水稻广陆矮四段的染色体荧光原位杂交分析

水稻广陆矮四段的染色体荧光原位杂交分析

在细节中，通过混合微标签。就像报告一样，在大多数微列表中，可以看到提供微列表的微列表。在细节中，微列表中的微列表是不需要注册的。数量和特征的数量是区域微列表。微列表中的特征数量是区域微列表中的特征数量。微列表中的特征数量和特征数量是区域微列表中的特征数量。微列表中的特征数量和特征数量是区域微列表中的特征数量。微列表中的特征数量和特征数量是区域微列表中的特征数量。微列表中的特征数量和特征数量是区域微列表中的特征数量。微列表中的特征数量是区域微列表中的特征数量。微列表中的特征数量是区域微列表中的特征数量。微列表中的特征数量是区域微列表中的特征数量。微列表中的特征数量是区域微列表中的特征数量。对于任何事情，都必须知道，微列表中的碳灰粉可以是微扩张的。ExtendedDNAfiberFISHhasbeendevelopedrapidlyinplants.Thistechniqueofferedusanopportunityforquantitativeanalysisoftelomeresequencesinthegenomeofrice.Therefore,inthisstudy,inordertoverifythesizedifferencesoftelomeresequencesamongchromosomes,wehaveperformedbothpachytenechromosomeandextendedDNAfiberFISHandpresentedcomparativeresultsbetweenthesetwokindsofFISHinrice(Oryza,sativassp.indicacv.-GuangluaiNo.4).1杏仁杏仁1.1“ge麻黄”4.3index,gulagliga.4,3g.4.4.4AsoneofthetestedbreedsinRGP(RiceGenomeProgram)internationalconsortium,Rice(Oryza,sativassp.indicacv.)“GuangLuaiNo.4”wasusedforFISHanalysis.1.2非定型主义双重身份,英国驳岸,英国,日本,香港,日本,香港,日本,香港,香港,有机地发展了ClonepAtT4,containingthehigheukaryotictelomeresequence5′-CCCTAAA-3′isolatedfromArabdopsisthaliana,waskindlyprovidedbyDr.Richards(WashingtonUniversity,USA).Theprobeswerelabeledwithdigoxigenin-11-dUTPaccordingtotheinstructionsofthemanufacture(Sino-AmericanBiotechnologyCompany,China).Thelabelingwascarriedoutat15℃for3-4h,then0.5mol/LNa2EDTAwasadded,andthelabelingresultantwassubsequentlysubjectedtopassthroughSepharoseCL-6B(Sigma)columnforpurification.Dotblottingwasperformedtodetectthelabelingefficiency.1.3改性玉米因子keptYoungpaniclesofthericecontaininganthersatvariousstagesofmeiosiswasharvestedandfixedinCarnoy’ssolution[100%ethanol:glacialaceticacid(3∶1)].Thesuitableanther(0.2mm)weredivestedfromspikeletsandkeptformacerationintheenzymesolutioncontaining2%pectolyase(SERVA)and2%cellulase(SERVA)at28℃forapproximately1.5h.Thentheenzymesolutionwasremoved,adropofdistilledwaterwasadded,andtheanthersweresmashedbytipsandcentrifugedat2000r/minfor5min,removedthewaterandaddedfreshCarnoy’ssolution,finally,thesuspensionwasdroppedtoslidesanddriedonflame.Theslideswerekeptin-20℃forFISH.1.4u3000centriforda-ro内部t.u-案例Nucleiwereisolatedfromfreshleavesof2-week-oldriceaccordingtoLiuandWhittier.Inshort,2goffreshleaftissueweregroundintothefinepowderinapre-cooledmortarandpestleinliquidnitrogen.Thepowderwasthentransferredtoa50mLcentrifugetubecontaining20mLice-coldnucleiisolationbuffer(NIB∶10mmol/LTris-HClpH9.5,10mmol/LEDTA,100mmol/LKCl,0.5mol/Lsucrose,4.0mmol/Lspermidine,1.0mmol/Lspermine,0.1%(v/v)mercaptoethanol).Aftergentleshakenonicefor5min,thehomogenatewasfilteredorderlythroughalayerofgauze,nylonmeshfilters(48,30μm)onice.Weomittedthe22-μm-nylonmeshfiltrationstepinordertoensurehighconcentrationofnuclei.Subsequently,about1mLNIBcontaining10%(v/v)TrionX-100wasaddedandthesamplewasthencentrifugedat2000r/minfor10minat4℃.Thepelletwasre-suspendedin200μLNIBattheconcentrationabout5×105nucleipermL,supplementedwithanequalvolumeof100%glycerolandstoredat-20℃.1.5dropletssiga标准Briefly,foroneslide,1μLofthenucleisuspensionwascentrifugedatroomtemperatureinamicro-centrifugeat3600r/minfor5min.Thepelletwasgentlyre-suspendedin4μLofPBS(10mmol/LsodiumphosphatepH7.0,140mmol/LNaCl).2μLdropletsofsuspensionwerepipettedontooneendofaPoly-L-lysineslide(Sigma)andair-dried.Then,8μLoflysisbuffer(0.5%(w/v)SDS,5mmol/LEDTA,100mmol/LTris(pH7.0))wasplacedonthenucleiandtheslideswereincubatedatroomtemperaturefor4min.DraggingwithacleancoverslipextendedtheDNAfibers.Theslideswerecompletelyair-dried,fixedin100%ethanol/glacialaceticacid(3∶1)for2min,andfinally,bakedat60℃for30min.Theslideswerestoredinaslideboxforuptoseveralweeksatroomtemperatureorat-20℃.1.6igmaeTheFISHofpachytenechromosomewasperformedaccordingtoapublishedprotocol,Fiber-FISHwasfollowedaccordingtoJacksonetal.whichwasmodifiedslightly.ForpachyteneFISH,50μLhybridizationmixturesincluding50%deionizedformamide(Sigma),10%sodiumdextransulphate(Sigma),2×SSC,10μgofsalmonspermDNAand20-40nglabeledprobes,wereusedforeachslide.ForFiber-FISH,15μLhybridizationmixtureswereusedforeachslide.Thehybridizationmixturewasdenaturedat100℃for10min,chilledoniceandplacedfor10min.Thenthehybridizationmixturewasaddedtotheslideandcoveredwithcoverslip,andtheprobeandtargetDNAweredenaturedinanovenat80℃for3min.Hybridizationwasperformedovernightforchromosomesor2-3dforDNAfibersat37℃inawetchamber.Theslideswerethenwashedin20%formamide,2×SSCand0.1×SSC,at42℃for3×15mineachstep,andsubsequentlywashedin0.1%ofTritonX-100,PBSatroomtemperature,5mineachstep.1.7-diamidwelling,6-diamid政府dapo和6-diamid底-2-phenyli数据soTheslidewascoveredwithmouseanti-dig(Roche),andsubsequentlytreatedwithDiganti-mouse(Roche).ThenRhodamineanti-dig(Roche)wasadded.Thesignalswereamplifiedbyimmunologicalreactionat37℃for30mineachandtheslideswerewashedwithPBS3×5minatintervals.Andfinallytheslideswerecounterstainedwith1μg/mL4’,6-diamidino-2-phenylindole(DAPI).ChromosomeswereobservedwithanOlympusBX60fluorescencemicroscopeequippedwithSensys1401ECCDcamera.Red,green,andblueimageswerecapturedinblackandwhitebackgroudswithdifferentfilters.TheimageswerecombinedandpesudocoloredinthecomputerusingsoftwareV++.2风险性能价值FISHtointerphasenucleusrevealedthatthetelomeresignalsweredispersedininterphasenucleus(Fig.1,A)withdifferentintensity.Fig.1Bdisplaysthat21of24endsoftwelvebivalentsshowedprominentsignaleachduringpachytenestage.Thedifferentsignalspotsonpachytenechromosomeswerenotthesameinsizeandintensity.WealsoknowintheRGBcolorscheme,thereare256possiblevaluesforeachofthered,green,andbluecomponentsofacolorpixelonacomputerscreen.InRGBnotation,eachcolorvaluerangesfrom0(nocolor)to255(fullcolor).Thevalue0indicatesthatthepixeliscompletelytransparent.Thevalue255indicatesthatthepixelisopaque.Thesemeanthatthevalue0indicatesnocontributionfromthisprimarycolorandthevalue255indicatesthemaximumintensityofthiscolorcomponent.Sotheredcolorvaluecanbeusedtorepresentthesignalintensity.Inthesamephoto,theredcolorvalueofthelargestsignalwas255(pixelintensity)andthesmallestwas60(pixelintensity).Thelargestonewas4.25timesofthesmallestoneinsizeandintensity.Mostofthesignalspotsvariedfrom94to222(pixelintensity)andtheaveragevalueofthesignalspotswas164.2±57.1.Itwasalsofoundthatthesignalsizewasnotrelatedtochromosomelength.Thelargesignalspotsshowedoneitherlongerorshorterchromosomes.Sodidthesmallsignalspots(Fig.1,B).Fig.1Cpresentsthatthesignalsappearedanextendedstringofbeadscontainingthreethrougheightbeadsrespectively.Thelongeststringofbeadsis6.55μm,whiletheshortestoneis1.82μmlong,whichareequalto16.44and4.56kbcorrespondinglybasedonastretchingfactorof2.51kb/μm.Theaveragevalueofsignallengthwas3.62±1.32μm,i.e.9.09±3.31kb.Basedonthesizeofthebasicunitoftelomeresequences[TTTAGGG]n,7bp,itcouldbeestimatedthatthesizeoflongestandtheshortestsignalsaswellastheaveragevaluewereequalto2349,651and1298±473respectivelyincopynumber.3whileindrain,while.3,3.4,4.3.4.3.4与3.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.ThelengthofthetelomereDNAsequencevariesnotonlyindifferentspeciesbutalsoindifferentvarietiesofonespecies.Forexample,Arabidopsisthalianatelomerelengthaverage2-4kb,tobaccotelomeresextendupto130kb,theaveragelengthsofthetelomererepeatswereestimatedat10.4kbforindicacv.IR8and3.14kbforjaponicacv.Nipponbarebasedonfiber-FISHresults.Inthepresentstudy,indicacv.GuangluaiNo.4telomeresaverageabout9.09±3.31kb,whichisdifferentfromindicacv.IR8.Itwasnotedthatthetelomererepeatarraywasdramaticallyvariedinawiderangeindifferentcultivars.Thesameresultswereshowedin22inbredlinesofmaize,thelengthoftelomererepeattractinleavesvariedmorethan20-fold,from1.8kbintheWF9lineto40kbintheCM37line.AccordingtotheresultsreportedbyJiangetal.,42of48chromosomeendsshowedsignaleach,thesignalswerenotdetectedonlyat6chromosomeends,whileinthisstudy,althoughthepachytenechromosomesweremorediscondensedandeasiertobehybridized,nosignalsweredetectedforafewendsofthebivalents.Itdemonstratedthatthereasonforafewchromosomeendsshowingnosignalsmainlywasnottheerrorinducedbyhybridizationtechniques,probablywasduetothecopynumberoftelomeresequenceswhichwastoolowtobedetectedbyroutineFISHtechniqueonthesechromosomeends.Asmentionedabove,thelargestsignalspotwas4.25timesofthesmallestoneinsizeandintensity,whilethehighestcopynumberwas3.6timesofthelowestonefortelomeresequences