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Cloning

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Cloning

    Cloning

Availableonlineat,Il,ww.sciencedirect.com

    ;,

    ;鲁秽ScienceDirect

    ;J.Genet.Genomics35(2008)7783

    ;JOURNAL

    ;GENEmCSAND

    ;GEN0MICS

    ;www.jgenetgenormcs.org

    ;Cloningandcharacterizationofnanosgeneinsilkworm

    ;Bombyxmori

    ;GuoliZhao,KepingChen,,QinYaoaWeihuaWangb

    ;InstituteofLireSciences,JiangsuUniversity,Zhenjiang212013,China ;CollegeofLifeScience,HenanNormalUniversity,Xinxiang453007,China ;Receivedforpublication9July2007;revised24July2007;accepted24July2007

    ;Abstract

    ;GenenartosisamaternalposteriorgroupgenerequiredfornormaldevelopmentofabdominalsegmentsandthegermlineinDroso.

    ;phila.Expressionofnanos.relatedgenesisassociatedwiththegermlineinabr

oadvarietyofothertaxa.Inthisstudy,the5.RACE

    ;methodandtheinsilicocloningmethodareusedtoisolatethenewnanos.1ikegeneofBombyx?nor/andthegeneobtainedisanalyzed

    ;withbioinformaticstools.TheputativeproteinisexpressedinEscherichiacoliandtheantiserumhasbeenproducedinNewZealand

    ;whiterabbits.TheresultshowsthatthenanoscDNAis1,913bpinfu11lengthandcontaiasa954bpopenreadingframe.Thededuced

    ;proteinhas317aminoacidresidues,withapredictedmolecularweightof351a,isoelectricpointof5.38.andcontainsaconserved

    ;nanosRNAbindingdomain.Theconservedregionofthededucedproteinshares73%homologywiththenanosproteinconservedregion

    ;ofHoneybee(Apismellifera).TllisgenehasbeenregisteredintheGenBankundertheaccessionnumberEF647589.Oneencodingse-

    ;quenceofthenanosfragmenthasbeensuccessfullyexpressedinE.coli.Westernblottinganalysisindicatesthathomemadeantiserum

    ;canspecificallydetectnanosproteinexpressedinprokaryoticcells. ;Keywords:Bombyxmori;bioinformatics;nanos;primordialgermceils;RACE

    ;Introduction

    ;Nanosgenesencodezincfingertranscriptionfactors

    ;thathavebeenshowntobeexpressedintheprimordial

    ;germcells(PGCs)ofDiptera(Drosophilamelano-gaster)

    ;(Curtiseta1.,1995:Kobayashieta1.,1996;Calvoeta1., ;2005),Caenorhabditiselegans(Schanereta1.,2003), ;Cnidarians(Torrasetal.,2004;Extavouretal.,2005), ;leech(Helobdellarobusta)(Kangetal.,2002),mice(Mus ;musculu)(Tsudaetal.,2003),andhumans(Homosapiens) ;(Jaruzelskaeta1..2003).InDrosophila,nanosisrequired ;forprimordialgermcellmigrationandfate(Kobayashiet ;al..1996)andactstorepresssomaticcel1fateinPGCs.by ;repressingdifferentiation(Hayashietal.,2004:Wangand ;Lin,2004).Inzebrafish(Daniorerio),nanosisrequired ;formigrationandsurvivalofPGCs(Koprunnereta1.. ;Correspondingauthor.

    ;E?mailaddress:kpchen@ujs.edu.crl

    ;2001).Micehavethreenanos.1ikegenes.twoofwhichare ;expressedinPGCsandrequiredfortheirmaintenance ;(Tsudaeta1.,2003).

    ;Nanosgenesplayaroleinspecilyingtheposteriorre

    ;gionsoftheinsectembryos(kisheta1.,1989;Wangand ;Lehmann,1991;Curtiseta1.,1995;LaUeta1.,2003;Calvo ;etal2005).InDrosophila,nanosRNAislocalizedtothe ;posteriorregionoftheembryo,andnanostranslationis ;repressedintherestoftheembryo,bythebindingofthe

    ;smaugproteintoanRNAsecondarystructureinthe ;3,_UTR(untranslatedregion)ofthenanosmRNA ;fDahanukareta1..1999).Nanosproteinregulates ;Hunchback(Hb)translationbyrecruitingacofactor, ;pumilio.whichbindsa”nanosresponseelement”inthe3

    ;UTRofthenchbackmRNA.thusrestrictingHbprotein ;expressiontotheanterioroftheembryo(Irisheta1.,1989) ;Thistranslationrepressionactivityintheposteriorofthe ;insectembryosaDpearstobeconservedintheorthopteran ;SchistocercaAmericana(Lal1eta1.,2003),wherenanos ;

    ;

    ;78GuoliZhaoeta1./JournalofGeneticsandGenomics35f2008)7783

    ;RNAisalsoposteriorlylocalized.Posteriorlylocafized ;nanosexpressionisalsofoundinmosquitoembryos ;(C~voeta1.,2005).However,thereisrarelyarelatedre- ;portaboutnanosinsilkworm(Bombyxmori).

    ;Expressedsequencetag(EST)databasesarevaluable ;resourcesfordiscoveringnovelgenesthroughffico ;cloning(Prigenteta1.,1999;Schultzeta1.,2000;Rinner ;andMorgenstem,2002).Inthisstudy,repeatedEST ;searching.multipleseqtIencecomparisons,andother

    ;data.miningtechniquesareemployedtoobtainnanosgene ;fromB.mori.C:ertainbioinformatictoolsarealsousedto ;analyzegenomicorganizationandtheencodedproteinof ;theisolatednanosgene.ToverifythenanosgeneinBom- ;byxmori.onefragmentofnanoswasexpressedinEs- ;cherichiacoliandtheantiserumwasproduced.Theresults ;0fmisstudyprovideusefulinformationforfuturestudies ;ontheB.morinanosgene.

    ;Materialsandmethods

    ;Materials

    ;TheC108strainofB.moriwasusedforal1experiments. ;RneasyMiniKitwaspurchasedfromQIAGEN(VENLO. ;Netherlands).5.FllURACECoreSetPCRreagentsand ;pM18.TvectorwereobtainedfromTaKaCompany

    ;(Dalian,China).Otherreagentswerepurchasedfrom ;ShanghaiSangonBiotechnologyCorporation(Shanghai, ;China).

    ;DataextractionofcDNAsequenceofB.morinanosgene ;TheNCBI’s(http://www.ncbi.nlm.nih.gov/)ESTdata.

    ;baseisapopularstartingpointforidentifyingexpressed ;sequencetagsofdifferentspecies.andmorethan188.119 ;B.moriESTsequencesarecurrentlyavailableintheGen.

    ;Bank.AnothersilkwormcDNAdatabaseBGI(http: ;//silkworm.genomics.org.cn0wasalsousedtocompare ;nanosandthegenomeinthisstudy.ThenanosmRNA ;sequencefGenBankaccessionno.DQ288392)ofhoney

    ;bee(Apismellifera1wasused.tosearchforthecDNAse. ;quenceoftheB.morinano3gene,byrepeatedcyclesof ;blasting.

    ;RT.PCRand5t.RACE

    ;Theovarywasdissectedfromthelarvaeonthefifthday ;ofthefifthinstar.frozenwith1iquidnitrogen.andground ;intopowder.TotalRNAwasextractedbyusingthe ;RneasyMiIliKitaccordingt0theusermanua1.Finally,the ;totalRNAwasinspectedwithGenespecHIfNakaInstru. ;mentsCo..Ltd.)andstoredat-70.Cforfurtheruse. ;Weused2IxgtotalRNAasatemplateinthefirst-strand ;cDNAsynthesis.Meanwhile.another2ggtotalRNAwas ;usedasatemplate,withthegenespecificprimer(GSP),in ;thefirst.strandcDNAsynthesis.andthennestedPCRwas ;performedusingtheprimersthatweredesignedto ;5RACE.basedonthesequenceobtainedfromtheNCBI ;database(GenBankaccessionno.AB017535).PCRcy- ;clingwascarriedoutat94.Cfor30s,60.Cfor30s,72.C

    ;for1min.for35cycles.ThePCRproductswereexamined ;byelectrophoresisin1%agarosegelfollowingethidium ;bromidestaining.

    ;ThespecificfragmentwasligatedintopM18..Tvector ;andthentransformedintoEcolffDH5strain).The

    ;plasmidwaspurifiedandsequencingwasperformedusing ;anautomaticsequencerCEQ8000(Beckman.Fullerton. ;USA1.

    ;Nucleotidesequenceanalysis

    ;TheprimersusedtoperformRACEweredesignedwith ;thesoftwareprimer5.0.cDNAdatabasesearcheswere ;performedusingBLAST(Altschuleta1.,1997).cDNA ;sequenceanalysiswasperformedusingtheDNAstar ;(DNASTAR,Madison,USA)Software.

    ;Thefirst130nucleotidesof3.UTRwereanalyzedus. ;ingthealgorithmmfoldversion3.1athttp://www.bio. ;info.rpi.edu/zukerm/ma/fMathewseta1..1999;Zuker, ;2003).topredictthesecondarystructure.

    ;Toestablishthegenomicorganization,thecDNAse. ;quencewasblastedtothecontigsofB.morigenomeinthe ;GenBank.SIM4fhttp://pbil.univ.1yon1.fr/sim4.php)was ;usedtoalignthecDNAsequencewiththegenomicse.

;quencestosearchforpotentialintrons.

    ;Proteinpredictionandphylogeneticanalysis ;TheExPASyTranslatetool(http://au.expasy.or# ;tools/dna.html,wasusedtodeducethecDNA’samino

    ;acidsequence,andsimilarityanalysiswasperformedusing ;theBLASTtoolintheGenBankrblastx)andSIBBLAST ;NetworkServicefhttp://au.expasy.org/tools/BLAST/).T}le ;deducedaminoacidsequencewascomparedwiththeho. ;mologysoftwareofGENEDOC(NRBSChttp://www. ;nrbsc.orgY).Asthedivergencetimeinvolvingmanyspe. ;ciescomparisonsandtheconfoundingeffectsofmultiple ;substitutionsinnucleotidesequencescouldoccurover ;longperiodsofevolutionarytime,weusedthededuced ;aminoacidsequencestoconstructphylogenetictrees.The ;treeswereconstructedusingtheMinimumEvolutionfM1

    ;method.M[Esearcheswereconductedusingthecomputer ;programM[EGA3fKumareta1..2004).

    ;Proteinexpression,purification,andpolyclonalantiserum ;production

    ;Oneencodingfragment(699bp)ofthenanosprotein ;wasamplifiedfromtheovarycDNAtemplateusingthe ;

;GuoliZhaoeta1./JournalofGeneticsandGenomics35r2008)7783

    ;polymerasechainreaction(PCR).Asenseprimer ;5Latgaattcatgcgactcaatggcactg3(1cDRIsiteunderlined)

    ;andanantisenseprimer5’-ccctcgagttagttcatgctgaaacttgtc

    ;3(XhoIsiteunderlined)wereusedinconstructionofthe ;recombinantplasmid.ThePCRamplifiedproductswere ;clonedtothepMD18Tvector,afterbeingverifledbvdi

    ;gestingwithEcoRIandXhoIrestrictionenzymes.and ;sequencingusinganautomaticsequencerCEQ8000 ;(Beckman,Fullerton.USA).Thedigestedfragmentwas ;subclonedintothepET30aexpressionvector(Novagen, ;LAJolla.USA).Recombinantvectorswereconfirmedby ;restrictiondigestionwithEcoRIandXhoIrestrictionen

    ;zymes.

    ;Forexpressionoftherecombinantprotein.therecom

    ;binantplasmidnanos/PET30awastransformedtoEs

    ;cherichiacolistrainBL21(DE3),apositiveclonewas ;culturedintheLBmediumandsupplementedwithkana

    ;mycin(50gg/mL)overnight,at37~(2,withshaking.This ;culturewasaddedtofleshLBmediumandctdturedat

    ;37.CwithvigorousshakingtoA600atabout0.6.Thecul

    ;turewastheninducedwithIfrrGinthefinalconcentration

    ;of0.2inmo1/Landfurtherculturedforanother10h.The ;recombinantproteinswereseparatedin10%SDS(define)

    PAGEwasperformedonthe ;polyacrylamidege1.SDS

    ;MiniProteinsystem(BioRad.California.USA).Thegel ;wasstainedwithCoomassieBrilliantBlueR250,tovisu

    ;alizetheproteinbands.

    ;Topurifythenanos6Hisfusionprotein,theinduced ;bacterialcellpeHetwascollected,lysised,andpurified ;usingtheNiresin(Novagen,LAJolla,USA),andthepu

    ;rifledproteinwasverifiedbvSDSPAGE.Antibodies

    ;againstthenanos6HisfusionproteinwereraisedinNew ;Zealandwhiterabbits.

    ;Resuits

    ;DataextractionofcDNAsequence,5’-RACE,andnucleo

    ;tidesequenceanalysis

    ;AfterrepeatedcyclesofblastinginNCBI.usingthe ;honeybeenanosmRNAsequence,onenanosrelated ;cDNA(GenBankaccessionno.AB017535)ofB.moriwas ;obtained.APCRfragmentabout500bpinlengthwasob

    ;tainedfromthesecondPCRof5RACE(Fig.1).We

    ;clonedthefragmenttopMD18Tvectorandthense

    ;quencedit.Followingtheanalysisoftheobtainedse

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