基于PEG介导原生质体转化构建粉红聚端孢荧光标记
|
摘要
粉红聚端孢是多种植物的重要病原菌。本研究通过酶解粉红聚端孢幼嫩菌丝细胞壁获得原生质体,用PEG介导原生质体转化将携带GFP基因和博来霉素抗性基因的外源DNA随机插入粉红聚端孢基因组,共获得博来霉素抗性菌株90株,转化效率达18个/μg。选取22株转化子荧光观察,发现均可表达荧光,菌株间荧光强度不同,其中10株转化子荧光表达较强。与野生型相比,突变菌株TR45的菌落生长、产孢量和致病力等生物学特性均未改变,在不含博来霉素的培养皿中继代培养10代荧光仍能稳定表达。本研究构建的高效原生质体制备和PEG介导粉红聚端孢遗传转化方法,可用于该菌基因功能研究,绿色荧光标记菌株可用于病菌侵入、田间监测、侵染循环等发生规律研究。
Trichothecium roseum is an important pathogen of various crops. Protoplasts of T. roseum were obtained via digesting cell walls of young hyphae by use of enzymatic solutions, and exogenous plasmid DNA carrying GFP gene and zeocin resistant gene was inserted into T. roseum genome by PEG mediated protoplast transformation. Totally 90 transformants were obtained from zeocin resistant screening plates, and DNA transformation efficiency reached 18 protoplasts/μg. Twenty-two transformants were used for observing fluorescence signals. Transformants showed green fluorescent signals on various fluorescent intensities depended on transformant individuals, and ten of them displayed relatively stronger fluorescent signals. There was no difference of biological characteristics including colonial morphology, conidiation as well as pathogenicity between the representative transformant TR45 and the wild type strain. As for transformant TR45 the green fluorescence was still stable after subculturing ten generations on plates without zeocin stress.The protocols of efficient protoplast preparation and PEG mediated protoplast transformation used in this study could be applied to explore functional genes of T. roseum. The obtained GFP tagged strains could be used for further researches on pathogen penetration, field detection and infection cycles of related diseases caused by T. roseum.
Trichothecium roseum is an important pathogen of various crops. Protoplasts of T. roseum were obtained via digesting cell walls of young hyphae by use of enzymatic solutions, and exogenous plasmid DNA carrying GFP gene and zeocin resistant gene was inserted into T. roseum genome by PEG mediated protoplast transformation. Totally 90 transformants were obtained from zeocin resistant screening plates, and DNA transformation efficiency reached 18 protoplasts/μg. Twenty-two transformants were used for observing fluorescence signals. Transformants showed green fluorescent signals on various fluorescent intensities depended on transformant individuals, and ten of them displayed relatively stronger fluorescent signals. There was no difference of biological characteristics including colonial morphology, conidiation as well as pathogenicity between the representative transformant TR45 and the wild type strain. As for transformant TR45 the green fluorescence was still stable after subculturing ten generations on plates without zeocin stress.The protocols of efficient protoplast preparation and PEG mediated protoplast transformation used in this study could be applied to explore functional genes of T. roseum. The obtained GFP tagged strains could be used for further researches on pathogen penetration, field detection and infection cycles of related diseases caused by T. roseum.
引文
Balasubramanian N,Juliet GA,Srikalaivani P,Lalithakurnari D,2003.Release and regeneration of protoplasts from the fungus Trichothecium roseum.Canadian Journal of Microbiology,49:263-268
Ballance DJ,Buxton FP,Turner G,1983.Transformation of Aspergillus nidulans,by the orotidine-5’-phosphate decarboxylase gene of Neurospora crassa.Biochemical&Biophysical Research Communications,112:284-289
Beccari G,Covarelli L,Nicholson P,2011.Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum.Plant Pathology,60:671-684
Bi Y,Tian SP,Zhao J,Ge YH,2005.Harpin induces local and systemic resistance against Trichothecium roseum in harvested Hami melons.Postharvest Biology&Technology,38(2):183-187
Bluhm BH,Zhao X,Flaherty JE,Xu JR,Dunkle LD,2007.RAS2regulates growth and pathogenesis in Fusarium graminearum.Molecular Plant-Microbe Interactions,20:627-636
Case ME,Schweizer M,Kushner SR,Giles NH,1979.Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA.Proceedings of the National Academy of Sciences of the United States of America,76:5259-5263
Dai PB,Liang XF,Wang YJ,Gleason ML,Zhang R,Sun GY,2018.High humidity and age-dependent fruit susceptibility promote development of Trichothecium black spot on apple.Plant Disease,doi.org/10.1094/PDIS-05-18-0734-RE.
Ding SL,Mehrabi R,Koten C,Kang ZS,Wei YD,Seong K,Kistler HC,Xu JR,2009.Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum.Eucaryotic Cell,8:867-876
Gao J,Li YB,Ke XW,Kang ZS,Huang LL,2011.Development of genetic transformation system of Valsa mali of apple mediated by PEG.Acta Microbiologica Sinica,51(9):1194-1199(in Chinese)
Gao LL,Zhang Q,Sun XY,Jiang L,Zhang R,Sun GY,Zha YL,Biggs AR,2013.Etiology of moldy core,core browning,and core rot of Fuji apple in China.Plant Disease,97:510-516
Guo YZ,Sun GY,Gao BW,Li CY,Zhang PL,Lei XL,2005.Studies on the identification of pathogen and the biological characteristics of black-dot disease of bagged apple.Acta Agriculturae Boreali-occidentalia Sinica,14(3):18-21(in Chinese)
Hamid MI,Hussain M,Ghazanfar MU,Raza M,Liu XZ,2014.Trichothecium roseum causes fruit rot of tomato,orange,and apple in Pakistan.Plant Disease,98:1271
Han XL,Bai JK,Zhang W,Zhang R,Sun GY,2016.PEG-mediated transformation of Colletotrichum fructicola.Acta Agriculturae Boreali-occidentalia Sinica,25(3):442-449(in Chinese)
He SL,Liu ZL,Liu XM,Xie YX,Pu JJ,2011.A new disease in mango caused by Trichothecium roseum.Journal of Fruit Science,28(3):474-478(in Chinese)
Helber N,Requena N,2008.Expression of the fluorescence markers DsRed and GFP fused to a nuclear localization signal in the arbuscular mycorrhizal fungus Glomus intraradices.New Phytologist,177:537-548
Hinnen A,Hicks JB,Fink GR,1978.Transformation of yeast.Proceedings of the National Academy of Sciences of the United States of America,75:1929-1933
Huang HC,Kokko EG,1993.Trichothecium roseum,a mycoparasite of Sclerotinia sclerotiorum.Canadian Journal of Botany,71:1631-1638
Kwon JH,Shen SS,Kim J,2010.Occurrence of pink mold rot of strawberry caused by Trichothecium roseum in Korea.Plant Pathology Journal,26:296
Li L,Xue CY,Bruno K,Nishimura M,Xu JR,2004.Two PAKkinase genes,CHM1 and MST20,have distinct functions in Magnaporthe grisea.Molecular Plant-Microbe Interactions,17:547-556
Li YP,Zhang HL,Tang Q,Liu YJ,2016.First report of Trichothecium roseum causing trunk disease of Ginkgo biloba in China.Plant Disease,100:1234
Liu ZH,Friesen TL,2012.Polyethylene glycol(PEG)-mediated transformation in filamentous fungal pathogens.Humana Press,835:365-375
Mehrabi R,Ding SL,Xu JR,2008.MADS-box transcription factor Mig1 is required for infectious growth in Magnaporthe grisea.Eukaryotic Cell,7:791-799
Mochizuki Y,Nakanishi H,Kodera Y,Ito S,Yamamura Y,Kato T,Hibi K,Akiyama S,Nakao A,Tatematsu M,2004.TNF-αpromotes progression of peritoneal metastasis as demonstrated using a green fluorescence protein(GFP)-tagged human gastric cancer cell line.Clinical&Experimental Metastasis,21:39-47
Nguyen TV,Sch?fer W,Bormann J,2012.The stress-activated protein kinase FgOS-2 is a key regulator in the life cycle of the cereal pathogen Fusarium graminearum.Molecular Plant-Microbe Interactions,25:1142-1156
Oh SY,Nam KW,Yoon DH,2014.Identification of Acremonium acutatum and Trichothecium roseum isolated from grape with white stain symptom in Korea.Mycobiology,42:269-273
Pan YM,Gao ZM,Ji WF,Guo M,Dai HM,Wang XF,2005.Studies on effects of medium nutrients on mycelial growth and conidial production of Trichothecium roseum.Journal of Northwest Sci-Tech University of Agriculture and Forestry,33(s1):68-72(in Chinese)
Pliego C,Kanematsu S,Ruano-Rosa D,de Vicente A,Lopez-Herrera C,Cazorla FM,Ramos C,2009.GFP sheds light on the infection process of avocado roots by Rosellinia necatrix.Fungal Genetics&Biology,46:137-145
Ponappa T,Brzozowski AE,Finer JJ,1999.Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein.Plant Cell Reports,19:6-12
Sharma G,Maymon M,Freeman S,2016.First detailed report of Trichothecium roseum causing post-harvest pink rot of avocado in Israel.Plant Disease,100:856
Shen HM,Li C,Gao L,Liu TG,Liu B,Wan Q,2017.Research progress in transformation of fungi mediated by protoplasts.Plant Protection,43(2):25-28(in Chinese)
Sprague SJ,Watt M,Kirkegaard JA,Howlett BJ,2007.Pathways of infection of Brassica napus roots by Leptosphaeria maculans.New Phytologist,176:211-222
Stewart CN,Via LE,1993.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCRapplications.Biotechniques,14:748-750
Tang Q,Fang L,Liu YJ,2015.First report of Trichothecium roseum causing trunk disease of Acer buergerianum in China.Plant Disease,99:1864
Urbasch I,1992.Hyperparasitic attack of Trichothecium roseum on conidia of Pestalotiopsis funerea.Journal of Phytopathology,136:231-237
Xu QJ,Hu XP,Chen T,Zhao JX,2012.Protoplast transformation of Fusarium oxysporum f.sp.vasinfectum mediated by polyethylene glyol.Cotton Science,24(3):222-228(in Chinese)
Zhao H,Liang WQ,Yang YH,Zhang DB,2003.Green fluorescent protein and its application in plant molecular biology.Plant Physiology Communications,39(2):171-178(in Chinese)
高静,李艳波,柯希望,康振生,黄丽丽,2011.PEG介导的苹果腐烂病菌原生质体转化.微生物学报,51(9):1194-1199
郭云忠,孙广宇,高保卫,李春游,张沛力,雷新乐,2005.套袋苹果黑点病病原菌鉴定及其生物学特性研究.西北农业学报,14(3):18-21
韩小路,白静科,张玮,张荣,孙广宇,2016.PEG介导的苹果果生刺盘孢Colletotrichum fructicola原生质体转化.西北农业学报,25(3):442-449
何石兰,刘增亮,刘晓妹,谢艺贤,蒲金基,2011.粉红聚端孢引起的杧果新病害研究.果树学报,28(3):474-478
潘月敏,高智谋,纪文飞,郭敏,代海敏,汪小福,2005.培养基营养条件对棉铃红粉病菌菌丝生长和孢子形成的影响.西北农林科技大学学报(自然科学版),33(s1):68-72
沈慧敏,李超,高利,刘太国,刘博,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
徐齐君,胡小平,陈婷,赵俊兴,2012.PEG介导的棉花枯萎病菌原生质体转化体系的建立.棉花学报,24(3):222-228
赵华,梁婉琪,杨永华,张大兵,2003.绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯,39(2):171-178
Ballance DJ,Buxton FP,Turner G,1983.Transformation of Aspergillus nidulans,by the orotidine-5’-phosphate decarboxylase gene of Neurospora crassa.Biochemical&Biophysical Research Communications,112:284-289
Beccari G,Covarelli L,Nicholson P,2011.Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum.Plant Pathology,60:671-684
Bi Y,Tian SP,Zhao J,Ge YH,2005.Harpin induces local and systemic resistance against Trichothecium roseum in harvested Hami melons.Postharvest Biology&Technology,38(2):183-187
Bluhm BH,Zhao X,Flaherty JE,Xu JR,Dunkle LD,2007.RAS2regulates growth and pathogenesis in Fusarium graminearum.Molecular Plant-Microbe Interactions,20:627-636
Case ME,Schweizer M,Kushner SR,Giles NH,1979.Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA.Proceedings of the National Academy of Sciences of the United States of America,76:5259-5263
Dai PB,Liang XF,Wang YJ,Gleason ML,Zhang R,Sun GY,2018.High humidity and age-dependent fruit susceptibility promote development of Trichothecium black spot on apple.Plant Disease,doi.org/10.1094/PDIS-05-18-0734-RE.
Ding SL,Mehrabi R,Koten C,Kang ZS,Wei YD,Seong K,Kistler HC,Xu JR,2009.Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum.Eucaryotic Cell,8:867-876
Gao J,Li YB,Ke XW,Kang ZS,Huang LL,2011.Development of genetic transformation system of Valsa mali of apple mediated by PEG.Acta Microbiologica Sinica,51(9):1194-1199(in Chinese)
Gao LL,Zhang Q,Sun XY,Jiang L,Zhang R,Sun GY,Zha YL,Biggs AR,2013.Etiology of moldy core,core browning,and core rot of Fuji apple in China.Plant Disease,97:510-516
Guo YZ,Sun GY,Gao BW,Li CY,Zhang PL,Lei XL,2005.Studies on the identification of pathogen and the biological characteristics of black-dot disease of bagged apple.Acta Agriculturae Boreali-occidentalia Sinica,14(3):18-21(in Chinese)
Hamid MI,Hussain M,Ghazanfar MU,Raza M,Liu XZ,2014.Trichothecium roseum causes fruit rot of tomato,orange,and apple in Pakistan.Plant Disease,98:1271
Han XL,Bai JK,Zhang W,Zhang R,Sun GY,2016.PEG-mediated transformation of Colletotrichum fructicola.Acta Agriculturae Boreali-occidentalia Sinica,25(3):442-449(in Chinese)
He SL,Liu ZL,Liu XM,Xie YX,Pu JJ,2011.A new disease in mango caused by Trichothecium roseum.Journal of Fruit Science,28(3):474-478(in Chinese)
Helber N,Requena N,2008.Expression of the fluorescence markers DsRed and GFP fused to a nuclear localization signal in the arbuscular mycorrhizal fungus Glomus intraradices.New Phytologist,177:537-548
Hinnen A,Hicks JB,Fink GR,1978.Transformation of yeast.Proceedings of the National Academy of Sciences of the United States of America,75:1929-1933
Huang HC,Kokko EG,1993.Trichothecium roseum,a mycoparasite of Sclerotinia sclerotiorum.Canadian Journal of Botany,71:1631-1638
Kwon JH,Shen SS,Kim J,2010.Occurrence of pink mold rot of strawberry caused by Trichothecium roseum in Korea.Plant Pathology Journal,26:296
Li L,Xue CY,Bruno K,Nishimura M,Xu JR,2004.Two PAKkinase genes,CHM1 and MST20,have distinct functions in Magnaporthe grisea.Molecular Plant-Microbe Interactions,17:547-556
Li YP,Zhang HL,Tang Q,Liu YJ,2016.First report of Trichothecium roseum causing trunk disease of Ginkgo biloba in China.Plant Disease,100:1234
Liu ZH,Friesen TL,2012.Polyethylene glycol(PEG)-mediated transformation in filamentous fungal pathogens.Humana Press,835:365-375
Mehrabi R,Ding SL,Xu JR,2008.MADS-box transcription factor Mig1 is required for infectious growth in Magnaporthe grisea.Eukaryotic Cell,7:791-799
Mochizuki Y,Nakanishi H,Kodera Y,Ito S,Yamamura Y,Kato T,Hibi K,Akiyama S,Nakao A,Tatematsu M,2004.TNF-αpromotes progression of peritoneal metastasis as demonstrated using a green fluorescence protein(GFP)-tagged human gastric cancer cell line.Clinical&Experimental Metastasis,21:39-47
Nguyen TV,Sch?fer W,Bormann J,2012.The stress-activated protein kinase FgOS-2 is a key regulator in the life cycle of the cereal pathogen Fusarium graminearum.Molecular Plant-Microbe Interactions,25:1142-1156
Oh SY,Nam KW,Yoon DH,2014.Identification of Acremonium acutatum and Trichothecium roseum isolated from grape with white stain symptom in Korea.Mycobiology,42:269-273
Pan YM,Gao ZM,Ji WF,Guo M,Dai HM,Wang XF,2005.Studies on effects of medium nutrients on mycelial growth and conidial production of Trichothecium roseum.Journal of Northwest Sci-Tech University of Agriculture and Forestry,33(s1):68-72(in Chinese)
Pliego C,Kanematsu S,Ruano-Rosa D,de Vicente A,Lopez-Herrera C,Cazorla FM,Ramos C,2009.GFP sheds light on the infection process of avocado roots by Rosellinia necatrix.Fungal Genetics&Biology,46:137-145
Ponappa T,Brzozowski AE,Finer JJ,1999.Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein.Plant Cell Reports,19:6-12
Sharma G,Maymon M,Freeman S,2016.First detailed report of Trichothecium roseum causing post-harvest pink rot of avocado in Israel.Plant Disease,100:856
Shen HM,Li C,Gao L,Liu TG,Liu B,Wan Q,2017.Research progress in transformation of fungi mediated by protoplasts.Plant Protection,43(2):25-28(in Chinese)
Sprague SJ,Watt M,Kirkegaard JA,Howlett BJ,2007.Pathways of infection of Brassica napus roots by Leptosphaeria maculans.New Phytologist,176:211-222
Stewart CN,Via LE,1993.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCRapplications.Biotechniques,14:748-750
Tang Q,Fang L,Liu YJ,2015.First report of Trichothecium roseum causing trunk disease of Acer buergerianum in China.Plant Disease,99:1864
Urbasch I,1992.Hyperparasitic attack of Trichothecium roseum on conidia of Pestalotiopsis funerea.Journal of Phytopathology,136:231-237
Xu QJ,Hu XP,Chen T,Zhao JX,2012.Protoplast transformation of Fusarium oxysporum f.sp.vasinfectum mediated by polyethylene glyol.Cotton Science,24(3):222-228(in Chinese)
Zhao H,Liang WQ,Yang YH,Zhang DB,2003.Green fluorescent protein and its application in plant molecular biology.Plant Physiology Communications,39(2):171-178(in Chinese)
高静,李艳波,柯希望,康振生,黄丽丽,2011.PEG介导的苹果腐烂病菌原生质体转化.微生物学报,51(9):1194-1199
郭云忠,孙广宇,高保卫,李春游,张沛力,雷新乐,2005.套袋苹果黑点病病原菌鉴定及其生物学特性研究.西北农业学报,14(3):18-21
韩小路,白静科,张玮,张荣,孙广宇,2016.PEG介导的苹果果生刺盘孢Colletotrichum fructicola原生质体转化.西北农业学报,25(3):442-449
何石兰,刘增亮,刘晓妹,谢艺贤,蒲金基,2011.粉红聚端孢引起的杧果新病害研究.果树学报,28(3):474-478
潘月敏,高智谋,纪文飞,郭敏,代海敏,汪小福,2005.培养基营养条件对棉铃红粉病菌菌丝生长和孢子形成的影响.西北农林科技大学学报(自然科学版),33(s1):68-72
沈慧敏,李超,高利,刘太国,刘博,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
徐齐君,胡小平,陈婷,赵俊兴,2012.PEG介导的棉花枯萎病菌原生质体转化体系的建立.棉花学报,24(3):222-228
赵华,梁婉琪,杨永华,张大兵,2003.绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯,39(2):171-178