向日葵锈菌弹性金属蛋白酶基因克隆及生物信息学分析
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摘要
为了研究向日葵锈病的致病机制,将向日葵锈菌转录组中表达量较高的一个基因(KU994904)中成熟部分进行生物信息学分析及基因克隆。利用Interpro对蛋白ORF区域进行分析,确定其成熟区域,通过生物信息学相关软件对其进行结构预测及预测分析,从向日葵锈菌中提取总RNA,并利用RT-PCR方法扩增此蛋白酶成熟基因并克隆。结果显示:该蛋白酶成熟基因序列全长1 335 bp,编码445个氨基酸,分子质量49.5 ku,其与M36家族中5条序列具有较高相似性,并发现同胞外金属蛋白酶基因序列相似度达到53.77%。因此,推断其为弹性金属蛋白酶。
In order to study the pathogenic mechanism of Puccinia helianthi Schw, bioinformatics method was used analyze the mature part of KU994904 gene that had a high expression level in the transcriptome of Puccinia helianthi Schw. The protein ORF region was analyzed by Interpro which can determine its mature region. Using the bioinformatical databases and softwares, its function was analyzed and predicted. Total RNA was extracted from Puccinia helianthi Schw and used to amplify the mature gene of the metalloproteinase by RT-PCR. Finally, the gene sequence of mature protein was cloned. The results showed that the sequence full-length of the gene was 1 335 bp, coding 445 amino acids. And the protein molecular weight was 49.5 ku, it was found that the gene had high similarity with five sequences of M36 family and the gene had 53.77% similarity with the extracellular metalloproteinase(mep). So it was deduced that the gene coded an elastinolytic metalloproteinase.
In order to study the pathogenic mechanism of Puccinia helianthi Schw, bioinformatics method was used analyze the mature part of KU994904 gene that had a high expression level in the transcriptome of Puccinia helianthi Schw. The protein ORF region was analyzed by Interpro which can determine its mature region. Using the bioinformatical databases and softwares, its function was analyzed and predicted. Total RNA was extracted from Puccinia helianthi Schw and used to amplify the mature gene of the metalloproteinase by RT-PCR. Finally, the gene sequence of mature protein was cloned. The results showed that the sequence full-length of the gene was 1 335 bp, coding 445 amino acids. And the protein molecular weight was 49.5 ku, it was found that the gene had high similarity with five sequences of M36 family and the gene had 53.77% similarity with the extracellular metalloproteinase(mep). So it was deduced that the gene coded an elastinolytic metalloproteinase.
引文
[1] 林晓红.向日葵NBS型抗锈病相关基因的克隆与分析[D].呼和浩特:内蒙古农业大学,2014.Lin X H.Molecular cloning and characterrization of NBS orthologues of rust resistance gene from sunflower[D].Hohhot:Inner Mongolia Agricultural University,2014.
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[3] 王丽芳.向日葵抗锈病生理机制及超微结构的研究[D].呼和浩特:内蒙古农业大学,2008.Wang L F.Physiology mechanism and the ultrastructure studies on the resistance to sunflower rust[D].Hohhot:Inner Mongolia Agricultural University,2008.
[4] Kolattukudy P E.Biopolyester membranes of plants:cutin and suberin[J].Science,1980,208(4447):990-1000.doi:10.1126/science.208.4447.990.
[5] Kolattukudy P E.Enzymatic penetration of the plant cuticle by fungal pathogens[J].Annu Rev Phytopathol,1985,23(4):223-250.doi:10.1146/annurev.py.23.090185.001255.
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[7] Starcher B C.Elastin and the lung[J].Thorax,1986,41(8):577-585.doi:10.1136/thx.41.8.577.
[8] Markaryan A,Morozova I,Yu H,Kolattukudy P E.Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung[J].Infect and Immun,1994,62(6):2149-2157.
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[12] Monod M,Togni G,Rahalison L,Fren K E.Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus[J].Journal of Medical Microbiology,1991,35(1):23-28.doi:10.1099/00222615-35-1-23.
[13] Ramesh M V,Sirakova T,Kolattukudy P E.Isolation,characterization,and cloning of cDNA and the gene for anelastinolytic serine protease from Aspergillus flavus[J].Infect and Immun,1994,62(1):79-85.doi:10.1007/BF01793577.
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[15] 郭丹丹.向日葵锈菌分泌蛋白预测及内参基因筛选[D].呼和浩特:内蒙古农业大学,2016.Guo D D.Secretory protein prediction and reference gene screening of Puccinia helianthi[D].Hohhot:Inner Mongolia Agricultural University,2016.
[16] 刘信丹,张英,吴孟华,曹晖.广藿香醇合成酶基因Pc-PTS1的克隆和生物信息学分析[J].中药材,2018,49(2):302-308.doi:10.13863/j.issn1001-4454.2018.02.010.Liu X D,Zhang Y,Wu M H,Cao H.Cloning and bioinformatics analysis of patchoulol synthase gene Pc-PTS1 from Pogostemon cablin[J].Journal of Chinese Medicinal Materials,2018,49(2):302-308.
[17] 张胜博,刘景辉,李立军,赵宝平.燕麦AsNAC1耐盐基因的克隆及初步验证[J].北方农业学报,2016,44(5):1-7.doi:10.3969/j.issn.2096-1197.2016.05.01.Zhang S B,Liu J H,Li L J,Zhao B P.Cloning and preliminary validation of the AsNAC1 gene for salt tolerance from oat[J].Journal of Northern Agriculture,2016,44(5):1-7.
[18] 张美祥.卵菌胞内效应子研究进展[J].南京农业大学学报,2018,41(1):18-25.doi:10.7685/jnau.201706100.Zhang M Y.Recent research progress on oomycete cytoplasmic effectors[J].Journal of Nanjing Agricultural University,2018,41(1):18-25.
[19] Kolattukudy P E,Sirakova T D.233 Fungalysin[J].Handbook of Proteolytic Enzymes,2004(1):792-793.doi:10.1016/C2009-1-60990-4.
[20] Ueda M,Yamaoto N,Kusuda M,Nakazawa M,Takenaka S,Miyatake K,Ouchi K,Sakaguchi M,Inouye K.Purification and characterization of a new fungalysin-like metallopeptidase from the culture filtrate of a plant worm,Nomuraea atypicola[J].Process Biochemistry,2013,48(1):190-194.doi:10.1016/j.procbio.2012.11.005.
[21] Kilaru S,Hoegger P J,Majcherczyk A.Expreesion of laccase gene Iccl in Coprinopsis cinerea under control of various basidiomycetous promoters[J].Appl Microbiol Biotechnol,2006,71(2):200-210.doi:10.1007/s00253-005-0128-1.
[22] Kim J S,Kim J E,Choi S E,Kumar P,Kim S S,Lee H H,Kim C S,Yeal,Kim M K,Kim Y S,Kim S J.Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia[J].Mycol Res,2008,112(8):990-998.doi:10.1016/j.mycres.2008.01.029.
[23] Ahn J H,Pan J G,Rhee J S.Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1[J].Journal of Bacteriology,1999,181(6):1847-1852.doi:10.1134/S0869593807040016.
[24] Dufour D,Nicodeme M,Perrin C,Driou A,Brusseaux E,Humbert G,Gaillard J,Dary A.Molecular typing of industrial strains of Pseudomonas spp.isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them[J].International Journal of Food Microbiology,2008,125(2):188-196.doi:10.1016/j.ijfoodmicro.2008.04.004.
[25] Zhang W W,Hu Y H,Wang H L,Sun L.Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain[J].Veterinary Microbiology,2009,139(1-2):183-188.doi:10.1016/j.vetmic.2009.04.026.
[26] Hase C C,Finkelstein R A.Bacterial extracellular zinc-containing metalloproteases[J].Microbiol Rev,1993,57(4):823-837.doi:10.1006/mpat.1993.1096.
[27] Wu J W,Chen X L.Extracellular metalloproteases from bacteria[J].Appl Microbiol Biotechnol,2011,92(2):253-262.doi:10.1007/s00253-011-3532-8.
[28] Baumann U.Crystal structure of the 50kDa metallo protease from Serratia marcescens[J].Journal of Molecular Biology,1994,242(3):244-251.doi:10.1006/jmbi.1994.1576.
[29] Ishii K,Adachi T,Hamamoto H,Sekimizu K.Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells[J].Journal of Biological Chemistry,2014,289(9):5876-5888.doi:10.1074/jbc.M113.544536.
[30] Chen C M,Liu J J,Chou C W,Lai C H,Wu L T.RpA,an extracellular protease similar to the metalloprotease of serralysin family,is required for pathogenicity of Ralstonia pickettii[J].Journal of Applied Microbiology,2015,119(4):1101-1111.doi:10.1111/jam.12903.
[31] Kalisz H M,Wood D A,Moore D.Production,regulation and release of extracellular proteinase activity in basidiomycete fungi[J].Transactions of the British Mycological Society,1987,88:221-227.doi:10.1016/s0007-1536(87)80218-8.
[32] 王佳,游松,周丽娜.倍半萜生物转化的研究进展[J].沈阳药科大学学报,2012,29(2):156-164.doi:10.14066/j.cnki.cn21-1349/r.2012.02.006.Wang J,You S,Zhou L N.Advances in research on sesquiterpene biotransformation[J].Journal of Shenyang Pharmaceutical University,2012,29(2):156-164.
[2] 曹娜.向日葵锈病初步研究[D].长春:吉林农业大学,2011.Cao N.Preliminary studies on sunflower Rust[D].Changchun:Jilin Agricultural University,2011.
[3] 王丽芳.向日葵抗锈病生理机制及超微结构的研究[D].呼和浩特:内蒙古农业大学,2008.Wang L F.Physiology mechanism and the ultrastructure studies on the resistance to sunflower rust[D].Hohhot:Inner Mongolia Agricultural University,2008.
[4] Kolattukudy P E.Biopolyester membranes of plants:cutin and suberin[J].Science,1980,208(4447):990-1000.doi:10.1126/science.208.4447.990.
[5] Kolattukudy P E.Enzymatic penetration of the plant cuticle by fungal pathogens[J].Annu Rev Phytopathol,1985,23(4):223-250.doi:10.1146/annurev.py.23.090185.001255.
[6] Kolattukudy P E,Lee J D,Rogers L M,Zimmerman P,Ceselski S,Fox B,Stein B,Copelan E A.Evidence for possible involvement of an elastolytic serine protease in aspergillosis[J].Infect and Immun,1993,61(6):2357-2368.doi:10.1016/0928-8244(93)90047-8.
[7] Starcher B C.Elastin and the lung[J].Thorax,1986,41(8):577-585.doi:10.1136/thx.41.8.577.
[8] Markaryan A,Morozova I,Yu H,Kolattukudy P E.Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung[J].Infect and Immun,1994,62(6):2149-2157.
[9] 荆谷,冯静,孔健,马桂荣.微生物金属蛋白酶的研究进展[J].生物工程进展,2002(1):61-63,56.doi:10.13523/j.cb.20020114.Jing G,Feng J,Kong J,Ma G R.Research progress on microbial source metalloproteinases[J].Progress in bioengineering,2002(1):61-63,56.
[10] Sirakova T D,Markaryan A,Kolattukudy P E .Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli[J].American Society for Microbiology,1994,62(10):4208-4218.doi:10.1016/0167-5699(94)90199-6.
[11] Kothary M,Chase T,Macmillan J M.Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice[J].Infect and Immun,1984,43(1):320-325.doi:10.1007/BF01641732.
[12] Monod M,Togni G,Rahalison L,Fren K E.Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus[J].Journal of Medical Microbiology,1991,35(1):23-28.doi:10.1099/00222615-35-1-23.
[13] Ramesh M V,Sirakova T,Kolattukudy P E.Isolation,characterization,and cloning of cDNA and the gene for anelastinolytic serine protease from Aspergillus flavus[J].Infect and Immun,1994,62(1):79-85.doi:10.1007/BF01793577.
[14] Monod M,Paris S,Sanglard D,Jaton-Ogay K,Bille J,Latge J P.Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatusl[J].Journal of Medical Microbiology,Infect and Immun,1993,61(10):4099-4104.doi:10.1099/00222615-35-1-23.
[15] 郭丹丹.向日葵锈菌分泌蛋白预测及内参基因筛选[D].呼和浩特:内蒙古农业大学,2016.Guo D D.Secretory protein prediction and reference gene screening of Puccinia helianthi[D].Hohhot:Inner Mongolia Agricultural University,2016.
[16] 刘信丹,张英,吴孟华,曹晖.广藿香醇合成酶基因Pc-PTS1的克隆和生物信息学分析[J].中药材,2018,49(2):302-308.doi:10.13863/j.issn1001-4454.2018.02.010.Liu X D,Zhang Y,Wu M H,Cao H.Cloning and bioinformatics analysis of patchoulol synthase gene Pc-PTS1 from Pogostemon cablin[J].Journal of Chinese Medicinal Materials,2018,49(2):302-308.
[17] 张胜博,刘景辉,李立军,赵宝平.燕麦AsNAC1耐盐基因的克隆及初步验证[J].北方农业学报,2016,44(5):1-7.doi:10.3969/j.issn.2096-1197.2016.05.01.Zhang S B,Liu J H,Li L J,Zhao B P.Cloning and preliminary validation of the AsNAC1 gene for salt tolerance from oat[J].Journal of Northern Agriculture,2016,44(5):1-7.
[18] 张美祥.卵菌胞内效应子研究进展[J].南京农业大学学报,2018,41(1):18-25.doi:10.7685/jnau.201706100.Zhang M Y.Recent research progress on oomycete cytoplasmic effectors[J].Journal of Nanjing Agricultural University,2018,41(1):18-25.
[19] Kolattukudy P E,Sirakova T D.233 Fungalysin[J].Handbook of Proteolytic Enzymes,2004(1):792-793.doi:10.1016/C2009-1-60990-4.
[20] Ueda M,Yamaoto N,Kusuda M,Nakazawa M,Takenaka S,Miyatake K,Ouchi K,Sakaguchi M,Inouye K.Purification and characterization of a new fungalysin-like metallopeptidase from the culture filtrate of a plant worm,Nomuraea atypicola[J].Process Biochemistry,2013,48(1):190-194.doi:10.1016/j.procbio.2012.11.005.
[21] Kilaru S,Hoegger P J,Majcherczyk A.Expreesion of laccase gene Iccl in Coprinopsis cinerea under control of various basidiomycetous promoters[J].Appl Microbiol Biotechnol,2006,71(2):200-210.doi:10.1007/s00253-005-0128-1.
[22] Kim J S,Kim J E,Choi S E,Kumar P,Kim S S,Lee H H,Kim C S,Yeal,Kim M K,Kim Y S,Kim S J.Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia[J].Mycol Res,2008,112(8):990-998.doi:10.1016/j.mycres.2008.01.029.
[23] Ahn J H,Pan J G,Rhee J S.Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1[J].Journal of Bacteriology,1999,181(6):1847-1852.doi:10.1134/S0869593807040016.
[24] Dufour D,Nicodeme M,Perrin C,Driou A,Brusseaux E,Humbert G,Gaillard J,Dary A.Molecular typing of industrial strains of Pseudomonas spp.isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them[J].International Journal of Food Microbiology,2008,125(2):188-196.doi:10.1016/j.ijfoodmicro.2008.04.004.
[25] Zhang W W,Hu Y H,Wang H L,Sun L.Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain[J].Veterinary Microbiology,2009,139(1-2):183-188.doi:10.1016/j.vetmic.2009.04.026.
[26] Hase C C,Finkelstein R A.Bacterial extracellular zinc-containing metalloproteases[J].Microbiol Rev,1993,57(4):823-837.doi:10.1006/mpat.1993.1096.
[27] Wu J W,Chen X L.Extracellular metalloproteases from bacteria[J].Appl Microbiol Biotechnol,2011,92(2):253-262.doi:10.1007/s00253-011-3532-8.
[28] Baumann U.Crystal structure of the 50kDa metallo protease from Serratia marcescens[J].Journal of Molecular Biology,1994,242(3):244-251.doi:10.1006/jmbi.1994.1576.
[29] Ishii K,Adachi T,Hamamoto H,Sekimizu K.Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells[J].Journal of Biological Chemistry,2014,289(9):5876-5888.doi:10.1074/jbc.M113.544536.
[30] Chen C M,Liu J J,Chou C W,Lai C H,Wu L T.RpA,an extracellular protease similar to the metalloprotease of serralysin family,is required for pathogenicity of Ralstonia pickettii[J].Journal of Applied Microbiology,2015,119(4):1101-1111.doi:10.1111/jam.12903.
[31] Kalisz H M,Wood D A,Moore D.Production,regulation and release of extracellular proteinase activity in basidiomycete fungi[J].Transactions of the British Mycological Society,1987,88:221-227.doi:10.1016/s0007-1536(87)80218-8.
[32] 王佳,游松,周丽娜.倍半萜生物转化的研究进展[J].沈阳药科大学学报,2012,29(2):156-164.doi:10.14066/j.cnki.cn21-1349/r.2012.02.006.Wang J,You S,Zhou L N.Advances in research on sesquiterpene biotransformation[J].Journal of Shenyang Pharmaceutical University,2012,29(2):156-164.