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    ;J.Genet.Genomics35(2008)91-95

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    ;AssociationofpolymorphismsofNramp1genewithimmunefunction ;andproductionperformanceoflargewhitepig

    ;HongmeiWu,DuxueCheng,LixianWang

    ;InstituteofAnimalScience,ChineseAcademyofAgriculturalScience,Beijing100094,China

    ;Receivedforpublication4September2007;revised8October2007;accepted18October2007

    ;Abstraet

    ;Thepresentresearchwasdesignedtostudytheassociationofpolyrnorphismofnaturalresistanceassociatedmacrophageproteinl

    ;(NrampI)withsomeimmunefunctionandtheproductionperformanceinLar

geW1litepig.ThePCR—RFLPtechniquewasappliedto

    ;analyzethecorrelationbetweenthepolymo~hismsofNramplgeneandimmunefunctionIvalueofPolymorphonuclearLeukocytes

    ;(PMN)obtainedbyNitroblueTetrazolium(ribT)ReductionandeffectofCytotoxininMonocyte1andproductionperformanceinl65

    ;Largeitepigs.TheresultsshowedthattherewasoneNdeIrestrictionlocusinLargeWhitepig.andbOmvaluesofPMNbvNBT

    ;ReductionandeffectofCytotoxininMonocyteingenotypeBBwerehigherthanthoseingenotypeAB(P<0.05).Simultaneously,the

    ;weightof180一

    dayoldpigswithgenotypeBBwashigherthanthatwithgenotypeAB(P<0.05).Theresultsindicatedthattherewasa

    ;significantcorrelationbetweendifferentgenotypesofNramplgeneandIi/lliltinefunctionandproductionperformance,anditcanbere—

    ;gardedasacandidategeneofdiseaseresistance.Alltheseresultsprovidevaluablereferencetofurtherstudiesofpigdiseaseresistance. ;Keywords:LargeWhitepig;Nramplgene;immunefunction;productionperformance

    ;Introduction

    ;Lossesduetoanimaldiseases,especiallyinfectious

    ;diseases.continuetoimpedethefivestockindustries.

    ;Thereexistsometraditionaldiseasecontrolmeasuressuch

    ;asstrengtheningbreedingmanagement,andpreventing ;andcuringdiseaseswithveterinarydrugsandusageof ;vaccines.However,antigenicdrift,appearanceofsuper ;virulentstrain,andinterferenceofmother-originantibody ;oftenleadtofailureofvaccineinoculationandpotential ;dangerofrecoveringstrongertoxicityoflowvirulent ;strain.Massiveutilizationofantibioticsmakesiteasierto ;speedupvariationofpathogenicmicroorganism,andthell ;drugresistanceoccurs,whichmakesitmoredi~cultto ;controldiseases.Moreover,overrelyonantibioticsin. ;creasesdrugresidueinanimalproducts,whichisharmful ;tohumanhealth.AUtheseseveresituationspromoteto ;explorenewmethodstopreventdiseases.Recently.witIl ;Correspondingauthor.

    ;E-maifaddress:iaswlx@263.net

    ;thefastdevelopmentofmoleculargeneticsandthedis—

    ;coveryofsomegenesorgeneticmarkersassociatedwith ;diseaseresistance,therearesomeeffectivesolutionsto ;solvetheseproblems,suchasimprovingthegeneticdis—

    ;easeresistance.Oneofthemaintasksistodiscoverthe ;maineffectivegenesorexcellentcandidategenescontro1. ;1inggeneraldiseaseresistantcapability(animalnaturaland

    ;non.etiologicalspecificgeneraldefensivecapability). ;Themembersofnaturalresistanceassociatedmacro- ;phageprotein(Nrarnp)genefamilywerefirstfoundin ;Musmusculus,andthenBelouchinamedthesegenesasthe ;Nrampgenefamily(Belouchieta1.,1995).Asoneofthe ;genefamily,NrampIencodesphosphoglycoproteinwith ;wholemembraneandhasthecharacteristicsoftransport ;activityandionchannelWidaleta1..1996).Nrampl ;mainlyexistsinreticuloendothelialeellsandorgans,such ;asouterwhitebloodcells,spleen,andlung,andcanresist ;severalintracellularpathogenicmicroorganismsrBlack- ;well,1996;Supeketa1.,1996).Atpresent,Nramplgene ;inswineislocatedonq23.26ofchromosome15(Sunet ;a1.,1998),andthewholelengthofNramplinswineis ;about15kb,containing15extronsand14intronsashu. ;覆一

    ;

    ;HongmeiWueta1./JournalofGeneticsandGenomics35(2008)9l__9s ;manbeingsandMusmusculus(Govonieta1.,1995;Mar- ;queteta1.,2000;W_ueta1.,2007).Tuggleeta1.(1997) ;reportedthatthewholesequenceofNramplinswineca- ;codes539aminoacids.whichhas87%similaritywitll

    ;humanbeings.NrampIgeneisspeciallyexpressedin ;phagocyticcells,suchasmacrophagus,neutrophilic ;granulocyte,andouterbloodcells.Therefore,itnotonly ;affectsanimalinherentimmunityandhasarelationship ;withtheresistanceeffectofseveralintracellular ;autoeciousnesspathogenicbacteria,butalsohas ;non—etiologicalspecifcdiseaseresistance.Aboveall,the ;Nramplgeneisbecomingoneoftheexcellentcandidate ;genesofgeneraldiseaseresistanceinswine. ;Atpresent,researchesonthediseaseresistanceof ;Nramplgenemainlyconcentrateonmouse,fowl,cow, ;anddog;however,thestudyofthecorrelationbetweenthe ;functionalgenestructureandthediseaseresistanceof ;NrampIinswineareseldom.Itissignificanttoanalyze ;therelationshipamongNramplgenepolymorphism,dis—

    ;easeresistance.andproductionperformanceinswine.,?u

    ;eta1.f2007)studiedthepolymorphismofthesixthintron ;ofNrampIamongdifferentpigbreeds.However,hedid ;notstudythecorrelationbetweengenepolymorphismand ;diseaseresistanceofNramplasfunctiongene.Thepresent ;studywasconductedtoresearchthecorrelationamong ;PCR—RF’LPpolymorphismofthesixthintronNdeIen—

;zymeandimmunefunction【Cytotoxineffectofmononu—

    ;clearmacrophage(CM),PMNbyNBTReduction(PN)】

    ;andproductionperformanceusing165Largetepigs ;asexperimentalanimals,aimingatrevealingthemolecular ;basisofdifferentimmuneresponsesandprovidingrefer- ;enceforswinediseaseresistantbreedingbymarker-based ;selection.

    ;Materialsandmethods

    ;Materia/s

    ;Onehundredandsixty—fiveLargetepigswere

    ;randomlyselectedfromswinebreedingstationofInstitute ;ofAnimalScience(IAS),ChineseAcademyofAgricul—

    ;turalSciences(CAAS)atrandom.AIlexperimentalpigs ;werefed,managed,andepidemicpreventedundersame ;conditionsaccordingtotheroutineproductionrequestan—

    ;derthesameconditions,andthediscrepancyofbirthtime ;wascontrolledwithin20days.weanedat30mday,and ;inoculatedat60thdaytoeliminatethediseaseresistant ;differencebeforeandaftervaccination.Sixmicrolitersof ;bloodsamplesfromeachpigweredrawnfromthepre—

    ;cavalvein(10%EDTAtoanticoagulateblood110days ;aftervaccination,andwereaveragelyseparatedinto2por-

    ;tions.Oneportionwasusedtodetecttheimmunityindex, ;andtheotherwasusedtoseparatethemonocyteandPN. ;Theimmunityindexwasdetectedimmediatelyafterblood ;collection.Theweightsoftheexperimentalpigswerere? ;cordedat30th,70th,and180thdays,respectively. ;Swinebloodgenomewasextractedusingtheconven? ;tionalphenol/chloroformmethodanddissolvedinTE ;buffer(SambrookandRussell,2001).

    ;P—,,l已rdesignandPCR-RFLPanalysis

    ;Primerwasdesignedaccordingtothesixthintron ;sequenceofswineNramplgenefGenBankaccession ;no.AY368470),andtheamplifiedsegmentisabout483bp ;inlength.ThesequencesoftheprimersareForward: ;5’-GCCAGCTTCCACAGTCTCCAG一3:Reverse:5.G—

    ;GGGG1CAAAGGGGAA(}AAG.3.A25LLIPCRmix.

    ;turecontained2.5oflOxbuffer,2of2.5mmol/L ;dNTP,0.2Lof5U/LTaqDNApolymerase(Bering ;TianweiAgeTechnologyLimitedCompanyofTsinghua ;Unive~ity),1of10pmoI/Lforwardandreverseprim. ;erseach,2LofgenomicDNA(about50ng),and

    ;16.3uLofddH20.Aml:llificationofDNAwasconducted ;for36cyclesfollowingtheconditionsasbelow:

    ;preliminarydegenerationat94.Cfor5min,degeneration ;at94.Cfor30s.annealingat64.Cfor30s,extensionat ;72.Cfor90s.followedbyafinalextensionat72.Cfor ;8min.Twomicrolitersof6xbromophenolbluebufferwei’e

    ;addedinto6止ofPCRproducts,whichwereloadedinto ;the1.5%sepharosegel(including0.O5%EB,.Electro- ;phoresiswascarriedoutatavoltageof5V/cmfor1h.and ;theelectrophoresisresultswereobservedbytheultraviolet ;lampandphotographed.

    ;PCRproductwasdigestedat37.CovernightusingNde ;Iina15reactionmixtureconsistingof8止amplified

    ;product,and4Urestrictionenzymeand1.5止buffer.The

    ;digestedproductwaselectrophoresisedina2%agarose ;gelatavoltageof5|cmfor2-3handwasthenviewed ;underultravioletlamp.

    ;Cellseparation

    ;Lymphocyteseparatingmediumwithspecificdensity ;1.077:t0.001wasusedtoseparatetherequiredcellsaccord—

    ;ingtodifferentcellulardensity(ShenandZhou,2002). ;Theprincipleandmethodoftheimmunityindexdetection ;TheprincirIleanddetectionofthevalueofPAbyNBT ;Reductionrefersto’’thefunctiondetectionofPMN’’ref.

    ;erence(WuandLiang,20021.MTT(methylthiazolyltetra- ;zolium)chronmtometrywasusedtodetectthepercentage ;ofCytotoxineffectofMonocyteonhumanleukemiccell ;K56203asicMedicineInstituteofPekingUIlionMedical ;College)asdescribedpreviously(WuandLiang,2002). ;

    ;Statisticalanalyticmethod

    ;HongmeiWuetal./JournalofGeneticsandGenomics35(2oo8)91-95 ;AVONAwasusedtoanalyzeone—factoranalysisof

    ;variancetobothimmunityindexandproductperform—

    ;anCe.

    ;Results

    ;TheamplificationresultsofPCRproduct

    ;AUtheswinegenomesoftheexperimentalgroupswere ;amplified,andthePCRproductsweredetectedwim1.5% ;agarosege1.Theproductobtainedwasthesameasthe ;targetfragmentandtheproductstraOwasclearwithout ;mixedstr印.Thus,theproductwasamplifiedwithfine ;stabilityandspecificity,andcanbecleavagedbyrestric—

    ;tionenzymedirectly(Fig.1.

    ;bp

    ;500

;300

    ;Ml2345678

    ;Fig.1.PCRproductofNramplgene.M:100bpDNAladdermarker; ;lanes1-8:individualPCRproduct.

    ;TheenzymecleavageresultsofPCRproduct

    ;ThePCRproductwascleavagedwithNdeI.andthe ;resultsshowedthatthereexistsNdeIcleavagepolymor—

    ;phicsites,andtherewerethreegenotypes,whichwere ;inducedbyenzymeincisesitesabsence(shownasA,and ;presence(shownasB,.TheAAgenotypeshowedone ;strap(483bp),theBBgenotypedisplayedtwostraps(373 ;bpand110bp),andtheheterozygoteABgenotypedis—

    ;playedthreestraps(483bp,373bp,andl10bp,respec—

    ;tively)(Fig.2).TheABtypewasthemost,andtheBB ;typewasmorethantheAAtype,andthefrequencyofA ;waslowerthanB(Table1.

    ;Genepolymorphismandimmunitycharactercorrelation ;analysisofNrampl

    ;Theimmunitycharacterdataofswinewereremedied ;byleastsquaretoanalyzethecorrelationbetweenpoly. ;morphismandimmunityofNramp1.Theresultsofvari= ;anceanalysisofindividualswinewimdifferentgeno—