?fer W,2005.Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marke.FEMS Yeast Research,5(6-7):653-662
Niu XM,Chen L,Yue Q,Wang BL,Zhang JX,Zhu CY,Zhang KQ,Bills GF,An ZQ,2014.Characterization of thermolide biosynthetic genes and a new thermolide from sister thermophilic fungi.Organic Letters,16(14):3744-3747
Peng YN,Wang PY,Gong XF,Zhu Y,Yang H,Wang ZY,Zhou JP,2017.Preparation and regeneration of protoplasts from Pythium oligandrum.Mycosystema,36(6):679-690(in Chinese)
Pan YY,Li EW,Che YS,Liu G,2015.Research progresses and trends of secondary metabolites in filamentous fungi.Mycosystema,34(5):890-899(in Chinese)
Pudake RN,Kumari M,Sahu BB,Sultan E,2017.Targeted gene disruption tools for fungal genomics.In:Varma A,Sharma AK(eds.)Modern tools and techniques to understand microbes.Springer,Cham.81-102
Schiestl RH,Zhu JIE,Petes TD,1994.Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.Molecular and Cellular Biology,14(7):4493-4500
Shen HM,Li C,Gao L,Liu TG,Liu B,Chen WQ,2017.Research progress in transformation of fungi mediated by protoplasts.Plant Protection,43(2):25-28(in Chinese)
Tian L,Chen JY,Wang JN,Wang JL,Dai XF,2011.High efficient gene knockout in Verticillium dahliae.Acta Microbiologica Sinica,51(7):906-913(in Chinese)
Wubie AJ,Hu Y,Li W,Huang J,Guo Z,Xu S,Zhou T,2014.Factors analysis in protoplast isolation and regeneration from a chalkbrood fungus,Ascosphaera apis.International Journal of Agriculture&Biology,16(1):89-96
Xu J,Li JG,Lin LC,Liu Q,Sun WL,Huang BQ,Tian CG,2015.Development of genetic tools for Myceliophthora thermophila.BMC Biotechnology,15(1):35
Xu Y,Tu Z,2007.Application and progress of filamentous fungi gene targeting.Journal of Food Science and Biotechnology,26(1):120-126(in Chinese)
Yang F,Gong YF,Liu G,Zhao SM,Wang J,2015.Enhancing cellulase production in thermophilic fungus Myceliophthora thermophila ATCC42464 by RNAinterference of cre1 gene expression.Journal of Microbiology and Biotechnology,25(7):1101-1107
Yang H,2017.The disruption,complementary and function analysis of CAP(adenylate cyclase-associated protein)in Thermomyces lanuginosus.Master Thesis,Jilin University,Changchun.43(in Chinese)
Yao TT,Wang ZX,2006.Preparation,regeneration and transformation of Aspergillus niger protoplasts.Journal of Food Science and Biotechnology,25(4):116-120(in Chinese)
Yu JH,Hamari Z,Han KH,Seo JA,Reyes-Domínguez Y,Scazzocchio C,2004.Double-joint PCR:a PCR-based molecular tool for gene manipulations in filamentous fungi.Fungal Genetics and Biology,41(11):973-981
Zhang LG,Luo SF,Zhou HS,Hu HL,Li PX,2018.Establishment of genetic transformation system of Epicoccum nigrum using PEG-mediated method.Mycosystema,37(2):205-214(in Chinese)
Zhang YP,Luo SC,Huang YP,Tang YP,Yi KX,Xie BY,Chen GY,2014.Isolation and regeneration of Phomopsis asparagi protoplast.Journal of Plant Protection,41(2):182-186(in Chinese)
Zhao CQ,Li DC,2007.A review of taxanomic research on thermophilic fungi.Mycosystema,26(4):601-606(in Chinese)
韩华,2012.疏绵状嗜热丝孢菌遗传转化体系的建立及蛋白激酶定位和敲除载体的构建.山东农业大学硕士论文,济南.46
李彩红,李志芳,冯自力,赵丽红,师勇强,王玲飞,刘义杰,朱荷琴,2013.农杆菌介导的基因敲除技术在丝状真菌基因功能研究中的应用.棉花学报,25(3):262-268
彭轶楠,王沛雅,巩晓芳,祝英,杨晖,王治业,周剑平,2017.生防真菌寡雄腐霉原生质体的制备及再生.菌物学报,36(6):679-690
潘园园,李二伟,车永胜,刘钢,2015.丝状真菌次级代谢产物的研究现状与发展趋势.菌物学报,34(5):890-899
沈慧敏,李超,高利,刘太国,刘博,陈万权,2017.原生质体法介导真菌遗传转化的研究进展.植物保护,43(2):25-28+42
田李,陈捷胤,汪佳妮,王金龙,戴小枫,2011.高效大丽轮枝菌(Verticillium dahliae)基因敲除体系的构建.微生物学报,51(7):906-913
许杨,涂追,2007.丝状真菌基因敲除技术研究进展.食品与生物技术学报,26(1):120-126
杨欢,2017.疏绵状嗜热丝孢菌中CAP的敲除、互补及功能研究.吉林大学硕士论文,长春.43
姚婷婷,王正祥,2006.黑曲霉原生质体的制备、再生及转化条件.食品与生物技术学报,25(4):116-120
张雷刚,罗淑芬,周宏胜,胡花丽,李鹏霞,2018.PEG介导的黑附球菌遗传转化体系的建立.菌物学报,37(2):205-214
张岳平,罗绍春,黄燕萍,汤泳萍,易克贤,谢丙炎,陈光宇,2014.芦笋茎枯病菌原生质体的制备及再生.植物保护学报,41(2):182-186
赵春青,李多川,2007.嗜热真菌的分类研究概况.菌物学报,26(4):601-606