华癸根瘤菌urtB基因突变株的构建与共生固氮表型分析
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摘要
【目的】尿素ABC转运体透性酶亚基编码基因urtB可能参与尿素代谢及支链氨基酸转运;本文旨在获得实验证据阐明urtB基因对华癸根瘤菌结瘤和固氮的影响,为深入研究其功能机制提供一定的科学依据。【方法】利用生物信息学分析urtB基因的结构特征及生物学功能,通过荧光定量检测urtB基因在自生和共生条件下的时空表达特征和启动子原位表达技术检测urtB基因组织表达特征,采用插入突变构建urtB突变株,通过植物盆栽并结合添加氮素处理,检测与分析突变体的共生固氮表型变化。【结果】分析表明urtB基因对于氮素转运非常重要,在共生条件下的表达水平比自生培养条件下显著上调表达;在成熟根瘤的固氮区中大量表达;正确构建和筛选获得了根瘤菌urtB突变株;接种urtB突变株与野生型菌株7653R相比较,突变体根瘤发育异常;植株地上部分生物量和根瘤固氮酶活性显著降低;添加氮素可恢复其共生缺陷表型。【结论】华癸中慢生根瘤菌urtB基因可能通过影响根瘤中氮转运或同化,进而在根瘤发育与共生固氮中发挥重要作用。
[Objective] Urea ABC-transporter permease subunit UrtB may be involved in urea metabolism and branched-chain amino acid transport. This study aimed to obtain experimental evidence to clarify the effect of urtB gene on the nodulation and nitrogen fixation of Mesorhizobium huakuii and provide scientific basis for further study of its functional mechanism. [Methods] The structure characteristics and biological functions of UrtB protein were analyzed by bioinformatics, the expression pattern of urtB gene under free-living growth and symbiosis conditions were detected by fluorescent quantitative real-time PCR, and in situ expression of urtB gene in nodule tissue during nodulation process were detected by promoter fusion with β-glucuronidase activity. The urtB gene mutant was constructed by site-directed insertion, pot plant experiments and in combination with nitrogen addition treatment were carried out to identify symbiotic phenotypes and restoration. [Results] It showed that the urtB gene was anticipated to be involved in nitrogen transport of rhizobia. The expression level of urtB gene under symbiotic conditions was significantly up-regulated compared to that under free-living conditions, and urtB gene was highly expressed in the nitrogen-fixing zone of the mature nodules; an urtB mutant of 7653 R was screened and obtained. Compared with the wild-type strain 7653 R, the 7653R△urtB mutant inoculated with host plant resulted in an abnormal development of the nodule, and the above ground biomass of plants and nodule nitrogenase activity were significantly decreased. Adding nitrogen in the pot experiment could restore its symbiotic defective phenotype. [Conclusion] The urtB gene of Mesorhizobium huakuii may affect nitrogen transport or assimilation in root nodules, and therefore it plays an important role in root nodule development and symbiotic nitrogen fixation.
[Objective] Urea ABC-transporter permease subunit UrtB may be involved in urea metabolism and branched-chain amino acid transport. This study aimed to obtain experimental evidence to clarify the effect of urtB gene on the nodulation and nitrogen fixation of Mesorhizobium huakuii and provide scientific basis for further study of its functional mechanism. [Methods] The structure characteristics and biological functions of UrtB protein were analyzed by bioinformatics, the expression pattern of urtB gene under free-living growth and symbiosis conditions were detected by fluorescent quantitative real-time PCR, and in situ expression of urtB gene in nodule tissue during nodulation process were detected by promoter fusion with β-glucuronidase activity. The urtB gene mutant was constructed by site-directed insertion, pot plant experiments and in combination with nitrogen addition treatment were carried out to identify symbiotic phenotypes and restoration. [Results] It showed that the urtB gene was anticipated to be involved in nitrogen transport of rhizobia. The expression level of urtB gene under symbiotic conditions was significantly up-regulated compared to that under free-living conditions, and urtB gene was highly expressed in the nitrogen-fixing zone of the mature nodules; an urtB mutant of 7653 R was screened and obtained. Compared with the wild-type strain 7653 R, the 7653R△urtB mutant inoculated with host plant resulted in an abnormal development of the nodule, and the above ground biomass of plants and nodule nitrogenase activity were significantly decreased. Adding nitrogen in the pot experiment could restore its symbiotic defective phenotype. [Conclusion] The urtB gene of Mesorhizobium huakuii may affect nitrogen transport or assimilation in root nodules, and therefore it plays an important role in root nodule development and symbiotic nitrogen fixation.
引文
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[5]Hosie AHF,Allaway D,Galloway CS,Dunsby HA,Poole PS.Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters(Bra/LIV)of the ABC family.Journal of Bacteriology,2002,184(15):4071-4080.
[6]McAllister LJ,Tseng HJ,Ogunniyi AD,Jennings MP,McEwan AG,Paton JC.Molecular analysis of the PSApermease complex of Streptococcus pneumoniae.Molecular Microbiology,2004,53(3):889-901.
[7]Jessberger N,Lu Y,Amon J,Titgemeyer F,Sonnewald S,Reid S,Burkovski A.Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis.BMCResearch Notes,2013,6(10):482-524.
[8]Li YX,Li F,Zhou XL,Xie FL,Li YG.Screening proteins interacting with the outer membrane protein opa22 in Mesorhizobium huakuii 7653R with bacterial two-hybrid library.Journal of Huazhong Agricultural University,2014,33(2):28-33.(in Chinese)李一星,李芳,周鑫兰,谢福莉,李友国.利用细菌双杂交文库筛选华癸中慢生根瘤菌7653R中与外膜蛋白Opa22互作的蛋白.华中农业大学学报,2014,33(2):28-33.
[9]Li YX,Wang JY,Shi XF,Chen DS,Li YG.Activity in vitro and function of a non-specific lipid transfer protein AsIB259from Astragalus sinicus in symbiotic nitrogen fixation.Journal of Huazhong Agricultural University,2014,33(2):19-25.(in Chinese)李一星,王建云,石晓峰,陈大松,李友国.紫云英非特异性转脂蛋白AsIB259的体外酶活性及在共生固氮中的功能.华中农业大学学报,2014,33(2):19-25.
[10]Peng JL,Hao BH,Liu L,Wang SM,Ma BG,Yang Y,Xie FL,Li YG.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells.PLoS One,2014,9(4):e93626.
[11]Heil JR,Cheng J,Charles TC.Site-specific bacterial chromosome engineering:Ф31 integrase mediated cassette exchange(IMCE).Journal of Visualized Experiments,2012,(61):e3698.
[12]van den Eede G,Deblaere R,Goethals K,van Montagu M,Holsters M.Broad host range and promoter selection vectors for bacteria that interact with plants.Molecular Plant Microbe Interact,1992,5(3):228-234.
[13]Wang C,Zhu H,Jin LP,Chen T,Wang LX,Kang H,Hong ZJ,Zhang ZM.Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus.Plant Molecular Biology,2013,82(1/2):97-111.
[14]Tang GR,Li NN,Liu YM,Yu LM,Yu LL,Yan JH,Luo L.Sinorhizobium meliloti glutathione reductase is required for both redox homeostasis and symbiosis.Applied and Environmental Microbiology,2018,84(3).DOI:10.1128/AEM.01937-17.
[15]Li YN,Zeng XB,Zhou XJ,Li YG.Cloning,mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R.Acta Microbiologica Sinica,2016,56(12):1901-1911.(in Chinese)李亚楠,曾小波,周雪娟,李友国.华癸中慢生根瘤菌7653R3个脂质转运蛋白基因的克隆、突变及共生表型.微生物学报,2016,56(12):1901-1911.
[16]Kuai JL,Liu XJ,Li WQ.Effects of different nitrogen levels on the growth characteristics of alfalfa inoculated with rhizobium.Grassland and Turf,2011,31(3):56-59,63.(in Chinese)蒯佳林,刘晓静,李文卿.不同氮素水平对接种根瘤菌紫花苜蓿生长特性的影响.草原与草坪,2011,31(3):56-59,63.
[17]Li YX,Zhou L,Li YG,Chen DS,Tan XJ,Lei L,Zhou JC.Anodule-specific plant cysteine proteinase AsNODF32 is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus.New Phytologist,2008,180(1):185-192.
[18]Estrada-Navarrete G,Cruz-Mireles N,Lascano R,Alvarado-Affantranger X,Hernández-Barrera A,Barraza A,Olivares JE,Arthikala M,Cárdenas L,Quinto C,Sanchez F.An autophagy-related kinase is essential for the symbiotic relationship between Phaseolus vulgaris and both Rhizobia and Arbuscular Mycorrhizal Fungi.The Plant Cell,2016,28(9):2326-2341.
[19]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408.
[20]Wang SM,Hao BH,Li JR,Gu HL,Peng JL,Xie FL,Zhao XY,Frech C,Chen NS,Ma BG,Li YG.Whole-genome sequencing of Mesorhizobium huakuii 7653R provides molecular insights into host specificity and symbiosis island dynamics.BMC Genomics,2014,15:440.
[21]Prell J,Poole P.Metabolic changes of rhizobia in legume nodules.Trends in Microbiology,2006,14(4):161-168.
[22]Schafer A,Tauch A,J?ger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[23]Abd-ElKarem Y,Elbers T,Reichelt R,Steinbüchel A.Heterologous expression of Anabaena sp.PCC7120cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium(Ensifer)meliloti 1021.Applied Microbiology and Biotechnology,2011,89(4):1177-1192.
[24]Karunakaran R,Ramachandran VK,Seaman JC,East AK,Mouhsine B,Mauchline TH,Prell J,Skeffington A,Poole PS.Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca.Journal Bacteriology,2009,191(12):4002-4014.
[25]Krusell L,Krause K,Ott T,Desbrosses G,Kr?mer U,Sato S,Nakamura Y,Tabata S,James EK,Sandal N,Stougaard J,Kawaguchi M,Miyamoto A,Suganuma N,Udvardi MK.The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.The Plant Cell,2005,17(5):1625-1636.
[2]Young JP,Crossman LC,Johnston AW,Thomson NR,Ghazoui ZF,Hull KH,Wexler M,Curson AR,Todd JD,Poole PS,Mauchline TH,East AK,Quail MA,Churcher C,Arrowsmith C,Cherevach I,Chillingworth T,Clarke K,Cronin A,Davis P,Fraser A,Hance Z,Hauser H,Jagels K,Moule S,Mungall K,Norbertczak H,Rabbinowitsch E,Sanders M,Simmonds M,Whitehead S,Parkhill J.The genome of Rhizobium leguminosarum has recognizable core and accessory components.Genome Biology,2006,7(4):R34-R89.
[3]Sachs G,Kraut JA,Wen Y,Feng J,Scott DR.Urea transport in bacteria:acid acclimation by gastric Helicobacter spp.The Journal of Membrane Biology,2006,212(2):71-82.
[4]Valladares A,Montesinos ML,Herrero A,Flores E.An ABC-type,high-affinity urea permease identified in Cyanobacteria.Molecular Microbiology,2002,43(3):703-715.
[5]Hosie AHF,Allaway D,Galloway CS,Dunsby HA,Poole PS.Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters(Bra/LIV)of the ABC family.Journal of Bacteriology,2002,184(15):4071-4080.
[6]McAllister LJ,Tseng HJ,Ogunniyi AD,Jennings MP,McEwan AG,Paton JC.Molecular analysis of the PSApermease complex of Streptococcus pneumoniae.Molecular Microbiology,2004,53(3):889-901.
[7]Jessberger N,Lu Y,Amon J,Titgemeyer F,Sonnewald S,Reid S,Burkovski A.Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis.BMCResearch Notes,2013,6(10):482-524.
[8]Li YX,Li F,Zhou XL,Xie FL,Li YG.Screening proteins interacting with the outer membrane protein opa22 in Mesorhizobium huakuii 7653R with bacterial two-hybrid library.Journal of Huazhong Agricultural University,2014,33(2):28-33.(in Chinese)李一星,李芳,周鑫兰,谢福莉,李友国.利用细菌双杂交文库筛选华癸中慢生根瘤菌7653R中与外膜蛋白Opa22互作的蛋白.华中农业大学学报,2014,33(2):28-33.
[9]Li YX,Wang JY,Shi XF,Chen DS,Li YG.Activity in vitro and function of a non-specific lipid transfer protein AsIB259from Astragalus sinicus in symbiotic nitrogen fixation.Journal of Huazhong Agricultural University,2014,33(2):19-25.(in Chinese)李一星,王建云,石晓峰,陈大松,李友国.紫云英非特异性转脂蛋白AsIB259的体外酶活性及在共生固氮中的功能.华中农业大学学报,2014,33(2):19-25.
[10]Peng JL,Hao BH,Liu L,Wang SM,Ma BG,Yang Y,Xie FL,Li YG.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells.PLoS One,2014,9(4):e93626.
[11]Heil JR,Cheng J,Charles TC.Site-specific bacterial chromosome engineering:Ф31 integrase mediated cassette exchange(IMCE).Journal of Visualized Experiments,2012,(61):e3698.
[12]van den Eede G,Deblaere R,Goethals K,van Montagu M,Holsters M.Broad host range and promoter selection vectors for bacteria that interact with plants.Molecular Plant Microbe Interact,1992,5(3):228-234.
[13]Wang C,Zhu H,Jin LP,Chen T,Wang LX,Kang H,Hong ZJ,Zhang ZM.Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus.Plant Molecular Biology,2013,82(1/2):97-111.
[14]Tang GR,Li NN,Liu YM,Yu LM,Yu LL,Yan JH,Luo L.Sinorhizobium meliloti glutathione reductase is required for both redox homeostasis and symbiosis.Applied and Environmental Microbiology,2018,84(3).DOI:10.1128/AEM.01937-17.
[15]Li YN,Zeng XB,Zhou XJ,Li YG.Cloning,mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R.Acta Microbiologica Sinica,2016,56(12):1901-1911.(in Chinese)李亚楠,曾小波,周雪娟,李友国.华癸中慢生根瘤菌7653R3个脂质转运蛋白基因的克隆、突变及共生表型.微生物学报,2016,56(12):1901-1911.
[16]Kuai JL,Liu XJ,Li WQ.Effects of different nitrogen levels on the growth characteristics of alfalfa inoculated with rhizobium.Grassland and Turf,2011,31(3):56-59,63.(in Chinese)蒯佳林,刘晓静,李文卿.不同氮素水平对接种根瘤菌紫花苜蓿生长特性的影响.草原与草坪,2011,31(3):56-59,63.
[17]Li YX,Zhou L,Li YG,Chen DS,Tan XJ,Lei L,Zhou JC.Anodule-specific plant cysteine proteinase AsNODF32 is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus.New Phytologist,2008,180(1):185-192.
[18]Estrada-Navarrete G,Cruz-Mireles N,Lascano R,Alvarado-Affantranger X,Hernández-Barrera A,Barraza A,Olivares JE,Arthikala M,Cárdenas L,Quinto C,Sanchez F.An autophagy-related kinase is essential for the symbiotic relationship between Phaseolus vulgaris and both Rhizobia and Arbuscular Mycorrhizal Fungi.The Plant Cell,2016,28(9):2326-2341.
[19]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408.
[20]Wang SM,Hao BH,Li JR,Gu HL,Peng JL,Xie FL,Zhao XY,Frech C,Chen NS,Ma BG,Li YG.Whole-genome sequencing of Mesorhizobium huakuii 7653R provides molecular insights into host specificity and symbiosis island dynamics.BMC Genomics,2014,15:440.
[21]Prell J,Poole P.Metabolic changes of rhizobia in legume nodules.Trends in Microbiology,2006,14(4):161-168.
[22]Schafer A,Tauch A,J?ger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[23]Abd-ElKarem Y,Elbers T,Reichelt R,Steinbüchel A.Heterologous expression of Anabaena sp.PCC7120cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium(Ensifer)meliloti 1021.Applied Microbiology and Biotechnology,2011,89(4):1177-1192.
[24]Karunakaran R,Ramachandran VK,Seaman JC,East AK,Mouhsine B,Mauchline TH,Prell J,Skeffington A,Poole PS.Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca.Journal Bacteriology,2009,191(12):4002-4014.
[25]Krusell L,Krause K,Ott T,Desbrosses G,Kr?mer U,Sato S,Nakamura Y,Tabata S,James EK,Sandal N,Stougaard J,Kawaguchi M,Miyamoto A,Suganuma N,Udvardi MK.The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.The Plant Cell,2005,17(5):1625-1636.
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