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2006,12(1):128130ChinJApplEnvironBiol=ISSN10062687X200620222531,23313311,214(1;2030006)(3310029;4650106),,,RNAPCRnorthern,.412;RNA;;CLCQ343.3EstablishmentofSystematicalMethodsforTranscriptionalExpressioninPlantMitochondria3PEIYanxi1,233,HAOJianping1,CHENZhujun3,CAOJiashu3,MAWenli1,LIUXiaohui1,2,FANSanhong1&CHENXuejun4(1CollegeofLifeSciencesandTechnology,2InstituteofBiotechnology,ShanxiUniversity,Taiyuan030006,China)(3CollegeofAgricultureandBiotechnology,ZhejiangUniversity,Hangzhou310029,China)(4KeyLabofBiotechnology,AcademyofYunnanTobaccoScience,Kunming650106,China)AbstractUsingtubermustardcytoplasmicmalesterilelineasstudymaterial,routinemethodsfortranscriptionalexpressionresearchweremodifiedandimprovedbasedontheexistinginformation.Themethodsforplantmitochodriaisolation,RNAex2traction,reversetranscription,PCRandNorthernblotwerecombinedsystematically,andthosemethodswereallappliedtostudygenetranscriptionalexpressioninplantmitochondria.Fig4,Ref12Keywordsplantmitochondria;RNA;reversetranscription;cytoplasmicmalesterileCLCQ343.3(CMS),.CMS,.,[1,2]..,RNARNA.,.,[36];,RNA,[7].RNAK[8],Tr2izolÒ(Gibco2BRL)RNAgents(Promega).,,.,,.1:2004212215:20052032173(No.30571195)SupportedbytheNa2tionalNaturalScienceFoundationofChina33Correspondingauthor(E2mail:peiyanxi@sxu.edu.cn)1.126710d,.1.2RNATrizolÒSuperscriptTMGibico/BRL;Taq2DNAdNTPs6N.232P2dATP;MILLIPORE;.1.3KembleRJ[4]()[9].RNaseDNaseI;[3],,:(2%,400mmol/Ll00mmol/LNaH2PO4,pH7.4),lh.17000g5min,(400mmol/L,100mmol/LNaH2PO4,pH7.4),17000g,5min,3.1%,.1%2h,50%70%80%85%90%100%10min.ØEpon8123Ø12Ø11h2h;Epon812,,JEM21200EX,H2600.RNATrizolÒ,12h..1.4RNARNA,100ngL-1Oligod(T)2L,100ngL-16N3LRNA5L(200ng),755min;5RT6L,0.1molL-1DTT3L,20nmolL-1dNTPs3L,2LMMLV(200U/L),RNasin1L,6LDEPC2H2O30L,422h,65.Oligod(T)6N.1.5cDNAPCRcDNA5LT[10]PCR.945min;9430s,5845s,7290s,38;7210min.P1P2,P1P3,1170bp830bp.1.6NorthernT830bp,RNANorthern.Northern[8].Typhone8600.22.1RNACMS(1),,,.RNA(2).1Fig.1Electronmicroscropicphotoofmitochondriaintubermustard2CMSRNAFig.2MitochondrialRNAoftubermistardCMSline2.2RT2PCRCMSRNA,Oligod(T)6N.,P1P21170bp,P1P3830bp(3),.RT2PCR,.3cDNAFig.3ResultofamplificationusingmicochondrialcDNAastemplatebydifferentprimerpairs1:P1P21170bp;2:P1P3830bp1:PCRproduct,1170bp,usingP1P2asprimerpair;2:PCRproduct,830bp,usingP1P3asprimerpair2.3TRNANorthernT,RNANorthernblot(4).,,,RNA,.4CMSRNANorthernFig.4NorthernblotanalysisofCMSmitochondrialRNA3,,.,,,,.,;RNA,,,.,,DnaseI,RnaseDnaseI;RNATE(DEPC),12h,RNA.RNA,,.,.mRNA,poly(A),mRNApoly(A)[11,12].RNA,Oligod(T)6N,,,RNA9211:.,T[10].RNAT,.,Oligod(T)6N,.References1AbadAR,MehrtensBJ,MackenzieSA.Specificexpressioninrepro2ductivetissuesandfateofamitochondrialsterility2associatedproteinincytoplasmicmalesterilebean.PlantCell,1995,7:271852BonhommeS,BudarF,LancelinD.SequencesandtranscriptanalysisoftheNcoOgura2specificfragmentarecorrelatedwithcytoplasmicmalesterilityinBrassicahybrids.MolGenGenet,1992,235:340483ParasonsDF,BonnerWD,VerboonJG.Electronmicroscopyofisolatedplantmitochondrialandusingboththethin2sectionandnegative2stainingtechniques.CanaJBot,1965,43:6476554KembleRJ.Aradid,singleleafnucleicacidassayfordeterminingthecytoplasmicorganellecomplementofrapeseedandrelatedbrassicaspe2cies.TheorApplGenet,1987,73:3643705MignounaH,VerminSS,BriquetM.MitochondrialDNAmodificationsassociatedwithcytoplasmicmalesterilityinrice.TheroApplGenet,1987,74:6666696LeaverCJ,HackE,FordeBG.Proteinsynthesisbyisolatedplantmito2chondria.MethEnzymol,1983,97:4764847Jean2ClaudeFarr,AlejandroAraya.RNAsplicinginhigherplantmito2chondria:determinationoffunctionalelementsingroupIIintronfromachimericcoxIIgeneinelectroporatedwheatmitochondria.PlantJ,2002,29(2):2032108SambrookJ,FritschEF,ManiatisT.MolecularCloning.NewYork:ColdSpringHarborLaboratoryPress,19899ChenXJ(),ChenZJ(),ZhangYZ().Ex2tractionofmitochondrialDNAfromcytoplasmicmalesterileandmaintai2nerlineoftubermustard.JZhejiangUniv(AgriLifeSci)((),2000,26(3):32132410PeiYX(),ChenZJ(),CaoJS().Cytoplas2micmalesterilityoftubermustardisassociatedwiththealternativesplicedmitochondrialTgenetranscripts.ChinSciBull(),2004,49(23):2312231711PhilippeG,AxelB.Fromgenetoproteininhigherplantmitochondri2a.CRAcadSci,2001,324:20921712GagliardiD,LeaverC.PolyadenylationacceleratesthedegradationofthemitochondrialmRNAassociatedwithcytoplasmicmalesterilityinsunflower.EMBOJ,1999,18:37573766031ChinJApplEnvironBiol12