铬铜和镍对人工湿地硝化细菌与反硝化细菌群落结构分布的影响靳振江

2013,22(12):[e-mail](413610544116300341273142)(2011GXNSFF018003)(2011GXNFSA0180062010GXNSFA013018)(0701K012)KH2012ZD004(1974)[e-mail]*1963E-mail:[e-mail]2013-06-19*1.5410042.5410043.54100430~15cmamoAnirK(PCR)PCR–(PCR–DGGE)amoAnirK3(8.60±2.75)×106·g–1137.76±42.16×106·g–115amoA73a8nirK3IIIamoA(r-0.870-0.884-0.898)amoA-(r0.8920.940)amoA(r=0.887)nirK(r-0.818-0.820-0.860nirK(r=0.917)X171.5A1674-5906201312-1936-09.[J].,2013,22(12):1936-1944.JINZhenjiang,HUANGHaitao,LIUJie,LINHua,ZHANGXuehong.EffectofCr,CuandNiondistributionsofnitrifyinganddenitrifyingbacteriacommunitiesinaconstructedwetland[J].EcologyandEnvironmentalSciences,2013,22(12):1936-1944.[1-2][3][4][5-6][5-7][7][8][9-10][11-12][13-14][11,15-18]3(LeersiahexandraSwartz)1937[5]amoAnirK(realtimePCR)PCR–(PCR–denaturinggradinentelectrophoresis,PCR–DGGE)(wavysubsurfaceconstructedwetland,W–SFCW)[5]××=2.00m×1.00m×0.70m0.20m1.80m30.60m0.40m0.10m0.30mpH6.9w()28.0g·kg–1b()11.9cmol·kg–1w(K)97.8mg·kg–1w(N)150.0mg·kg–1w(P)9.5mg·kg–11(S1)2(S2)3(S3)1aCrCuNi82.0%96.8%94.0%3(1)320~15cm2mmMoBioDNADNA(genomicDNA)iCyclerIQ5ThermocyclerBio–rad,Hercules,amoAnirKDNA(BioPhotometer,Eppendorff,)260nm25μLPCR2×SYBRGreenrealtimePCRmastermix(Biouniquer,)12.5μL9.5μLDNA(genomicDNA15ng·μL–1)1μL20μm·μL–11μLamoARotthauwe[19](ammoniamonooxygenase)332349802822amoA–1F5′-GGGGTTTCTACTGGTGGT-3′amoA-2R-5′CCCCTCKGSAAAGCCTTCTTC-3′KGTSGCamoA94℃2mins3094℃1min,60℃1min72℃1min72℃5minamoADNA1.96×1013.06×10553amoA98.2%r20.95nirKHenry[20](nitritereductase)nirKnirK-8765′-ATYGGCGGVAYGGCGA–3′nirK-1040(5′-GCCTCGATCAGRTTRTGGTT-3′)YCTVGCARGAnirKamoAnirKDNA3.2×1015.02×10553nirK96.3%r20.99amoAnirK90.589.5℃2·g–1Table1Changeofheavymetalsinthreestagesofconstructedwetlandw()/(mg·kg–1)w()/(mg·kg–1)/%CrCuNiCrCuNiCrCuNiS1169.10(98.02)a47.78(10.71)b56.79(0.03)b3.64(2.43)a6.56(5.31)ab3.15(0.57)a2.09(0.23)b17.37(0.85)a5.54(1.00)aS2202.30(36.85)a57.00(1.56)a78.36(0.66)a5.85(1.74)a8.46(0.14)a3.96(1.32)a2.86(0.34)a14.83(0.65)a5.04(1.64)aS354.94(4.61)b29.83(0.32)c29.24(0.41)c0.23(0.05)b6.97(0.48)b2.22(0.05)b0.42(0.05)c23.37(1.37)a7.61(0.29)a3(P0.05)19382212201312amoAamoA-1/amoA-2RnirKnirK-876/nirK-104040GC5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3′[21]amoA-1FnirK-8765′PCR25μL12.5μL2×MasterMix20μm·μL–1GC1μLDNA15ngPCR94℃2min3094℃1min55℃1min72℃1min72℃5minPCR1.5%3%Bio-RAD475Model475GradientDeliverySystemamoA45%~65%100%7M40%8%37.5160℃140V8.5hnirK50%~70%60℃160V5h3min10min23minBio-RADGelDoc-2000QuantityOne(Bio-RadLaboratories,U.S.A.)(operationaltaxonomicunit,OUT)–Margalef[2][22]–(H′)=1-lnsiiipp=Margalef(DMg)=()NSln1−(E)=SHln′(Ds)=1-=siip12S(OperationalTaxonomicUnits,OTUs)NOUTpi=ni/NiOUTDGGEDNA1.5mL4℃DNAPCRPCRPCRDNA(EZgenecyclepurekitBiomiga)(pEASY–T3cloningkitTransGen)SangonNCBI[23]Chu[10]nirKThrobäck[24]MEGA4.1neighbor–joiningmethodJTTmatrix-based1000amoANCBIGenBankJQ689969-JQ689982nirK200bpGenBankMicrosoftExcel20033SPSS13.0OneWayANOVA(P0.05)Pearson(P0.05)2amoA3(8.60±2.75)×106·g–11Table2AbundancesofamoAandnirKinconstructedwetlanddeterminedbyreal–timePCRw(DNA)/(μg·g–1)amoAnirK(DNA×102)/(·ng–1)(×106)/(·g–1)(DNA×103)/(·ng–1)(×106)/(·g–1)S14.56(2.18)b4.99(1.20)b2.15(0.54)b4.36(1.40)b19.59(7.57)bS29.73(2.23)a1.65(0.49)c1.32(0.43)c0.58(0.13)b5.78(1.15)cS39.10(1.81)a7.87(2.07)a8.60(2.75)a14.97(1.65)a137.75(42.16)a1939(2.15±0.54)×106·g–12(1.32±0.43)×106·g–13amoAr–0.870–0.884–0.898P0.0240.0190.01531122323amoA3232321~435~9210~13114335~911~1273a1411~41013623amoAMargalef3–32amoA–(r0.8920.940P0.0170.005)(r=0.887P=0.018)2nirK3(137.75±42.16)×106·g–11(19.59±7.57)×106·g–12(5.78±1.15)×106·g–13nirK(r–0.818–0.828–0.860P0.0470.0460.028)43nirK232nirK32353232eg3a~df2h3(6)dVI7II3nirK3-Margalef12nirK(r=0.917P=0.010)S1-1S1-2S2-1S2-2S3-1S3-2S1S2S32Fig.1DGGEprofileofamoAgeneinthreestagesofconstructedwetlandFig.2ClusteranalysisofamoADGGEprofile19402212201312图3用邻接法把研究中得到amoA序列翻译为氨基酸序列后构建的进化树，每个节点基于1000次重复，大于50%的值显示在图上，黑体的克隆是本研究中产生的序列，括号中为登录号Fig.3Neighbor-joiningphylogenetictreesofpartialamoAgenestranslatedintoaminoacidsequencesfromDGGEbands.Percentbootstrapvaluessupportingmorethan500(of1000)iterationsareshownatthenodes.Clonesfromthisstudyareshowninbold.AccessionnumbersfortheamoAgenesareshowninparenthesesNitrosospirasp.L13,AJ238542Nitrosospiramultiformis,U15733w709,AF353253Band12(JQ689980)Band7(JQ689975)Band8(JQ689976)Band11(JQ689979)Band6(JQ689974)Band5(JQ689973)Band9(JQ689977)siEASt25c27,AY177967KMSt37c18,AY249662AF056067Nitrosospirasp,AJ298685Herbi100-90-13,AY010094Nitrosospirasp.N120,AJ298719Band3(JQ689971)w2613,AF354830RR60H5,AJ238544Band4(JQ689972)Band13(JQ689981)RR60-H6,AJ238545RR90-7,Z97840W627,AF353250OMSLFt30b24,AY249747W923,AF353259PabII3.2,AJ388582Nitrosospirasp.O13,AJ298722Nitrosospirasp.O4,AJ298723Nitrosospirasp.L115,AJ298698NAB-8-C11,AF056069Nitrosospirasp.Nsp12,AJ298716Nitrosospirasp.40KI,AJ298687Nitrosospirasp.III2,AJ298694Nitrosospirasp.NpAV,AF016003,U20644RR90-3,Z97838RR45-4,Z978