苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能
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摘要
【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。
[Objective] Cytochrome P450(CYP) plays a significant role in mycotoxin metabolism of necrotrophic fungus and may be related to pathogenicity. The objective of the study was to reveal the function of CYP genes in infection process. [Methods] The knockout cassette was constructed using Double-joint PCR and mutants were obtained and confirmed by PEG-mediated transformation of protoplasts, PCR and Southern blotting analysis. Gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple twigs was used to detect the pathogenicity. Salient differences were analyzed by SPSS and q RT-PCR was used to detect the expression of melanin gene cluster. [Results] Compared with the original strain 03-8, the colony color of deletion mutant changed from light yellow to white, and the amount of propagulum reduced by 51.3%. The relative expression level of Vmcyp5-knockout mutant was assayed by q RT-PCR, and mutant's melanin gene cluster was down-regulated. More importantly, the pathogenicity of deletion mutant was decreased by 24.5%. Complementation mutant was almost back to the level of the original strain 03-8 in colony color, propagulum and pathogenicity. [Conclusion] Vmcyp5 may be related to melanin biosynthesis and propagulum formation, and participates in pathogen process of Valsa mali.
[Objective] Cytochrome P450(CYP) plays a significant role in mycotoxin metabolism of necrotrophic fungus and may be related to pathogenicity. The objective of the study was to reveal the function of CYP genes in infection process. [Methods] The knockout cassette was constructed using Double-joint PCR and mutants were obtained and confirmed by PEG-mediated transformation of protoplasts, PCR and Southern blotting analysis. Gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple twigs was used to detect the pathogenicity. Salient differences were analyzed by SPSS and q RT-PCR was used to detect the expression of melanin gene cluster. [Results] Compared with the original strain 03-8, the colony color of deletion mutant changed from light yellow to white, and the amount of propagulum reduced by 51.3%. The relative expression level of Vmcyp5-knockout mutant was assayed by q RT-PCR, and mutant's melanin gene cluster was down-regulated. More importantly, the pathogenicity of deletion mutant was decreased by 24.5%. Complementation mutant was almost back to the level of the original strain 03-8 in colony color, propagulum and pathogenicity. [Conclusion] Vmcyp5 may be related to melanin biosynthesis and propagulum formation, and participates in pathogen process of Valsa mali.
引文
[1]Wang XL,Wei JL,Huang LL,Kang ZS.Re-evaluation of pathogens causing Valsa canker on apple in China.Mycologia,2011,103(2):317–324.
[2]Ke XW,Huang LL,Han QM,Gao XN,Kang ZS.Histological and cytological investigations of the infection and colonization of apple bark by Valsa mali var.mali.Australasian Plant Pathology,2013,42(1):85–93.
[3]Li ZP,Gao XN,Du ZT,Hu Y,Kang ZS,Huang LL.Survey of apple Valsa canker in Weibei area of Shaanxi province.Acta Agriculturae Boreali-Occidentalis Sinica,2013,22(1):174–178.(in Chinese)李正鹏,高小宁,杜战涛,胡杨,康振生,黄丽丽.陕西渭北地区苹果树腐烂病发生情况调查.西北农业学报,2013,22(1):174–178.
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[5]Qi GF,Yang B,Ye JR.Research advances on the toxin of the plant pathogenic fungus.Journal of Nanjing Forestry University,2000,24(2):66–70.(in Chinese)祁高富,杨斌,叶建仁.植物病原真菌毒素研究进展.南京林业大学学报,2000,24(2):66–70.
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[8]Ehrlich KC,Chang PK,Yu JJ,Cotty PJ.Aflatoxin biosynthesis cluster gene cyp A is required for G aflatoxin formation.Applied and Environmental Microbiology,2004,70(11):6518–6524.
[9]Proctor RH,Plattner RD,Desjardins AE,Busman M,Butchko RAE.Fumonisin production in the maize pathogen Fusarium verticillioides:genetic basis of naturally occurring chemical variation.Journal of Agricultural and Food Chemistry,2006,54(6):2424–2430.
[10]Bhatnagar D,Ehrlich KC,Cleveland TE.Molecular genetic analysis and regulation of aflatoxin biosynthesis.Applied Microbiology and Biotechnology,2003,61(2):83–93.
[11]Yu JJ,Chang PK,Ehrlich KC,Cary JW,Bhatnagar D,Cleveland TE,Payne GA,Linz JE,Woloshuk CP,Bennett JW.Clustered pathway genes in aflatoxin biosynthesis.Applied and Environmental Microbiology,2004,70(3):1253–1262.
[12]Desjardins AE,Hohn TM.Mycotoxins in plant pathogenesis.Molecular Plant-Microbe Interactions,1997,10(2):147–152.
[13]Kimura M,Tokai T,Takahashi-Ando N,Ohsato S,Fujimura M.Molecular and genetic studies of Fusarium trichothecene biosynthesis:pathways,genes,and evolution.Bioscience,Biotechnology,and Biochemistry,2007,71(9):2105–2123.
[14]Wen Y,Hatabayashi H,Arai H,Kitamoto HK,Yabe K.Function of the cyp X and mox Y genes in aflatoxin biosynthesis in Aspergillus parasiticus.Applied and Environmental Microbiology,2005,71(6):3192–3198.
[15]Siewers V,Viaud M,Jimenez-Teja D,Collado IG,Gronover CS,Pradier JM,Tudzynsk B,Tudzynski P.Functional analysis of the cytochrome P450 monooxygenase gene bcbot1of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor.Molecular Plant-Microbe Interactions,2005,18(6):602–612.
[16]Yin ZY,Liu HQ,Li ZP,Ke XW,Dou DL,Gao XN,Song N,Dai QQ,Wu YX,Xu JR,Kang ZS,Huang LL.Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark.New Phytologist,2015,208(4):1202–1216.
[17]Yu JH,Hamari Z,Han KH,Seo JA,Reyes-Domínguez Y,Scazzocchio C.Double-joint PCR:a PCR-based molecular tool for gene manipulations in filamentous fungi.Fungal Genetics and Biology,2004,41(11):973–981.
[18]Gao J,Li YB,Ke XW,Kang ZS,Huang LL.Development of genetic transformation system of Valsa mali of apple mediated by PEG.Acta Microbiologica Sinica,2011,51(9):1194–1199.(in Chinese)高静,李艳波,柯希望,康振生,黄丽丽.PEG介导的苹果腐烂病菌原生质体转化.微生物学报,2011,51(9):1194–1199.
[19]Song N,Dai QQ,Huang LL,Han QM.Construction of knockout vector of GTP cyclohydrolase II gene and mutant’s biological characteristics of Valsa mali.Scientia Agricultura Sinica,2014,47(15):2980–2989.(in Chinese)宋娜,戴青青,黄丽丽,韩青梅.苹果树腐烂病菌GTP-环化水解酶II基因敲除载体构建及其突变体的表型分析.中国农业科学,2014,47(15):2980–2989.
[20]Zheng DW.Construction of gene knock-out approach for Ustilaginoidea virens and the function analysis of HOG1 in Ustilaginoidea virens and Fusarium graminearum.Doctor Dissertation of Northwest A&F University,2015.(in Chinese)郑大伟.稻曲病菌基因敲除体系的建立及HOG1基因在稻曲病菌和禾谷镰刀菌中功能验证.西北农林科技大学博士学位论文,2015.
[21]Zang R,Huang LL,Kang ZS,Wang XL.Biological characteristics and pathogenicity of different isolates of Cytospora spp.isolated from apple trees in Shaanxi province.Acta Phytopathologica Sinica,2007,37(4):343–351.(in Chinese)臧睿,黄丽丽,康振生,王旭丽.陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究.植物病理学报,2007,37(4):343–351.
[22]Xu CJ,Wu YX,Dai QQ,Li ZP,Gao XN,Huang LL.Function of polygalacturonase genes Vmpg7 and Vmpg8 of Valsa mali.Scientia Agricultura Sinica,2016,49(8):1489–1498.(in Chinese)许春景,吴玉星,戴青青,李正鹏,高小宁,黄丽丽.苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能.中国农业科学,2016,49(8):1489–1498.
[23]Yin ZY,Ke XW,Huang DX,Gao XN,Voegele RT,Kang ZS,Huang LL.Validation of reference genes for gene expression analysis in Valsa mali var.mali using real-time quantitative PCR.World Journal of Microbiology and Biotechnology,2013,29(9):1563–1571.
[24]Bashyal BM,Chand R,Kushwaha C,Sen D,Prasad LC,Joshi AK.Association of melanin content with conidiogenesis in Bipolaris sorokiniana of barley(Hordeum vulgare L.).World Journal of Microbiology and Biotechnology,2010,26(2):309–316.
[25]Farkas J,Schricker R,Briza P,Eckerstorfer M,Breitenbach M.The enzymatic properties of Dit2p(CYP56)from Saccharomyces cerevisiae.FASEB Journal,1997,11(9):A827.
[26]Tsitsigiannis DI,Keller NP.Oxylipins as developmental and host-fungal communication signals.Trends in Microbiology,2007,15(3):109–118.
[27]B?mke C,Rojas MC,Gong F,Hedden P,Tudzynski B.Isolation and characterization of the gibberellin biosynthetic gene cluster in Sphaceloma manihoticola.Applied and Environmental Microbiology,2008,74(17):5325–5339.
[28]Koch A,Kumar N,Weber L,Keller H,Imani J,Kogel KH.Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species.Proceedings of the National Academy of Sciences of the United States of America,2013,110(48):19324–19329.
[29]Ichinose H.Molecular and functional diversity of fungal cytochrome P450s.Biological and Pharmaceutical Bulletin,2012,35(6):833–837.
[2]Ke XW,Huang LL,Han QM,Gao XN,Kang ZS.Histological and cytological investigations of the infection and colonization of apple bark by Valsa mali var.mali.Australasian Plant Pathology,2013,42(1):85–93.
[3]Li ZP,Gao XN,Du ZT,Hu Y,Kang ZS,Huang LL.Survey of apple Valsa canker in Weibei area of Shaanxi province.Acta Agriculturae Boreali-Occidentalis Sinica,2013,22(1):174–178.(in Chinese)李正鹏,高小宁,杜战涛,胡杨,康振生,黄丽丽.陕西渭北地区苹果树腐烂病发生情况调查.西北农业学报,2013,22(1):174–178.
[4]Li ZP.Study on the biological control of Saccharothrix yanglinggensis Hhs.015 on apple Valsa canker.Master Dissertation of Northwest A&F University,2012.(in Chinese)李正鹏.杨凌糖丝菌Hhs.015对苹果树腐烂病的生物防治研究.西北农林科技大学硕士学位论文,2012.
[5]Qi GF,Yang B,Ye JR.Research advances on the toxin of the plant pathogenic fungus.Journal of Nanjing Forestry University,2000,24(2):66–70.(in Chinese)祁高富,杨斌,叶建仁.植物病原真菌毒素研究进展.南京林业大学学报,2000,24(2):66–70.
[6]Mc Lean KJ,Leys D,Munro AW.Microbial cytochromes P450//de Montellano PRO.Cytochrome P450:Structure,Mechanism,and Biochemistry.Switzerland:Springer International Publishing,2015:261–407.
[7]?re?nar B,Petri??.Cytochrome P450 enzymes in the fungal kingdom.Biochimica et Biophysica Acta(BBA)-Proteins and Proteomics,2011,1814(1):29–35.
[8]Ehrlich KC,Chang PK,Yu JJ,Cotty PJ.Aflatoxin biosynthesis cluster gene cyp A is required for G aflatoxin formation.Applied and Environmental Microbiology,2004,70(11):6518–6524.
[9]Proctor RH,Plattner RD,Desjardins AE,Busman M,Butchko RAE.Fumonisin production in the maize pathogen Fusarium verticillioides:genetic basis of naturally occurring chemical variation.Journal of Agricultural and Food Chemistry,2006,54(6):2424–2430.
[10]Bhatnagar D,Ehrlich KC,Cleveland TE.Molecular genetic analysis and regulation of aflatoxin biosynthesis.Applied Microbiology and Biotechnology,2003,61(2):83–93.
[11]Yu JJ,Chang PK,Ehrlich KC,Cary JW,Bhatnagar D,Cleveland TE,Payne GA,Linz JE,Woloshuk CP,Bennett JW.Clustered pathway genes in aflatoxin biosynthesis.Applied and Environmental Microbiology,2004,70(3):1253–1262.
[12]Desjardins AE,Hohn TM.Mycotoxins in plant pathogenesis.Molecular Plant-Microbe Interactions,1997,10(2):147–152.
[13]Kimura M,Tokai T,Takahashi-Ando N,Ohsato S,Fujimura M.Molecular and genetic studies of Fusarium trichothecene biosynthesis:pathways,genes,and evolution.Bioscience,Biotechnology,and Biochemistry,2007,71(9):2105–2123.
[14]Wen Y,Hatabayashi H,Arai H,Kitamoto HK,Yabe K.Function of the cyp X and mox Y genes in aflatoxin biosynthesis in Aspergillus parasiticus.Applied and Environmental Microbiology,2005,71(6):3192–3198.
[15]Siewers V,Viaud M,Jimenez-Teja D,Collado IG,Gronover CS,Pradier JM,Tudzynsk B,Tudzynski P.Functional analysis of the cytochrome P450 monooxygenase gene bcbot1of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor.Molecular Plant-Microbe Interactions,2005,18(6):602–612.
[16]Yin ZY,Liu HQ,Li ZP,Ke XW,Dou DL,Gao XN,Song N,Dai QQ,Wu YX,Xu JR,Kang ZS,Huang LL.Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark.New Phytologist,2015,208(4):1202–1216.
[17]Yu JH,Hamari Z,Han KH,Seo JA,Reyes-Domínguez Y,Scazzocchio C.Double-joint PCR:a PCR-based molecular tool for gene manipulations in filamentous fungi.Fungal Genetics and Biology,2004,41(11):973–981.
[18]Gao J,Li YB,Ke XW,Kang ZS,Huang LL.Development of genetic transformation system of Valsa mali of apple mediated by PEG.Acta Microbiologica Sinica,2011,51(9):1194–1199.(in Chinese)高静,李艳波,柯希望,康振生,黄丽丽.PEG介导的苹果腐烂病菌原生质体转化.微生物学报,2011,51(9):1194–1199.
[19]Song N,Dai QQ,Huang LL,Han QM.Construction of knockout vector of GTP cyclohydrolase II gene and mutant’s biological characteristics of Valsa mali.Scientia Agricultura Sinica,2014,47(15):2980–2989.(in Chinese)宋娜,戴青青,黄丽丽,韩青梅.苹果树腐烂病菌GTP-环化水解酶II基因敲除载体构建及其突变体的表型分析.中国农业科学,2014,47(15):2980–2989.
[20]Zheng DW.Construction of gene knock-out approach for Ustilaginoidea virens and the function analysis of HOG1 in Ustilaginoidea virens and Fusarium graminearum.Doctor Dissertation of Northwest A&F University,2015.(in Chinese)郑大伟.稻曲病菌基因敲除体系的建立及HOG1基因在稻曲病菌和禾谷镰刀菌中功能验证.西北农林科技大学博士学位论文,2015.
[21]Zang R,Huang LL,Kang ZS,Wang XL.Biological characteristics and pathogenicity of different isolates of Cytospora spp.isolated from apple trees in Shaanxi province.Acta Phytopathologica Sinica,2007,37(4):343–351.(in Chinese)臧睿,黄丽丽,康振生,王旭丽.陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究.植物病理学报,2007,37(4):343–351.
[22]Xu CJ,Wu YX,Dai QQ,Li ZP,Gao XN,Huang LL.Function of polygalacturonase genes Vmpg7 and Vmpg8 of Valsa mali.Scientia Agricultura Sinica,2016,49(8):1489–1498.(in Chinese)许春景,吴玉星,戴青青,李正鹏,高小宁,黄丽丽.苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能.中国农业科学,2016,49(8):1489–1498.
[23]Yin ZY,Ke XW,Huang DX,Gao XN,Voegele RT,Kang ZS,Huang LL.Validation of reference genes for gene expression analysis in Valsa mali var.mali using real-time quantitative PCR.World Journal of Microbiology and Biotechnology,2013,29(9):1563–1571.
[24]Bashyal BM,Chand R,Kushwaha C,Sen D,Prasad LC,Joshi AK.Association of melanin content with conidiogenesis in Bipolaris sorokiniana of barley(Hordeum vulgare L.).World Journal of Microbiology and Biotechnology,2010,26(2):309–316.
[25]Farkas J,Schricker R,Briza P,Eckerstorfer M,Breitenbach M.The enzymatic properties of Dit2p(CYP56)from Saccharomyces cerevisiae.FASEB Journal,1997,11(9):A827.
[26]Tsitsigiannis DI,Keller NP.Oxylipins as developmental and host-fungal communication signals.Trends in Microbiology,2007,15(3):109–118.
[27]B?mke C,Rojas MC,Gong F,Hedden P,Tudzynski B.Isolation and characterization of the gibberellin biosynthetic gene cluster in Sphaceloma manihoticola.Applied and Environmental Microbiology,2008,74(17):5325–5339.
[28]Koch A,Kumar N,Weber L,Keller H,Imani J,Kogel KH.Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species.Proceedings of the National Academy of Sciences of the United States of America,2013,110(48):19324–19329.
[29]Ichinose H.Molecular and functional diversity of fungal cytochrome P450s.Biological and Pharmaceutical Bulletin,2012,35(6):833–837.