荷花LEAFY基因的克隆及表达分析刘艺平

PlantPhysiologyJournalPlantPhysiologyJournal2014,50(2):203~2082014,50(2):203~2082032013-09-222013-11-21(072102150001)*(E-mail:[e-mail];Tel:[phone])LEAFY1,1,1,2,1,1,*1,450002;2,451100:LEAFY(LFY),‘’,LFY,RT-PCRRACELFYcDNA,NnLFYNnLFYcDNA1494bp,(ORF)1173bp,390,44517.1DaLFY,LFYLFYPCR,LFY,,:;LFY;PCRCloningandExpressionAnalysisofaLEAFYGeneFromLotus(NelumbonuciferaGaertn.)LIUYi-Ping1,DONGJi-Xiu1,ZHANGMan1,LIChuang2,LINa1,KONGDe-Zheng1,*1CollegeofForestry,HenanAgriculturalUniversity,Zhengzhou450002,China;2XinzhengNanlongLakeHabitableEducationParkManagementCommittee,MunicipalConstructionEnvironmentalProtectionBureau,Xinzheng,Henan451100,ChinaAbstract:LEAFY(LFY)genesareaclassofinflorescencemeristemidentitygenewhichplayimportantrolesintheprocessofflowering.Inthisstudy,ahomologueofLFY,designatedNnLFY,wasisolatedfromNelumbonucifera‘Hongxiamantian’byreversetranscriptase-polymerasechainreaction(RT-PCR)andrapidamplificationofcDNAends(RACE).ThecDNAsequenceofNnLFYwas1494bplongandcontainedanopenreadingframe(ORF)of1173bpencodingaproteinof390aminoacids.Itsmolecularmasswas44517.1Da.TheNnLFYsharedhighesthomologywiththeLFYfrommelonaccordingtothehomologyandphylogeneticanalysesoftherelationshipofNnLFYwithsomeknownLFYgenesfromotherplants.Real-timequantitativePCRanalysisindicatedthattheNnLFYwasubiquitouslyexpressedintheroot,stem,leafandflowerinthedifferentperiodsandthehighestexpressionwasdetectedinthebudperiod.Keywords:lotus;LFY;real-timequantitativePCRLEAFY(LFY),,,(Mouradov2002;Amasino2004)ABCLFY,CoenMeyerowitz(1991),LFY,LFY,,,LFY(ShannonMeeks-Wagner1993)(MandelYanofsky1995)(MizukamiMa1997),LFY,(1999),LFY,(Coup-land1995),,(Arabidopsisthaliana)(Weigel1992)(Ginkgobiloba)(2002)(Phalaenopsisamabilis)(2012)204(Litchichinensis)(2013)LFY,LFYRT-PCRRACE,LFYcDNA,LFYPCR,1‘’(NelumbonuciferaGaertn.‘Hongxiamantian’),,,–80cDNA(RevertAidTMFirstStrandcDNASynthesisKit)Fermentas;5'-RACE(SMARTerTMRACEcDNAAmpli-ficationKit)(TaKaRa);RT-PCR(Power2×SYBRReal-timePCRPremixture)BioTeke;DNA;TaqDL1000DL2000DNAmarker(TaKaRa);DH5α;pUCm-T();()22.1RNARNACTAB(2009),A260/A280A260/A230RNA,,AlphaImagerPE(AlphaInnotech)2.2LFYcDNALFYcDNA(GenBankHQ328497.1),3'-RACELP-F1LP-F25'-RACELP-R1LP-R2(1),B26(5'GACTCGAG-TCGACATCGATTTTTTTTTTTTTTTTT3')UPM(5'CTAATACGACTCACTATAGGGCAAGCAGTG-GTATCAACGCAGAGT3')RevertAidTMFirstStrandcDNASynthesisKit(Fermentas)SMARTERTMRACEcDNAAmplificationKit(Contech)3'-RACE-ReadycDNA5'-RACEcDNAPCR20μL,,pUCm-T,DH5α,PCR,(ORF)LFY-F(ORF)LFY-R(ORF)(1)2.3LFYDNAMAN5'-3'-RACE,,;NCBIBLAST()ORF;Primer5.0;ProtParam1LFYTable1PrimersequencesusedingenecloningandexpressionanalysesofN.nuciferaLFY(5'→3')3'-RACELP-F1GCCGAAGATGCGACACTA5230sLP-F2GTATCAATGGCAGCACGAC5'-RACELP-R1TTCATCCAGGCAATGCAGC5530sLP-R2CAGATGGAATAGATAATCAAGCCCGTcDNALFY-F(ORF)ATGGATCCTGAAGCTTTCACCGCAAGCTTGTTC5630sLFY-R(ORF)TTAGAAGGTAGAGGGGGGAGCGATGGTAGCF-18SCCATAAACGATGCCGAC5520sR-18SCACCACCCATAGAATCAAGALFYLFY-F1TGGTATCAATGGCAGCAC5520sLFY-R1GAGCAGGCAGAAGCAGA:LEAFY205()LFY;LFYNCBICDD(ConservedDomainDatabase);DNAMAN6.0LFY2.4LFYRNAcDNALFYcDNA,RT-PCR18SrRNA,R-18SF-18SLFY,LFY-F1LFY-R1cDNAPCR,BioTekePower2×SYBRReal-timePCRPremixtureiQ5MulticolorReal-TimePCRDetectionSystemcDNA,2-ΔΔCT,31LFYcDNAORFLFY3'-RACE5'-RACE435837bp(1),3'-5'-LFY,3'-23A(polyA)DNAMANLFY3'-RACE5'-RACEcDNA,LFYcDNA,1494bp,GenBankKC202819.1,PCR(1),LFYcDNA,ORF1173bp,5'-69bp,3'-252bp,ATG,TAANCBIBLAST,LFY90%2LFYLFY(Solanumlycopersicum;AAF66099)(Juglansregia;ACZ48701)(Cucumismelo;FJ971628)(Chrysan-themumlavandulifolium;AAT51708)(Nymphaeaodorata;AAF77609)(Liliumlongiflorum;ABR13015)(Oryzasativa;BAA21547)LFY(2),,LFY,(82%),(56%),LFYLFY,,(3)3LFYLFYNCBICDD,FLO_LFY,LFYLFYcDNA,390,ProtParam44517.1Da,C1946H3041N595O577S16,20,Arg(10.8%),1LFYFig.1ElectrophoresispatternofN.nuciferaLFYS1:3'-RACE;S2:5'-RACE;S3:LFYORF;M1:DL1000DNAmarker;M2:DL2000DNAmarker206Ala(9.2%),(Asp+Glu)48,(Arg+Lys)63DNA-MANLFY(4),,,,50.00%,,41.79%,,8.21%4LFYPCR,LFY(5),LFY5,LFY;,,,;,LFY,LFY,2LFYFig.2MultiplealignmentofpredictedaminoacidsequencesofLFYgenesfromdifferentplants:LEAFY207,,LFYmRNA1.6kb(Weigel1992),GenBankFLO,LFY(2005)LFYcDNA1494bp,390,FLO_LFY,LFY(2004),LFY,LFY,LFY,,Blazquez(1997),LFY,,,JrLFY,,(2011),LFY,ELF1(Southerton1998)GinNdlyGinlfyLFY,(2005)(2003)3LFYFig.3PhylogeneticanalysisofLFYgenesfromdifferentplants5LFYFig.5TherelativeexpressionlevelsofLFYgeneduringdifferentperiodsindifferentpartsofN.nucifera4LFYFig.4Predictionofsecondstuctureforsequenceproteinoflotus208‘’2LFYAFL1AFL2,AFL2,AFL1(2004)LFYDFL,,LFY,LFY(2011),LFY,,,,,,(2003).LEAFYcDNA.,30(3):267~271,,,,,(2012).LFY.,(2):65~68,,,,(2005).LEAFY.,27(2):241~244(2011).LFY[].:,,,,(2013).LEAFY.,34(1):57~61,,,,,(2011).LEAFY.,31(1):197~203,,(2005).LEAFY.,22(5):605~613,,,,(2004).:DFL.,2(4):597~599,,,(1999).LFYcDNA.,41(3):268~271,,,(2004).LEAFY.,26(1):137~142,,,,(2009).RNA.,27(10):1226~1228,,,(2002).LEAFY.,38(4):167~170AmasinoR(2004).Vernalization,competence,andtheepigeneticmemoryofwinter.PlantCel1,16(10):2553~2559BlazquezMA,SoowalLN,LeeL,WeigelD(1997).LEAFYexpressionandflow