生物克隆论文克隆技术的论文

201029610191025GenomicsandAppliedBiology,2010,Vol.29,No.6,10191025AnArticleCloningandBioinformaticAnalysisofPotatoαAmylaseGeneamyA1WangYuliWangYonggangMaJianzhong*WangQianTaoPengfeiSuYiSchoolofLifeScienceandEngineering,LanzhouUniversityofTechnology,Lanzhou,730050*Correspondingauthor,[e-mail],weamplifiedacDNAforpotatoαamylasebyRT-PCRandclonedit.Sequenceanal-ysisshowedthatthecDNAhada1224bpopenreadingframeandwasreferredtoasamyA1,whichencodesforanαamylasewith407aminoacidresidues(GenBankaccessionnumber:GQ406048.1)withtheMW46.40kD.Afterthatweusedsemi-quantitativeRT-PCRassaytodetectivetheexpressionoftheamyA1geneinpotatoleavesandstems.Theresultshowedthattheexpressioninstemsisalittlestrongerthaninleaves.Thenweanalyzedtheaminoacidsequencebioinformatically,includingitscodonusagebias,physicalandchemicalproperties,subcellu-larlocalization,andconservedstructures.29αamylasegenesfromsameordifferentspeciesweretakenfromtheGenBankforconstructingaphylogenetictree.Thebioinformaticalanalysesshowedthattheputativeproteinshared98%identitywithapublishedpotatoαamylase(GenBankaccessionnumber:M79328.1)attheaminoacidlevel.Thedeductedαamylasealsocontainsacatalyticdomain(PF00128、SM00624)between20to348andaC-terminalbeta-sheetdomain(PF07821)between349~407,whicharesimilartoonesoftheamylasefamily13.Thepostulatedeight-strandedalpha/betabarrelwasalsofoundintheenzyme,whichwasthoughtasanactivesiteofαamylase.Accordingtothephylogenetictree,thetwogenesfrompotatopresentsmoreclosehomologytothosefromcassavaandapplethanfrombarley,riceandmaize.KeywordsPotato,αamylase,Clone,Bioinformaticanalysisα*,,730050*,[e-mail]:10.3969/gab.029.001019RT-PCR(Solanumtuberosum)RNA、cDNA。cDNA1224bp407、46.40kD、。αamyA1(NCBI:GQ406048.1)。RT-PCRamyA1、。amyA1amyA1、、。NCBI29α。NCBIα(NCBI:M79328.1)98%。2034813(PF00128、SM00624)349407αCβ(PF07821)。(β/α)8。2αProgramfund:ThisresearchwassupportedbygrantsfromtheTechnicalBureau,Gansu(No.070244)andtheDegreeCoursesonBuildingLanZhouUniversityofTechnology(No.201011)GenomicsandAppliedBiology、、。,α,,αamylasefamilyisthelargestglycosidehydrol-ysefamily,whichcontainsmorethan30members,in-cludinghydrolase,transferaseandisomerase,andmostofwhichbelongtoglycosidehydrolasefamily13(GH13)thatexistsinthenature.Currently,variesofamylasegenescDNAsequencehavebeenclonedfromanimals,plants,fungiandbacteriabygeneticengineer-ingtechnology.Basedondifferentorigins,itcanbegenerallydividedintobacteriaamylase,fungiamylase,plantamylaseandanimalamylase,etc.Atthesametime,peoplehaveclonedmanyextremelyresistantαamylasegenebytechniquessuchasmutation,whichhasextendedtheapplicationinthefieldofindustry(Fitter,2005).Withtheconsumptionofnonrenewableresourcessuchaspetrolandtheincreasingdemandforenergyinthedevelopmentofhumanity,findingnewrenewableresourcesasthesubstituteinthepost-petroltimeisim-minent.Asthethirdhighestyieldeconomicalcropoftheworld,potatoispotentiallyadvantageousasthema-terialtoproduceethanol(Ghangetal.,2007).Fromcloningpotatoselfamylase-encodinggene,atransgenicamylaseengineeringyeastisconstructed,usingpotatoastherawmaterial.Producingfuelethanolbybiologi-calfermentationcanincreasetheaddedvalueofpotatoandsolvetheproblemofenergycrisis.Inthisresearch,bycloningαamylasegeneindifferenttissuesofpota-to,westudiedthedistributionintissuesinthecourseofbourgeonandgrowth.Accordingtobioinformationanalysis,thestructureandfunctionofαamylasegenewasbetterunderstood(MacGregoretal.,2001).Thisprovidedtheoreticalaccordancefortheexpressionofpotatoαamylasegeneindifferentexpressionsystemssuchasyeast.1Results1.1Extractionoftotal-RNAandamplificationofgenebyRT-PCRTotal-RNAofleafandstemfrompotatocultureseedlingwasextractedaccordingtoTrizolRNAextrac-tionmanufacturer'sinstruction.AbsorbanceofRNAso-lutionoriginatedfromleafstemwasexaminedbyCary50under280nmand260nmUVspectrophotometer,separately.A260/A280(ratio,R)were1.92and1.97sepa-rately.Rvaluerangedfrom1.80~2.0.ThequalityofRNAmettherequirements.Meanwhile,theintegrityofRNAwasexaminedbyagarosegelelectrophoresis.Bands28Sand18Swasbrightandclear,andtheillu-minationof28Sbandwastwiceasmuchasthatof18S(Figure1).ThequalityofRNAwasveryfine.ThefirstchainofcDNAwassynthesizedbyre-versetranscriptionfrontotal-RNA,andaccordingtowhichαamylasegenewasamplifiedbyPCR.PCRproductwasexaminedby1.0%agarosegelelectrophore-sis.TheproductcouldbeamplifiedfromcDNAbotho-riginatedfromleafandstem.Itwas1.2kbandwasconsistentasexpectedandwasnamedαamylase,ab-breviatedamyA(Figure2).1.2Analysisofαamylasegeneexpressionindiffer-enttissuesDuringthegerminationofpotatotuber,theactivityofαamylasegenewasmuchrelevanttoitsgrowth.Itcouldfacilitatethegerminationofthetuberandthegrowthofearlyseedlings.Also,expressionαamylasegenewasfoundinothertissuesandcellsduringtheFigure2RT-PCRresultsofTotal-RNAfrompotatoleaf(1,2)andfrompotatostem(3,4)Note:M:DL2000DNAMarkerFigure1Total-RNAfrompotatoleaf(1)andfrompotatostem(2)1020growth.Analysisbysemi-quantitativeRT-PCRshowedthat(Figure2)expressionofαamylasegenewasfoundinbothleafandstem,andtheabundanceofwhichwashigherinstemtissue.Furtherclone