棘孢木霉天冬氨酸蛋白酶家族基因特性研究
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摘要
本研究旨对棘孢木霉基因组20个天冬氨酸蛋白酶家族基因(Asp)特性进行分析。分别进行了保守区及家族预测、氨基酸多序列比较及功能区预测、进化关系分析及其在不同培养条件下棘孢木霉中的差异表达分析。20个Asp均属Asp蛋白酶家族(PF00026),均为酸性(p I<7)。有18个Asps含有信号肽。多序列比对发现Asps含有2个特征性催化指纹"DTGS/A"和"DT/SGS/Y/T/A/N",根据第二个"DTGT"、"DSGT"、"DTGA/N"和"DSGY/T/S"特征性催化指纹,将20个Asps分为4个分支。进一步在C-hungry胁迫下,10个Asps基因上调,Asp54.2上调最高为3222。在N-hungry胁迫下,8个Asps基因上调,Asp50.9上调最高为33100。山新杨(Tas-Pd Pap)叶粉诱导后,13个Asps基因上调,Asp50.9上调最高为21078。说明棘孢木霉促杨树生长及与病原菌拮抗过程中,Asp起了非常重要的作用。
The study aims at analyzing the characteristics of 20 aspartic protease family genes(ASP) in Trichoderma asperellum genome. Their conserved domains and family, the amino acid sequences alignment,functional domains are predicted, and the evolutionary relationship and the differential expression of Asp genesof T. asperellum under different culture conditions are analyzed. All 20 ASPs belong to the Asp family(PF00026) and are all acidic protease(p I<7). 18 of 20 Asps have signal peptides. The alignment analysis findsthat they contain two special catalytic characteristic fingerprints"DTGS/A"and"DT/SGS/Y/T/A/N"and the20 Asps are divided into 4 branches according to the second catalytic fingerprints"DTGT","DSGT","DTGA/N", and"DSGY/T/S"respectively. Further, under C-hungry stress, the expression of 10 Asps is up-regulated,and the Asp54.2 is up-regulated to the highest value of 3222. Under N-hungry stress, the expression of 8 Aspsare up-regulated, particularly the Asp50.9 is up-regulated to the highest value of 33100. After induced withleaf powder of Poplar(Tas + Pd Pap), the expression of 13 Asp genes of T. asprellum are up-regulated,especially the Asp50.9 is up-regulated to the highest value of 21078. It indicates that the aspartic proteasegenes play important roles in the process of T. asperellum promoting poplar growth and interacting with the pathogen.
The study aims at analyzing the characteristics of 20 aspartic protease family genes(ASP) in Trichoderma asperellum genome. Their conserved domains and family, the amino acid sequences alignment,functional domains are predicted, and the evolutionary relationship and the differential expression of Asp genesof T. asperellum under different culture conditions are analyzed. All 20 ASPs belong to the Asp family(PF00026) and are all acidic protease(p I<7). 18 of 20 Asps have signal peptides. The alignment analysis findsthat they contain two special catalytic characteristic fingerprints"DTGS/A"and"DT/SGS/Y/T/A/N"and the20 Asps are divided into 4 branches according to the second catalytic fingerprints"DTGT","DSGT","DTGA/N", and"DSGY/T/S"respectively. Further, under C-hungry stress, the expression of 10 Asps is up-regulated,and the Asp54.2 is up-regulated to the highest value of 3222. Under N-hungry stress, the expression of 8 Aspsare up-regulated, particularly the Asp50.9 is up-regulated to the highest value of 33100. After induced withleaf powder of Poplar(Tas + Pd Pap), the expression of 13 Asp genes of T. asprellum are up-regulated,especially the Asp50.9 is up-regulated to the highest value of 21078. It indicates that the aspartic proteasegenes play important roles in the process of T. asperellum promoting poplar growth and interacting with the pathogen.
引文
[1]Benítez T,Rincón A M,Limón M C,et al.Biocontrol mechanisms of Trichoderma strains[J].International Microbiology the Official Journal of the Spanish Society for Microbiology,2004,7(4):249-260.
[2]Yang X,Cong H,Song J,et al.Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris[J].World J Microbiol Biotechnol.,2013,29(11):2087-2094.
[3]Geremia R A,Goldman G H,Jacobs D,et al.Molecular characterization of the proteinase-encoding gene,prb1,related to mycoparasitism by Trichoderma harzianum[J].Mol Microbiol.,1993,8(3):603-613.
[4]Flores A,Chet I,Herrera-Estrella A.Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinaseencoding gene prb1[J].Curr Genet.,1997,31(1):30-37.
[5]Suarez M B,Vizcaino J A,Llobell A,et al.Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the Tricho EST functional genomics approach[J].Curr Genet.,2007,51(5):331-342.
[6]Seidl V,Song L,Lindquist E,et al.Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey[J].BMC Genomics,2009,10(1):567.
[7]Rubio V,González V,Angeles Portal M D L,et al.Biological control of damping-off on pine(Pinus spp.)with a new fungal species,Ceratobasidium albasitensis isolated in Albacete(Spain).[J].IOBC/WPRS Bull.,2001,24(3).
[8]De Marco J L,Felix C R.Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches'broom disease[J].BMC Biochem.,2002,3(1):3.
[9]Kapat A,Zimand G,Elad Y.Effect of two isolates of Trichoderma harzianumon the activity of hydrolytic enzymes produced by Botrytis cinerea[J].Physiological&Molecular Plant Pathology,1998,52(2):127-137.
[10]Elad Y,Kapat A.The Role of Trichoderma harzianum Protease in the Biocontrol of Botrytis cinerea[J].European Journal of Plant Pathology,1999,105(2):177-189.
[11]Viterbo A,Harel M,Chet I.Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots[J].Fems Microbiology Letters.2004,238(1):151-158.
[12]Qualhato T F,Lopes F A,Steindorff A S,et al.Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi:evaluation of antagonism and hydrolytic enzyme production.[J].Biotechnology Lettersm,2013,35(9):1461-1468.
[13]Tchameni S N,Mel N,Bad B,et al.Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease[J].Crop Protection,2011,30(10):1321-1327.
[14]Kakvan N,Heydari A,Zamanizadeh H R,et al.Development of new bioformulations using Trichoderma and Talaromyces fungal antagonists for biological control of sugar beet damping-off disease[J].Crop Protection.2013,53(Nov.):80-84.
[15]Wijesinghe C J,Rsw W,Jkrr S,et al.Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by(Thielaviopsis paradoxa)[J].Crop Protection.2011,30(3):300-306.
[16]Huang Y,Mijiti G,Wang Z,et al.Functional analysis of the class II hydrophobin gene HFB2-6 from the biocontrol agent Trichoderma asperellum ACCC30536.[J].Microbiological Research,2015,171:8.
[17]刘文萍,韩玉琴,南相日,等.山新杨组织培养快繁技术研究[J].中国农学通报,2005,21(2):101-102.
[18]Druzhinina I S,Seidl-Seiboth V,Herrera-Estrella A,et al.Trichoderma:the genomics of opportunistic success.Nat Rev Microbiol 9:749-759[J].Nature Reviews Microbiology.2011,9(10):749-759.
[19]Dou K,Wang Z,Zhang R,et al.Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536[J].Microbiological Research,2014,169(12):915-923.
[20]Oh S,Chang W,Suh J.An aspartic protease analogue:intermolecular catalysis of peptide hydrolysis by carboxyl groups.[J].Bioorganic&Medicinal Chemistry Letters,2001,1(11):1469.
[21]Delgado-Jarana J,Rincon A M,Benitez T.Aspartyl protease from Trichoderma harzianum CECT 2413:cloning and characterization[J].Microbiology,2002,148(Pt 5):1305-1315.
[22]Suarez M B,Sanz L,Chamorro M I,et al.Proteomic analysis of secreted proteins from Trichoderma harzianum.Identification of a fungal cell wall-induced aspartic protease[J].Fungal Genet Biol.2005,42(11):924-934.
[23]陈德强.木霉防治香蕉镰刀菌枯萎病研究[D].海口:海南大学,2012.
[24]吕晨.哈茨木霉Rac基因的过表达及其功能初步分析[D].哈尔滨:哈尔滨工业大学,2013.
[2]Yang X,Cong H,Song J,et al.Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris[J].World J Microbiol Biotechnol.,2013,29(11):2087-2094.
[3]Geremia R A,Goldman G H,Jacobs D,et al.Molecular characterization of the proteinase-encoding gene,prb1,related to mycoparasitism by Trichoderma harzianum[J].Mol Microbiol.,1993,8(3):603-613.
[4]Flores A,Chet I,Herrera-Estrella A.Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinaseencoding gene prb1[J].Curr Genet.,1997,31(1):30-37.
[5]Suarez M B,Vizcaino J A,Llobell A,et al.Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the Tricho EST functional genomics approach[J].Curr Genet.,2007,51(5):331-342.
[6]Seidl V,Song L,Lindquist E,et al.Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey[J].BMC Genomics,2009,10(1):567.
[7]Rubio V,González V,Angeles Portal M D L,et al.Biological control of damping-off on pine(Pinus spp.)with a new fungal species,Ceratobasidium albasitensis isolated in Albacete(Spain).[J].IOBC/WPRS Bull.,2001,24(3).
[8]De Marco J L,Felix C R.Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches'broom disease[J].BMC Biochem.,2002,3(1):3.
[9]Kapat A,Zimand G,Elad Y.Effect of two isolates of Trichoderma harzianumon the activity of hydrolytic enzymes produced by Botrytis cinerea[J].Physiological&Molecular Plant Pathology,1998,52(2):127-137.
[10]Elad Y,Kapat A.The Role of Trichoderma harzianum Protease in the Biocontrol of Botrytis cinerea[J].European Journal of Plant Pathology,1999,105(2):177-189.
[11]Viterbo A,Harel M,Chet I.Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots[J].Fems Microbiology Letters.2004,238(1):151-158.
[12]Qualhato T F,Lopes F A,Steindorff A S,et al.Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi:evaluation of antagonism and hydrolytic enzyme production.[J].Biotechnology Lettersm,2013,35(9):1461-1468.
[13]Tchameni S N,Mel N,Bad B,et al.Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease[J].Crop Protection,2011,30(10):1321-1327.
[14]Kakvan N,Heydari A,Zamanizadeh H R,et al.Development of new bioformulations using Trichoderma and Talaromyces fungal antagonists for biological control of sugar beet damping-off disease[J].Crop Protection.2013,53(Nov.):80-84.
[15]Wijesinghe C J,Rsw W,Jkrr S,et al.Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by(Thielaviopsis paradoxa)[J].Crop Protection.2011,30(3):300-306.
[16]Huang Y,Mijiti G,Wang Z,et al.Functional analysis of the class II hydrophobin gene HFB2-6 from the biocontrol agent Trichoderma asperellum ACCC30536.[J].Microbiological Research,2015,171:8.
[17]刘文萍,韩玉琴,南相日,等.山新杨组织培养快繁技术研究[J].中国农学通报,2005,21(2):101-102.
[18]Druzhinina I S,Seidl-Seiboth V,Herrera-Estrella A,et al.Trichoderma:the genomics of opportunistic success.Nat Rev Microbiol 9:749-759[J].Nature Reviews Microbiology.2011,9(10):749-759.
[19]Dou K,Wang Z,Zhang R,et al.Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536[J].Microbiological Research,2014,169(12):915-923.
[20]Oh S,Chang W,Suh J.An aspartic protease analogue:intermolecular catalysis of peptide hydrolysis by carboxyl groups.[J].Bioorganic&Medicinal Chemistry Letters,2001,1(11):1469.
[21]Delgado-Jarana J,Rincon A M,Benitez T.Aspartyl protease from Trichoderma harzianum CECT 2413:cloning and characterization[J].Microbiology,2002,148(Pt 5):1305-1315.
[22]Suarez M B,Sanz L,Chamorro M I,et al.Proteomic analysis of secreted proteins from Trichoderma harzianum.Identification of a fungal cell wall-induced aspartic protease[J].Fungal Genet Biol.2005,42(11):924-934.
[23]陈德强.木霉防治香蕉镰刀菌枯萎病研究[D].海口:海南大学,2012.
[24]吕晨.哈茨木霉Rac基因的过表达及其功能初步分析[D].哈尔滨:哈尔滨工业大学,2013.