小麦赤霉菌FGSG＿05656基因敲除及功能分析
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摘要
小麦赤霉菌FGSG_05656基因在MGV1敲除突变体中显著下调,为了进一步研究其生物学功能,本研究采用Split-Marker基因敲除技术,构建了含有潮霉素抗性基因(hph)的基因缺失盒。通过聚乙二醇(PEG)法转化野生型原生质体。潮霉素(Hygromycin)选择培养基筛选转化子后,PCR正负筛选鉴定敲除突变体。基于缺失突变体的表型和感染能力的变化来分析FGSG_05656基因的生物学功能。与野生型PH-1相比,敲除突变体的生长速率和分生孢子数显著减少,表明FGSG_05656基因可能参与菌丝体的生长和分生孢子的发生。植物感染试验显示缺失突变体与野生型之间没有显著差异。分生孢子是病原菌感染寄主的主要器官,它的减少可以显著减弱病原菌的扩散,FGSG_05656基因敲除突变体分生孢子显著减少,这一结果对于进一步研究分生孢子发生的分子机理具有重要意义,同时为小麦抗病分子育种提供依据。
To further study the biological function of FGSG_05656 gene in Fusarium graminearum, which was significantly down-regulated in the MGV1 deletion mutant, the Split-Marker gene knockout strategy was applied in this study to construct the gene deletion cassettes containing a hygromycin resistance gene(hph) and then the cassettes were transformed into protoplast of wild type mediated by polyethylene glycol(PEG). After the transformants were screened on hygromycin selective medium, the deletion mutants were screened and confirmed by PCR using positive and negative primer. The biological function of FGSG_05656 gene was analyzed based on the changes of phenotype and infection ability of the deletion mutants. Compared with the wild type PH-1, the growth rate and the number of the conidia of the knock-out mutants were significantly reduced, suggesting that the FGSG_05656 gene might be involved in the mycelium growth and the conidia generation. Plant infection assay revealed that there was no significant difference between the deletion mutants and wild type. The conidia were the major organs of pathogens to infect host-cell, the reduction of which could significantly weaken the spread of pathogens. The conidia of the FGSG_05656 deletion mutant significant decreased, which was of great significance for further study of the molecular mechanism of conidiogenesis and provide guidance for molecular breeding of wheat disease resistance at the same time.
To further study the biological function of FGSG_05656 gene in Fusarium graminearum, which was significantly down-regulated in the MGV1 deletion mutant, the Split-Marker gene knockout strategy was applied in this study to construct the gene deletion cassettes containing a hygromycin resistance gene(hph) and then the cassettes were transformed into protoplast of wild type mediated by polyethylene glycol(PEG). After the transformants were screened on hygromycin selective medium, the deletion mutants were screened and confirmed by PCR using positive and negative primer. The biological function of FGSG_05656 gene was analyzed based on the changes of phenotype and infection ability of the deletion mutants. Compared with the wild type PH-1, the growth rate and the number of the conidia of the knock-out mutants were significantly reduced, suggesting that the FGSG_05656 gene might be involved in the mycelium growth and the conidia generation. Plant infection assay revealed that there was no significant difference between the deletion mutants and wild type. The conidia were the major organs of pathogens to infect host-cell, the reduction of which could significantly weaken the spread of pathogens. The conidia of the FGSG_05656 deletion mutant significant decreased, which was of great significance for further study of the molecular mechanism of conidiogenesis and provide guidance for molecular breeding of wheat disease resistance at the same time.
引文
Catlett N.L.,Lee B.,Yoder O.C.,and Turgeon B.G.,2002,Splitmarker recombination for efficient targeted deletion of fungal genes,Fungal Genetics Newsletter,50(49):9-11
Chang Y.M.,and Hou Z.M.,2015,Research on gene knockout and function of FgPDE1 in Fusarium graminearum,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),35(8):59-67(常玉梅,侯占铭,2015,禾谷镰刀菌中Fg PDE1基因的敲除及其功能的研究,中国生物工程杂志,35(8):59-67)
Chen H.J.,Lu Z.,Ding Y.,Yang Z.H.,Song Z.,and Feng L.Z.,2018,identification and bioinformatics analysis of MAPKof Calonectria pseudoreteaudii,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(1):24-35(陈慧洁,陆芝,丁奕,杨泽慧,宋漳,冯丽贞,2018,桉树焦枯病菌MAPK的鉴定与生物信息学分析,基因组学与应用生物学,37(1):24-35)
Cuomo C.A.,Güldener U.,Xu J.R.,Trail F.,Turgeon B.G.,Di Pietro A.,Walton J.D.,Ma L.J.,Baker S.E.,Rep M.,Adam G.,Antoniw J.,Baldwin T.,Calvo S.,Chang Y.L.,Decaprio D.,Gale L.R.,Gnerre S.,Goswami R.S.,Hammond-Kosack K.,Harris L.J.,Hilburn K.,Kennell J.C.,Kroken S.,Magnuson J.K.,Mannhaupt G.,Mauceli E.,Mewes H.W.,Mitterbauer R.,Muehlbauer G.,Münsterk觟tter M.,Nelson D.,O'donnell K.,Ouellet T.,Qi W.H.,Quesneville H.,Roncero M.I.G.,Seong K.Y.,Tetko I.V.,Urban M.,Waalwijk C.,Ward T.J.,Yao J.Q.,Birren B.W.,and Kistler H.C.,2007,The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization,Science,317(5843):1400-1402
Gale L.R.,Chen L.F.,Hernick C.A.,Takamura K.,and Kistler H.C.,2002,Population analysis of Fusarium graminearum from wheat fields in Eastern China,Phytopathology,92(12):1315-1322
Guo X.F.,and Hou Z.M.,2018,Knockout and function research of FGSG_08948 gene in Fusarium graminearum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2409-2414(郭雪芳,侯占铭,2018,小麦赤霉菌FGSG_08948基因敲除及功能研究,分子植物育种,16(8):2409-2414)
Hou Z.M.,Xue C.Y.,Peng Y.L.,Katan T.,Kistler H.C.,and Xu J.R.,2002,A mitogen-activated protein kinase gene(MGV1)in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection,Mol.Plant-Microbe In.,15(11):1119-1127
Liu J.L.,Hu Z.Q.,Li H.P.,and Liao Y.C.,2016,Screening and identification of the mimic epitope of the cell wall proteins of Fusarium L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(2):235-240(刘锦龙,胡祖权,李和平,廖玉才,2016,镰刀菌细胞壁蛋白抗原模拟表位的筛选和鉴定,基因组学与应用生物学,35(2):235-240)
Ma L.J.,van der Does H.C.,Borkovich K.A.,Coleman J.J.,Daboussi M.J.,Pietro A.D.,Dufresne M.,Freitag M.,Grabherr M.,Henrissat B.,Houterman P.M.,Kang S.,Shim W.B.,Woloshuk C.,Xie X.H.,Xu J.R.,Antoniw J.,Baker S.E.,Bluhm B.H.,Breakspear A.,Brown D.W.,Butchko R.A.E.,Chapman S.,Coulson R.,Coutinho P.M.,Danchin E.G.J.,Diener A.,Gale L.R.,Gardiner D.M.,Goff S.,Hammond-Kosack K.E.,Hilburn K.,Hua-Van A.,Jonkers W.,Kazan K.,Kodira C.D.,Koehrsen M.,Kumar L.,Lee Y.H.,Li L.D.,Manners J.M.,Miranda-Saavedra D.,Mukherjee M.,Park G.,Park J.,Park S.Y.,Proctor R.H.,Regev A.,Ruiz-Roldan M.C.,Sain D.,Sakthikumar S.,Sykes S.,Schwartz D.C.,Turgeon B.G.,Wapinski I.,Yoder O.,Young S.,Zeng Q.D.,Zhou S.G.,Galagan J.,Cuomo C.A.,Kistler H.C.,and Rep M.,2010,Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium,Nature,464(7287):367-373
McMullen M.,Jones R.,and Gallenberg D.,1997,Scab of wheat and barley:A re-emerging disease of devastating impact,P-lant Disease,81(12):1340-1348
Parry D.W.,Jenkinson P.,and Mcleod L.,1995,Fusarium ear blight(scab)in small grain cereals-a review,Plant Pathol.,44(2):207-238
Peng H.L.,and Hou Z.M.,2017,Research on knockout and function of FGSG_04871 gene in Fusarium graminearum,Neimenggu Shifan Daxue Xuebao(Journal of Inner Mongolia N-ormal University),46(3):394-400(彭海丽,侯占铭,2017,小麦赤霉菌FGSG_04871基因敲除及功能研究,内蒙古师范大学学报,46(3):394-400)
Wiemann P.,Sieber C.M.K.,von Bargen K.W.,Studt L.,Niehaus E.M.,Espino J.J.,HuβK.,Michielse C.B.,Albermann S.,Wagner D.,Bergner S.V.,Connolly L.R.,Fischer A.,Reuter G.,Kleigrewe K.,Bald T.,Wingfield B.D.,Ophir R.,Freeman S.,Hippler M.,Smith K.M.,Brown D.W.,Proctor R.H.,Münsterk觟tter M.,Freitag M.,Humpf H.U.,Güldene U.,and Tudzynski B.,2013,Deciphering the cryptic genome:genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites,PLoS Pathogens,9(6):e1003475
Windels C.E.,2000,Economic and social impacts of Fusarium head blight:changing farms and rural communities in the northern great plains,Phytopathology,90(1):17-21
Wu B.,2011,Characterization of FgAC1 gene of Fusarium graminearum,Thesis for M.S.,Inner Mongolia Normal University,Supervisor:Hou Z.M.,pp.11-13(吴彬,2011,小麦赤霉菌FgAC1基因敲除及功能研究,硕士学位论文,内蒙古师范大学,导师:侯占铭,pp.11-13)
Zhao X.H.,Mehrabi R.,and Xu J.R.,2007,Mitogen-activated protein kinase pathways and fungal pathogenesis,Eukaryotic Cell,6(10):1701-1714
Chang Y.M.,and Hou Z.M.,2015,Research on gene knockout and function of FgPDE1 in Fusarium graminearum,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),35(8):59-67(常玉梅,侯占铭,2015,禾谷镰刀菌中Fg PDE1基因的敲除及其功能的研究,中国生物工程杂志,35(8):59-67)
Chen H.J.,Lu Z.,Ding Y.,Yang Z.H.,Song Z.,and Feng L.Z.,2018,identification and bioinformatics analysis of MAPKof Calonectria pseudoreteaudii,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(1):24-35(陈慧洁,陆芝,丁奕,杨泽慧,宋漳,冯丽贞,2018,桉树焦枯病菌MAPK的鉴定与生物信息学分析,基因组学与应用生物学,37(1):24-35)
Cuomo C.A.,Güldener U.,Xu J.R.,Trail F.,Turgeon B.G.,Di Pietro A.,Walton J.D.,Ma L.J.,Baker S.E.,Rep M.,Adam G.,Antoniw J.,Baldwin T.,Calvo S.,Chang Y.L.,Decaprio D.,Gale L.R.,Gnerre S.,Goswami R.S.,Hammond-Kosack K.,Harris L.J.,Hilburn K.,Kennell J.C.,Kroken S.,Magnuson J.K.,Mannhaupt G.,Mauceli E.,Mewes H.W.,Mitterbauer R.,Muehlbauer G.,Münsterk觟tter M.,Nelson D.,O'donnell K.,Ouellet T.,Qi W.H.,Quesneville H.,Roncero M.I.G.,Seong K.Y.,Tetko I.V.,Urban M.,Waalwijk C.,Ward T.J.,Yao J.Q.,Birren B.W.,and Kistler H.C.,2007,The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization,Science,317(5843):1400-1402
Gale L.R.,Chen L.F.,Hernick C.A.,Takamura K.,and Kistler H.C.,2002,Population analysis of Fusarium graminearum from wheat fields in Eastern China,Phytopathology,92(12):1315-1322
Guo X.F.,and Hou Z.M.,2018,Knockout and function research of FGSG_08948 gene in Fusarium graminearum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2409-2414(郭雪芳,侯占铭,2018,小麦赤霉菌FGSG_08948基因敲除及功能研究,分子植物育种,16(8):2409-2414)
Hou Z.M.,Xue C.Y.,Peng Y.L.,Katan T.,Kistler H.C.,and Xu J.R.,2002,A mitogen-activated protein kinase gene(MGV1)in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection,Mol.Plant-Microbe In.,15(11):1119-1127
Liu J.L.,Hu Z.Q.,Li H.P.,and Liao Y.C.,2016,Screening and identification of the mimic epitope of the cell wall proteins of Fusarium L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(2):235-240(刘锦龙,胡祖权,李和平,廖玉才,2016,镰刀菌细胞壁蛋白抗原模拟表位的筛选和鉴定,基因组学与应用生物学,35(2):235-240)
Ma L.J.,van der Does H.C.,Borkovich K.A.,Coleman J.J.,Daboussi M.J.,Pietro A.D.,Dufresne M.,Freitag M.,Grabherr M.,Henrissat B.,Houterman P.M.,Kang S.,Shim W.B.,Woloshuk C.,Xie X.H.,Xu J.R.,Antoniw J.,Baker S.E.,Bluhm B.H.,Breakspear A.,Brown D.W.,Butchko R.A.E.,Chapman S.,Coulson R.,Coutinho P.M.,Danchin E.G.J.,Diener A.,Gale L.R.,Gardiner D.M.,Goff S.,Hammond-Kosack K.E.,Hilburn K.,Hua-Van A.,Jonkers W.,Kazan K.,Kodira C.D.,Koehrsen M.,Kumar L.,Lee Y.H.,Li L.D.,Manners J.M.,Miranda-Saavedra D.,Mukherjee M.,Park G.,Park J.,Park S.Y.,Proctor R.H.,Regev A.,Ruiz-Roldan M.C.,Sain D.,Sakthikumar S.,Sykes S.,Schwartz D.C.,Turgeon B.G.,Wapinski I.,Yoder O.,Young S.,Zeng Q.D.,Zhou S.G.,Galagan J.,Cuomo C.A.,Kistler H.C.,and Rep M.,2010,Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium,Nature,464(7287):367-373
McMullen M.,Jones R.,and Gallenberg D.,1997,Scab of wheat and barley:A re-emerging disease of devastating impact,P-lant Disease,81(12):1340-1348
Parry D.W.,Jenkinson P.,and Mcleod L.,1995,Fusarium ear blight(scab)in small grain cereals-a review,Plant Pathol.,44(2):207-238
Peng H.L.,and Hou Z.M.,2017,Research on knockout and function of FGSG_04871 gene in Fusarium graminearum,Neimenggu Shifan Daxue Xuebao(Journal of Inner Mongolia N-ormal University),46(3):394-400(彭海丽,侯占铭,2017,小麦赤霉菌FGSG_04871基因敲除及功能研究,内蒙古师范大学学报,46(3):394-400)
Wiemann P.,Sieber C.M.K.,von Bargen K.W.,Studt L.,Niehaus E.M.,Espino J.J.,HuβK.,Michielse C.B.,Albermann S.,Wagner D.,Bergner S.V.,Connolly L.R.,Fischer A.,Reuter G.,Kleigrewe K.,Bald T.,Wingfield B.D.,Ophir R.,Freeman S.,Hippler M.,Smith K.M.,Brown D.W.,Proctor R.H.,Münsterk觟tter M.,Freitag M.,Humpf H.U.,Güldene U.,and Tudzynski B.,2013,Deciphering the cryptic genome:genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites,PLoS Pathogens,9(6):e1003475
Windels C.E.,2000,Economic and social impacts of Fusarium head blight:changing farms and rural communities in the northern great plains,Phytopathology,90(1):17-21
Wu B.,2011,Characterization of FgAC1 gene of Fusarium graminearum,Thesis for M.S.,Inner Mongolia Normal University,Supervisor:Hou Z.M.,pp.11-13(吴彬,2011,小麦赤霉菌FgAC1基因敲除及功能研究,硕士学位论文,内蒙古师范大学,导师:侯占铭,pp.11-13)
Zhao X.H.,Mehrabi R.,and Xu J.R.,2007,Mitogen-activated protein kinase pathways and fungal pathogenesis,Eukaryotic Cell,6(10):1701-1714