烟草科技TobaccoScience

/TobaccoScience&Technology/TobaccoAgronomy200811(256):(2006GG22):(1972-),,:,3:E2mail:[e-mail]:2008-06-05:E2mail:[e-mail]:03712676726501,2,1,2,1,131.,26500912.(),650202:;;;:K326,,:;,,,,,:TS444;TS414:A:1002-0861(2008)11-0051-05IdentificationandPhylogenesisAnalysisofCultivableMicroorganismsonTobaccoLeafSurfaceDuringAgingYANGJIN2KUI(1),DUANYAN2QING(2),CHENCHUN2MEI(1),LIQING2HUA(2),HUANGJING2WEN(1),andZHANGKE2QIN(1)1.LaboratoryforConservationandUtilizationofBio2resources,YunnanUniversity,Kunming650091,China2.TechnologyCentreofHongyunTobacco(Group)Co.,Ltd.,Kunming650202,ChinaKeywords:Flue2curedtobacco;Aging;Microorganism;PhylogenesisAbstract:Thecultivablemicroorganismswereisolatedfromtheleafsurfaceofflue2curedtobaccocv.K326sampledatdifferentagingstages,andthesystematicstatusofthesemicroorganismsandtheirphylogeneticrelationshipwereanalyzedwithmolecularbiologicalmethod.Theresultsshowedthattheamountandracesofcultivablemicroorganismsonleafsurfaceobviouslydecreasedwiththeprolongationofagingtime.ThemajorbacteriaareBacillussp.andEnterobactersp.;andBacillussp.isthedominant.Moreover,thereiscertainrelativitybetweentheamountofbacteriaandthestalkpositionoftobaccoleaf,namely,theamountofbacteriaonmiddleleafislargerthanthatonlowerleaf,andthatonupperleafisthesmallest.However,thereisnoobviousrelativitybetweentheamountandraceoffungiandthelengthofagingtimeorstalkpositionoftobaccoleaf.,,,,,[122],[326],,,15,,16SrRNA(ITS)[728],,[4,9212]K326,,,11.11.1.120073(),11.1.2PfuDNApolymeraseTaqDNApolymerase(Takara);DNA();();3730(ABI);BP2211D(0101g,);()1.1.3:E.coliDH5(Takara);:pMD182T(Takara)1.21.2.1[13],,3,DNADNAPCR1()()S1B3F6S6X3F18S2C3F6S7B3F30S3X3F6S8C3F30S4B3F18S9X3F30S5C3F18:;K326,16SrRNA(ITS)PCR,[728]16SrRNA20F(5-AGAGTTTGATCCTGGCTCAG-3)1500R(5-GGTTACCTTGTTACGACT-3)[14];(ITS)ITS4(5$TCCTCCGCTTATTGATATGC$3)ITS5(5$GGAAGTAAAAGTCGTAACAAGG$3)[15]PCRpMD18-T,E.coliDH5,PCR,1.2.216SrRNAITS,GenBank(BLASTsearch),ITS16SrRNAClustalX[16]ITS16SrRNAITS16SrRNA,MEGA4.0[17]Neighbor-Joining[18],1000Bootstrap[19]22.1,9K326DNA,20F1500R16SrRNA,PCR1500bp(1A)ITS4ITS5ITS,PCR500bp(1B)16SrRNAITSpMD18-T,DNAman16SrRNAITS,16SrRNAITSGenBank,,9(Bacillussp.)(Paenibacillussp.)(Enterobactersp.)(Citrobactersp.)(Erwiniasp.)(Pantoeasp.)6,(Aspergillussp.)(Trichodermasp.)(Rhizopussp.)25(Fusariumsp.)4,2,9,K32697.5%,(6)33,181,K326,[9],(6),,(2),6618,,430,18,4,,618;9,,,K326,,,(2)6,(2.5%),3,,,,18,,30,K326:A16SrRNAPCR,MDNA,12PCR;BITSPCR,1,2,3,45PCR1(16SrRNA)(ITS)PCR2K326(/g)(/g)(/g)S11.12105B(5.5104);P(2.8104);C(1.8104);Er(9.4103)R(0.9103)S23.31105B(1.2105);P(9.3104);E(5.9104);C(3.9104);Er(2104)R(1.0103)S31.95105B(8.5104);P(4.4104);Er(1.5104);Pa(5.1104)A(0.9103);T(0.1103)S41.79104B(9103);P(3103);E(3103);Er(2.4103)F(4.5102)S52.42104B(1.8104);P(6.2103)S62.15104B(1.5104);E(6.7103)S71.58104B(9103);P(4.9103);E(1.8103)S82.31104B(1.5104);P(4.5103);E(3.2103)S92.23104B(1.2104);P(6103);E(2.7103);C(1.6103):B.(Bacillussp.);P.(Paenibacillussp.);E.(Enterobactersp.);C.(Citrobactersp.);Er.(Erwiniasp.);Pa.(Pantoeasp.);F.(Fusariumsp.);R.(Rhizo2pussp.);A.(Aspergillussp.);T.(Trichodermasp.)35,2.2GenBank,16SrRNAITS,Clustal[14],K326,23(2),();(),4(3),,,3K326,,,K326,[10211],,,,[426,10212,20],,,,,,,,,,,,,[9210],[9210],,[1].[M].,.:,1993.[2],,,.[J].,2003(6):63266.[3]FrankenburgWC.Studyonthefermentationofcigarleaf45tobacco[J].ArchBiochem,1947(14):1572181.[4],,,.[J].,1998(1):6.[5],.[J].,2007,23(1):3142318.[6].[J].,2001(4):729.[7],.ITS[J].,2007,35(13):378523786.[8],,.16SrRNA[J].,2006,27(11):15218.[9],,,.[J].,2000(3):14217.[10],,,.[J].,2001(2):26230.[11],,,.[J].,1997(4):13214.[12],,,.[J].,1998,20:5092512.[13].[M].:,2006.[14]WeisburgWG,BarnsMS,PelletierAD,etal.16Sribo2somalDNAamplificationforphylogeneticstudy[J].JBacteriol,1991,173:6972703.[15]WhiteTJ,BrunsT,LeeS,etal.AmplificationanddirectsequencingoffungalribosomalRNAgenesforphylogenetics[M].AcademicPress,SanDiego,1990:3152322.[16]ThompsonJD,GibsonTJ,PlewniakF,etal.TheClastalXwindowsinterface:flexiblestrategiesformultiplesequencealignmentaidedbyqualityanalysistools[J].NucleicAcidsRes,1997,24:487624882.[17]TamuraK,DudleyJ,NeiM,etal.MEGA4:Molecularevolutionarygeneticsanalysis(MEGA)softwareversion4.0[J].MolBiolEvol,2007,24:159621599.[18]SaitouN,NeiM.Theneighbor2joiningmethod:anewmethodforreconstructingphylogenetictrees[J].MolBiolEvol,1987(4):4062425.[19]FelsensteinJ.Confidencelimitsonphylogenies:Anapproachusingthebootstrap[J].Evolution,1985,39:7832791.[20],,,.[J].,2006,22:66270.2009()1957,:(CA)(TA)CAJ-CD(),,,(),20072564,10,23,1240::,,,,,,,:16,,20,,:ISSN1002-0861,:CN41-1137/TS,:36-33:41000040000602009,,,,:2:450001::0371-67672669:0371-67672668E2mail:[e-mail]55