11(1):1～7,2002
ChromosomalLocalizationofGenesbz1,bz2
inMaizebyUsingUltra2sensitiveFISHwithTyramideSignalAmplification(TSA2FISH)
LIZong2yun,NINGShun2bin,HANYong2hua,LIULi2hua,SONGYun2chun①
(KeyLaboratoryofMOEforPlantDevelopmentalBiology,WuhanUniversity,Wuhan430072,China)
Abstract:Ithasbeenreportedthatendospermundergoesprogrammedcelldeath(PCD)duringmaizekerneldevelopment.Bothbz1(bronze)andbz2areanthocyaninbiosyntheticgenes,andrelatedtode2velopmentofaleuroniclayerofmaizeseeds.Tyramidesignalamplificationfluorescenceinsituhybridiza2tion(TSA2FISH)isanovelandhighsensitiveFISHtechnique,whichissuitableforroutineapplicationinplantcytogeneticresearch.Usingthistechnique,wephysicallymappedthebz1geneontotheshortarmofchromosome9andthelongarmofchromosome1;thepercentagedistancesfromcentromeretohy2bridizationsitewere40.2,75.4respectively,andthebz2ontothelongarmofchromosome1andtheshortarmofchromosome5;thepercentagedistancesfromcentromeretohybridizationsitewere21.6,15.3separately.TheTSA2FISHtechniquesofsmalllowcopyDNAsequencesforplantsarediscussed.Keywords:programmedcelldeath(PCD);bronzegenes;tyramidesignalamplificationfluorescencein
situhybridization(TSA2FISH);maize
1 IntroductionProgrammedcelldeath(PCD)isanintegralpartofthedevelopmentofmulticellularorgan2ismsandischaracterizedbyageneticallydeterminedprogramthatcanorchestratecelldeath.Inplants,PCDhasbeenshowntooccurindiversetissues,inthediseaseresistanceinresponsetopathogenandpestattack,inmanyaspectsofplantreproductionanddevelopment.Inpreviousstudy,ithasbeenreportedthatendospermundergoesPCDduringmaizekernelorwheatseeddevelopment
[2]
[1]
.Cerealendospermiscomposedofthestarchyendospermandaleuroniclayer,and
functionsasstoragetissueinwhichthemajorityofstarchandseedstorageproteinsaresynthesized.Inthematurekernel,thestarchyendospermisanon2livingtissuewhereasthealeuroniccellsareviablebutundergoPCDwhengerminationistriggered
[2]
.
Received:2002-04-03;Accepted:2002-05-12
TheprojectweresupportedbytheNationalNaturalScienceFoundationofChina(No.30070376)andtheResearchFundforDoctoralProgramofHigherEducation(No.207980112)
①Correspondingauthor.E2mail:ycsong@whu.edu.cn
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
2Developmental&ReproductiveBiology Vol.11 No.1 2002
Bronze1,encodesUDPglucoseflavonoidglucosyl2transferase(UFGT),ananthocyaninbiosyn2theticenzymeinthealeuroniclayeroftheseed,whichmodifiespurplealeuroneandplantcolortopaleorreddishbrown,anthersyellow2fluorescent.Thebz1geneisarecombinationhotspotinthemaizegenome,andatleast100timesmorerecombinogenicthantheaveragesegmentofthemaizegenomeandrelatedtotransposonelementsgenesandmyc2likegenes
[7]
[3～6]
Ithasbeendemonstratedthatbz1expressioninem2
bryotissuesisdependentonC1andanR2scalleleofRthatsharehomologytothemybproto2onco2
.TheconsensusMYBrecognitionsequence(TAACTG)isalsofoundin
[8]
themaizebronze21promoterandothers.Bronze2,isanotheranthocyaninbiosyntheticgene,en2codesbz2productinmaize,it’spotentialfunctionareflavonoidacylation,glycosylation,transport,ordeposition,whichperformsthelastgeneticallydefinedstepinanthocyaninbiosynthesis,resultinginthedepositionofredandpurplepigmentsinthevacuolesofmaizetissues.ItisdemonstratedthataglutathioneS2transferaseinvolvedinvacuolartransferwasencodedbythemaizegenebz2
[9]
.
PhysicalmappingplaysamajorroleinthecloningofgenesandFISHisimportanttosuchstudies.Todate,variousDNAsequences,includingtotalgenomicDNA,rDNAgenes,repetitivese2quences,retrotransposonsequencesandlow2copysequences,YAC(yeastartificialchromosome),BAC(bacterialartificialchromosome),havebeenphysicallymappedonplantchromosomebyFISHtechnique.Inthepastdecade,theFISHtechnologyhasdevelopedsignificantlywithbothimprovedresolutionandsensitivity.Toloworsinglecopygenes,thedetectionraterelatedpositivelytothesizeofthecorrespond2ingprobe.Thedetectionrateswere10%～20%whenthesizesofprobeswerearound1Kb.Toim2proveFISHresolution,bothhigh2resolutionmicroscopyandfurtherdecondensationofchromatinhavebeenpursued,sopachytene2FISH,interphase2FISH,fiber2FISHtechniqueshavebeendevel2opedinrecentyears.ToimproveFISHsensitivity,differentapproacheshavealsobeendeveloped.Thefirstistheuseofcooledcharge2coupleddevice(CCD)camera.Withthistechnique,thedetec2tionsensitivitycanbeincreasedabout302fold.ThesecondisprimedinsituDNAlabeling(PRINS),whichisanalternativetoFISHwhereunlabeledoligonucleotideshybridizedtothetargetsequencesservedasprimersforchainelongationinsitucatalyzedbyaDNA2polymeraseinthepr2esenceoflabelednucleotidesplantchromosomes
[11]
[10]
.ThismethodhasbeenusedinlocalizationofDNAsequenceson
.ButthedetectionlimitforPRINSisabout20kbpertargetandthistechnique
hasahighbackground.ThethirdisTyramide2FISH,thismethodbasedonthedepositionoffluoro2chrome2labeledtyramidebyhorseradishperoxides(HRP).Horseradishperoxidase(HRP)reactswithhydrogenperoxideandthephenolicpartoflabeledtyramidetoproduceaquinine2likestructurebearingaradicalontheC2group,this“activated”tyramidethencovalentlybindstotyrosineresi2duesinclonevicinityoftheHRP,thusdepositingmanylabeledtyramidecloselytotheprobethatcarriestheHRPreporter,directlyorindirectly
[12]
.
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
LIZong2yunetal:Chromosomallocalizationofgenesbz1,bz2...3
[12]
IntroductionofTSAinimmunocytochemistryrepresentsanimportantmilestone.In1995,
[13]
RaapreportedgreatincreaseinFISHsignalintensityfollowingvariousTSAstrategies
[14]
.Since
then,Tyr2FISHtechniquewassuccessfullyusedinhumancDNAcloneandsingle2copygenedetection
.Anexpressedsequencetags(ESTs)fromsixgenesweresuccessfullylocalizedbyfi2
[14]
[15]
.VanGijlswijketal(1997)demon2
ber2FISHbyuseofsensitivetyramide2baseddetection
[16]
stratedthatthedetectionsensitivityofTyr2FISHcouldbeincreasedupto100timescomparedtosomeexistedtechniques.Khrustalevaetal
.(2001)demonstratedthatthistechniquewaspossi2
bletodetecttargetT2DNAsequencesonplantmetaphasechromosomesassmallas710bpwithoutusingacooledCCDcamera.TheyalsoindicatedthattheTyr2FISHmethodwassuitableforroutineapplicationinplantcytogeneticresearchduetoitsshortdetectiontime,highsensitivity,andlowbackground.Hereweusedultra2sensitiveFISHwithtyramidesignalamplification(TSA2FISH)forlocatingbz1andbz2geneinmaize.
2 MaterialsandMethods
2.1 Plantmaterialandprobes
Maize(ZeamaysL.)inbredlineHuangZao4,whichderivedfromanativecultivarinChi2na,wasusedasthetestedplantmaterial.SeedswereprovidedbyprofessorSongJiancheng,Shan2dongAgriculturalUniversity,Shandongprovince.Bothofthetestedprobesbz1,bz2wereclonedinvectorpUC19,kindlyprovidedbyProf.Coe(UniversityofMissouri,USA).Bothare1500bpand300bpDNAfragmentsinsizerespectivelyandtherestrictionlociwerepst1.Theprobeswerela2beledwithBio2112dUTPfollowingNicktranslationprotocolofferedbythekit(Sino2AmericanBio2technologyCompany).
2.2 Preparationofmetaphasechromosomes
Theroottipsof1～2mmlengthfromvigorouslygrownmaizeandfixedimmediatelyinethanol2aceticacid(3∶1)at4℃forovernight.Aftercompletelywashingwithdistilledwater,theroottipsweretreatedwithamixtureof2%pectinase(SERVA)and2%cellulase(SERVA)at28℃forapproximately2.5～3h.Finally,thetreatedroottipsweresubjectedtoahypotonictreatmentindistilledwater,andthensquashedonslidesandflame2driedfreezerbeforeuse.
2.3 Fluorescenceinsituhybridization(Tyr2FISH)
InsituhybridizationwasperformedaccordingtotheproceduredescribedbySongetal
[17]
[17]
.Theslideswerekeptin-20℃
,
withsomemodifications.Theslidepreparationsweresubmergedin1%H2O2inPBSfor30min,and
μthenpretreatedwith100gΠmlRNasein2×SSC(0.3Msodiumchlorideplus0.03molΠLsodiumcitrate)at37℃for1h,rinsedbrieflyin2×SSC.ChromosomalDNAwasthendenaturedbyim2mersingtheslidesin70%deionizedformamidein2×SSCat70℃for5min.Afterdehydrationin
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
4Developmental&ReproductiveBiology Vol.11 No.1 2002
μanice2cold70%,95%and100%ethanolseries,50lhybridizationmixturewith50%deionizedformamide(Sigma),10%sodiumdextransulphate(Sigma),2×SSC,10μgofsalmonspermDNA,and20～40nglabeledprobes,wasusedforeachslide.Thehybridizationmixturewasdena2turedat100℃for10min,chilledoniceandplacedfor10min.Hybridizationwasperformedover2nightat37℃.Theslideswerethenwashedin20%formamide,2×SSC,0.1×SSC,at42℃for3
×15mineachstep,andsubsequentlywashedin0.1%ofTritonX2100,PBSatroomtemperature,5mineachstep.
2.4 Visualizationofhybridizedprobes
Thebiotin2labeledprobesweredetectedwithstreptavidin2HRPaccordingtotheprotocoloftheTSA2directkit(NENLifeScienceProducts,Boston,USA).SlideswerewashedinfreshTNTbuffer(0.1molΠLTrisHCl,0.15molΠLNaClpH7.5,0.05%Tween20)atRTfor5min,andthenincu2batedinTNBbuffer(0.1molΠLTrisHCl,0.15molΠLNaCl,0.5%blockingreagent)atRTfor30mininahumidchamber.Afteraboveblockingstep,theslideswereincubatedinstreptavidin2HRPfor30minatRT.Thetyramidedetectionsolutionwaspreparedbythoroughlymixinga1∶50tyramide2CY3stocksolutioninamplificationdiluents(NENLifeScienceProducts,Boston,USA).
μThe100ltyrmide2CY3solutionwaspipettedontotheslides,incubatedatRTfor3～7min,andthenwashedthreetimesfor5mininTNTatRT.Followingabriefwashin2×SSC,andfinallythe
μslideswerecounterstainedwith1gΠml4′,62diamidino222phenylindole(DAPI)containing.Chro2mosomeswereobservedwithanOlympusBX60fluorescencemicroscopeequippedwithSensys1401ECCDcamera.Redandblueimageswerecapturedinblackandwhitewithdifferentfilters.TheimageswerecombinedandpesudocoloredinthecomputerusingsoftwareV
++
.
3 Results
AftercounterstainedbyCY3andDAPI,thechromosomesappearedblueandthesignalsshowedred.Thetestedprobesbz1andbz2bothshowedtwodifferenthybridizationsites.Thetestedprobebz1washybridizedontothemiddleregionof9S(theshortarmofchromosome9)andthedi2stalendof1L(thelongarmofchromosome1)(Fig.1A,B),andthesignalofprobebz2couldbedetectedontheregionnearthecentromereof1L(thelongarmofchromosome1)andthedistalendof5S(Fig.1C,D).Thepercentagedistancefromthesignaltocentromerewasmeasured.Thecal2culationoftheaveragesoftheparameterforeachdetectedchromosomewaspresentedasTable1.
Table1 TheresultsofFISHofbz1andbz2inmaize
Probe
bz1bz2
Chromosomearm
9S1L1L5SNo.ofdetection
160136129108Detectionrate(%)
48.630.931.320.0Percentdistancefromthecentromere(FL%)
40.275.421.615.3 S:Shortarm;L:Longarm
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
LIZong2yunetal:Chromosomallocalizationofgenesbz1,bz2...5
Fig.1 TheFISHsitesinmaizechromosomeswithgenesbz1(A,B),bz2(C,D).
A,B:Twosites(redsignals)ofgenebz1aredetectedontheshortarmofchromosome9(arrow)andthelongarmofchromosome1(arrowhead)inmaize;C,D:Twosites(redsignals)ofgenebz2arede2tectedonthelongarmofchromosome1(arrow)andtheshortarmofchromosome5(arrowhead)inmaize.Bluefluorescenceshowsthechromosomesstainedwith
DAPI.Redsignalsshowgenesbz1,bz2sitesinmaizechromosomes.
4 Discussion
Thephysicalpositionsofbz1in9Sandbz2in1Lcoincidewiththoseintheirgeneticmappattern
[5]
.Thesignalsofbz1andbz2werealsodetectedon1Land5Srespectively.Itmeansthat
theyareduplicatedsequences.Thedetectionratesarenotthesameamongthesignalsofbz1and
bz2aseachother(Table1).Thedetectionrateofbz1ishigherthanthatofbz2.Thismaybedue
tothelengthofprobebz1islongerthanthatofbz2.Generallyspeaking,themoreisthehomology,thehigherisdetectionrate
[18]
.Thedetectionrateofbz1in9Sishigherthanin1L,andthedetec2
tionrateofbz2in1Lishigherthanin5S.Theseresultsindicatethatbz1andbz2havehomologoussequenceson1Land5S,andthedegreeofhomologyofprobesin9Sand1Lishigherthanthatin
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
6Developmental&ReproductiveBiology Vol.11 No.1 2002
1Land5S.
Themaizegenomecontainsextensivechromosomalduplication.Ithasbeenshownthatdupli2catedsequencesinmaizegenomearearrangedonchromosomesfollowingthemodesof52129,32826and102227,whichmeanschromosome1sharessimilaritywithbothchromosomes5and9,etc
[19,20]
.
Inpresentstudy,bothbz1andbz2showtwodifferenthybridizationsites.Thismeansthattheyareduplicatedsequences,IthasbeendemonstratedthatmanyRFLPmarkersareduplicatedatleastoncesomewhereelseinthemaizegenome.Comparativemappingstudieshaveidentifiedroughly10
[21,22]
duplicate(orhomologous)chromosomalregionsinmaize.Theextentofchromosomalduplica2
tionandpatternofcolinearitysuggeststhatmaizehasapolyploidoriginandcanbeusedtobetterunderstandtheorganizationandevolutionofthemaizegenome
[20,23]
.
Inthisstudy,wewerealsoabletodetecta300bptargetsequencewithafrequencyof31.3%byCCDcamerawiththismethod.Itindicatesthatthedetectionleveloflow2andsingle2copyhy2bridizationsignalscanbesignificantlyincreasedbyTyr2FISHmethod.References:
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利用高灵敏的TSA2FISH在玉米
中定位bz1、bz2基因
李宗芸,宁顺斌,韩永华,刘立华,宋运淳
(武汉大学植物发育生物学教育部重点实验室,武汉430072)
①
摘 要:植物中,程序性细胞死亡(PCD)发生在植物生殖和发育的许多方面,已有的研究表明,在玉米种子的发育过程中,胚乳组织经历了程序性细胞死亡的过程。bz1(bronze)和bz2是与种子的糊粉层发育相关的花青素生物合成基因,在玉米基因组中,bz1基因所在区域是重组热点,bz2与类黄酮的酰化、糖基化、转运、沉积等有关,基因的物理定位有利于基因的分离和克隆。TSA2FISH(Tyramidesignalamplificationfluorescenceinsituhybridization)是一种新颖的高灵敏度的荧光原位杂交技术,它的主要反应原理是辣根过氧化物酶催化过氧化氢和标记的酪胺分子(tyramide)的苯环部分反应,使荧光标记的酪胺分子在直接带有或间接带有
HRP报告分子的探针周围沉积,信号因此得以极大的放大,从而大大提高了荧光原位杂交技
术的灵敏度,90年代中期开始引入动物和人类组织化学和细胞遗传学研究中,2001年才应用于植物细胞遗传学的研究。利用这一技术,我们将bz1基因定位于玉米的第9染色体的短臂和第1染色体的长臂上,其信号点距着丝粒的百分距离分别为40.2,75.4;bz2基因定位于玉米的第1染色体的长臂和第5染色体的短臂上,其信号点距着丝粒的百分距离分别为
21.6,15.3。本文讨论了TSA2FISH技术在植物中小的、低拷贝的DNA序列定位上的应用。
关键词:程序性细胞死亡(PCD);bronzegene;TSA2FISH(酪胺信号放大的荧光原位杂交Tyra2
midesignalamplificationfluorescenceinsituhybridization);玉米
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