一株耐硒壶瓶碎米荠内生菌分离、鉴定及其体外硒代谢研究
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摘要
【背景】壶瓶碎米荠对硒具有超积累能力,并主要以硒代胱氨酸的形式存在,与已有的硒超积累植物显著不同,其硒超积累机制不明。【目的】从硒超积累植物壶瓶碎米荠(Cardamine hupingshanensis)体内分离耐硒内生菌,并对其进行鉴定和体外硒代谢特征研究,为壶瓶碎米荠超积累硒的机制研究提供参考。【方法】从壶瓶碎米荠新鲜叶片中分离纯化耐硒内生菌株,对其进行生理生化特征及16S rRNA基因序列分析鉴定,并对其进行亚硒酸钠培养代谢。【结果】获得一株耐硒内生菌CSN-1,被鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus),培养液中硒含量低(Se 1.5 mg/L)时其吸光度值较对照组高,硒含量高(Se 10 mg/L)时其吸光度值较对照组低;代谢后的上清液中硒主要以Se~(4+)存在,而菌体中硒主要是硒代胱氨酸(SeCys_2)。【结论】硒超积累植物壶瓶碎米荠叶片体内存在甲基营养型芽孢杆菌(Bacillus methylotrophicus)CSN-1,具有将亚硒酸钠转化为硒代胱氨酸的能力,低浓度的硒对该内生菌的生长具有一定的促进作用,而高浓度的硒则会抑制该内生菌的生长。
[Background] Cardamine hupingshanensis is a novel selenium(Se)-hyperaccumulating plant in China with the predominate Se species as selenocystine(SeCys_2), which is different with other Se-hyperaccumulating plants in USA, eg. Astragalus bisulcatus, Stanleya pinnata. [Objective]To explore the role of microorganism during Se-hyperaccumulation in Cardamine hupingshanensis,isolation, identification and Se metabolism in vitro of a Se-tolerant endophyte from C.hupingshanensis were conducted. [Methods] A Se-tolerant endophyte was isolated and purified from fresh leaves of C. hupingshanensis as CSN-1. The physiological and biochemical analysis and 16 S rRNA gene sequence analysis were carried out. Moreover, the endophyte CSN-1 was cultured in selenite medium to study its Se metabolism. [Results] The Se-tolerant endophyte CSN-1 was identified as Bacillus methylotrophicus. The selenite culture revealed that the medium absorbance was higher than that in controls under 1.5 mg Se/L medium, but lower under 10 mg Se/L medium.The metabolized Se speciation was Se~(4+) in supernants, but SeCys_2 in bacteria deposits. [Conclusion]The Se-tolerant endophyte Bacillus methylotrophicus CSN-1 presented in Se-hyperaccumulating plant, Cardamine hupingshanensis, and could transform selenite into SeCys_2. The low concentration of Se could promote the growth of Bacillus methylotrophicu CSN-1, but negative effects could be observed at high Se levels.
[Background] Cardamine hupingshanensis is a novel selenium(Se)-hyperaccumulating plant in China with the predominate Se species as selenocystine(SeCys_2), which is different with other Se-hyperaccumulating plants in USA, eg. Astragalus bisulcatus, Stanleya pinnata. [Objective]To explore the role of microorganism during Se-hyperaccumulation in Cardamine hupingshanensis,isolation, identification and Se metabolism in vitro of a Se-tolerant endophyte from C.hupingshanensis were conducted. [Methods] A Se-tolerant endophyte was isolated and purified from fresh leaves of C. hupingshanensis as CSN-1. The physiological and biochemical analysis and 16 S rRNA gene sequence analysis were carried out. Moreover, the endophyte CSN-1 was cultured in selenite medium to study its Se metabolism. [Results] The Se-tolerant endophyte CSN-1 was identified as Bacillus methylotrophicus. The selenite culture revealed that the medium absorbance was higher than that in controls under 1.5 mg Se/L medium, but lower under 10 mg Se/L medium.The metabolized Se speciation was Se~(4+) in supernants, but SeCys_2 in bacteria deposits. [Conclusion]The Se-tolerant endophyte Bacillus methylotrophicus CSN-1 presented in Se-hyperaccumulating plant, Cardamine hupingshanensis, and could transform selenite into SeCys_2. The low concentration of Se could promote the growth of Bacillus methylotrophicu CSN-1, but negative effects could be observed at high Se levels.
引文
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[11]Rodriguez RJ,Henson J,van Volkenburgh E,et al.Stress tolerance in plants via habitat-adapted symbiosis[J].The ISME Journal,2008,2(4):404-416
[12]Bai HF,Chen LB,Liu KM,et al.A new species of Cardamine(Brassicaceae)from Hunan,China[J].Novon:A Journal for Botanical Nomenclature,2008,18(2):135-137
[13]Yuan LX,Zhu YY,Lin ZQ,et al.A novel selenocystine-accumulating plant in selenium-mine drainage area in Enshi,China[J].PLo S One,2013,8(6):e65615
[14]Pickering IJ,Wright C,Bubner B,et al.Chemical form and distribution of selenium and sulfur in the selenium hyperaccumulator Astragalus bisulcatus[J].Plant Physiology,2003,131(3):1460-1467
[15]Freeman JL,Tamaoki M,Stushnoff C,et al.Molecular mechanisms of selenium tolerance and hyperaccumulation in Stanleya pinnata[J].Plant Physiology,2010,153(4):1630-1652
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[21]Liu XY.The research on the effect of selenium-enrich antistaling agent on Navel Orange[J].Journal of Sichuan Normal University(Natural Science),2011,34(4):565-568(in Chinese)刘兴艳.富硒功能保鲜剂在脐橙上的富硒效果研究[J].四川师范大学学报:自然科学版,2011,34(4):565-568
[22]Li X,Liu Y,Peng YS,et al.Microcalorimetric study of the antibacterial action of four kinds ofα-amino dialkyl phosphonate ester containing selenomorpholine[J].Journal of Wuhan University of Technology,2001,23(11):4-7(in Chinese)李曦,刘义,彭云山,等.含硒的膦酸酯化合物抑菌作用的微量热研究[J].武汉理工大学学报,2001,23(11):4-7
[23]Oremland RS,Herbel MJ,Blum JS,et al.Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria[J].Applied and Environmental Microbiology,2004,70(1):52-60
[24]Ghosh A,Mohod AM,Paknikar KM,et al.Isolation and characterization of selenite-and selenate-tolerant microorganisms from selenium-contaminated sites[J].World Journal of Microbiology and Biotechnology,2008,24(8):1607-1611
[25]Li ZG,Luo YM,Teng Y.The Research Method of Soil and Environmental Microorganism[M].Beijing:Science Press,2008:52-61(in Chinese)李振高,骆永明,腾应.土壤与环境微生物研究法[M].北京:科学出版社,2008:52-61
[26]Sura-de Jong M,Reynolds RJ,Richterova K,et al.Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties[J].Frontiers in Plant Science,2015,6:113
[27]Dong XZ,Cai MY.Common Bacterial Identification System Hand[M].Beijing:Science Press,2001:267-295(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:267-295
[28]Losi ME,Frankenberger WT.Reduction of selenium oxyanions by Enterobacter cloacae SLD1a-1:isolation and growth of the bacterium and its expulsion of selenium particles[J].Applied and Environmental Microbiology,1997,63(8):3079-3084
[29]Dungan RS,Yates SR,Frankenberger Jr WT.Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pondsediment[J].Environmental Microbiology,2003,5(4):287-295
[30]Kessi J,Hanselmann KW.Similarities between the abiotic reduction of selenite with glutathione and the dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli[J].Journal of Biological Chemistry,2004,279(49):50662-50669
[31]Oremland RS,Hollibaugh JT,Maest AS,et al.Selenate reduction to elemental selenium by anaerobic bacteria in sediments and culture:biogeochemical significance of a novel,sulfate-independent respiration[J].Applied and Environmental Microbiology,1989,55(9):2333-2343
[32]Kessi J,Ramuz M,Wehrli E,et al.Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum[J].Applied and Environmental Microbiology,1999,65(11):4734-4740
[33]Roux M,Sarret G,Pignot-Paintrand I,et al.Mobilization of selenite by Ralstonia metallidurans CH34[J].Applied and Environmental Microbiology,2001,67(2):769-773
[34]van Fleet-Stalder V,Chasteen TG,Pickering IJ,et al.Fate of selenate and selenite metabolized by Rhodobacter sphaeroides[J].Applied and Environmental Microbiology,2000,66(11):4849-4853
[35]Wang DL,Xiao M,Qian W,et al.Progress in research of the product of the red elemental selenium reduced from selenium oxyanions by bacteria[J].Acta Microbiologica Sinica,2007,47(3):554-557(in Chinese)王东亮,肖敏,钱卫,等.细菌还原氧化态硒产生红色单质硒的研究进展[J].微生物学报,2007,47(3):554-557
[36]Staicu LC,Ackerson CJ,Cornelis P,et al.Pseudomonas moraviensis subsp.stanleyae,a bacterial endophyte of hyperaccumulator Stanleya pinnata,is capable of efficient selenite reduction to elemental selenium under aerobic conditions[J].Journal of Applied Microbiology,2015,119(2):400-410
[37]Li WC,Ye ZH,Wong MH.Effects of bacteria on enhanced metal uptake of the Cd/Zn-Hyperaccumulating plant,Sedum alfredii[J].Journal of Experimental Botany,2007,58(15/16):4173-4182
[38]Abou-Shanab RAI,van Berkum P,Angle JS.Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale[J].Chemosphere,2007,68(2):360-367
[39]Belimov AA,Hontzeas N,Safronova VI,et al.Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard(Brassica juncea L.Czern.)[J].Soil Biology and Biochemistry,2005,37(2):241-250
[2]Feng YJ,Song W.Endophytic bacteria[J].Ziran Zazhi,2001,23(5):249-252(in Chinese)冯永君,宋未.植物内生细菌[J].自然杂志,2001,23(5):249-252
[3]Hilszczańska D.Endophytes-characteristics and possibilities of application in forest management[J].Forest Research Papers,2016,77(3):276-282
[4]Wang ZW,Ji YL,Chen YG.Studies and biological significances of plant endophytes[J].Microbiology China,2015,42(2):349-363(in Chinese)王志伟,纪燕玲,陈永敢.植物内生菌研究及其科学意义[J].微生物学通报,2015,42(2):349-363
[5]Mercado-Blanco J,Prieto P.Bacterial endophytes and root hairs[J].Plant and Soil,2012,361(1/2):301-306
[6]Lodewyckx C,Mergeay M,Vangronsveld J,et al.Isolation,characterization,and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp.calaminaria[J].International Journal of Phytoremediation,2002,4(2):101-115
[7]Li HY,Wei DQ,Shen M,et al.Endophytes and their role in phytoremediation[J].Fungal Diversity,2012,54(1):11-18
[8]Jacobson CB,Pasternak JJ,Glick BR.Partial purification and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2[J].Canadian Journal of Microbiology,1994,40(12):1019-1025
[9]Glick BR,Cheng ZY,Czarny J,et al.Promotion of plant growth by ACC deaminase-producing soil bacteria[J].European Journal of Plant Pathology,2007,119(3):329-339
[10]Du YJ,Wang Q,Han LB.The effect of Neotyphodium typhinum infected on photosynthetic characteristics of tall fescue[J].Ecology and Environmental Sciences,2009,18(2):590-594(in Chinese)杜永吉,王祺,韩烈保.内生真菌Neotyphodium.typhinum感染对高羊茅光合特性的影响[J].生态环境学报,2009,18(2):590-594
[11]Rodriguez RJ,Henson J,van Volkenburgh E,et al.Stress tolerance in plants via habitat-adapted symbiosis[J].The ISME Journal,2008,2(4):404-416
[12]Bai HF,Chen LB,Liu KM,et al.A new species of Cardamine(Brassicaceae)from Hunan,China[J].Novon:A Journal for Botanical Nomenclature,2008,18(2):135-137
[13]Yuan LX,Zhu YY,Lin ZQ,et al.A novel selenocystine-accumulating plant in selenium-mine drainage area in Enshi,China[J].PLo S One,2013,8(6):e65615
[14]Pickering IJ,Wright C,Bubner B,et al.Chemical form and distribution of selenium and sulfur in the selenium hyperaccumulator Astragalus bisulcatus[J].Plant Physiology,2003,131(3):1460-1467
[15]Freeman JL,Tamaoki M,Stushnoff C,et al.Molecular mechanisms of selenium tolerance and hyperaccumulation in Stanleya pinnata[J].Plant Physiology,2010,153(4):1630-1652
[16]Stolz JF,Basu P,Oremland RS.Microbial transformation of elements:the case of arsenic and selenium[J].International Microbiology,2002,5(4):201-207
[17]Stolz JF,Basu P,Santini JM,et al.Arsenic and selenium in microbial metabolism[J].Annual Review of Microbiology,2006,60(1):107-130
[18]Turner RJ,Weiner JH,Taylor DE.Selenium metabolism in Escherichia coli[J].Bio Metals,1998,11(3):223-227
[19]Liu Y,Yan CN,Song ZH,et al.Thermochemical studies of selenium——IV.Microcalorimetric study of the inhibiting action of Na2Se O3 on growth metabolism of microbes[J].Chinese Journal of Applied Chemistry,1998,15(1):29-32(in Chinese)刘义,颜承农,宋昭华,等.硒的热化学研究——IV.微量热法研究Na2Se O3对微生物生长代谢的抑制[J].应用化学,1998,15(1):29-32
[20]Wang DL.Study on selenium transformation of photosynthetic bacteria[D].Jinan:Master’s Thesis of Shandong University,2007(in Chinese)王东亮.光合细菌硒转化的研究[D].济南:山东大学硕士学位论文,2007
[21]Liu XY.The research on the effect of selenium-enrich antistaling agent on Navel Orange[J].Journal of Sichuan Normal University(Natural Science),2011,34(4):565-568(in Chinese)刘兴艳.富硒功能保鲜剂在脐橙上的富硒效果研究[J].四川师范大学学报:自然科学版,2011,34(4):565-568
[22]Li X,Liu Y,Peng YS,et al.Microcalorimetric study of the antibacterial action of four kinds ofα-amino dialkyl phosphonate ester containing selenomorpholine[J].Journal of Wuhan University of Technology,2001,23(11):4-7(in Chinese)李曦,刘义,彭云山,等.含硒的膦酸酯化合物抑菌作用的微量热研究[J].武汉理工大学学报,2001,23(11):4-7
[23]Oremland RS,Herbel MJ,Blum JS,et al.Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria[J].Applied and Environmental Microbiology,2004,70(1):52-60
[24]Ghosh A,Mohod AM,Paknikar KM,et al.Isolation and characterization of selenite-and selenate-tolerant microorganisms from selenium-contaminated sites[J].World Journal of Microbiology and Biotechnology,2008,24(8):1607-1611
[25]Li ZG,Luo YM,Teng Y.The Research Method of Soil and Environmental Microorganism[M].Beijing:Science Press,2008:52-61(in Chinese)李振高,骆永明,腾应.土壤与环境微生物研究法[M].北京:科学出版社,2008:52-61
[26]Sura-de Jong M,Reynolds RJ,Richterova K,et al.Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties[J].Frontiers in Plant Science,2015,6:113
[27]Dong XZ,Cai MY.Common Bacterial Identification System Hand[M].Beijing:Science Press,2001:267-295(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:267-295
[28]Losi ME,Frankenberger WT.Reduction of selenium oxyanions by Enterobacter cloacae SLD1a-1:isolation and growth of the bacterium and its expulsion of selenium particles[J].Applied and Environmental Microbiology,1997,63(8):3079-3084
[29]Dungan RS,Yates SR,Frankenberger Jr WT.Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pondsediment[J].Environmental Microbiology,2003,5(4):287-295
[30]Kessi J,Hanselmann KW.Similarities between the abiotic reduction of selenite with glutathione and the dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli[J].Journal of Biological Chemistry,2004,279(49):50662-50669
[31]Oremland RS,Hollibaugh JT,Maest AS,et al.Selenate reduction to elemental selenium by anaerobic bacteria in sediments and culture:biogeochemical significance of a novel,sulfate-independent respiration[J].Applied and Environmental Microbiology,1989,55(9):2333-2343
[32]Kessi J,Ramuz M,Wehrli E,et al.Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum[J].Applied and Environmental Microbiology,1999,65(11):4734-4740
[33]Roux M,Sarret G,Pignot-Paintrand I,et al.Mobilization of selenite by Ralstonia metallidurans CH34[J].Applied and Environmental Microbiology,2001,67(2):769-773
[34]van Fleet-Stalder V,Chasteen TG,Pickering IJ,et al.Fate of selenate and selenite metabolized by Rhodobacter sphaeroides[J].Applied and Environmental Microbiology,2000,66(11):4849-4853
[35]Wang DL,Xiao M,Qian W,et al.Progress in research of the product of the red elemental selenium reduced from selenium oxyanions by bacteria[J].Acta Microbiologica Sinica,2007,47(3):554-557(in Chinese)王东亮,肖敏,钱卫,等.细菌还原氧化态硒产生红色单质硒的研究进展[J].微生物学报,2007,47(3):554-557
[36]Staicu LC,Ackerson CJ,Cornelis P,et al.Pseudomonas moraviensis subsp.stanleyae,a bacterial endophyte of hyperaccumulator Stanleya pinnata,is capable of efficient selenite reduction to elemental selenium under aerobic conditions[J].Journal of Applied Microbiology,2015,119(2):400-410
[37]Li WC,Ye ZH,Wong MH.Effects of bacteria on enhanced metal uptake of the Cd/Zn-Hyperaccumulating plant,Sedum alfredii[J].Journal of Experimental Botany,2007,58(15/16):4173-4182
[38]Abou-Shanab RAI,van Berkum P,Angle JS.Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale[J].Chemosphere,2007,68(2):360-367
[39]Belimov AA,Hontzeas N,Safronova VI,et al.Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard(Brassica juncea L.Czern.)[J].Soil Biology and Biochemistry,2005,37(2):241-250