环境因子对脱氯功能念珠藻Nostoc PD-2降解多氯联苯过程中基因表达的影响
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摘要
以分离筛选自多氯联苯污染水稻田中的脱氯功能蓝藻Nostoc PD-2为材料,两种典型的三氯代多氯联苯PCB28和PCB30为目标污染物,在不同的氮源、碳源和培养温度条件下,研究了脱氯功能藻种Nostoc PD-2中双加氧酶基因和细胞色素b6f复合体铁硫蛋白基因的表达情况及两种基因对脱氯作用的影响.结果显示,以硝酸钠作为氮源时,与氮气氮源组相比,PCB28和PCB30降解组中的两种基因均显著上调表达,双加氧酶基因的上调倍数分别为1.9和5.7倍,铁硫蛋白基因的上调倍数分别为1.1和1.7倍;添加碳酸钠时,与对照相比,双加氧酶基因最高上调了2.2倍,铁硫蛋白基因最高上调了3.4倍;提高温度对双加氧酶基因和铁硫蛋白基因的表达均有促进作用.相较于铁硫蛋白,双加氧酶基因相对表达量与PCB28和PCB30的脱氯百分比之间的相关系数分别为0.872和0.832,表明双加氧酶基因活性与功能藻种PD-2脱氯降解PCBs的相关性更高.本研究结果表明,不同环境因子可引起脱氯功能藻种Nostoc PD-2降解PCBs过程中双加氧酶基因和铁硫蛋白基因的差异表达,添加碳源对降解效果的促进作用最明显,脱氯功能藻种在工程应用中采用优化的环境条件有利于提高降解效率.
Cyanbacterium Nostoc PD-2 isolated from polychlorinated biphenyl( PCB)-contaminated paddy soils was used to biodegrade two kinds of typical trichlorodiphenyls( 2,4,4'-trichlorobiphenyl and 2,4,6-trichlorobiphenyl). The expressions of dioxygenase genes and cytochrome b6 f complex Fe-S protein genes in Nostoc PD-2 during the PCB-degradation were determined under the conditions of different nitrogen sources,carbon sources,and cultural temperatures. In addition,the relationships between both genes expressions and PCB-dechloriantion efficiencies were also analyzed. Results showed that,the expressions of both genes in the sodium nitrate group were significantly up-regulated comparing to that in the nitrogen group. The upexpression folds of dioxygenase gene under the exposure of PCB28 group and PCB30 group were 1.9 and 5.7,respectively. While the up-expression folds of Fe-S protein gene were 1.1 and 1.7,respectively. Compared to the control group,the maximum up-regulated folds of dioxygenase gene and Fe-S protein gene in the sodium carbonate group were 2. 2 and 3. 4,respectively. Elevated temperature could facilitate the expressions of dioxygenase gene andcytochrome b6 f complex Fe-S protein gene. The expression of dioxygenas gene more significantly correlated with the dechlorination percentages of both PCB congeners than that of Fe-S protein gene. The correlation coefficients between dioxygenase gene expressions and the dechlorination percentages of both PCBs congeners were 0. 872 and 0. 832,respectively. Such findings can demonstrate a new insight to clarify the molecular mechanism of PCBbiodegradation by Nostoc PD-2. In addition,using sodium carbonate as carbon source can promote the efficiencies of PCB-dechlorination by Nostoc PD-2.Our study can provide the new application of cyanobacteria in bioremediation engineering.
Cyanbacterium Nostoc PD-2 isolated from polychlorinated biphenyl( PCB)-contaminated paddy soils was used to biodegrade two kinds of typical trichlorodiphenyls( 2,4,4'-trichlorobiphenyl and 2,4,6-trichlorobiphenyl). The expressions of dioxygenase genes and cytochrome b6 f complex Fe-S protein genes in Nostoc PD-2 during the PCB-degradation were determined under the conditions of different nitrogen sources,carbon sources,and cultural temperatures. In addition,the relationships between both genes expressions and PCB-dechloriantion efficiencies were also analyzed. Results showed that,the expressions of both genes in the sodium nitrate group were significantly up-regulated comparing to that in the nitrogen group. The upexpression folds of dioxygenase gene under the exposure of PCB28 group and PCB30 group were 1.9 and 5.7,respectively. While the up-expression folds of Fe-S protein gene were 1.1 and 1.7,respectively. Compared to the control group,the maximum up-regulated folds of dioxygenase gene and Fe-S protein gene in the sodium carbonate group were 2. 2 and 3. 4,respectively. Elevated temperature could facilitate the expressions of dioxygenase gene andcytochrome b6 f complex Fe-S protein gene. The expression of dioxygenas gene more significantly correlated with the dechlorination percentages of both PCB congeners than that of Fe-S protein gene. The correlation coefficients between dioxygenase gene expressions and the dechlorination percentages of both PCBs congeners were 0. 872 and 0. 832,respectively. Such findings can demonstrate a new insight to clarify the molecular mechanism of PCBbiodegradation by Nostoc PD-2. In addition,using sodium carbonate as carbon source can promote the efficiencies of PCB-dechlorination by Nostoc PD-2.Our study can provide the new application of cyanobacteria in bioremediation engineering.
引文
Ahmed M,Focht D D.1973.Degradation of polychlorinated biphenyls bytwo species of Achromobacter[J].Canadian Journal ofMicrobiology,19(1):47-52
Aken B,Correa P,Schnoor J.2010.Phytoremediation of polychlorinatedbiphenyls:new trends and promises[J].Environmental Science&Technology,44(8):2767-2776
Barriault D,Plante M M,Sylvestre M.2002.Family shuffling of atargeted bphA region to engineer biphenyl dioxygenase[J].Journal ofBacteriology,184(14):3794-3800
Bedard D L,Pohl E A,Bailey J J,et al.2005.Characterization of thePCB substrate range of microbial dechlorination process LP.Environmental Science&Technology,39(17):6831-6838
Brühlmann F,Chen W.1999a.Transformation of polychlorinatedbiphenyls by a novel BphA variant through the meta-cleavagepathway[J].FEMS Microbiology Letters,179(2):203-208
Brühlmann F,Chen W.1999b.Tuning biphenyl dioxygenase for extendedsubstrate specificity[J].Biotechnology and Bioengineering,63(5):544-551
Cui H,Wang Y,Qin S.2012.Genomewide analysis of carotenoidcleavage dioxygenases in unicellular and filamentous cyanobacteria[J].Comparative and Functional Genomics,doi:10.1155/2012/164690
CˇvancˇarováM,KrˇesinováZ,FilipováA,et al.2012.Biodegradation ofPCBs by ligninolytic fungi and characterization of the degradationproducts[J].Chemosphere,88(11):1317-1323
Emmett E A,Marco M M,Schmith J M,et al.1988.Studies oftransformer repair workers exposed to PCBs:I study design,PCBconcentrations,questionnaire,and clinical examination results[J].American Journal of Industrial Medicine,13(4):415-427
Furukawa K,Suenaga H,Goto M.2004.Biphenyl dioxygenases:functional versatilities and directed evolution[J].Journal ofBacteriology,186(16):5189-5196
Furusawa Y,Nagarajan V,Tanokura M,et al.2004.Crystal structure ofthe terminal oxygenase component of biphenyl dioxygenase derivedfrom Rhodococcus sp.strain RHA1[J].Journal of MolecularBiology,342(3):1041-1052
Goris J,Vos P D,Caballero-Mellado J,et al.2004.Classication of thebiphenyl-and polychlorinated biphenyl-degrading strain LB400Tandrelatives as Burkholderia xenovorans sp.nov[J].InternationalJournal of Systematic and Evolutionary Microbiology,54(5):1677-1681
Ilg A,Beyer P,Al-Babili S.2009.Characterization of the rice carotenoidcleavage dioxygenase 1 reveals a novel route for geranial biosynthesis[J].FEBS Journal,276(3):736-747
Jaward F M,Meijer S N,Steinnes E,et al.2004.Further studies on thelatitudinal and temporal trends of persistent organic pollutants inNorwegian and U.K.background air[J].Environmental Science&Technology,38(9):2523-2530
金相灿,储昭升,杨波,等.2008.温度对水华微囊藻及孟氏浮游蓝丝藻生长、光合作用及浮力变化的影响[J].环境科学学报,28(1):50-55
Kaneko T,Nakamura Y,Wolk C P,et al.2001.Complete genomicsequence of the filamentous nitrogen-fixing cyanobacterium Anabaenasp.strain PCC 7120[J].DNA Research,8(5):205-213
Kumamaru T,Suenaga H,Mitsuoka M,et al.1998.Enhanceddegradation of polychlorinated biphenyls by directed evolution ofbiphenyl dioxygenase[J].Nature Biotechnology,16(7):663-666
Kuritz T,Wolk C P.1995.Use of filamentous cyanobacteria forbiodegradation of organic pollutants[J].Applied EnvironmentalMicrobiology,61(1):234-238
Kuritz T,Bocanera L V,Rivera N S.1997.Dechlorination of lindane bythe cyanobacterium Anabaena sp.strain PCC7120 depends on thefunction of the nir operon[J].Journal of Bacteriology,179(10):3368-3370
Kuritz T.1999.Cyanobacteria as agents for the control of pollution bypesticides and chlorinated organic compounds[J].Journal ofApplied Micorbiology,85(S1):186-192
Lambo A J,Patel T R.2006.Cometabolic Degradation of polychlorinatedbiphenyls at low temperature by psychrotolerant bacteriumHydrogenophaga sp.IA3-A[J].Current Microbiology,53(1):48-52
Li R,Carmichael W W,Brittain S,et al.2001.Isolation andidentification of the cyanotoxin cylindrospermopsin and deoxycylindrospermopsin from a Thailand strain of Cylindrospermopsisraciborskii(cyanobacteria)[J].Toxicon,39(7):973-980
李鑫,胡洪营,张玉平.2011.无机碳源对栅藻LX1生长及油脂积累特性的影响[J].环境科学,32(8):2260-2266
Manodori L,Gambaro A,Ivo Moret,et al.2006.Seasonal evolution ofgas-phase PCB concentrations in the Venice Lagoon area[J].Chemosphere,62(3):449-458
Meiger S N,Ockenden W A,Sweetman A,et al.2003.Globaldistribution and budget of PCBs and HCB in background surfacesoils:Implications for sources and environmental processes[J].Environmental Science&Technology,37(4):667-672
Morris P J,Mohn W W,Quensen J F,et al.1992.Establishment ofapolychlorinated biphenyl degrading enrichment culture withpredominantly meta dechlorination[J].Applied and EnvironmentalMicrobiology,58(9):3088-3094
Mouhamadou B,Faure M,Sage L,et al.2013.Potential ofautochthonous fungal strains isolated from contaminated soils fordegradation of polychlorinated biphenyls[J].Fungal Biology,117(4):268-274
Ohtsubo Y,Nagata Y,Kimbara K,et al.2000.Expression of the bphgenes involved in biphenyl/PCB degradation in Pseudomonas sp.KKS102 induced by the biphenyl degradation intermediate,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid[J].Gene,256(1/2):223-228
Ohtsubo Y,Kudo T,Tsuda M,et al.2004.Strategies for bioremediationof polychlorinated biphenyls[J].Applied Microbiology andBiotechnology,65(3):250-258
Pieper D H.2005.Aerobic degradation of polychlorinated biphenyls[J].Applied Microbiology and Biotechnology,67(2):170-191
Rippka R,Deruelles J,Waterbury J B,et al.1979.Genericassignments,strain histories and properties of pure cultures ofcyanobacteria[J].Microbiology,111(1):1-61
Sakai M,Ezaki S,Suzuki N,et al.2005.Isolation and characterizationof a novel polychlorinated biphenyl-degrading bacterium,Paenibacillus sp.KBC101[J].Applied Microbiology andBiotechnology,68(1):111-116
Seto M,Kimbara K,Shimura M,et al.1995.A novel transformation ofpolychlorinated biphenyls by Rhodococcus sp.strain RHA1[J].Applied Microbiology and Biotechnology,61(9):3353-3358
Sierra I,Valera J L,Marina M L,et al.2003.Study of thebiodegradation process of polychlorinated biphenyls in liquid mediumand soil by a new isolated aerobic bacterium(Janibacter sp.)[J].Chemosphere,53(6):609-618
Triscari-Barberi T,Simone D,Calabrese F M,et al.2012.Genomesequence of the polychlorinated-biphenyl degrader Pseudomonaspseudoalcaligenes KF707[J].Journal of Bacteriology,194(16):4426-4427
Wang J X,Chen L,Huang S Q,et al.2012.RNA-seq basedidentification and mutant validation of gene targets related to ethanolresistance in cyanobacterial Synechocystis sp.PCC 6803[J].Biotechnology for Biofuels,5:89
王立柱,温皓成,邹渝,等.2010.产油微藻的分离、筛选及自养培养氮源、碳源的优化[J].微生物学通报,37(3):336-341
魏中青,刘丛强,梁小兵,等.2007.贵州红枫湖地区水稻土多氯联苯和有机氯农药的残留[J].环境科学,28(2):255-260
Williams W A.1994.Microbial reductive dechlorination oftrichlorobiphenyls in anaerobic sediment slurries[J].EnvironmentalScience&Technology,28(4):630-635
Yu M L,Guo Y L,Hsu C C,et al.2000.Menstruation and reproductionin women with polychlorinated biphenyl(PCB)poisoning:longterm follow-up interviews of the women from the Taiwan Yuchengcohort[J].International Journal of Epidemiology,29(4):672-677
Zall D M,Fisher D,Garner M Q.1956.Photometric determination ofchlorides in water[J].Analytical Chemistry,28(11):1665-1668
Zhang H J,Hu C M,Jia X Y,et al.2012.Characteristics ofγ-hexachlorocyclohexane biodegradation by a nitrogen-fixingcyanobacterium,Anabaena azotica[J].Journal of AppliedPhycology,24(2):221-225
郑洪立,高振,张齐,等.2011.无机碳源对小球藻自养产油脂的影响[J].生物工程学报,27(3):436-444
Aken B,Correa P,Schnoor J.2010.Phytoremediation of polychlorinatedbiphenyls:new trends and promises[J].Environmental Science&Technology,44(8):2767-2776
Barriault D,Plante M M,Sylvestre M.2002.Family shuffling of atargeted bphA region to engineer biphenyl dioxygenase[J].Journal ofBacteriology,184(14):3794-3800
Bedard D L,Pohl E A,Bailey J J,et al.2005.Characterization of thePCB substrate range of microbial dechlorination process LP.Environmental Science&Technology,39(17):6831-6838
Brühlmann F,Chen W.1999a.Transformation of polychlorinatedbiphenyls by a novel BphA variant through the meta-cleavagepathway[J].FEMS Microbiology Letters,179(2):203-208
Brühlmann F,Chen W.1999b.Tuning biphenyl dioxygenase for extendedsubstrate specificity[J].Biotechnology and Bioengineering,63(5):544-551
Cui H,Wang Y,Qin S.2012.Genomewide analysis of carotenoidcleavage dioxygenases in unicellular and filamentous cyanobacteria[J].Comparative and Functional Genomics,doi:10.1155/2012/164690
CˇvancˇarováM,KrˇesinováZ,FilipováA,et al.2012.Biodegradation ofPCBs by ligninolytic fungi and characterization of the degradationproducts[J].Chemosphere,88(11):1317-1323
Emmett E A,Marco M M,Schmith J M,et al.1988.Studies oftransformer repair workers exposed to PCBs:I study design,PCBconcentrations,questionnaire,and clinical examination results[J].American Journal of Industrial Medicine,13(4):415-427
Furukawa K,Suenaga H,Goto M.2004.Biphenyl dioxygenases:functional versatilities and directed evolution[J].Journal ofBacteriology,186(16):5189-5196
Furusawa Y,Nagarajan V,Tanokura M,et al.2004.Crystal structure ofthe terminal oxygenase component of biphenyl dioxygenase derivedfrom Rhodococcus sp.strain RHA1[J].Journal of MolecularBiology,342(3):1041-1052
Goris J,Vos P D,Caballero-Mellado J,et al.2004.Classication of thebiphenyl-and polychlorinated biphenyl-degrading strain LB400Tandrelatives as Burkholderia xenovorans sp.nov[J].InternationalJournal of Systematic and Evolutionary Microbiology,54(5):1677-1681
Ilg A,Beyer P,Al-Babili S.2009.Characterization of the rice carotenoidcleavage dioxygenase 1 reveals a novel route for geranial biosynthesis[J].FEBS Journal,276(3):736-747
Jaward F M,Meijer S N,Steinnes E,et al.2004.Further studies on thelatitudinal and temporal trends of persistent organic pollutants inNorwegian and U.K.background air[J].Environmental Science&Technology,38(9):2523-2530
金相灿,储昭升,杨波,等.2008.温度对水华微囊藻及孟氏浮游蓝丝藻生长、光合作用及浮力变化的影响[J].环境科学学报,28(1):50-55
Kaneko T,Nakamura Y,Wolk C P,et al.2001.Complete genomicsequence of the filamentous nitrogen-fixing cyanobacterium Anabaenasp.strain PCC 7120[J].DNA Research,8(5):205-213
Kumamaru T,Suenaga H,Mitsuoka M,et al.1998.Enhanceddegradation of polychlorinated biphenyls by directed evolution ofbiphenyl dioxygenase[J].Nature Biotechnology,16(7):663-666
Kuritz T,Wolk C P.1995.Use of filamentous cyanobacteria forbiodegradation of organic pollutants[J].Applied EnvironmentalMicrobiology,61(1):234-238
Kuritz T,Bocanera L V,Rivera N S.1997.Dechlorination of lindane bythe cyanobacterium Anabaena sp.strain PCC7120 depends on thefunction of the nir operon[J].Journal of Bacteriology,179(10):3368-3370
Kuritz T.1999.Cyanobacteria as agents for the control of pollution bypesticides and chlorinated organic compounds[J].Journal ofApplied Micorbiology,85(S1):186-192
Lambo A J,Patel T R.2006.Cometabolic Degradation of polychlorinatedbiphenyls at low temperature by psychrotolerant bacteriumHydrogenophaga sp.IA3-A[J].Current Microbiology,53(1):48-52
Li R,Carmichael W W,Brittain S,et al.2001.Isolation andidentification of the cyanotoxin cylindrospermopsin and deoxycylindrospermopsin from a Thailand strain of Cylindrospermopsisraciborskii(cyanobacteria)[J].Toxicon,39(7):973-980
李鑫,胡洪营,张玉平.2011.无机碳源对栅藻LX1生长及油脂积累特性的影响[J].环境科学,32(8):2260-2266
Manodori L,Gambaro A,Ivo Moret,et al.2006.Seasonal evolution ofgas-phase PCB concentrations in the Venice Lagoon area[J].Chemosphere,62(3):449-458
Meiger S N,Ockenden W A,Sweetman A,et al.2003.Globaldistribution and budget of PCBs and HCB in background surfacesoils:Implications for sources and environmental processes[J].Environmental Science&Technology,37(4):667-672
Morris P J,Mohn W W,Quensen J F,et al.1992.Establishment ofapolychlorinated biphenyl degrading enrichment culture withpredominantly meta dechlorination[J].Applied and EnvironmentalMicrobiology,58(9):3088-3094
Mouhamadou B,Faure M,Sage L,et al.2013.Potential ofautochthonous fungal strains isolated from contaminated soils fordegradation of polychlorinated biphenyls[J].Fungal Biology,117(4):268-274
Ohtsubo Y,Nagata Y,Kimbara K,et al.2000.Expression of the bphgenes involved in biphenyl/PCB degradation in Pseudomonas sp.KKS102 induced by the biphenyl degradation intermediate,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid[J].Gene,256(1/2):223-228
Ohtsubo Y,Kudo T,Tsuda M,et al.2004.Strategies for bioremediationof polychlorinated biphenyls[J].Applied Microbiology andBiotechnology,65(3):250-258
Pieper D H.2005.Aerobic degradation of polychlorinated biphenyls[J].Applied Microbiology and Biotechnology,67(2):170-191
Rippka R,Deruelles J,Waterbury J B,et al.1979.Genericassignments,strain histories and properties of pure cultures ofcyanobacteria[J].Microbiology,111(1):1-61
Sakai M,Ezaki S,Suzuki N,et al.2005.Isolation and characterizationof a novel polychlorinated biphenyl-degrading bacterium,Paenibacillus sp.KBC101[J].Applied Microbiology andBiotechnology,68(1):111-116
Seto M,Kimbara K,Shimura M,et al.1995.A novel transformation ofpolychlorinated biphenyls by Rhodococcus sp.strain RHA1[J].Applied Microbiology and Biotechnology,61(9):3353-3358
Sierra I,Valera J L,Marina M L,et al.2003.Study of thebiodegradation process of polychlorinated biphenyls in liquid mediumand soil by a new isolated aerobic bacterium(Janibacter sp.)[J].Chemosphere,53(6):609-618
Triscari-Barberi T,Simone D,Calabrese F M,et al.2012.Genomesequence of the polychlorinated-biphenyl degrader Pseudomonaspseudoalcaligenes KF707[J].Journal of Bacteriology,194(16):4426-4427
Wang J X,Chen L,Huang S Q,et al.2012.RNA-seq basedidentification and mutant validation of gene targets related to ethanolresistance in cyanobacterial Synechocystis sp.PCC 6803[J].Biotechnology for Biofuels,5:89
王立柱,温皓成,邹渝,等.2010.产油微藻的分离、筛选及自养培养氮源、碳源的优化[J].微生物学通报,37(3):336-341
魏中青,刘丛强,梁小兵,等.2007.贵州红枫湖地区水稻土多氯联苯和有机氯农药的残留[J].环境科学,28(2):255-260
Williams W A.1994.Microbial reductive dechlorination oftrichlorobiphenyls in anaerobic sediment slurries[J].EnvironmentalScience&Technology,28(4):630-635
Yu M L,Guo Y L,Hsu C C,et al.2000.Menstruation and reproductionin women with polychlorinated biphenyl(PCB)poisoning:longterm follow-up interviews of the women from the Taiwan Yuchengcohort[J].International Journal of Epidemiology,29(4):672-677
Zall D M,Fisher D,Garner M Q.1956.Photometric determination ofchlorides in water[J].Analytical Chemistry,28(11):1665-1668
Zhang H J,Hu C M,Jia X Y,et al.2012.Characteristics ofγ-hexachlorocyclohexane biodegradation by a nitrogen-fixingcyanobacterium,Anabaena azotica[J].Journal of AppliedPhycology,24(2):221-225
郑洪立,高振,张齐,等.2011.无机碳源对小球藻自养产油脂的影响[J].生物工程学报,27(3):436-444