棉花尖孢镰刀菌绿色荧光蛋白的转化
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摘要
棉花枯萎病是棉花的主要病害之一,严重威胁棉花产量和品质。绿色荧光蛋白(GFP)是发光水母(Aequorea victoria)体内的一种蛋白,作为报告基因拥有诸多优点,目前已被广泛应用于植物研究当中。为研究枯萎菌浸染棉花的过程及植病互作分子机理,本研究利用PEG介导的原生质体转化方法,将绿色荧光蛋白(GFP)基因转入到棉花枯萎病菌中,创制转报告基因株系。结果表明:转化子连续转接2代后仍能够稳定遗传,且荧光强度良好。经荧光共聚焦显微镜观察和PCR验证GFP基因已成功转入到菌株中,转化所得的菌株将为后续可视化检测和分析枯萎病菌对棉花的侵染等研究提供帮助。
Cotton Fusarium wi lt is one of the main diseases of cotton and seriously threatens cotton yield and quality. Green fluorescent protein(GFP) is a kind of protein in Aequorea victoria. As a reporter gene, it has many advantages and has been widely used in plant research. In order to study the process of cotton infected by Fusarium oxysporum and the molecular mechanism of plant-pathology interaction, the method of PEG mediated protoplast transformation was applied to transform the GFP genes into Fusarium oxysporum and transgenic reporter lines were created. The results showed that the transformants with GFP could be inherited steadily and their fluorescence signal was kept strongly after more than two successive generations. It was confirmed that the GFP genes had been successfully transferred into the strain of Fusarium oxysporum by observation of fluorescence confocal microscopy and PCR verification. The acquisition of the transformants with GFP would lay a solid foundation for subsequent study of the visual detection and analysis about the infection of cotton by Fusarium oxysporum.
Cotton Fusarium wi lt is one of the main diseases of cotton and seriously threatens cotton yield and quality. Green fluorescent protein(GFP) is a kind of protein in Aequorea victoria. As a reporter gene, it has many advantages and has been widely used in plant research. In order to study the process of cotton infected by Fusarium oxysporum and the molecular mechanism of plant-pathology interaction, the method of PEG mediated protoplast transformation was applied to transform the GFP genes into Fusarium oxysporum and transgenic reporter lines were created. The results showed that the transformants with GFP could be inherited steadily and their fluorescence signal was kept strongly after more than two successive generations. It was confirmed that the GFP genes had been successfully transferred into the strain of Fusarium oxysporum by observation of fluorescence confocal microscopy and PCR verification. The acquisition of the transformants with GFP would lay a solid foundation for subsequent study of the visual detection and analysis about the infection of cotton by Fusarium oxysporum.
引文
Horowitz S.,Freeman S.,and Sharon A.,2002,Use of green fluorescent protein-transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum,Phytopathology,92(7):743-749
Huang G.C.,Zhu S.W.,Dong Y.M.,and Sun J.S.,1998,Green fluorescent protein and its application in plant research,Zhiwu Xuebao(Chinese Bulletin of Botany),15(5):24-30(黄国存,朱生伟,董越梅,孙敬三,1998,绿色荧光蛋白及其在植物研究中的应用,植物学报,15(5):24-30)
Li Y.D.,2010,Transformation of green fluorescent protein gene into Fusarium oxysporum f.sp.cubense and cloning its beta-glucosidase gene,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisors:Xu W.Y.,and Liu B.,pp.32(李燕丹,2010,香蕉枯萎病菌尸标记和β-葡萄糖甘酶基因克隆,硕士学位论文,福建农林大学,导师:许文耀,刘波,pp.32)
Liu C.Y.,Zhao P.J.,Wang J.,and Huang J.S.,2009,Research progress of genetic transformation methods of biological control fungi,Zhongguo Zhibao Daokan(China Plant Protection),29(1):13-16(刘昌燕,赵培静,汪军,黄俊生,2009,生防真菌遗传转化方法研究进展,中国植保导刊,29(1):13-16)
Lu X.R.,Jia X.Y.,and Niu J.H.,2018,The present situation and prospects of cotton industry development in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),51(1):26-36(卢秀茹,贾肖月,牛佳慧,2018,中国棉花产业发展现状及展望,中国农业科学,51(1):26-36)
LüZ.X.,Li J.D.,and Zhu Z.Q.,2001,Klebsiella oxytoca SG-11labeling by green fluorescent protein(GFP)and its colonization in rice seedind roots,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),9(1):13-18(吕泽勋,李久蒂,朱至清,2011,用绿色荧光蛋白基因(GFP)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖,农业生物技术学报,9(1):13-18)
NahaLkova J.,and Fatehi J.,2003,Red fluorescent protein(Ds R-ed2)as a novel reporter in Fusarium oxysporum f.sp.lycopersici,FEMS Microbiol.Lett.,225(2):305-309
Nonomura T.,Tajima H.,and Kitagawa Y.,2003,Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum,strains simultaneously colonizing root surfaces,J.Gen.Plant Pathol.,69(1):45-48
Nurkiyanova K.M.,Ryabov E.V.,and Commandeur U.,2000,Tagging potato leafroll virus with the jellyfish green fluorescent protein gene,J.Gen.Virol.,81(3):617-626
Shen H.M.,Li C.,Gao L.,Liu T.G.,Liu B.,and Chen W.Q.,2017,Research progress in transformation of fungi mediated by protoplasts,Zhiwu Baohu(Plant Protection),43(2):25-28(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)
Shimomura O.,Johnson F.H.,and Saiga Y.,1962,Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea,J.Cell Comp.Physiol.,59(3):223-239
Tong Z.G.,Cai B.H.,Zhang Z.,Wang F.R.,Zhang K.C.,Yan G.H.,and Zhou Y.,2008,Advances in application research on genetic transformation of fruit trees via particle bombardment,Guoshu Xuebao(Journal of Fruit Science),25(3):389-395(佟兆国,蔡斌华,章镇,王富荣,张开春,闫国华,周宇,2008,果树中基因枪法遗传转化的研究进展,果树学报,25(3):389-395)
Vain P.,Worland B.,and Kohli A.,1998,The green fluorescent protein(GFP)as a vital screenable marker in rice transformation,Theor.Appl.Genet.,96(2):164-169
Wang L.K.,2013,Protopplasts formation and preliminary trial of PEG-mediated ransformation of Tulasnella calospora,Thesis for M.S.,Beijing Forestry University,Supervisor:Liu H.X.,pp.9(王丽琨,2013,美孢胶膜菌(Tulasnella calospora)原生质体制备与CaCl2-PEG转化初步试验,硕士学位论文,北京林业大学,导师:刘红霞,pp.9)
Wang X.G.,2011,Pathogenic differentiation and genetic diversity of Fusarium oxysporum f.sp.vasinfection,Thesis for M.S.,Agricultural University of Hebei,Supervisor:Ma P.,pp.14(王晓光,2011,棉花枯萎病菌致病力分化及遗传多样性研究,硕士学位论文,河北农业大学,导师:马平,pp.14)
Wang Y.,Yang C.D.,Xue L.,Zhang Z.F.,Feng H.Z.,and Zhang J.L.,2017,Bacillus mojavensis ZA1 labeling with GFP and its functional stability,Zhiwu Baohu Xuebao(Journal of Plant Protection),44(4):657-663(王颖,杨呈德,薛莉,张振粉,冯红中,张俊莲,2017生防菌株ZA1的GFP基因标记及其功能稳定性测定,植物保护学报,44(4):657-663)
Xiao R.F.,Zhu Y.J.,Li Y.D.,Huang S.F.,and Li B.,2009,Green fluorescent protein gene transformation on Fusariumox ysporum f.sp.niveum strain,FOV-135,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),24(6):521-524(肖荣凤,朱育菁,李燕丹,黄素芳,刘波,2009,西瓜尖孢镰刀菌FOV-135的绿色荧光蛋白基因转化,福建农业学报,24(6):521-524)
Yu S.X.,Fan S.L.,Wang H.T.,Wei H.L.,and Pao C.Y.,2016,Progresses in research on cotton high yield breeding in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(18):3465-3476(喻树迅,范术丽,王寒涛,魏恒玲,庞朝友,2016,中国棉花高产育种进展,中国农业科学,49(18):3465-3476)
Zhou L.H.,Li G.Q.,Wang Z.X.,and Zhu G.J.,2005,Preparation and regeneration of protoplasts from Monascus purpureus and genetic transformation system,Yichuan(Hereditas),27(3):423-428(周礼红,李国琴,王正祥,诸葛健,2005,红曲霉原生质体的制备、再生及其遗传转化系统,遗传,27(3):423-428)
Huang G.C.,Zhu S.W.,Dong Y.M.,and Sun J.S.,1998,Green fluorescent protein and its application in plant research,Zhiwu Xuebao(Chinese Bulletin of Botany),15(5):24-30(黄国存,朱生伟,董越梅,孙敬三,1998,绿色荧光蛋白及其在植物研究中的应用,植物学报,15(5):24-30)
Li Y.D.,2010,Transformation of green fluorescent protein gene into Fusarium oxysporum f.sp.cubense and cloning its beta-glucosidase gene,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisors:Xu W.Y.,and Liu B.,pp.32(李燕丹,2010,香蕉枯萎病菌尸标记和β-葡萄糖甘酶基因克隆,硕士学位论文,福建农林大学,导师:许文耀,刘波,pp.32)
Liu C.Y.,Zhao P.J.,Wang J.,and Huang J.S.,2009,Research progress of genetic transformation methods of biological control fungi,Zhongguo Zhibao Daokan(China Plant Protection),29(1):13-16(刘昌燕,赵培静,汪军,黄俊生,2009,生防真菌遗传转化方法研究进展,中国植保导刊,29(1):13-16)
Lu X.R.,Jia X.Y.,and Niu J.H.,2018,The present situation and prospects of cotton industry development in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),51(1):26-36(卢秀茹,贾肖月,牛佳慧,2018,中国棉花产业发展现状及展望,中国农业科学,51(1):26-36)
LüZ.X.,Li J.D.,and Zhu Z.Q.,2001,Klebsiella oxytoca SG-11labeling by green fluorescent protein(GFP)and its colonization in rice seedind roots,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),9(1):13-18(吕泽勋,李久蒂,朱至清,2011,用绿色荧光蛋白基因(GFP)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖,农业生物技术学报,9(1):13-18)
NahaLkova J.,and Fatehi J.,2003,Red fluorescent protein(Ds R-ed2)as a novel reporter in Fusarium oxysporum f.sp.lycopersici,FEMS Microbiol.Lett.,225(2):305-309
Nonomura T.,Tajima H.,and Kitagawa Y.,2003,Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum,strains simultaneously colonizing root surfaces,J.Gen.Plant Pathol.,69(1):45-48
Nurkiyanova K.M.,Ryabov E.V.,and Commandeur U.,2000,Tagging potato leafroll virus with the jellyfish green fluorescent protein gene,J.Gen.Virol.,81(3):617-626
Shen H.M.,Li C.,Gao L.,Liu T.G.,Liu B.,and Chen W.Q.,2017,Research progress in transformation of fungi mediated by protoplasts,Zhiwu Baohu(Plant Protection),43(2):25-28(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)
Shimomura O.,Johnson F.H.,and Saiga Y.,1962,Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea,J.Cell Comp.Physiol.,59(3):223-239
Tong Z.G.,Cai B.H.,Zhang Z.,Wang F.R.,Zhang K.C.,Yan G.H.,and Zhou Y.,2008,Advances in application research on genetic transformation of fruit trees via particle bombardment,Guoshu Xuebao(Journal of Fruit Science),25(3):389-395(佟兆国,蔡斌华,章镇,王富荣,张开春,闫国华,周宇,2008,果树中基因枪法遗传转化的研究进展,果树学报,25(3):389-395)
Vain P.,Worland B.,and Kohli A.,1998,The green fluorescent protein(GFP)as a vital screenable marker in rice transformation,Theor.Appl.Genet.,96(2):164-169
Wang L.K.,2013,Protopplasts formation and preliminary trial of PEG-mediated ransformation of Tulasnella calospora,Thesis for M.S.,Beijing Forestry University,Supervisor:Liu H.X.,pp.9(王丽琨,2013,美孢胶膜菌(Tulasnella calospora)原生质体制备与CaCl2-PEG转化初步试验,硕士学位论文,北京林业大学,导师:刘红霞,pp.9)
Wang X.G.,2011,Pathogenic differentiation and genetic diversity of Fusarium oxysporum f.sp.vasinfection,Thesis for M.S.,Agricultural University of Hebei,Supervisor:Ma P.,pp.14(王晓光,2011,棉花枯萎病菌致病力分化及遗传多样性研究,硕士学位论文,河北农业大学,导师:马平,pp.14)
Wang Y.,Yang C.D.,Xue L.,Zhang Z.F.,Feng H.Z.,and Zhang J.L.,2017,Bacillus mojavensis ZA1 labeling with GFP and its functional stability,Zhiwu Baohu Xuebao(Journal of Plant Protection),44(4):657-663(王颖,杨呈德,薛莉,张振粉,冯红中,张俊莲,2017生防菌株ZA1的GFP基因标记及其功能稳定性测定,植物保护学报,44(4):657-663)
Xiao R.F.,Zhu Y.J.,Li Y.D.,Huang S.F.,and Li B.,2009,Green fluorescent protein gene transformation on Fusariumox ysporum f.sp.niveum strain,FOV-135,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),24(6):521-524(肖荣凤,朱育菁,李燕丹,黄素芳,刘波,2009,西瓜尖孢镰刀菌FOV-135的绿色荧光蛋白基因转化,福建农业学报,24(6):521-524)
Yu S.X.,Fan S.L.,Wang H.T.,Wei H.L.,and Pao C.Y.,2016,Progresses in research on cotton high yield breeding in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(18):3465-3476(喻树迅,范术丽,王寒涛,魏恒玲,庞朝友,2016,中国棉花高产育种进展,中国农业科学,49(18):3465-3476)
Zhou L.H.,Li G.Q.,Wang Z.X.,and Zhu G.J.,2005,Preparation and regeneration of protoplasts from Monascus purpureus and genetic transformation system,Yichuan(Hereditas),27(3):423-428(周礼红,李国琴,王正祥,诸葛健,2005,红曲霉原生质体的制备、再生及其遗传转化系统,遗传,27(3):423-428)