··研究论文
GeneCloningandSequenceAnalysisof
inTomatoFruit*
YUBian-Yun
ZHUBen-Zhong
LUOYun-Bo**
and
(CollegeofFoodScienceandNutritionalEngineering,ChinaAgriculturalUniversity,Beijing100083,China)
Ethylene-responsiveelementbindingproteins(EREBPs)aremembersofafamilyofplanttranscriptionfactors,which
containahighlyconservedDNAbindingdomainknownastheERF(ethylene-responsivefactor)domain.Apairofdegenerateprimersweredesignedintheconserveddomain(ERFdomain)whichbasedonthealignmentofEREBPsgenefamilyintomato(
)andotherplantspecies.
Server.Thesequencesimilarityofof
to
and
tomato(
gene
Twonewgenes
to
and
and
wereisolatedbyusing114bpfragmentamplifiedbywas35%attheaminoacidlevel.Theseguencesimilarity
and
cDNAsequencesweredepositedin
gene
RT-PCRasaprobeforscreeningtomatofruit(pinkstage)cDNAlibraryandidentifiedasmembersofEREBPsfamilybyBLAST
was46%attheaminoacidlevel.The
Genbank(GenBankaccessionnumberAY077626andAY275554respectively).
);ethylene-responsiveelemenbindingproteins;screeninglibrary;
番茄果实中基因的克隆及序列分析*
蔚变云朱本忠罗云波**
（中国农业大学食品学院，北京100083）
摘要：乙烯反应元件结合蛋白属于植物特有的一个转录因子家族，这个家族保守的DNA结合域称为ERF结构域。根据对番茄(
在保守区（ERF区域）)和其它植物物种中EREBP基因家族的同源性比较，设计合成一对简
和和
和与基因；和基因
[1]
[3]
并引物，通过RT-PCR扩增得到一个114bp的片段，用此片段作探针筛选番茄cDNA文库(粉红期)，得到两个基因
。通过BLAST工具在GenBank中搜索表明,
在氨基酸水平上的序列相似性为35%，基因。
和
关键词：番茄；乙烯反应元件结合蛋白；筛库;
基因属于EREBP家族，与
在氨基酸水平上的序列相似性为46%，是新的
的核酸序列在GenBank发表，登录号分别为AY077626和AY275554。
EREBPs(ethylene-responsiveelementbinding
proteins)havebeenkeyresearchsubjectswhichwerefirstisolatedasGCCboxbindingproteinsfromtobacco[1].TheGCCboxisaconservedcis-elementinlargenumberofthepromoterregioninPRgenes.AnumberofEREBPsgenefamilyhavebeenisolatedfromdiverseplantspecies,suchastobacco
(No.39825118)andNationalScienceFundationofChina(No.30270934).YuBianyun:Female,ph.Dstudent,E-mail: , [2] ,tomato [4] ,,soybean [5][6],tobacco,DREbindingprotein,[7][8],and.EREBPscontain ahighlyconservedDNAbindingdomainnamedastheERF(ethylene-responsivefactor)domain,consistingof58or59aminoacids[1,9],whichhasnovelcharacters *SupportedbytheStateKeyBasicResearchandDevelopmentPlan(973)ofChina(No.G1999011701),NationalOutstandingYouthofChina PDF 文件使用 \"pdfFactory Pro\" 试用版本创建 www.fineprint.cn 第2期YUBian-Yunetal:GeneCloningandSequenceAnalysisofandinTomatoFruit133 bothinitsstructureandinitsmodeofDNArecognition,representalargemultigenefamilywithmanymembersinplantsandareanewclassoftranscriptionfactorsuniquetoplantswithoutsequencehomologywithknowntranscriptionfactorsorDNAbindingproteins.AlthoughanethylenesignaltransductionpathwayfromEIN3toERF1toPR ,asimilarproteinshasbeenestablishedin routefromEIN3toEREBPstofruitripeningandsenescencehasnotbeenelucidatedintomato[10],whichisamodelplantinfruitandvegetableripeningandsenescenceresearches.Tofurtherunderstandtheethylenesignaltransductionandmechanismoffruitripening, twonewmembersofEREBPsfamily,andwereisolatedfromtomatofruit. sequenceofthealignment,degenerateprimersforward 5'GC(A/G)GA(G/A)ATTCG(C/T/G)GA(C/T)CC(A/G)AC3'andreverse5'ACT(C/T/A)CCTCT(G/A)AG(C/T)CTAAA(T/A)GC3'weredesignedtoamplifytheERFdomainfromtomatofruitviaRT-PCR. TotalRNAusedforRT-PCRwaspreparedfrompinktomatofruit[11].ReversetranscriptionreactionwasconductedaccordingtotheinstructionofPromega(USA).PCRwasperformed94益,53益,72益for1minforeachof35cyclesfollowinga5minincubationat94益,andcompletedwith10minincubationat72益.ThePCRproductwasrecoveredwithDNARecoveryKit(Sangon,Shanghai)andlabeledby琢-32PwithRandomprimerDNALabelingKit(TaKaRa,Dalian). Inthisstudy,wedescribedtheisolationandsequence and.characteristicof 1MaterialsandMethods The琢-32PlabeledPCRfragmentcontainingthe codingsequenceforERFdomainwasusedasahybridizationprobetoscreenthecDNAlibrary.HybridizationreactionwasperformedaccordingtoChurchandGilbertmethods[12].Afterhybridization,themembraneswerewashedtwicewith2伊SSC,0.1%SDSat65益for15min,oncewith1伊SSC,0.1%SDSat65益for10min.FilterswereexposedtoX-rayfilmwithanintensifyingscreenat原70益for2~3days.Afterthreecyclesofscreening,positiveplaqueswereexcisedwiththehelperphageandrecircularizedtogenerateasubcloneinthepBK-CMVphagemidvector.TheentirenucleotidesequenceofthecDNAwassequencedbyBioAsiaBiotechnologyCompany.ThesequencesobtainedwereeditedtoremoveanyvectorsequenceviaVecscreen(www.ncbi.nlm.nih.gov).DNAsequencedatawereanalyzedusingDNAMAN(Version4.0)andNCBIBLASTserver(www.ncbi.nlm.nih.gov/blast).AtomatoZAPExpresscDNAlibrarywasconstructedfromtomato()fruit(pinkstage)RNA(constructedbyInstituteofGeneticsandDevelopmentalBiology,CAS).pGEM-Teasyvector,ReverseTranscriptionKit,T4ligase,Restrictionenzyme(RⅠ,Ⅰ)andDNApolymerasewerepurchasedfromPromega(USA).DNARecoveryKitwastheproductofSangon(Shanghai)BiotechnologyCompanyandRandomprimerDNALabelingKitwaspurchasedfromTaKaRaCompany. NucleotideacidsequencesofcDNAscontainingtheconservedEREBPDNA-bindingdomain(ERFdomain)isolatedfromseveralplantspeciesweresearchedviaBLAST(www.ncbi.nlm.nih.gov/blast)andalignedwiththeDNAMAN(Version4.0)program.Thesesequencesincludetobacco (GenBankaccessionNo.D38123,D38126,D38124,D38125respectively),tomato(GenBankaccessionNo.U89255,U89256,U89257respectively) (GenBankaccessionNo.and AB008103,AB008104,AB008105,AB008106,AB008107respectively).Fromtheconsensus 2Results A114bpPCRfragmentwasobtainedwith degenerateprimerstotheconservedERFdomainofEREBPsfromdiverseplants.ThePCRfragmentwas PDF 文件使用 \"pdfFactory Pro\" 试用版本创建 www.fineprint.cn 134 农业生物技术学报2004年 ligatedtopGEM-Teasyvector,thenscreenedbyblueandwhitecolonies.Thepositivecolonywasculturedforplasmidextraction.Theplasmidwasidentifiedby RⅠdigest(Fig.1).ThePCRproductwassequencedbyBioAsiaBiotechnologyCompany.ThesequencewasidentifiedcorrectlybybeingalignedwiththecodingsequenceforERFdomain WiththePCRfragmentamplifiedaboveasaprobe,aZAPExpressvectorclonedcDNA(pinktomatofruit)librarywasscreened.Afterthreecyclesofscreening,twopositiveplaqueswereobtained(Fig.2).ThepositiveplaqueswereexcisedwiththehelperphageandrecircularizedtogenerateasubcloneinthepBK-CMVphagemidvector.Subsequentlyinsertcharacterizationinaplasmidsystemwasdoneby RⅠandⅠdigest(RⅠandⅠadapterwereaddedtothecDNAendswhenlibrarywasconstructed).Insertsizeofoneclonewasapproximately1.1kbwhichwasnamed.Theotherinsertionapproximately2.8kbwasnamed (Fig.3).Thededucedtranslationproductsofand containedtheERFdomain(Fig.4).ThecDNAseemedtobefulllengthbecause(i)it containedanopenreadingframeinitiatedbyanATGand(ii)itwasterminatedbyapoly(A)tail.Toolong3'-untranslatedregionofthecDNAmightbetheresultofrecombinationofandtheothercDNA.Thecompletesequenceofconsistedofapredictedopenreadingframefor204aminoacids Fig.1.RT-PCRamplificationandidentificationoftheconserved fragment M,DL2000marker;1,PCRproduct(templatecDNAfrompinktomatofruit);2,PCRproduct(templateDNAfrompGEM-TeasywithPCRproduct);3,pGEM-TeasyinsertedwithPCRproduct;4,pGEM-Teasy (insertedwithPCRproduct)digestedby RⅠ. Fig.2.PositiveplaqueinscreeningcDNAlibrary A.Firstscreening;B.Secondscreening;C.Tertiaryscreening Fig.3.Identificationof by RⅠ(A)anddigest M,DL2000marker;1,pBK-CMV-digestedbypBK-CMV-digestedby andⅠ(B) RⅠ;2,plasmid;3,pBK-CMV-RⅠ;4,plasmid .pBK-CMV- PDF 文件使用 \"pdfFactory Pro\" 试用版本创建 www.fineprint.cn 第2期YUBian-Yunetal:GeneCloningandSequenceAnalysisofandinTomatoFruit135 withapredictedmolecularmassof23.2kDandpIof8.96.Thepredictedtranslationproductofofopenreadingframecontained210aminoacidswithapredictedmolecularmassof23.0kDandpIof8.57.AsillustratedbythesequencealignmentinFig.5,eachof andhadaconserved58aminothe acidsERFdomainwithahighdegreeofsimilaritytotheERFdomainofEREBPsfromtheotherplantspecies. andhadtwohighlyconservedmotifsreferredtotheYRGandRAYDelementsintheERF domainrespectively.TheWAAIERDmotifintheYRGelementmightberesponsibleforDNAbindingsequencespecificity.TheRAYDelementcontainedaconservedcoreregionwhichwaspredictedtoformanamphipathic琢-helix.Howtheamphipathic琢-helixinvolvesinthe DNAbindingwasuncertain[13].AlthoughtheERFdomainswerehighlyconservedinplantspecies,theaminoacidsequencesflankingtheERFdomainswerehighlydivergentwhichmightberesponsibleforthespecificfunction.TheERFdomainscontainedshortconservedsequenceelementsthatwereconsistentwiththeirfunctionastranscriptionfactors.Thesesequenceelementsincludedaputativenuclearlocalizationsignal(NLS)whichwascomposedofashortclusterofthe andbasicaminoacidresiduesRK(R/K)[14]. alsocontainedaserine-richsequenceand acidicdomain,bothofwhichwerecharacteristicoftranscriptionactivationdomain. 3Discussion Fig.4.cDNAsequenceanddeducedaminoacidsequenceof (A)and (B) TheERFdomainisindicatedbygraybox.TheUnderlinesshowtheputativenuclearlocalizationsignals.Theacidicdomainandserine-rich domainaremarkedbydashes. AnumberofEREBPshavebeenidentifiedfromseveralplantspecies.Theseincludetobacco~[1],~[2],tomato~[3], [4][5],soybean,tobacco [6] ,DREbindingproteinsDREB1andDREB2[7],and [8] .Theycontainahighlyconserved DNAbindingdomain―ERFdomainwhichisnovelbothinstructureandinitsmodeofDNArecognition.Itiscomposedofa茁sheetandan琢helixandthe茁sheetinteractingmonomericallywiththetargetDNA[15].ThesequencesimilarityoftheseEREBPsisrestrictedintheERFdomain.ThelimitedsimilarityoutsidetheERFdomainsuggeststhepossibilityofdifferentmodeofactivationanddifferentrecognitionsequenceofeach PDF 文件使用 \"pdfFactory Pro\" 试用版本创建 www.fineprint.cn 136 农业生物技术学报2004年 Fig.5.AlignmentoftheERFdomainsofandwithERFdomainsfromotherplantEREBPs SequenceswerealignedwithDNAMANprogram.Blackboxesindicateaminoacidresiduesidenticaltotheconsensussequenceshownbelowthealignment.Dotsindicategapsusedtooptimizealignment.ThealignmentproteinsincludedtomatoPTI4/516(GenBankaccessionNo.U89255,U89256 andU89257respectively),tobaccoEREBP4(GenBankaccessionNo.D38125), ERF1(GenBankaccessionno.AF076278),tomato DDTFR10/A(GenBankAccessionNo.AF204784) EREBP[1].IthasbeenreportedthattheERFdomainiscloselyrelatedtotheAP2domain[16].However,ithasbeenindicatedthattheAP2andERFdomainsbelongtodistinctfamilies.AP2containstwoDNAbindingdomains,whereasERFcontainsonebindingdomain.TheAP2domainislikelytopossessamodeofDNArecognitiondistinctfromthatoftheERFdomainaswellasadifferentDNAtargetsequence[2].Inthispaper,andwereisolatedfrompinktomatofruit.ThesetwogenesbelongedtotheEREBPsfamily,becausetheencodedproteinscontainedoneERFdomain,incontrasttoAP2-likeproteinswhichcontainedtwoDNAbindingdomains.ButtheERFdomainistheonlypartofthetwoproteinsthatexhibitsignificantsequencehomologywithmanyknownproteins.Moreover,andcontaintheWAAIERDmotif,whichisnotfoundinAP2-likeproteins[17]. AllknownAP2membersareinvolvedinplantdevelopment[13],whereastheEREBPsarelikelytobeinvolvedinresponsestobioticandabioticstresses[17~19].Analysisofthepromoterregionsofethylene-induciblePRgeneshasledtotheidentificationofanethylene-reponsiveelement(ERE),whichcontainsaconservedAGCCGCCmotif,designatedastheGCCbox[9].AmongtheEREBPs,tobacco~[1], [4]~[2],,tomato~[3], [5] andsoybeanhavebeenshowntohaveGCCboxspecificbindingactivity.SomeEREBPsbind specifically to the DRE/CRT element (dehydration-responsiveelement)andrepeatsequencecontainingthecoresequencePuCCGAC[7]whichisinvolvedinregulatinggeneexpressioninresponsetolowtemperature,highsaltordroughtstresses.ThesesequencesresembletheGCCboxandcontainCCGNCasacommoncoresequence.TheseEREBPsinclude [7,20] .，，and However,tobaccocanbindspecificallytotheGCCandDREsequences[6].FurtherstudiesareneededtodeterminewhetherandbindtotheGCCboxand/orDREelementortheothercis-element.Intomato,organ-anddevelopment-speci-ficregulationofethyleneresponsesmayoccurattheleveloftheEREBPtranscriptionfactors[21].Itisnotknownwhetherandhaveapossiblemechanismfordifferentialregulationofethyleneresponses.Whetherandhavefunctionsthatarespecifictotomatofruitripeningandsenescencestillneedtobeidentified. 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