应用16srdna克隆文库技术研究长期稻草还田对水稻土细菌多样性的影响

2010,30(13):38653874ActaEcologicaSinica(40771115);(KZCX22YW2BR201);(2008BADA7B07):2009205229;:20092072273Correspondingauthor.E2mail:[e-mail]16SrDNA1,2,1,1,1,1,1,1,3(1.,,410125;2.,430070):(CK)(OM),16SrDNADNA,16SrDNA,,(CK),(OM),(CK)(OM),;,16SrDNA,,2:16SrDNA;;;;Effectoflong2termfertilizationbytheapplicationofricestrawonbacterialdiversityinpaddysoilWANGXia1,2,CHENZhe1,YUANHongzhao1,WUMinna1,WEIWenxue1,WUJinshui1,QINHongling1,31KeyLaboratoryforAgro2ecologicalProcessesinSubtropicalRegion,InstituteofSubtropicalAgriculture,CAS,Hunan410125,China2CollegeofResourcesandEnvironment,HuazhongAgriculturalUniversity,Wuhan430070,ChinaAbstract:Paddysoilbacterialcommunitystructurewasstudiedthrough16SrDNAgene.Thesoilsampleswerecollectedfromthelong2termfertilizationexperimentfieldattheTaoyuanAgro2ecosystemResearchStation.TotalmicrobialDNAwasdirectlyextractedfrombothtreatmentsincludingamendedwithricestraw(OM)andCK.Theclonelibrarieswereconstructedaftercloningandsequencing.Theresultshowedthatthebacterialcommuntiycompositionofthepaddysoilamendedwithricestraw(OM)waschangedsignificantlycomparedwithCK,bothShannonindexandevennessdecreasedindicatingitsbacterialdiversitywasdecreased.Proteobacteriawerepredominatedbothinricestraw(OM)andCKtreatments,followedbyAcidobacteria.ApplicationofricestrawinducedobviousdecreaseofthegenotypesofProteobacteria,Verrucomicrobia,ChloroflexiandChlorobicomparedtoCK,inthecontrast,thegenotypesofNitrospiraincreased.Thephylogenetictreebasedonthe16SrDNAsequencesshowedthatapplicationofricestrawvariedthedistributionofAcidobacteriagreatest,followedbyVerrucomicrobiaandDelta2Proteobacteria.Therefore,applicationofricestrawhadanimportantimpactonbacterialcommunitystructure.KeyWords:16SrDNA;ricestraw;bacterialdiversity;phylogeneticanalysis[1],[224],[5][627],1945Dawson[8],2008BIOLOG[9],,[10],,,,1%10%[11212],16SrDNA,,[13214][15]16SrDNA2RFLP,[16]16SrRNA,,,,DNA,,,DNA,16SrRNA,,11.1(11072E,2852N),16.15,1447.9mm,1531.4h,102.14.187kJ/cm2,199015.4gkg-1,1.88gkg-1,0.60gkg-1,12.8gkg-1,16.2mgkg-1,74.3mgkg-1,pH5.74,24207:1)NPK(CK);2)NPK+(OM)OM1990,10cm,4221kg/hm2,5005kg/hm23,,4.1m8.1m19902006NPK1119902006(kg/hm2)Table1Fertilizerapplicationlevelsindifferenttreatmentsduring19902006TreatmentFertilizerNPK2)RiceStrawNPKCK72.9(112.5)1)39.3(26.2)70.2(56.0)OM63.5(112.5)39.3(26.2)70.2(56.0)42.28.7120.6CK109.4(150.0)0(13.09)127.0(80.9)OM118.8(150.0)0(13.09)127.0(80.9)46.210.4128.0:1)19901996,1996;2)NPK1990200620073(),,5,2cm,215cm,,(200g),-80,;(500g),2mm4,2668330(g/kg)37.21b47.16aSoilAvailablePhosphorus/(mg/kg)15.00b22.15aSoilAvailablePotassium/(mg/kg)43.01b152.90aSoilAvailableNitrogen/(mg/kg)132.42b182.09aMBC/(mg/kg)1026.2b1232.94aMBN/(mg/kg)79.45b116.13aTotalNitrogen/(g/kg)1.90b2.40aTotalPhosphorus/(g/kg)0.62b0.74apH5.10a5.05a00.25mm1.162.21Contentofdry0.250.5mm0.691.27sievedaggregate0.51mm1.933.5212mm1.11.5625mm7.811.515mm87.3279.92:P0.05LSD,1.2DNAProteous[17][18],SDS2GITC2PEG,0.5g2mL,20,215000g,10min1.3PCR16SrDNA,3510(Agrobacteri2um,Alcaligenes,Azospirillum,Azotobacter,Bacillus,Flavobacterium,Paracoccus,Propionibacterium,Pseudo2monas,Thiobacillus)ClustalWAlignment,:52ACAYTGGDACTGAGACACGG2352GATTACTAGCGATTCCRRCTTC231050bp50LPCR10PCR(plusMg2+)5L;10mmol/LdNTP2L,10mol/L2L;TaqDNA2U;100ng;50LPCR:95,5min;40:9530s,5045s,721min;7210minPCREB1%1.4(WizardSVgelandPCRclean2upsystem,Promega)16SrDNA,pGEM2TVectorSystem(Promega),(Escherichiacoli)(Ampicillin)/IPTG/X2GalLB,371624h,4,PCRT7SP6PCR,DNA,1.5NCBI(),,CHECK2CHIMERA[19]16SrDNA,ClustalX1.83Mega4.0,(Neighbor-Joining)16SrDNAGenBank,FJ2650802FJ2655281.6Shannon2Wiener:H=-(Pi)(lnPi);:E=H/Hmax(Hmax=lnS)C=1-(n1/N)[20]LIBSHUFF,(),95%,P0.05,,P0.05[21]768313:16SrDNA(Operationaltaxonomicunit,OTU)[22](CK)(OM)15016SrDNA,134111OTU16SrDNA(3),(CK)(OM)Shannon4.654.56,0.990.97,316SrDNATable3Diversityindicesofbacterial16SrDNAclonelibrariesfromCKandOMTreatmentCloneOTUsShannonShannonIndicesEvennessIndicesCK1501344.650.99OM1501114.560.97Libshuff,16SrDNA(CK)(OM)(P0.01),2.216SrDNA416SrDNATable4Sequenceanalysisof16SrDNAclonesfromCKandOMItemCKcloneNo/%OMcloneNo/%Proteobacteria634270472Proteobacteria171132212Proteobacteria149642Proteobacteria53642Proteobacteria27182617Acidobacteria29193121Verrucomicrobia171185firmicutes1491913Chloroflexi7521Gemmatimonadetes4321Actinobacteria1111Bacteroidetes3253Nitrospirae2143Planctomycetes3243Chlorobi2100Unclassified5343Total15010015010016SrDNAGenBank,(4),(CK)(OM)(Proteobacteria),42%47%(Acidobacteria)(Proteobacteria),,(CK)(OM)(Acidobacteria)19%21%,(Acidobacteria)2%,(CK)10%(Verrucomicrobia),11%,(OM)10%(Firmicutes),13%(Chloroflexi),(Gemmatimonadetes),(Actinobacteria),(Bacteroidetes),(Nitrospirae),(Planctomycetes)(Chlorobi)(CK)(OM),10%,(Chlorobi)(OM),(CK)(OM)3%GenBank,,4,5%,210%,25%,,6%,,,,4%,4%868330(CK)(O