寄生于南方根结线虫卵的长梗木霉几丁质酶基因TlChi46的克隆
|
摘要
为进一步筛选高效寄生线虫真菌和阐明其寄生线虫卵的机理,本研究从湖北省烟草南方根结线虫雌虫分离到1株具高效生防潜力的菌株HBF1。形态学、rDNA-ITS和翻译延长因子tef1-α序列分析鉴定该菌为长梗木霉菌Trichoderma longibrachiatum,其对南方根结线虫卵第10 d寄生率为80.45%。通过简并引物设计和RACE技术克隆其几丁质酶基因,分析该基因序列及与其他几丁质酶的同源性。该菌是1株可以产生几丁质酶的南方根结线虫卵寄生真菌,第10 d几丁质酶的活性达到高峰,为24.88μmol/h/mL。本研究首次从长梗木霉HBF1菌株中克隆到1个几丁质酶基因TlChi46,该基因DNA全长1 793 bp,含3个内含子和1个1 272 bp的开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.23。同源性比对表明和昆虫寄生菌几丁质酶的亲缘关系较远。从长梗木霉HBF1中克隆得到的几丁质酶基因编码的几丁质酶的功能域可供进一步研究,高效产几丁质酶并有效寄生南方根结线虫卵的长梗木霉对南方根结线虫具有良好的生防潜力。
A fungus named HBF1 was isolated from female of M.incognita on tobacco in Hubei province,China and was identified as Trichoderma longibrachiatum based on morphological characteristics and molecular analysis of rDNA-ITS coupled with translation elongation factor 1-alpha.To identify virulent factors and elucidate the parasitic mechanism on M.incognita eggs,the egg parasitism of M.incognita eggs by T.longibrachiatum HBF1 was investigated in vitro.The percentage of parasitized eggs was 80.45%at the 10 th day after inoculation.From this isolate,the novel genomic and cDNA clones encoding chitinase have been cloned using the degenerate PCR primers and RACE techniques.The analysis of the gene and the deduced amino acid sequence along with its phylogeny analysis was by biology software.Results showed that HBF1 was able to penetrate nematode egg and produce chitinase,maximum activity of chitinase was 24.88 μmol/h/mL recorded at the tenth day after inoculation.We have cloned a novel chitinase gene TlChi46 from T.longibrachiatum HBF1 for the first time.The gene is 1 793 bp in length and contains three putative introns.The ORF of TlChi46 is 1 272 bp in size with encoding protein of 423 aa,molecular mass of 45.9 kDa and pI of 5.23.Phylogeny analysis reveals that this chitinase and other reported Trichoderma spp.chitinases are clustered together and are phylogenetically distant from other entomopathogenic fungi.These results demonstrated that the amino acid sequence coded by chitinase gene TlChi46 from T.longibrachiatum HBF1 possesses promising functional domains for the further research,T.longibrachiatum HBF1 with the the strong chitinolytic and egg-parasitic activity has a great biocontrol potential against M.incognita.
A fungus named HBF1 was isolated from female of M.incognita on tobacco in Hubei province,China and was identified as Trichoderma longibrachiatum based on morphological characteristics and molecular analysis of rDNA-ITS coupled with translation elongation factor 1-alpha.To identify virulent factors and elucidate the parasitic mechanism on M.incognita eggs,the egg parasitism of M.incognita eggs by T.longibrachiatum HBF1 was investigated in vitro.The percentage of parasitized eggs was 80.45%at the 10 th day after inoculation.From this isolate,the novel genomic and cDNA clones encoding chitinase have been cloned using the degenerate PCR primers and RACE techniques.The analysis of the gene and the deduced amino acid sequence along with its phylogeny analysis was by biology software.Results showed that HBF1 was able to penetrate nematode egg and produce chitinase,maximum activity of chitinase was 24.88 μmol/h/mL recorded at the tenth day after inoculation.We have cloned a novel chitinase gene TlChi46 from T.longibrachiatum HBF1 for the first time.The gene is 1 793 bp in length and contains three putative introns.The ORF of TlChi46 is 1 272 bp in size with encoding protein of 423 aa,molecular mass of 45.9 kDa and pI of 5.23.Phylogeny analysis reveals that this chitinase and other reported Trichoderma spp.chitinases are clustered together and are phylogenetically distant from other entomopathogenic fungi.These results demonstrated that the amino acid sequence coded by chitinase gene TlChi46 from T.longibrachiatum HBF1 possesses promising functional domains for the further research,T.longibrachiatum HBF1 with the the strong chitinolytic and egg-parasitic activity has a great biocontrol potential against M.incognita.
引文
[1]Rich J R,Dunn R,Noling J.Nematicides:past and present uses[A].Chen S Y,Dickson D W.Nematology advances and perspectivies(vol 2),Nematode management and utilization[M].Oxfordshire:CABI Publishing,2004.1041-1082.
[2]Wharton D.Nematode egg-shells[J].Parasitology,1980,81(2):447-463.
[3]Ubhayasekera W.Structure and function of chitinases from glycoside hydrolase family 19[J].Polymer International,2011,60(6):890-896.
[4]Kim D,Riggs R,Kim K.Ultrastructure of Heterodera glycines parasitized by Arkansas fungus 18[J].Phytopathology,1992,82(4):429-433.
[5]Khan A,Williams K,Molloy M P,et al.Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels[J].Protein Expression and Purification,2003,32(2):210-220.
[6]Lsek H,KrajcíD.Penetration of ovicidal fungus Verticillium chlamydosporium through the Ascaris lumbricoides egg-shells[J].Folia Parasitologica,1987,34(1):57-60.
[7]Tikhonov V E,Lopez-Llorca L V,Salinas J,et al.Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V.suchlasporium[J].Fungal Genetics and Biology,2002,35(1):67-78.
[8]Khan A,Williams K L,Nevalainen H K M.Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles[J].Biological Control,2004,31(3):346-352.
[9]Gan Z,Yang J,Tao N,et al.Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita[J].Applied M icrobiology and Biotechnology,2007,76(6):1309-1317.
[10]Dong L Q,Yang J K,Zhang K Q.Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus[J].Journal of Applied M icrobiology,2007,103(6):2476-2488.
[11]Harman G E.Overview of mechanisms and uses of Trichoderma spp.[J].Phytopathology,2006,96(2):190-194.
[12]Howell C.Mechanisms employed by Trichoderma species in the biological control of plant diseases:the history and evolution of current concepts[J].Plant Disease,2003,87(1):4-10.
[13]Suarez B,Rey M,Castillo P,et al.Isolation and characterization of PRA1,a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413displaying nematicidal activity[J].Applied M icrobiology and Biotechnology,2004,65(1):46-55.
[14]Meyer S L F,Massoud S I,Chitwood D J,et al.Evaluation of Trichoderma virens and Burkholderia cepacia for antagonistic activity against root-knot nematode,Meloidogyne incognita[J].Nematology,2000,2(8):871.
[15]Sharon E,Bar-Eyal M,Chet I,et al.Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum[J].Phytopathology,2001,91(7):687-693.
[16]Sharon E,Chet I,Viterbo A,et al.Parasitism of Trichoderma on Meloidogyne javanica and role of the gelatinous matrix[J].European Journal of Plant Pathology,2007,118(3):247-258.
[17]SzabóM,Csepregi K,Gálber M,et al.Control plantparasitic nematodes w ith Trichoderma species and nematode-trapping fungi:The role of chi18-5 and chi18-12 genes in nematode egg-parasitism[J].Biological Control,2012,63(2):121-128.
[18]Viterbo A,Haran S,Friesem D,et al.Antifungal activity of a novel endochitinase gene(chit36)from Trichoderma harzianum Rifai TM[J].FEM S M icrobiology Letters,2001,200(2):169-174.
[19]Seidl V,Huemer B,Seiboth B,et al.A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases[J].The FEBS Journal,2005,272(22):5923-5939.
[20]Kovacs K,Szakacs G,Pusztahelyi T,et al.Production of chitinolytic enzymes w ith Trichoderma longibrachiatum IM I 92027 in solid substrate fermentation[J].Applied Biochemistry and Biotechnology,2004,118(1-3):189-204.
[21]Imoto T.A simple activity measurement of lysozyme[J].Agricultural and Biological Chemistry,1971,35:1154-1156.
[22]Cantarel B L,Coutinho P M,Rancurel C,et al.The carbohydrate-active enzymes database(CAZy):an expert resource for glycogenomics[J].Nucleic Acids Research,2009,37(suppl.1):D233-D238.
[23]Synstad B,Gseidnes S,Van Aalten D M,et al.M utational and computational analysis of the role of conserved residues in the active site of a family 18chitinase[J].European Journal of Biochemistry,2004,271(2):253-262.
[24]Li H,Greene L H.Sequence and structural analysis of the chitinase insertion domain reveals tw o conserved motifs involved in chitin-binding[J].PLo S One,2010,5(1):e8654.
[25]Orikoshi H,Baba N,Nakayama S,et al.Molecular analysis of the gene encoding a novel cold-adapted chitinase(Chi B)from a marine bacterium,Alteromonas sp.strain O-7[J].Journal of Bacteriology,2003,185(4):1153-1160.
[26]Verma M,Brar S K,Tyagi R D,et al.Antagonistic fungi,Trichoderma spp.:Panoply of biological control[J].Biochemical Engineering Journal,2007,37(1):1-20.
[27]Lin S,Wu X,Cao J Z,et al.Biocontrol potential of chitinase-producing nematophagous fungus Acremonium implicatum against Meloidogyne incognita(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2013,43(5):509-517.
[28]Seidl V.Chitinases of filamentous fungi:a large group of diverse proteins w ith multiple physiological functions[J].Fungal Biology Review s,2008,22(1):36-42.
[29]Terwisscha van Scheltinga A C,Kalk K H,Beintema J J,et al.Crystal structures of hevamine,a plant defence protein w ith chitinase and lysozyme activity,and its complex w ith an inhibitor[J].Structure,1994,2(12):1181-1189.
[30]Ihrmark K,Asmail N,Ubhayasekera W,et al.Comparative molecular evolution of Trichoderma chitinases in response to mycoparasitic interactions[J].Evolutionary Bioinformatics Online,2010,6:1-26.
[2]Wharton D.Nematode egg-shells[J].Parasitology,1980,81(2):447-463.
[3]Ubhayasekera W.Structure and function of chitinases from glycoside hydrolase family 19[J].Polymer International,2011,60(6):890-896.
[4]Kim D,Riggs R,Kim K.Ultrastructure of Heterodera glycines parasitized by Arkansas fungus 18[J].Phytopathology,1992,82(4):429-433.
[5]Khan A,Williams K,Molloy M P,et al.Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels[J].Protein Expression and Purification,2003,32(2):210-220.
[6]Lsek H,KrajcíD.Penetration of ovicidal fungus Verticillium chlamydosporium through the Ascaris lumbricoides egg-shells[J].Folia Parasitologica,1987,34(1):57-60.
[7]Tikhonov V E,Lopez-Llorca L V,Salinas J,et al.Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V.suchlasporium[J].Fungal Genetics and Biology,2002,35(1):67-78.
[8]Khan A,Williams K L,Nevalainen H K M.Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles[J].Biological Control,2004,31(3):346-352.
[9]Gan Z,Yang J,Tao N,et al.Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita[J].Applied M icrobiology and Biotechnology,2007,76(6):1309-1317.
[10]Dong L Q,Yang J K,Zhang K Q.Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus[J].Journal of Applied M icrobiology,2007,103(6):2476-2488.
[11]Harman G E.Overview of mechanisms and uses of Trichoderma spp.[J].Phytopathology,2006,96(2):190-194.
[12]Howell C.Mechanisms employed by Trichoderma species in the biological control of plant diseases:the history and evolution of current concepts[J].Plant Disease,2003,87(1):4-10.
[13]Suarez B,Rey M,Castillo P,et al.Isolation and characterization of PRA1,a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413displaying nematicidal activity[J].Applied M icrobiology and Biotechnology,2004,65(1):46-55.
[14]Meyer S L F,Massoud S I,Chitwood D J,et al.Evaluation of Trichoderma virens and Burkholderia cepacia for antagonistic activity against root-knot nematode,Meloidogyne incognita[J].Nematology,2000,2(8):871.
[15]Sharon E,Bar-Eyal M,Chet I,et al.Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum[J].Phytopathology,2001,91(7):687-693.
[16]Sharon E,Chet I,Viterbo A,et al.Parasitism of Trichoderma on Meloidogyne javanica and role of the gelatinous matrix[J].European Journal of Plant Pathology,2007,118(3):247-258.
[17]SzabóM,Csepregi K,Gálber M,et al.Control plantparasitic nematodes w ith Trichoderma species and nematode-trapping fungi:The role of chi18-5 and chi18-12 genes in nematode egg-parasitism[J].Biological Control,2012,63(2):121-128.
[18]Viterbo A,Haran S,Friesem D,et al.Antifungal activity of a novel endochitinase gene(chit36)from Trichoderma harzianum Rifai TM[J].FEM S M icrobiology Letters,2001,200(2):169-174.
[19]Seidl V,Huemer B,Seiboth B,et al.A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases[J].The FEBS Journal,2005,272(22):5923-5939.
[20]Kovacs K,Szakacs G,Pusztahelyi T,et al.Production of chitinolytic enzymes w ith Trichoderma longibrachiatum IM I 92027 in solid substrate fermentation[J].Applied Biochemistry and Biotechnology,2004,118(1-3):189-204.
[21]Imoto T.A simple activity measurement of lysozyme[J].Agricultural and Biological Chemistry,1971,35:1154-1156.
[22]Cantarel B L,Coutinho P M,Rancurel C,et al.The carbohydrate-active enzymes database(CAZy):an expert resource for glycogenomics[J].Nucleic Acids Research,2009,37(suppl.1):D233-D238.
[23]Synstad B,Gseidnes S,Van Aalten D M,et al.M utational and computational analysis of the role of conserved residues in the active site of a family 18chitinase[J].European Journal of Biochemistry,2004,271(2):253-262.
[24]Li H,Greene L H.Sequence and structural analysis of the chitinase insertion domain reveals tw o conserved motifs involved in chitin-binding[J].PLo S One,2010,5(1):e8654.
[25]Orikoshi H,Baba N,Nakayama S,et al.Molecular analysis of the gene encoding a novel cold-adapted chitinase(Chi B)from a marine bacterium,Alteromonas sp.strain O-7[J].Journal of Bacteriology,2003,185(4):1153-1160.
[26]Verma M,Brar S K,Tyagi R D,et al.Antagonistic fungi,Trichoderma spp.:Panoply of biological control[J].Biochemical Engineering Journal,2007,37(1):1-20.
[27]Lin S,Wu X,Cao J Z,et al.Biocontrol potential of chitinase-producing nematophagous fungus Acremonium implicatum against Meloidogyne incognita(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2013,43(5):509-517.
[28]Seidl V.Chitinases of filamentous fungi:a large group of diverse proteins w ith multiple physiological functions[J].Fungal Biology Review s,2008,22(1):36-42.
[29]Terwisscha van Scheltinga A C,Kalk K H,Beintema J J,et al.Crystal structures of hevamine,a plant defence protein w ith chitinase and lysozyme activity,and its complex w ith an inhibitor[J].Structure,1994,2(12):1181-1189.
[30]Ihrmark K,Asmail N,Ubhayasekera W,et al.Comparative molecular evolution of Trichoderma chitinases in response to mycoparasitic interactions[J].Evolutionary Bioinformatics Online,2010,6:1-26.