烟草靶斑病菌内切多聚半乳糖醛酸酶基因endoPGs的克隆及表达特征分析

doi:10.3864/j.issn.0578-1752.2014.10.007

摘要/Abstract

摘要： 【目的】内切多聚半乳糖醛酸酶（endo polygalacturonases，endoPGs）是重要的致病因子之一。论文从烟草靶斑病菌（Rhizoctonia solani）强致病力菌株YC-9和弱致病菌株LF-2中克隆该致病基因，分析比较该基因的序列特征、进化关系和表达特性，研究其在与烟草互作过程的致病作用。【方法】通过比较GenBank中不同植物病原真菌的endoPGs序列设计简并引物，首先获得菌株YC-9及LF-2的endoPGs基因片段，再采用RACE技术克隆获得其cDNA全长序列；生物信息学分析比较其保守结构域及序列特征；采用MEGA 4.0软件构建endoPGs的系统进化树；通过real-time RT-PCR技术对强致病力菌株YC-9及弱致病力菌株LF-2的endoPGs与烟草K326互作的表达特性进行研究。【结果】克隆获得了烟草靶斑病菌强致病力菌株YC-9和弱致病力菌株LF-2的endoPGs的cDNA全长序列，分别命名为endoPG1和endoPG2，开放阅读框（ORF）长度均为1 086 bp，编码361个氨基酸；比较分析表明endoPG1和endoPG2的推测蛋白均具有PLNO3003基因家族保守结构域，其跨膜结构间存在差异；成功构建了该基因系统进化树，结果表明来自烟草靶斑病菌的endoPGs构成独立分支，且烟草靶斑病菌endoPG1及endoPG2同源关系最近；real-time RT-PCR表达特性分析结果显示，靶斑病菌endoPG1和endoPG2与烟草互作（接种）之后该基因的表达与未互作（不接种）的对照样品相比均呈明显上调趋势，同时endoPG1表达迅速且高于endoPG2。【结论】烟草靶斑病菌强致病力菌株YC-9及弱致病力菌株LF-2的内切多聚半乳糖醛酸酶基因均具有PLNO3003基因家族的保守结构域，其推测蛋白的跨膜结构间存在差异，该推测蛋白与烟草靶斑病菌同源关系最近，且endoPG1和endoPG2的推测蛋白的同源性最高；内切多聚半乳糖醛酸酶基因的表达受与烟草互作的诱导，在不同致病力菌株中存在明显差异。

关键词: 烟草靶斑病菌 , 内切多聚半乳糖醛酸酶基因 , 克隆 , 基因表达

Abstract: 【Objective】 Endo polygalacturonases (endoPGs) is considered to be one of the important pathogenic factors. The objectives of this study are to clone and compare endoPGs from the strong pathogenic strain YC-9 and the weak pathogenic strain LF-2 from Rhizoctonia solani, analyze the sequence features, evolutionary relationships and expression characteristics of endoPG1 and endoPG2, and to provide a theoretical foundation for clarifying molecular mechanism of the pathogenicity. 【Method】 Degenerate primers were developed based on the sequence of different endoPGs from plant pathogens in GenBank, and partial cDNA fragments of the strains YC-9 and LF-2 were firstly acquired, then the full-length cDNA sequences were cloned through RACE techniques, and the conserved domains and sequence features of the genes were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 4.0 software, and the relative expression characters of endoPG1 and endoPG2 were also analyzed by real-time RT-PCR. 【Result】 Two endo polygalacturonases genes were cloned and named as endoPG1 and endoPG2. The genes possessed the conserved domains of PLNO3003 gene superfamily. The open reading frame (ORF) of the full length cDNA was 1 086 bp, encoding a protein of 361 amino acid residues. Differences between endoPG1 and endoPG2 were indicated by comparing the full-length cDNA sequences and transmembrane regions of prediction protein. The PLNO3003 subunit phylogenetic tree was constructed, which showed that the corresponding amino acid sequences of endoPGs from R. solani formed an independent branch, and that of endoPG1 and endoPG2 had the highest homology. Real-time RT-PCR demonstrated that the expression of endoPG1 and endoPG2 were more obviously up-regulated in hyphae of inoculated tobacco leaves than in hyphae of non-inoculated ones, and the expression quantity of endoPG1 was higher than that of endoPG2. 【Conclusion】 The full cDNA sequences of endoPG1 and endoPG2 were successfully cloned from tobacco target spot disease of R. solani, and both have conserved domains of PLNO3003 gene superfamily, and it has obviously differences between transmembrane regions of prediction protein of endoPG1 and endoPG2. Phylogenetic analysis showed that the corresponding amino acid sequences of endoPG1 and endoPG2 have a highest homology relationship. Finally the endoPG1 and endoPG2 gene expression can be obviously induced by interaction with tobacco and the strong and weak pathogenicity strains in YC-9 and LF-2 have obvious differences.

Key words: Rhizoctonia solani , endoPGs , cloning , gene expression

赵艳琴1, 2, 吴元华1, 赵秀香1, 陈建光1, 伏颖1, 安梦楠1. 烟草靶斑病菌内切多聚半乳糖醛酸酶基因endoPGs的克隆及表达特征分析[J]. 中国农业科学, 2014, 47(10): 1939-1946.

ZHAO Yan-Qin-1, 2 , WU Yuan-Hua-1, ZHAO Xiu-Xiang-1, CHEN Jian-Guang-1, FU Ying-1, AN Meng-Nan-1. Cloning and Expression Analysis of the Endo Polygalacturonases Gene endoPGs in Rhizoctonia solani Causing Tobacco Target Spot[J]. Scientia Agricultura Sinica, 2014, 47(10): 1939-1946.

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参考文献

[1]Shew H D, Main C E. Rhizoctonia leaf spot of flue-cured tobacco in North Carolina. Plant Disease, 1985, 69: 901-903.

[2]Shew H D, Main C E. Infection and development of target spot of flue-cured tobacco caused by Thanatephorus cucumeris. Plant Disease, 1990, 74: 109-113.

[3]吴元华, 王左斌, 刘志恒, 赵秀香, 梁景颐. 我国烟草新病害-靶斑病. 中国烟草学报, 2006, 12(6): 22-23.

Wu Y H, Wang Z B, Liu Z H, Zhao X X, Liang J Y. New tobacco disease: Target spot of flue-cured tobacco in China. Acta Tabacaria Sinica, 2006, 12(6): 22-23. (in Chinese)

[4]Wu Y H, Zhao Y Q, Fu Y, Zhao X X, Chen J G. First report of target spot of flue-cured tobacco caused by Rhizoctonia solani AG-3 in China. Plant Disease, 2012, 96(12): 1824.

[5]孙文秀. 大豆疫霉根腐病菌多聚半乳糖醛酸酶活性对其致病力的影响. 现代农业科学, 2008, 15(2): 42-43.

Sun W X. Effect of activities of polygalacturonase on pathogenicity in Phytophthora sojae. Modern Agricultural Sciences, 2008, 15(2): 42-43. (in Chinese)

[6]高洪敏, 陈捷, 何晶, 宁家林, 于兵, 刘君, 哈娟. 禾谷镰刀菌产生的细胞壁降解酶的特性研究. 玉米科学, 2005, 13(3): 112-113.

Gao H M, Chen J, He J, Ning J L, Yu B, Liu J, Ha J. The character of cell wall degradation enzyme produced by Fusarium gramimearum. Journal of Maize Sciences, 2005, 13(3): 112-113. (in Chinese)

[7]陈捷, 高洪敏, 纪明山, 宋佐衡. 玉米茎腐病菌产生的细胞壁降解酶的致病作用. 植物病理学报, 1998, 28(3): 221-226.

Chen J, Gao H M, Ji M S, Song Z H. On the pathogenicity of CWDEs produced from stalk rot pathogens in maize. Acta Phytopathologica Sinica, 1998, 28(3): 221-226. (in Chinese)

[8]冯晶, 高增贵, 薛春生, 庄敬华, 陈捷, 白淑英. 玉米弯孢霉叶斑病菌产生的细胞壁降解酶的致病作用研究. 杂粮作物, 2002, 22(3): 164-166.

Feng J, Gao Z G, Xue C S, Zhuang J H, Chen J, Bai S Y. The pathogenesis of the cell degrading enzymes produced by Curvularia lunata. Rain Fed Crops, 2002, 22(3): 164-166. (in Chinese)

[9]杨媚, 杨迎青, 郑丽, 李明海, 周而勋. 水稻纹枯病菌细胞壁降解酶组分分析、活性测定及其致病作用. 中国水稻科学, 2012, 26(5): 600-606.

Yang M, Yang Y Q, Zheng L, Li M H, Zhou E X. Component analysis, activity measurement and pathogenic effect of cell wall degarading enzymes from rice sheath blight pathogen Rhizoctonia solani. Chinese Journal of Rice Science, 2012, 26(5): 600-606. (in Chinese)

[10]孙文秀. 大豆疫霉菌多聚半乳糖醛酸酶pspg1基因的克隆及表达分析. 大豆科学, 2009, 28(5): 781-783, 790.

Sun W X. Molecular cloning and expression analysis of a polygalacturonase pspg1 from Phytophthora sojae. Soybean Science, 2009, 28(5): 781-783, 790. (in Chinese)

[11]Götesson A, Marshall J S, Jones D A, Hardham A R. Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophtora cinnamomi. Molecular Plant-Microbe Interactions, 2002, 15(9): 907-921.

[12]Di Pietro A, Roncero M I. Cloning, expression and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Molecular Plant- Microbe Interactions, 1998, 11(2): 91-98.

[13]Centis S, Guillas I, Sejalon N, Esquerré-Tugayé M T. Endopolygalacturonase genes from Colletotrichum lindemuthianum: cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of fungus. Molecular Plant-Microbe Interactions, 1997, 10(6): 769-775.

[14]Wagner F, Kusserow H, Schafer W. Cloning and targeted disruption of two polygalacturonase genes in Penicillium olsonii. FEMS Microbiology Letters, 2000, 186: 293-299.

[15]de Vries R P, Visser J. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiology and Molecular Biology Reviews, 2001, 65(4): 497-522.

[16]Li R G, Rimmer R, Buchwalolt L, Sharpe A G, Séguin-Swartz G, Hegedus D D. Interaction of Sclerotinia sclerotionum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes. Fungal Genetics and Biology, 2004, 41: 754-765.

[17]Reignault P, Mercier M, Bompeix G, Boccara M. Pectin methylesterase from Botrytis cinerea: physiological biochemical and immunochemical studies. Microbiology, 1994, 140: 3249-3255.

[18]ten Have A, Mulder W, Visser J, van Kan J. The endopolygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Molecular Plant-Microbe Interactions, 1998, 11(10): 1009-1016.

[19]Leone G, Schoffelmeer E A M, Van den Heuvel J. Purification and characterization of a constitutive polygalacturonase associated with the infection process of French bean leaves by Botrytis cinerea. Canadian Journal of Botany, 1990, 68: 1921-1930.

[20]杜可欣. 玉米纹枯病菌内切多聚半乳糖醛酸酶的基因克隆、表达及致病性分析[D]. 泰安: 山东农业大学, 2009.

Du K X. Gene cloning, expression and pathogenicity of endo- polygalacturonase from Rhizoctonia solani Kühn AG1-IA[D]. Taian: Shandong Agricultural University, 2009. (in Chinese)

[21]韩爽. 玉米纹枯病菌内切多聚半乳糖醛酸酶基因的克隆表达及致病性分析[D]. 泰安: 山东农业大学, 2011.

Han S. Gene cloning, expression and pathogenicity of endo- polygalacturonase from Rhizoctonia solani Kühn[D]. Taian: Shandong Agricultural University, 2011. (in Chinese)

[22]Oeser B, Heidrich P M, Muler U, Tudzynski P, Tenberge K B. Polygalacturonase is a pathogenicity factor in the Claviceps purpurea/rye interaction. Fungal Genetics and Biology, 2002, 36: 176-186.

[23]Isshiki A, Akimitsu K, Yamamoto M, Yamamoto H. Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternate. Molecular Plant-Microbe Interactions, 2001, 14(6): 749-757.

[24]王友德. 水稻纹枯病菌PG的分离纯化、理化性质及Rspg1基因的克隆与表达[D]. 扬州: 扬州大学, 2010.

Wang Y D. Isolation, purification and characterization of polygalacturonase of Rhizoctonia solani causing rice sheath blight and cloning and expression of Rspg1 gene of the pathogen[D]. Yangzhou: Yangzhou University, 2010. (in Chinese)