棉花GhFTL1基因的克隆及初步功能分析-东锐

CottonScience2011，23（6）：515~521GhFTL1，，，，*，*(，832003)：RT-PCRRACE，33FT，GhFTL1(：HM631972)。ORF（OpenReadingFrame）525bp，174，FT，FT。、、、、，。GhFTL1FT。18FT，GhFTL1MdFT1。GhFTL1，T1。GhFTL1。：GhFTL1；FloweringLocusT；；；：S562.035.3：A：1002-7807（2011）06-0515-07CloningandPrimaryAnalysisoftheFunctionofGhFTL1GeneinCotton(GossypiumhirsutumL.)DONGRui,YUANHai-ying,GUChao,ZHENGYin-ying,HUANGXian-zhong*,CUIBai-ming*(KeyLaboratoryofAgrobiotechnology,CollegeofLifeScience,ShiheziUniversity,Shihezi,Xinjiang832003,China)Abstract:AFT-likehomologwasisolatedfromGossypiumhirsutumL.CV.Xinluzao33throughRT-PCRandRACE,whichwasnamedGhFTL1gene(accessionnumber:HM631972).The525-bpopenreadingframeencodedaputativeproteinof174aminoacidswithhighsimilaritytoFTproteinsofdifferentplantswhichcontainstwocriticalresiduesandconservedmotif,bothofwhichidentifiedasdiagnosticoftrueFT.GhFTL1geneshowedconstitutiveexpressioninroot,stem,flower,leaf,fiber,andovule.Thelevelsoftranscriptinleafandovulewerehigherthanthatinothertissues.PhylogeneticanalysisattheaminoacidlevelconfirmedthattheisolatedsequenceisFT-like,andalsoshowedthatGhFTL1hadshortergeneticdistancewithMdFT1comparedwithother18FThomologsconfirmedtoinducingearlyfloweringfrom13differentplants.Over-expressionofGhFTL1inArabidopsisusingtheCaMV35Spromoterresultedinsignificantacceleratedfloweringcomparedwithwild-typeplants.Basedonourresults,weconcludedthatGhFTL1wasresponsibleforinducingfloweringandmaybeoneofimportantcomponentsforthefloweringpathwayincotton.Keywords:GhFTL1;FloweringLocusT;florigen;earlyflowering;cotton(GossypiumhirsutumL.)：2011-03-28：(1986-)，，，[e-mail]；*，，[e-mail]；，[e-mail]：(2008ZX08005-002)，。，。，，[1]。，，。FloweringLocusT(FT)，。FTCottonScience23CottonScience，FT528bp，PEBP(Phosphatidylethanolamine-Bind-ingProtein)[2-3]。35FT。，90%。，FT，FT，[2-8]。FT，，，FT()，FloweringLocusD(FD)，，Apetala1(AP1)、Leafy(LFY)，[9]。，、，、[10-11]。，。，FT。FloweringLocusT-like(FTL)，，。，FTGhFTL1，，。11.133。、、，、，，-80℃。(Col-0)。1.2RNAcDNARNACTAB，[12]。RNAcDNA，PromegaM-MLVReverseTranscriptase。1.31。GhFTL1GhFTL1FGhFTL1R。5′RACEGhFTL15GSP1GhFTL15GSP2。3′RACEGhFTL13GSP1GhFTL13GSP2。5′RACETaKaRa5′FullRaceKit，。3′RACEInvitrogen，。，GhFTL1ORFFGhFTL1ORFR，cDNA，TaKaRaExTaqRT-PCR，，：94℃3min94℃30s，60℃45s，72℃1.5min，35，72℃10min。PCR1%，，Promega，PromegapGEM-Teasy，DH5α，，，。1.4DNAstar，MEGA4.1(MolecularEvolutionaryGeneticsAnal-ysis,Version4.1)FT，ClustalW，Neighbor-Joining，1000Bootstrap[13]。1.5RT-PCR，GhFTL1RTFGhFTL1RTR，28。Ubiquitin7(：DQ116441)UBQ7FUBQ7R，25。PCR1%。1.6KpnXba，pCAMBIA2300-35S()，5166CottonScience22.1GhFTL1FT(AAF03926.1)(Query)，tBlastn()ExpressionSe-quenceTag(EST)EST(ES826802.1)，RT-PCRcD-NA。BLAST，FT，RACE5′3′cDNA，877bpcDNA，DNAstarcDNA，RT-PCR，，525bp，174(1A)，GhFTL1(FloweringLocusT-Like1，GenBankHM631972)。GhFTL1AtFT(AF152096)72%，AtFT(AAF03936)79%。FT/TFL16：AtFT(AAF03936)、AtTFL1(AAB41624.1)、AtTSF(AAF03937)、AtMFT(AAD37380)、AtBFT(NP201010)ATC(BAA75931)[15-19]，。6GhFTL1、GhTFL1a(ABW24963)GhTFL1b(ABW24964)[20](1A)，GhFTL1FT/TFL1(Y)、(Q)，14(LGRQTVYAPGWRQN)LYN/IYN[21-22]。9，GhFTL1AtFT、AtTSF(1B)，GhFTL1FT。2.2GhFTL1Ubiquitin7，RT-PCRGhFTL1、、、、21d3d，GhFTL1，(1C)，GhFTL1。p35S::GhFTL1。GV3101，。Col-020%Bleach，4℃3~4d，1/2MS，1%Glucose,2.2g·L-1Murashige&SkoogMedium(DuchefaBiochemie,Netherland),0.05%MES(Amersham,America),0.8%Agar,pH5.8。25℃，16h、8h，2~3，(1∶1)，16h、8h。[14]。1Table1PCRprimersusedinthisstudyPrimernameSequence(5′—3′)GhFTL1FGhFTL1RGhFTL15GSP1GhFTL15GSP1GhFTL13GSP1GhFTL13GSP2GhFTL1ORFFGhFTL1ORFRGhFTL1RTFGhFTL1RTRGhUBQ7FGhUBQ7RCTAGAGACAGAGATCCTTTGGTTGTTGGTCCTACGGCCACCGGATCCACTCTCCCGGGTCACTTGGACTTGGAGCATCAGGCAGACCACCTCTTGACCAAAGCGCTTTGGTCAAGAGGTGGTCTGCTACTTGGAAGGCAAACGGTGTACGCTCGGGGTACCATGCCTAGAGACAGAGATCCTTTGGTGCTCTAGATCATGTCCTACGGCCACCGGATCCACTGCAGACCACCTCTTGACCAAAGCAAGTAAGTAGCTGTGCGTATTCGTCATAAGAGGTCGAGTCTTCGGACAGCTTGATCTTCTTGGGCTTG：KpnXba。Note:TheunderlinesindicatedthesitesforKpnandXbarestrictiondigest,respectively.：GhFTL151723CottonScience2.3GhFTL1FT18，13。GenBank()，，，90%。18FT(2)，AtFT、GmFT5a、InFTFT，FT、。GhFTL1MdFT1，89.6%。、MdFT1，[8]。GhFTL1。2.4GhFTL1CaMV35Sp35S::GhFTL1，T0，8T1()，(3A，2)，RT-PCR(3B)，，，，(3C)。GhFTL1，。3FT/TFL1，，。TerminalFlower-ing1(TFL1)FT，FT/TFL1，TFL1[16]。83FT/TFL1，FT：A）9FT/TFL1；B）9FT/TFL1；C）GhFTL1。A“*”FT/TFL1，。Note:A）AminoacidsalignmentofninedifferentFT/TFL1homologs；B）PhylogentictreeofninedifferentFT/TFL1ho-mologs；C）ExpressionpatternofGhFTL1indifferenttissuesofcotton.AsterisksindicatedaminoacidsthatarecriticaltodefineFTorTFL1-likeproteins,andtheconcervedmotifswereindicatedusingaminoacidsunderlinedinA.19FT/TFL1，GhFTL1Fig.1Aminoacidsalignment,andphylogentictreeofninedifferentFT/TFL1homologs,andexpressionpatternofGhFTL1indifferenttissuesofcotton5186CottonScience“▲”GhFTL1。TheGhFTL1proteinusedinthisstudyismarkedwitha▲marker.218FTGhFTL1Fig.2Phylogenetictreeof18FT-homologusgenesofdifferentplantspeciesconfirmedtoinducingearlyfloweringwithGhFTL1A）p35S::GhFTL1T1(bar=1.5cm)，WT；B）RT-PCRGhFTL1；C）(*，P0.05，n=8)。A）p35S::GhFTL1intransgenicT1plantshowedearlyflowering,bar=1.5cm,WTrepresentswildtype;B）ExpressionofGhFTL1was