木霉菌T23胶毒素合成基因的生物信息学分析与克隆
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摘要
胶毒素是生防木霉菌重要的次生代谢产物之一。本研究以生防木霉菌T23为供试材料,旨在通过生物信息学技术及表达分析,挖掘木霉菌T23中胶毒素合成候选基因,探索木霉菌胶毒素合成的分子调控机制,可为新型生物农药的开发及应用提供理论依据。研究表明,木霉菌T23中胶毒素合成候选基因簇全长28 kb,簇内包含了8个基因,分别与烟曲霉胶毒素合成基因簇内的gliP、gliC、gliN、gliK、gliI、gliG、gliF、gliM高度同源。提取培养2 d、3 d、4 d、5 d的木霉菌T23菌丝的RNA,通过半定量RT-PCR技术探索各候选基因在木霉菌T23不同生长时期的表达情况,显示各基因在不同生长时期均有表达,属于组成型表达基因。成功克隆得到木霉菌T23中的gliP-T23基因并完成基因结构分析,该基因全长6 339 bp,由4个外显子和3个内含子组成,为后续的基因功能验证提供基础。
Gliotoxin is one of the important secondary metabolites of bio-control Trichoderma spp. In this paper,using bio-control Trichoderma T23 as tested material, gliotoxin biosynthetic candidate gene cluster in Trichoderma T23 was identified and the molecular regulation mechansim of Trichoderma gliotoxin biosynthesis was explored through bioinformatics technique and expression analysis, which could provide theoretical basis for the development and application of new biopesticides. The results showed that the full length of gliotoxin biosynthesis candidate gene cluster in Trichoderma T23 was 28 kb, including 8 genes, which were highly homologous to gliP,gliC, gliN, gliK, gliI, gliG, gliF, gliM located in gliotoxin biosynthesis gene cluster of Aspergillus fumigatus,respectively. The RNAs of Trichoderma T23 mycelium after 2 d, 3 d, 4 d and 5 d cultivation were extracted and the expression of each candidate gene in the different growth stages of Trichoderma T23 was explored through semi-quantitative RT-PCR. The result showed that each gene expressed in different growth stages, belonging to constitutive expression genes. gliP-T23 in Trichoderma T23 was cloned and obtained, and gene structure analysis was completed. The full length of gliP-T23 was 6 339 bp, including 4 exons and 3 introns, which could provide the basis for subsequent genetic function verification.
Gliotoxin is one of the important secondary metabolites of bio-control Trichoderma spp. In this paper,using bio-control Trichoderma T23 as tested material, gliotoxin biosynthetic candidate gene cluster in Trichoderma T23 was identified and the molecular regulation mechansim of Trichoderma gliotoxin biosynthesis was explored through bioinformatics technique and expression analysis, which could provide theoretical basis for the development and application of new biopesticides. The results showed that the full length of gliotoxin biosynthesis candidate gene cluster in Trichoderma T23 was 28 kb, including 8 genes, which were highly homologous to gliP,gliC, gliN, gliK, gliI, gliG, gliF, gliM located in gliotoxin biosynthesis gene cluster of Aspergillus fumigatus,respectively. The RNAs of Trichoderma T23 mycelium after 2 d, 3 d, 4 d and 5 d cultivation were extracted and the expression of each candidate gene in the different growth stages of Trichoderma T23 was explored through semi-quantitative RT-PCR. The result showed that each gene expressed in different growth stages, belonging to constitutive expression genes. gliP-T23 in Trichoderma T23 was cloned and obtained, and gene structure analysis was completed. The full length of gliP-T23 was 6 339 bp, including 4 exons and 3 introns, which could provide the basis for subsequent genetic function verification.
引文
Bertagnolli B.L.,Daly S.R.,and Sinclair J.B.,1998,Antimycotic compounds from the plant pathogen Rhizoctonia solani and its antagonist Trichoderma harzianum,Journal of Phytopathology,146(2-3):131-135
Bok J.W.,Chung D.W.,Balajee S.A.,Marr K.A.,Andes D.,Nielsen K.F.,Frisvad J.C.,Kirby K.A.,and Keller N.P.,2006,Gli Z,a transcriptional regulator of gliotoxin biosynthesis,contributes to Aspergillus fumigatus virulence,Infection and Immunity,74(12):6761-6768
Brian P.W.,and Hemming H.G.,1945,Gliotoxin,a fungistatic metabolic product of Trichoderma viride,Annals of Applied Biology,32(3):214-220
Cao Y.T.,2007,Screening anti-Trichoderma to several kind of pathogenic germs and improve strains,Thesis for M.S.,Sichuan Agricultural University,Supervisors:Yao G.,Wen C.J.,and Peng H.X.,pp.2(曹玉桃,2007,对几种病原菌拮抗木霉的筛选及菌种改良,硕士学位论文,四川农业大学,导师:姚革,文成敬,彭化贤,pp.2)
Chang S.L.,Chiang Y.M.,Yeh H.H.,Wu T.K.,and Wang C.C.,2013,Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase Gli C,Bioorganic and Medicinal Chemistry Letters,23(7):2155-2157
Ding A.M.,Li F.X.,Chen Y.Q.,Zong P.,Qu X.,Gong D.P.,Liu G.S.,and Sun Y.H.,2014,Homology-based cloning and expression analysis of Rf genes encoding PPR-containing proteins in tobacco,Genetics and Molecular Research,13(1):2310-2322
Dolan S.K.,O'keeffe G.,Jones G.W.,and Doyle S.,2015,Resistance is not futile:gliotoxin biosynthesis,functionality and utility,Trends in Microbiology,23(7):419-428
Eziashi E.I.,Uma N.U.,Adekunle A.A.,and Airede C.E.,2006,Effect of metabolites produced by Trichoderma species against Ceratocystis paradoxa in culture medium,African Journal of Biotechnology,5(9):703-706
Gardiner D.M.,and Howlett B.J.,2005,Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus,FEMS Microbiology Letters,248(2):241-248
Gough C.L.,Genin S.,Zischek C.,and Boucher C.A.,1992,Hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria,Molecular Plant-Microbe Interactions,5(5):384-389
Han Z.C.,2016,Advances in utilization of antagonistic Trichoderma spp.against plant pathogens,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),32(4):946-952(韩长志,2016,植物病原拮抗菌木霉属真菌的研究进展,江苏农业学报,32(4):946-952)
Howell C.R.,2006,Understanding the mechanisms employed by Trichoderma virens to effect biological control of cotton diseases,Phytopathology,96(2):178-180
Jin T.T.,Chen J.T.,Zhu L.Y.,Zhao Y.F.,Guo J.J.,and Huang Y.Q.,2015,Comparative mapping combined with homology-based cloning of the rice genome reveals candidate genes for grain zinc and iron concentration in maize,BMC Genetics,16(1):17
Kubicek C.P.,Herrera-Estrella A.,Seidl-Seiboth V.,Martinez D.A.,Druzhinina I.S.,Thon M.,Zeilinger S.,Casas-Flores S.,Horwitz B.A.,Mukheriee P.K.,Mukherjee M.,Kredics L.,Aacaraz L.D.,Antal Z.,Atanasova L.,Cervantes-Badillo M.G.,Challac ombe J.,Chertkov O.,McCluskey K.,Coulpier F.,Deshpande N.,von D觟hren H.,Ebbole D.J.,Esquivel-Naranjo E.U.,Fekete E.,Flipphi M.,Glaser F.,Gómez-Rodríquez E.Y.,Gruber S.,Han C.,Henrissat B.,Hermosa R.,Hernández-O觡ate M.,Karaffa L.,Kosti I.,Crom S.L.,Lindquist E.,Lucas S.,Lübeck M.,Lübeck P.S.,Margeot A.,Metz B.,Misra M.,Nevalainen H.,Omann M.,Packer N.,Perrone G,Uresti-Rivera EE,Salamov A,Schmoll M,Seiboth B,Shapiro H,Sukno S,Tamayo-Ramos J.A.,Tisch D.,Wiest A.,Wilkinson H.H.,Zhang M.,Coutinho P.M.,Kenerley C.M.,Monte E.,Baker S.E.,and Grigoriev I.V.,2011,Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma,Genome Biology,12(4):1-15
Lamdan N.L.,Shalaby S.,Ziv T.,Kenerley C.M.,and Horwitz B.A.,2015,Secretome of the biocontrol fungus Trichoderma virens co-cultured with maize roots:role in induced systemic resistance,Molecular and Cell Proteomics,14(4):1054-1063
Lin A.,Lee T.M.,and Rern J.C.,1994,Tricholin,a new antifungal agent from Trichoderma viride and its action in biological control of Rhizoctonia solani,The Journal of Antibiotics(Tokyo),47(7):799-805
Patron N.J.,Waller R.F.,Cozijnsen A.J.,Straney D.C.,Gardiner D.M.,Nierman W.C.,and Howlett B.J.,2007,Origin and distribution of epipoluthiodioxopiperazine(ETP)gene clusters in filamentous ascomycetes,BMC Evolutionary Biology,7(1):174
Sugui J.A.,Pardo J.,Chand Y.C.,Zarember K.A.,Nardone G.,Galvez E.M.,Müllbacher A.,Gallin J.I.,Simon M.M.,and Kwon-Chung K.J.,2007,Gliotoxin is a virulence factor of Aspergillus fumigatus:gli P deletion attenuates virulence in mice immunosuppressed with hydrocortisone,Eukaryotic Cell,6(9):1562-1569
Vinale F.,Sivasithamparam K.,Ghisalberti E.L.,Marra R.,Barbetti M.J.,Li H.,Woo S.L.,and Lorito M.,2008,A novel role for Trichoderma secondary metabolites in the interactions with plants,Physiological and Molecular Plant Pathology,72(1):80-86
Wang D.N.,Toyotome T.,Muraosa Y.,Watanabe A.,Wuren T.,Bunsupa S.,Aoyaqi K.,Yamazaki M.,Takino M.,and Kamei K.,2014,Gli A in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin,Medical Mycology,52(5):504-516
Wilhite S.E.,Lumsden R.D.,and Straney D.C.,1994,Mutational analysis of gliotoxin production by the biocontrol fungus Gliocladium virens in relation to suppression of pythium damping-off,Phytopathology,84(8):816-821
Wu X.Q.,Zhao Z.J.,Li Z.,Hu J.D.,Zhao X.Y.,Wang Y.L.,Huang Y.J.,Li J.S.,and Yang H.T.,2015,Impact of Trichoderma wettable powder application on winter wheat field growth,Shandong Kexue(Shangdong Science),28(6):35-42(吴晓青,赵忠娟,李哲,扈进冬,赵晓燕,王贻莲,黄玉杰,李纪顺,杨合同,2015,施加木霉可湿性粉剂对冬小麦田间生长的影响,山东科学,28(6):35-42)
Zeng H.L.,Ye P.S.,He L.,Li Q.F.,and Wei S.G.,2005a,Preliminary study on the control effect of Trichoderma spp.to root rot disease in Ligusticum Chuanxiong,Xinan Nongye Xue bao(Southwest China Journal of Agricultural Sciences),18(4):427-430(曾华兰,叶鹏盛,何炼,李琼芳,韦树谷,2005a,木霉菌防治川芎根腐病的初步研究,西南农业学报,18(4):427-430)
Zeng H.L.,Ye P.S.,Li Q.F.,He L.,and Yue F.L.,2005b,Effects of Trichoderma harzianum T23 on peanut yield,Yunnan Nongyedaxue Xuebao(Journal of Yunnan Agricultural University),20(1):145-146(曾华兰,叶鹏盛,李琼芳,何炼,岳福良,2005b,哈茨木霉T23对花生的促生增产作用,云南农业大学学报,20(1):145-146)
Zeng H.L.,Ye P.S.,He L.,Li Q.F.,Wei S.G.,Ye X.P.,and He Z.X.,2006,Ophiopgon japonicus root rot disease and Trichoderma spp.based biocontrol,Shijie Kexuejishu Zhongyiyao Xiandaihua(Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology),8(5):63-66(曾华兰,叶鹏盛,何炼,李琼芳,韦树谷,叶香平,何遵信,2006,利用木霉对麦冬根腐病的生物防治研究,世界科学技术-中医药现代化,8(5):63-66)
Zeng H.L.,Ye P.S.,and Li Q.F.,2002,Study on the control of medicinal herbs'soil-borne disease by Trichoderma spp.,Yunnan Nongyedaxue Xuebao(Journal of Yunnan Agricultural University),17(4):386-388(曾华兰,叶鹏盛,李琼芳,2002,中药材土传病害拮抗木霉的筛选与应用初探,云南农业大学学报,17(4):386-388)
Zhang G.Z.,Yang H.T.,Zhou H.Z.,Chen K.,and Wang Y.L.,2007,Competitive effects of fungicide on rhizosphere of Trichodema spp.in soil and maize growth,Nongye Huanjing Kexue Xuebao(Journal of Agro-Environment Science),26(S):229-231(张广志,杨合同,周红姿,陈凯,王贻莲,2007,土壤杀菌剂对木霉根际竞争的影响及木霉对玉米生长作用的初步研究,农业环境科学学报,26(增刊):229-231)
Bok J.W.,Chung D.W.,Balajee S.A.,Marr K.A.,Andes D.,Nielsen K.F.,Frisvad J.C.,Kirby K.A.,and Keller N.P.,2006,Gli Z,a transcriptional regulator of gliotoxin biosynthesis,contributes to Aspergillus fumigatus virulence,Infection and Immunity,74(12):6761-6768
Brian P.W.,and Hemming H.G.,1945,Gliotoxin,a fungistatic metabolic product of Trichoderma viride,Annals of Applied Biology,32(3):214-220
Cao Y.T.,2007,Screening anti-Trichoderma to several kind of pathogenic germs and improve strains,Thesis for M.S.,Sichuan Agricultural University,Supervisors:Yao G.,Wen C.J.,and Peng H.X.,pp.2(曹玉桃,2007,对几种病原菌拮抗木霉的筛选及菌种改良,硕士学位论文,四川农业大学,导师:姚革,文成敬,彭化贤,pp.2)
Chang S.L.,Chiang Y.M.,Yeh H.H.,Wu T.K.,and Wang C.C.,2013,Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase Gli C,Bioorganic and Medicinal Chemistry Letters,23(7):2155-2157
Ding A.M.,Li F.X.,Chen Y.Q.,Zong P.,Qu X.,Gong D.P.,Liu G.S.,and Sun Y.H.,2014,Homology-based cloning and expression analysis of Rf genes encoding PPR-containing proteins in tobacco,Genetics and Molecular Research,13(1):2310-2322
Dolan S.K.,O'keeffe G.,Jones G.W.,and Doyle S.,2015,Resistance is not futile:gliotoxin biosynthesis,functionality and utility,Trends in Microbiology,23(7):419-428
Eziashi E.I.,Uma N.U.,Adekunle A.A.,and Airede C.E.,2006,Effect of metabolites produced by Trichoderma species against Ceratocystis paradoxa in culture medium,African Journal of Biotechnology,5(9):703-706
Gardiner D.M.,and Howlett B.J.,2005,Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus,FEMS Microbiology Letters,248(2):241-248
Gough C.L.,Genin S.,Zischek C.,and Boucher C.A.,1992,Hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria,Molecular Plant-Microbe Interactions,5(5):384-389
Han Z.C.,2016,Advances in utilization of antagonistic Trichoderma spp.against plant pathogens,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),32(4):946-952(韩长志,2016,植物病原拮抗菌木霉属真菌的研究进展,江苏农业学报,32(4):946-952)
Howell C.R.,2006,Understanding the mechanisms employed by Trichoderma virens to effect biological control of cotton diseases,Phytopathology,96(2):178-180
Jin T.T.,Chen J.T.,Zhu L.Y.,Zhao Y.F.,Guo J.J.,and Huang Y.Q.,2015,Comparative mapping combined with homology-based cloning of the rice genome reveals candidate genes for grain zinc and iron concentration in maize,BMC Genetics,16(1):17
Kubicek C.P.,Herrera-Estrella A.,Seidl-Seiboth V.,Martinez D.A.,Druzhinina I.S.,Thon M.,Zeilinger S.,Casas-Flores S.,Horwitz B.A.,Mukheriee P.K.,Mukherjee M.,Kredics L.,Aacaraz L.D.,Antal Z.,Atanasova L.,Cervantes-Badillo M.G.,Challac ombe J.,Chertkov O.,McCluskey K.,Coulpier F.,Deshpande N.,von D觟hren H.,Ebbole D.J.,Esquivel-Naranjo E.U.,Fekete E.,Flipphi M.,Glaser F.,Gómez-Rodríquez E.Y.,Gruber S.,Han C.,Henrissat B.,Hermosa R.,Hernández-O觡ate M.,Karaffa L.,Kosti I.,Crom S.L.,Lindquist E.,Lucas S.,Lübeck M.,Lübeck P.S.,Margeot A.,Metz B.,Misra M.,Nevalainen H.,Omann M.,Packer N.,Perrone G,Uresti-Rivera EE,Salamov A,Schmoll M,Seiboth B,Shapiro H,Sukno S,Tamayo-Ramos J.A.,Tisch D.,Wiest A.,Wilkinson H.H.,Zhang M.,Coutinho P.M.,Kenerley C.M.,Monte E.,Baker S.E.,and Grigoriev I.V.,2011,Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma,Genome Biology,12(4):1-15
Lamdan N.L.,Shalaby S.,Ziv T.,Kenerley C.M.,and Horwitz B.A.,2015,Secretome of the biocontrol fungus Trichoderma virens co-cultured with maize roots:role in induced systemic resistance,Molecular and Cell Proteomics,14(4):1054-1063
Lin A.,Lee T.M.,and Rern J.C.,1994,Tricholin,a new antifungal agent from Trichoderma viride and its action in biological control of Rhizoctonia solani,The Journal of Antibiotics(Tokyo),47(7):799-805
Patron N.J.,Waller R.F.,Cozijnsen A.J.,Straney D.C.,Gardiner D.M.,Nierman W.C.,and Howlett B.J.,2007,Origin and distribution of epipoluthiodioxopiperazine(ETP)gene clusters in filamentous ascomycetes,BMC Evolutionary Biology,7(1):174
Sugui J.A.,Pardo J.,Chand Y.C.,Zarember K.A.,Nardone G.,Galvez E.M.,Müllbacher A.,Gallin J.I.,Simon M.M.,and Kwon-Chung K.J.,2007,Gliotoxin is a virulence factor of Aspergillus fumigatus:gli P deletion attenuates virulence in mice immunosuppressed with hydrocortisone,Eukaryotic Cell,6(9):1562-1569
Vinale F.,Sivasithamparam K.,Ghisalberti E.L.,Marra R.,Barbetti M.J.,Li H.,Woo S.L.,and Lorito M.,2008,A novel role for Trichoderma secondary metabolites in the interactions with plants,Physiological and Molecular Plant Pathology,72(1):80-86
Wang D.N.,Toyotome T.,Muraosa Y.,Watanabe A.,Wuren T.,Bunsupa S.,Aoyaqi K.,Yamazaki M.,Takino M.,and Kamei K.,2014,Gli A in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin,Medical Mycology,52(5):504-516
Wilhite S.E.,Lumsden R.D.,and Straney D.C.,1994,Mutational analysis of gliotoxin production by the biocontrol fungus Gliocladium virens in relation to suppression of pythium damping-off,Phytopathology,84(8):816-821
Wu X.Q.,Zhao Z.J.,Li Z.,Hu J.D.,Zhao X.Y.,Wang Y.L.,Huang Y.J.,Li J.S.,and Yang H.T.,2015,Impact of Trichoderma wettable powder application on winter wheat field growth,Shandong Kexue(Shangdong Science),28(6):35-42(吴晓青,赵忠娟,李哲,扈进冬,赵晓燕,王贻莲,黄玉杰,李纪顺,杨合同,2015,施加木霉可湿性粉剂对冬小麦田间生长的影响,山东科学,28(6):35-42)
Zeng H.L.,Ye P.S.,He L.,Li Q.F.,and Wei S.G.,2005a,Preliminary study on the control effect of Trichoderma spp.to root rot disease in Ligusticum Chuanxiong,Xinan Nongye Xue bao(Southwest China Journal of Agricultural Sciences),18(4):427-430(曾华兰,叶鹏盛,何炼,李琼芳,韦树谷,2005a,木霉菌防治川芎根腐病的初步研究,西南农业学报,18(4):427-430)
Zeng H.L.,Ye P.S.,Li Q.F.,He L.,and Yue F.L.,2005b,Effects of Trichoderma harzianum T23 on peanut yield,Yunnan Nongyedaxue Xuebao(Journal of Yunnan Agricultural University),20(1):145-146(曾华兰,叶鹏盛,李琼芳,何炼,岳福良,2005b,哈茨木霉T23对花生的促生增产作用,云南农业大学学报,20(1):145-146)
Zeng H.L.,Ye P.S.,He L.,Li Q.F.,Wei S.G.,Ye X.P.,and He Z.X.,2006,Ophiopgon japonicus root rot disease and Trichoderma spp.based biocontrol,Shijie Kexuejishu Zhongyiyao Xiandaihua(Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology),8(5):63-66(曾华兰,叶鹏盛,何炼,李琼芳,韦树谷,叶香平,何遵信,2006,利用木霉对麦冬根腐病的生物防治研究,世界科学技术-中医药现代化,8(5):63-66)
Zeng H.L.,Ye P.S.,and Li Q.F.,2002,Study on the control of medicinal herbs'soil-borne disease by Trichoderma spp.,Yunnan Nongyedaxue Xuebao(Journal of Yunnan Agricultural University),17(4):386-388(曾华兰,叶鹏盛,李琼芳,2002,中药材土传病害拮抗木霉的筛选与应用初探,云南农业大学学报,17(4):386-388)
Zhang G.Z.,Yang H.T.,Zhou H.Z.,Chen K.,and Wang Y.L.,2007,Competitive effects of fungicide on rhizosphere of Trichodema spp.in soil and maize growth,Nongye Huanjing Kexue Xuebao(Journal of Agro-Environment Science),26(S):229-231(张广志,杨合同,周红姿,陈凯,王贻莲,2007,土壤杀菌剂对木霉根际竞争的影响及木霉对玉米生长作用的初步研究,农业环境科学学报,26(增刊):229-231)