Analysis

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    ;J.Genet.Genomics35(2008)41-48

    ;AnalysisofDNAmethylationindi

    ;JOURNAL0

    ;GENE?CS

    ;GENOMCS

    ;www.jgenetgenomics.org

    ;rentmaizetissues

    ;YanliLu,TingzhaoRong,MojuCao

    ;MaizeResearchInstituteofSichuanAgricultureUniversity~KeyLaborator

    yofCropGeneticResourceandImprovement

    ;Ministo,ofEducation,Ya’an,625014,China

    ;Receivedforpublication26April2007;revised20July2007;accepted21July

    2007

    ;Abstract

    ;DNAmethylationplaysanimportantroleingeneexpressionregulationdurin

    gbiologicaldevelopmentandtissuedifferentiationin

;plants?Thisstudyadoptedmethylation—sensitiveAmplifiedfragmentleng

    thpolymorphism(AFLP)tocomparethelevelsofDNAcyto—

    ;sinemethylationatCCGGsitesintassel,bractealleaf,andearleaffrommaizeinbredlines,18Whiteand18Red,

    ;respective1v.anda1so

    ;examinedspecificmethylationpatternsofthethreetissues.Significantdifferencesincytosinemethylationlevelamongthethreetissues

    ;andthesamechangingtendencyintwo1nbredlinesweredetected. ;BothMSAPrmethylationsensitiveamplificationpolymorphism)ratio ;andfullmethylationlevelwerethehighestinbractealleaf,andthelowestintasse1.Meanwhile,differentmethylationlevelswereob—

    ;servedInthesametissuefromtheinbredlines,18Whiteand18Red. ;Fullmethylationofinternalcytosinewasthedominanttypeinthe ;maizegenome?lhedifterentialmethylationpatterns1nthethreetissueswereobserved.Inaddition.sequencingofninedifferentiallvme—

    ;thylatedfragmentsandthesubsequentblastsearchrevealedthatthecytosinemethylated5CCGG一3sequencesweredistributedinre—

    ;peatlngsequences,inthecodingandnoncodingregions.Southemhybridizationwasusedtoverifythemethylationpolymorphism.These

    ;resultsclearlydemonstratedthepoweroftheMSAPtechniqueforlarge—scal

    eDNAmethylationdetectioninthemaizegenome,andthe

    ;complexityofDNAmethylationchangeduringplantgrowthanddevelopmen

    t.Thedifferentmethylationlevelsmayberelatedtospecific ;geneexpressioninvarioustissues.

    ;Keywords:DNAmethylation;MSAP;isoschizomers ;Introduction

    ;DNAmethylationinvolvestransferofmethylgroups ;fromS—adenosine—L—methioninetocytosinesandade—

    ;nines,byDNAmethyltransferases,afterDNAduplica—

    ;tion.InlivingcellsDNAmethylationhasbeenreported ;asoneofthemostcommoncovalentmodificationsof ;DNA.andhasbothepigeneticandmutageniceffects ;causingspecificgeneexpression,celldifferentiation, ;chromatininactivation,embryogrowth,andcancer ;(Courtiereta1.,1995;GonzalgoandJones,1997;Yuanet ;a1.,2004).Inprokaryotes,DNAprotecttheirownfrom ;disturbancebyforeigngenesthroughmethylation,andin ;eukaryotesmethylationmainlyparticipatesingeneex—

    ;pressionregulationtoadiustplantgrowthanddevelop一

    ;Correspondingauthor.

    ;E—mailaddress:caomj@sicau.edu.cn

    ;ment(Suneta1.,1999).DNAmethylationisnecessary ;fornormalplantgrowth.Ifthemethylationlevelisin. ;sufficient.itwilldisplayabnormalitiesofbiologicalde—

;velopmentandphenotype(Ronemuseta1.,1996;Finne—

    ;ganeta1.,2000).Indifierentstagesofplantgrowth,the ;changesinDNAmethylationlevelplayanimportantrole ;inresponsetovariationofitsheredityandtheenviron—

    ;ment(Richards,1997;Holliday,1987).DNAmethyla—

    ;tionmaysuppressgeneexpression.Activelytranscribed ;sequencesareoftenfoundtobelessmethylatedthanthe ;promotersandcertaincodingregionsofsilentgenes ;(Finneganeta1.,1993).Demethylationmaymakesome ;silentgenesactiveandexpressive,forexample,plant ;vernalizationresultsinDNAdemethylationthatinduces ;oracceleratesflowering(ShermanandTalbert.2002; ;Huaeta1..2005).DNAmethylationisalsoconnected ;withtransgenesilencing.Lieta1.(2001hasdemon. ;stratedthatmethylationofthepromoterregionresultsin ;一

    ;

    ;42YanliLuetal/JournalofGeneticsandGenomics35(2008)4i8 ;uidAgenesilencingintransgenictobacco(Nicotiana ;tabacumL.).Meanwhile,asignificantdifierencein ;theDNAmethylationlevelexistsamongvarioustis—

    ;suetypesinseveralplantspecies,suchas,tomato

    ;(SolanumtuberosumL.)(Messeguereta1.,1991), ;maize(ZeamaysL.,(Lundeta1..1995).andrice ;(OryzasativaL.)(Dhareta1.,1990),implyingthat ;DNAmethylationmayalsofunctionintissueororgan ;diflferentiationandformation.

    ;MethodsfordetectingDNAmethylationhavebeen ;steadilyimprovingalongwiththestudyofmethyla. ;tion.Inrecenttimes,therehavebeenavarietyof ;techniquesusedtoanalyzeDNAmethylationsuchas ;highperformanceliquidchromatography(HPLC), ;bisulfitesmethod,methylationspecificPCR(MSP), ;sequencingofspecificgene,andmethylationsensi—

    ;tiveamplificationpolymorphism(MSAP)(Wuand ;Zhu,2004).MSAPcombinedwiththeappealing ;featuresofamplifiedfragmentlengthpolymorphism ;fAFLPisbasedontherestrictionenzymeandPCR ;amplification.andishighlyeflficientfor1arge.scale ;detectionofcytosinemethylationinthegenome. ;UsingtheMSAPmethod,Cerveraeta1.(2002)have ;estimatedtheextentofgenomicmethylationanddis—

    ;tinguishedtenecotypesinArabidopsisthaliana. ;Zhangeta1.(2006)havecomparedpatternsofDNA

    ;methylationbetweenhaploidsandcOrrespOnding ;diploidsinrice.andhavefoundthatmethylation ;mutationscoverthewholericegenomeandare

    specific.MSAPhasalsobeenusedbyLueta1.. ;site—

    ;(2005)toassessdynamicchangesofcytosineme. ;thylationduringrapeseed(BrassicanapusL.)ger- ;minationandtocomparethelevelofDNAmethyla—

    ;tioninvarioustissuesfromseedlingstage(Lueta1.. ;2005).Bothmethylationanddemethylationevents ;aredetectedduringseedgerminationanddifierent ;levelsofDNAmethylationareobservedinvarious ;tissues.Inaddition.MSAPisextensivelyappliedin ;manyareastoinvestigateassociationsbetweenme—

    ;thylationandplantphenotypicinstabilityunder ;variousconditions(Xueta1.,2000;Labraeta1.. ;2004).andperformancesofhybridsfXiongandXu ;1999:Yieta1..2005a).

    ;Inthisstudy,weanalyzedDNAcytosinemethyl—

    ;ationinCCGGsitesofdifierentmaizetissuesusing ;thetechniquesofMSAPW_ealsoisolated.sequenced. ;andverifiedsomefragmentsthataredifierentially ;methylatedamongdifierenttissues.Themaioraimof

;thisworkistoexplorethecharacterizatiOnandregu—

    ;larityofDNAmethylationduringplantgrowthand ;development.Theresultsofthisstudyalsopro. ;videevidenceforabetterunderstandingoftherole ;ofDNAmethylationinspecificgeneexpressionin ;varioustissues.

    ;Materialsandmethods

    ;PlantmaterialsandDNAextraction

    ;Tissuesoftasse1.bracteal1eaf,andearleafwereco1. ;1ectedseparatelyfromtwoinbredlines(i.e..18ite ;and18Redofmaize.whichweremainminedatthe ;MaizeResearchInstituteoftheSichuanAgriculture ;University.GenomicDNAwasextractedusingthe ;CTABmethod(Gu1998).

    ;MSAPanalysisofDNAmeth),lation

    ;Thisexperimentadoptedanimprovedtechnique. ;MSAPwhichwassimilartoAFLPexceptthatthe”fre—

    ;quentcutter”enzymeMseI.wasreplacedbyisoschi.

    ;zomersHpaIIandMspI.whichdisplayeddifferential ;sensitivitytoDNAmethylation(VosandHogers.1995: ;XiongandXu.1999).TheMSAPsystemconsistedof ;threemajorparts,digestionandligationreactions,pream-

    ;plificationandselectiveamplificationreactions,andde. ;tectionreactions.Thedesignedadaptersandprimersfor ;EcoRIandHpaII.MspIlistedinTab1e1wereasde. ;scribedbyXiongeta1.(1999),withsomemodifications. ;Digestionandligationreactions

    ;DNAsampleswereseparatelyrestrictedwithEcoR ;I/HpaIIandEcoRI/MspI(TaKaRa.Dalian.China).To ;minimizediscrepancycausedbyexperimentalfactors, ;digestionand1igationwereperformedsimultaneously. ;Thedigestion—ligationreactionwasperformedinavol—

    ;umeof25ULcontaining250ngDNAtemplate,3UEcoRI, ;3UHpaII(orMspI),1.5UT4DNAligase(TaKaRa. ;DalianChina)5pmolEcoRIadapter.50pmo1HpaII/ ;MspIadapter.and2.5BL10×T4.1igasebu虢The

    ;mixturewasincubatedat37.Covernight.inactivatedat ;65.Cfor10min.andstoredat-20.C.

    ;Preamplificationandselectiveamplification ;PreamplifiedPCRreactionswereperformedinafinal ;volumeof20Lcontaining2BLofligationproducts,40 ;ngofEO0andHM00Preamplifiedprimer(Table11,1U ;ofTaqpolymerase,1.6ULofdNTPs(2.5ml’noI/Leach),

    ;1.2LofMgC12(251TI1TIOI/L),and2Lof10×PCR

    ;buffer.DNAfragmentswereamplifiedfor25cyclesof ;94.Cfor90s,56.Cfor30s,and72.Cfor1min.priorto ;selectiveamplification.ThePCRproductfromthepre. ;amplificationwasdiluted1to20(v:v)withddH,O,and ;avolumeof1.5uLofthesedilutedsampleswasmixed ;withthePCRbu仟er,40ngofE00+3,andHM00+3se.

    ;1ectiveprimer,whichhadthreeadditionalselectivenu. ;cleotidebasescomparedtothepreamplifiedprimerf11a一

    ;

    ;YanliLueta1./JournalofGenericsandGenomics35r2008)41-钳

    ;ble1),1UofTaqpolymerase,1.6LLLofdNTPs(2.5 ;mmo1/Leach),l-2LLLofMgC12(25mmol/L),and2LLL ;of10×PCRbu佰erinafinalvolumeof20LLL.ThePCR ;conditionswereasfollows:l3cyclesat94.Cfor30s. ;65.Cfor30s(reducedbv0.7.Ceachcycle),72.Cfor ;1min;23cyclesat94.Cfor30s.56.Cfor30s.and ;72.Cfor1min.

    ;1.ab1e1

    ;Adapterandprimersequences

    ;Detectionassay

    ;Fivemicrolitersofselectiveamplifiedproducts, ;mixed1:1fv:v)witha1oadingbuffer,wereheatedat

    ;95.Cfor5minandquicklychilledonice.Theentire ;mixturewasloadedontoa4.5%denaturingpolyacryla—

    ;midege1.ElectrODhOresiswasperformedataconstant ;powerof75Wfor1.5h.Aftersilverstaindetection. ;statisticalanalysiswascarriedout.

    ;CloningandsequencingofMSAPfragments

    ;Ninefragmentswereremoveddirectlyfromdried ;polyacrylamidegelsontheplateusingarazorblade.The ;fragmentswererelaydratedwi山50LofddH2O.heatedat

    ;95.Cfor5min.andslowlycooleddowntoroomtem—

    ;perature.Thetubeswerecentrifugedat12,0o0gfor10 ;min.Aliquotsof5Lsupematantwereusedastemplates ;forreamplification.PCRreactionswereperformedwi山

    ;thesalTleprimercombinationsandreactionconditionsas ;usedinselectiveamplification.Aftercheckingthe1.5% ;agarosegel,thebandwasrecoveredwithagelextraction ;kit(Promega,Madison,USA),accordingtothemanu—

    ;facturer’sinstructions.Subsequently.theproductofre—

    ;coverywasligatedintothevectorpMD18一TfTaKaRa.

    ;Dalian.China).andtransfclrmedintoanEscherichiacolf ;strain,DH5a.Sequencedeterminationswerecarriedout ;atInvitrogen(Shanghai,China).Homologysearchand