核盘菌几丁质酶基因家族分析与表达特性研究
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摘要
核盘菌菌核以子实体形式萌发是作物菌核病病害循环中关键性的一环,研究菌核子实体萌发分子机理将为菌核病的安全控制提供线索.该研究通过生物信息学方法对核盘菌几丁质酶基因家族进行分析,并利用实时荧光RT-PCR对其在菌核子实体萌发阶段的表达进行探讨.生物信息学分析表明核盘菌中共有12个基因编码假定的几丁质酶,它们均具有GH18结构域,部分蛋白还具有几丁质结合结构域和纤维素结构域;除SS1G_11212外其余蛋白均为亲水性蛋白;亚细胞定位预测表明其中10个假定几丁质酶位于胞外,其余2个分别位于细胞核和细胞质中.通过构建系统发育树分析发现假定几丁质酶可以分为2大类,其中3个与酵母几丁质酶CTS1归为一类,其余9个与CTS2归为另一类.除SS1G_00773外,其余11个几丁质酶基因在核盘菌菌核子实体萌发过程中表达量均有变化,推测几丁质酶家族蛋白可能参与核盘菌菌核子实体萌发过程.
The carpogenic germination of sclerotia for Sclerotinia sclerotiorumis a key step of crop sclerotiniose infection cycle.Studying the molecular mechanism involved in the carpogenic germination of scleortia will provide clues to control the sclerotiniose safely.In this study,chitinase family proteins of S.sclerotiorum were analyzed with the bioinformation approach,and the expression levels of chitinase genes during the carpogenic germination of sclerotia were determined with RT-PCR method.Bioinformatics analysis showed that 12 genes were predicted to encode chitinase in S.sclerotiorum.All of them had GH18 domains and some also had a chitin-binding domain and a cellulose-binding domain.All the chitinases were hydrophilic except SS1G_11212.Ten chitinases were predicted to be located outside the cell,and the others were located in the nucleus cytoplasm.Phylogenetic tree analysis showed that the 12 chitinases could be divided into two categories,of which 3with yeast chitinase CTS1 were clustered together and the rest with CTS2 were clustered together.The expression level of the 11 chitinase genes changed during the carpogenic germination of sclerotia,except SS1G_00773,suggesting that chitinase family proteins are involved in the carpogenic germination of sclerotia for S.sclerotiorum.
The carpogenic germination of sclerotia for Sclerotinia sclerotiorumis a key step of crop sclerotiniose infection cycle.Studying the molecular mechanism involved in the carpogenic germination of scleortia will provide clues to control the sclerotiniose safely.In this study,chitinase family proteins of S.sclerotiorum were analyzed with the bioinformation approach,and the expression levels of chitinase genes during the carpogenic germination of sclerotia were determined with RT-PCR method.Bioinformatics analysis showed that 12 genes were predicted to encode chitinase in S.sclerotiorum.All of them had GH18 domains and some also had a chitin-binding domain and a cellulose-binding domain.All the chitinases were hydrophilic except SS1G_11212.Ten chitinases were predicted to be located outside the cell,and the others were located in the nucleus cytoplasm.Phylogenetic tree analysis showed that the 12 chitinases could be divided into two categories,of which 3with yeast chitinase CTS1 were clustered together and the rest with CTS2 were clustered together.The expression level of the 11 chitinase genes changed during the carpogenic germination of sclerotia,except SS1G_00773,suggesting that chitinase family proteins are involved in the carpogenic germination of sclerotia for S.sclerotiorum.
引文
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[2]BOLTON M D,THOMMA B P,NELSON B D.Sclerotinia sclerotiorum(Lib.)de Bary:Biology and Molecular Traits of a Cosmopolitan Pathogen[J].Molecular Plant Pathology,2006,7(1):1-16.
[3]杨新美.油菜菌核病(Sclerotinia sclerotiorum)在我国的寄主范围及生态特性的调查研究[J].植物病理学报,1959,5(2):111-121.
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[7]STEADMAN J R.Control of Plant Diseases Caused by Sclerotinia Species[J].Phytopathology,1979,69(8):904-907.
[8]YOUNG N,ASHFORD A E.Changes During Development in the Permeability of Sclerotia of Sclerotinia Minor to an Apoplastic Tracer[J].Protoplasma,1992,167(3/4):205-214.
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[11]胡燕梅,杨龙,李国庆.重寄生真菌盾壳霉产生几丁质酶的条件优化[J].中国生物防治学报,2010,26(2):167-173.
[12]胡仕凤,高必达,陈捷.木霉几丁质酶及其基因的研究进展[J].中国生物防治学报,2008,24(4):369-375.
[13]汤伟,夏伟,李雅华,等.棘孢木霉几丁质酶基因的克隆与生物信息学分析[J].中国生物化学与分子生物学报,2012,28(4):385-392.
[14]SEIDL V,HUEMER B,SEIBOTH B,et al.A Complete Survey of Trichoderma Chitinases Reveals Three Distinct Subgroups of Family 18Chitinases[J].FEBS Journal,2010,272(22):5923-5939.
[15]林毅,彭锟,关雄.苏云金杆菌entomocidus亚种几丁质酶基因的克隆与生物信息学分析[J].激光生物学报,2006,15(6):598-601.
[16]高颂,刘立会,刘仕平,等.家蚕GH18家族几丁质酶的系统进化和BmChi的时期表达分析[J].蚕业科学,2012,38(3):418-423.
[17]范晓军,宋志芳,仙笑笑,等.金纹细蛾几丁质酶基因生物信息学分析[J].基因组学与应用生物学,2013,32(1):91-96.
[18]李大琪,杜建中,张建琴,等.东亚飞蝗几丁质酶家族基因的表达特性与功能研究[J].中国农业科学,2011,44(3):485-492.
[19]XU F H,FAN C M,HE Y Q.Chitinases in Oryza sativa ssp.Japonica and Arabidopsis thaliana[J].Acta Genetica Sinica,2007,34(2):138-150.
[20]NICOL S.Life After Death for Empth Shells:Crustacean Fisheries Create a Mountain of Waste Shells,Made of a Strong Natural Polymer,Chitin.Now Chemists Are Helping to Put This Waste to Some Surprising Uses[J].New Scientist,1991,129(1):46-48.
[21]KURANDA M J,ROBBINS P W.Chitinase is Required for Cell Separation During Growth of Saccharomyces cerevisiae[J].Journal of Biological Chemistry,1991,266(29):19758-19767.
[22]LI D C.Review of Fungal Chitinases[J].Mycopathologia,2006,161(6):345-360.
[23]SEIDL V.Chitinases of Filamentous Fungi:a Large Group of Diverse Proteins with Multiple Physiological Functions[J].Fungal Biology Reviews,2008,22(1):36-42.
[24]YANAI K,TAKAYA N,KOJIMA N,et al.Purification of Two Chitinases from Rhizopus oligosporus and Isolation and Sequencing of the Encoding Genes[J].Journal of Bacteriology,1992,174(22):7398-7406.
[25]韩艺娟,林成增,王琴秋,等.稻瘟病菌假定几丁质酶家族基因的表达特点[J].热带作物学报,2013,34(8):1544-1551.
[26]RAMOT O,VITERBO A,FRIESEM D,et al.Regulation of Two Homodimer Hexosaminidases in Themycoparasitic Fungus Trichoderma asperellum by Glucosamine[J].Current Genetics,2004,45(4):205-213.
[27]TAKAYA N,YAMAZAKI D,HORIUCHI H,et al.Cloning and Characterization of a Chitinase-Encoding Gene(chiA)from Aspergillus Nidulans,Disruption of Which Decreases Germination Frequency and Hyphal Growth[J].Journal of the Agricultural Chemical Society of Japan,1998,62(1):60-65.
[28]YAMAZAKI H,YAMAZAKI D,TAKAYA N,et al.A Chitinase Gene,chiB,Involved in the Autolytic Process of Aspergillus nidulans[J].Current Genetics,2007,51(2):89-98.
[29]李培.通过RNA干扰方法研究稻瘟病菌几丁质酶家族基因功能[D].福州:福建农林大学,2014.
[30]DUNKLER A,JORDE S,WENDLAND J.An Ashbya Gossypii cts2Mutant Deficient in a Sporulation-Specific Chitinase Can be Complemented by Candida Albicans CHT4.Microbiological Research[J].2008,163(6):701-710.
[31]LIMON M C,MARGOLLES-CLARK E,BENTEZ T,et al.Addition of Substrate-Binding Domains Increases Substrate-Binding Capacity and Specific Activity of a Chitinase from Trichoderma harzianum[J].Fems Microbiology Letters,2001,198(1):57-63.
[32]KING L,BUTLER G.Ace2p,a Regulator of CTS1(Chitinase)Expression,Affects Pseudohyphal Production in Saccharomyces cerevisiae[J].Current Genetics,1998,34(3):183-191.
[33]PISHKO E J,KIRKLAND T N,COLE G T.Isolation and Characterization of Two Chitinase-Encoding Genes(ctsl,cts2)from the Fungus Coccidioides immitis[J].Gene,1995,167(1/2):173-177.
[2]BOLTON M D,THOMMA B P,NELSON B D.Sclerotinia sclerotiorum(Lib.)de Bary:Biology and Molecular Traits of a Cosmopolitan Pathogen[J].Molecular Plant Pathology,2006,7(1):1-16.
[3]杨新美.油菜菌核病(Sclerotinia sclerotiorum)在我国的寄主范围及生态特性的调查研究[J].植物病理学报,1959,5(2):111-121.
[4]CLARKSON J P,STAVELEY J,PHELPS K,et al.Ascospore Release and Survival in Sclerotinia sclerotiorum[J].Mycological Research,2003,107(2):213-222.
[5]SCHWARTZ H F,STEADMAN J R.Factors Affecting Sclerotium Populations of,and Apothecium Production by Sclerotinia sclerotiorum[J].Phytopathology,1978,68(3):383-388.
[6]ABAWI G S,GROGAN R G.Epidemiology of Diseases Caused by Sclerotinia Species[J].Phytopathology,1979,69(8):899-903.
[7]STEADMAN J R.Control of Plant Diseases Caused by Sclerotinia Species[J].Phytopathology,1979,69(8):904-907.
[8]YOUNG N,ASHFORD A E.Changes During Development in the Permeability of Sclerotia of Sclerotinia Minor to an Apoplastic Tracer[J].Protoplasma,1992,167(3/4):205-214.
[9]WILLETTS HJ,BULLOCK S.Developmental Biology of Sclerotia[J].Mycological Research,1992,96(10):801-816.
[10]WERR T C,SARR L W,CHEN M L.An Antifungal Chitinase Produced by Bacillus subtilis Using Chitin Waste as a Carbon Source[J].World Journal of Microbiol and Biotechnology,2010,26(5):945-950.
[11]胡燕梅,杨龙,李国庆.重寄生真菌盾壳霉产生几丁质酶的条件优化[J].中国生物防治学报,2010,26(2):167-173.
[12]胡仕凤,高必达,陈捷.木霉几丁质酶及其基因的研究进展[J].中国生物防治学报,2008,24(4):369-375.
[13]汤伟,夏伟,李雅华,等.棘孢木霉几丁质酶基因的克隆与生物信息学分析[J].中国生物化学与分子生物学报,2012,28(4):385-392.
[14]SEIDL V,HUEMER B,SEIBOTH B,et al.A Complete Survey of Trichoderma Chitinases Reveals Three Distinct Subgroups of Family 18Chitinases[J].FEBS Journal,2010,272(22):5923-5939.
[15]林毅,彭锟,关雄.苏云金杆菌entomocidus亚种几丁质酶基因的克隆与生物信息学分析[J].激光生物学报,2006,15(6):598-601.
[16]高颂,刘立会,刘仕平,等.家蚕GH18家族几丁质酶的系统进化和BmChi的时期表达分析[J].蚕业科学,2012,38(3):418-423.
[17]范晓军,宋志芳,仙笑笑,等.金纹细蛾几丁质酶基因生物信息学分析[J].基因组学与应用生物学,2013,32(1):91-96.
[18]李大琪,杜建中,张建琴,等.东亚飞蝗几丁质酶家族基因的表达特性与功能研究[J].中国农业科学,2011,44(3):485-492.
[19]XU F H,FAN C M,HE Y Q.Chitinases in Oryza sativa ssp.Japonica and Arabidopsis thaliana[J].Acta Genetica Sinica,2007,34(2):138-150.
[20]NICOL S.Life After Death for Empth Shells:Crustacean Fisheries Create a Mountain of Waste Shells,Made of a Strong Natural Polymer,Chitin.Now Chemists Are Helping to Put This Waste to Some Surprising Uses[J].New Scientist,1991,129(1):46-48.
[21]KURANDA M J,ROBBINS P W.Chitinase is Required for Cell Separation During Growth of Saccharomyces cerevisiae[J].Journal of Biological Chemistry,1991,266(29):19758-19767.
[22]LI D C.Review of Fungal Chitinases[J].Mycopathologia,2006,161(6):345-360.
[23]SEIDL V.Chitinases of Filamentous Fungi:a Large Group of Diverse Proteins with Multiple Physiological Functions[J].Fungal Biology Reviews,2008,22(1):36-42.
[24]YANAI K,TAKAYA N,KOJIMA N,et al.Purification of Two Chitinases from Rhizopus oligosporus and Isolation and Sequencing of the Encoding Genes[J].Journal of Bacteriology,1992,174(22):7398-7406.
[25]韩艺娟,林成增,王琴秋,等.稻瘟病菌假定几丁质酶家族基因的表达特点[J].热带作物学报,2013,34(8):1544-1551.
[26]RAMOT O,VITERBO A,FRIESEM D,et al.Regulation of Two Homodimer Hexosaminidases in Themycoparasitic Fungus Trichoderma asperellum by Glucosamine[J].Current Genetics,2004,45(4):205-213.
[27]TAKAYA N,YAMAZAKI D,HORIUCHI H,et al.Cloning and Characterization of a Chitinase-Encoding Gene(chiA)from Aspergillus Nidulans,Disruption of Which Decreases Germination Frequency and Hyphal Growth[J].Journal of the Agricultural Chemical Society of Japan,1998,62(1):60-65.
[28]YAMAZAKI H,YAMAZAKI D,TAKAYA N,et al.A Chitinase Gene,chiB,Involved in the Autolytic Process of Aspergillus nidulans[J].Current Genetics,2007,51(2):89-98.
[29]李培.通过RNA干扰方法研究稻瘟病菌几丁质酶家族基因功能[D].福州:福建农林大学,2014.
[30]DUNKLER A,JORDE S,WENDLAND J.An Ashbya Gossypii cts2Mutant Deficient in a Sporulation-Specific Chitinase Can be Complemented by Candida Albicans CHT4.Microbiological Research[J].2008,163(6):701-710.
[31]LIMON M C,MARGOLLES-CLARK E,BENTEZ T,et al.Addition of Substrate-Binding Domains Increases Substrate-Binding Capacity and Specific Activity of a Chitinase from Trichoderma harzianum[J].Fems Microbiology Letters,2001,198(1):57-63.
[32]KING L,BUTLER G.Ace2p,a Regulator of CTS1(Chitinase)Expression,Affects Pseudohyphal Production in Saccharomyces cerevisiae[J].Current Genetics,1998,34(3):183-191.
[33]PISHKO E J,KIRKLAND T N,COLE G T.Isolation and Characterization of Two Chitinase-Encoding Genes(ctsl,cts2)from the Fungus Coccidioides immitis[J].Gene,1995,167(1/2):173-177.