二化螟线粒体cox1基因的克隆、序列测定和分子系统学分析.doc

二化螟线粒体cox1基因的克隆、序列测定和分子系统学分析agriculturalscience&technology,2011,12(5):674677copyright2011,informationinstituteofhaasallrightsreservedcloning,sequencingandagriculturalbiotechnologymolecularphylogeneticanalysisofthemitochondrialcytochromeoxidaseigeneofchilosuppressaliswangaimin.honggui-yun一.weizhao?jun1.schoolofbiotechnologyandfoodengineering,hefeiuniversityoftechnology,hefei230009;2.departmentofenvironmentalengineer-ing,anhuiuniversityofarchitecture,hefei230601abstract0bjectivetheresearchaimedatcloningandanalyzingmitochondrialcytochromeoxidaseigene(cox1)ofesuppressalis.methodthemitochondrialcox1geneofcsuppressaliswasclonedwithpcrmethodandsequenced.then.cox1sequencesofother21lepidopteranspecieswereobtainedbyblastingthegenbankwithcox1genesequenceofc.suppressalis.finally.homologycomparisonandmolecularphylogeniticanalysisamongthe22lepidopteranspecieswereconducted.resulttheopenreadingframeofcox1genefr0mc.suppressaliscontained1531nucleotidesencodingaputativeproteinof510aminoacids.thecox1geneusedastartcodoncga.andanincom-pleteterminationcodoncomposedofonlyt.basedonlheaminoacidsequencesofcox1.themolecularphylogenetictreeoflepidopterawasreconstructedusingthemaximumlikelihoodfml,method.themolecularphylogenetictreewassimilartothemorphologicalphylogenetictreemainly.butalsoshowedsomedifferences.conclusionitheresultwillprovidereferenceforfurtherresearchonexpressionandapplicationofcox1genekeywordsmitochondrialdna:chilosuppressalis:cox1gene:phylogeneticanalysismitochondrionisthesiteforoxidativephosphorylation,fattyacidandproteinbiosynthesisincells.andisinvolvedincellmetabolism,growthandagingprocess.inaddition,itisasemiautonomousorganelle.thegenesinmitochondrialgenomearecloselyrelatedtomitochondrialoxidativephosphorylation,whichsuppliesmostoftheenergyincells.inrecentyears.ithasbeenfoundthatvariationinmitochondrialgenomeiscloselyconnectedtoavarietyofhumandiseasesandaging川.1nsectmitoch0ndrionisacovalentclosedcirculardoublestrandeddna.ofwhichthestructureisrelativelysimpleandstable.trons.repetitivesequencesandunequalexchangeinmitochondrialgenome.inaddition,thereplacementrateofmitochondriaigenomeis510timeshigherthannucleardnaandfollowsastrictmaternaiinherit-ancepattern.mitochondriaidnawithinanindividualshowsahighdegreeofuniformity,isnon-tissuespecificingeneral,easilypurifiedfromthetissuesandreproducible.whichfacilitatesanalysisofresults.becauseofthesecharacteristicsinstructureandevolution.mtdnahasbecomeanimportantmaterialfortheresearchesofmolecularphylogenyandevolution.generally.insectmitochondrialgenomeis16kbinlength.encodes37genes,including13proteincodinggenesacyto.chromebgene(cob),twoatpenzymesubunitgenes(atp6andatp8),threecytochromeoxidasesubunitgenes(cox1,cox2andcox3)andsevennadhcatabolicenzymegenes(na一6andnad4l)i,tworrnagenesand22trnagenes一.chilosuppressa帖.themostharmfuipestofrice.isareceived:april7,2011accepted:may24,2011supportedbynewcenturyprogramforexcellenttalentsofministryofeducationofchina(ncet-070251):talentsfoundationofanhuiprovince(08040106803).correspondingauthoremail?cnspeciesofpyralididae.alsoknownaszuanxinchonginchina.sofar.noreportaboutthemitochondriaidnafromc.sup.pressafishasbeenfound.therefore.inpresentresearch,thecompletecox1geneofc.suppressaliswasamplifiedandclonedwithpcrmethodandsequenced:thencox1se.quencesofother21lepidopteraspecieswereobtainedbyblastingthegenbankwiththesequencedcox1geneofc.suppressalis;finally,thehomologycomparisonandmo?iecularphylogeniticanalysiswereconducted.theresultiaidafoundationforfurtherinvestigationonexpressionandapplicationofcox1gene.materialsandmethodsmaterialsc.suppressaliswasprovidedbythehuazhongagricul-turaluniversity:thecloningvectorpmd18.twaspurchasedfromtakaracompany:hoststrainusedinpresentstudywasxl一1.methodspreparationofmitochondriaidnaofcsuppressalisthemitochondrialdnaofc.suppressaliswasextractedbyusingthegenomednaextractionkit(takara).amplificationandcloningofcox1genefromc.suppressalisaccordinqtothecox1sequenceofhelicoverpaa,ma.era.bombyxmoriandphflosamcyntharicini.apairofprimerswasdesignedasfollows:coxlf:5一acagtttatcgcttaacctcagcc-3andcox1r:5一cctttatagatggggtt_raaa.byusingthemitochondriaidnaofc.suppressa|lsasatemplate.pcrwasstartedwithpredenaturingat94for2min.followedby35cyclesof95for15s.58for2min:theamplificationwascompletedbyholdingthereactionmixtureat72ocfor10mintoallowcorn.pleteextensionofpcrproducts.subsequently,agarosegelelectrophoresiswascarriedouttorecoverythetargetfra.qment.whichwasthenlinkedintopmd18.tvectorbeforewangai-mineta1.cloning,sequencingandmolecularphylogeneticanalysisofthemitochondrialcytochromeoxidaseigeneofch.iosuppressalis675transformation.therecombinantwasselectedandidentifiedwithenzymedigestionmethod,andthensequencedbytaka-racompany.sequenceanalysisandmolecularphylogeneticanalysisofm_tochondriaicox1fromc.suppressalbasedonthemitochondriaicox1sequenceofc.suppressalis.thatofother21lepidopteraspecieswereobtainedfromgenbank.then.dnastarsoftwarewasadoptedforanalyzingtheorfsequenceandinferringtheaminoacidsequenceofthetargetgeneaccordingtothegeneticcodesofmitochondrialgenesfr0minvertebrate.moreover.thehomologyandmolecularge-nealogicairelationshipofthecox1genesbetweenc.sup-pressalisandother21lepidopteraspecieswereinvestigatedbyusingclustalxlsoftware.thecox1genesequencesandtheaminoacidse-quencesencodedbycox1genesoffouroutgroupsand22lepidopteraspeciesobtainedfr0mgenbankwerecomparedinclustalx1.83softwareusingdefaultparameters.insuchsequencealignment.thecodondamageandbaseshiftcausedbyalignmentcouldbeavoidwhenthegenelengthdiffersbetweendifferentspecies.theparameters(gop/gep)foralignmentwerethedefaultvalues,andthentheresultingsequencealignmentdocumentswereinputintosoftwaregblocks0.91bwithdefaultparameterstoselecttheconsewedaminoacidfragments.thealignmentdatasetcomposedof510nu-cleotideswasobtainedaccordingtotheaminoacidalignmentdocuments.thealignmentsequenceswereconvenedtophylipornexformatthroughdmabesoftwaretobeusedfortheconstructionofphylogenetictreewithmlmethod.resultsandanalysiscloningofmitochondrialcox1genefromcisuppressalisbyusingtheextractedmitochondriaigenomeasatern?plate.pcramplificationwascarriedoutinpresenceofprim-erscoxlfandcox1r.theresultingdnafragmentswerei-dentifiedthroughelectrophoreticanalysis,andthenabrightbandof1600bpwasshownffig.1).thefragmentwasrecoveredandclonedintopmd18-tvector.identifiedwithcolonypcr.thepositivecolonywassequenced.2.00kbl|00kb0.75kb0.5okb0.25kbo.10kbm:dnamarker:1:cox1pcrproduct.fig.1thepcrproductofcox1geneofc.suppressalissequenceanalysisofmitochondriaicox1genefrom/-/.armigeraaccordingtothesequenceanalysisresult,thefragment391568bpoftheresulting1618bpnucleotideswasthewholecox1gene.thenucleotidesequenceofcox1genewastranslatedintoaminoacidsequencebyreferringtothegeneticcodesofmitochondrialgenefrominvertebrate.theresultsshowedthatcox1geneofc.suppressaliscontained1531nucleotidesencodingaputativeproteinof510aminoacids.1tsstartcodonwascga.anditsterminationcodonwascomposedofonlytbase(fig.2).apolyawillbeaddedtotheincompletestopcodonintranscriptionprocesstof0rmacompletestopcodon.sequenceanalysisresultshowedthatthestartcodonofalithe22lepidopteraspecies(cga)wasnotthesameasthatofthenucleargenomegenes(atg).whichmavbethespecificfeatureofmitochondrialgenomeoflepidopteranspecies.molecularphylogeneticanalysisoflepidopteraspeciesbasedoncox1genesequencemitochondriaigenomesof21lepidopteranspeciesexceptgsuppressaliswereobtainedbyblastinggenbankwithcox1genesequenceofsuppressa/is(table1).inaddition,thedrosophilamelanogaster(drm),drosophilayakuba(dry),locustamigratoria(lom)andanophelesgambiae(ang)wereselectedasoutgroups.clustal1.8,dambe.meg3.1.phylid3.67andothersoftwarewereadoptedtoconstructphy-logenetictreeofthe22lepidopteraspeciesbasedontheami-noacidsequenceofcox1gene(fig.3).asshowninfig.3,c.suppressaliswasclusteredwithdiatraeasaccharalis.di-atraeasaccharalisandostrinianubilalisofpyralidae.whichwasexactlyconsistentwiththeresultoftaxonomy.accordingtotheclassicmorphologicalphylogenetictreeoflepidopteraproposedbykristenseneta1.,theadoxophyeshonmajoftortricoideawasthefarthestbranchinmorphologicaiphyloge-netictreeoflepidoptera【.buttheresultsofthisstudyshowedthatthespeciesinpapilionoideaandnymphalidaeweremostdistantlyrelatedtootherlepidopteraspecies.inaddition.previousstudyrevealedthatbombycidaeandsatmr-niidaesharedacloserrelationship,whichwasalsodifferentwiththeresultsofthisstudy.table1classificationof22insectsfromlepidopteraandtheirgenbankaccessionnumberagriculturalscience&technologyvo1.12.no.5.2011acagtttatcgcttaacctcagccattttattttattgcgaaaatgactttactcaacac0x1frkwlystaatcataaagatattggaactttatattttatttttggtatttgagcaggtataattgganhkd工gtlyf工fg工wagm工gaeatctcttagacttttaattcgtgctgaattaggaactceaggatctttaattggagattslsll工raelgtpgsl工gdgatcaaatttataatactattgttacagctcatgcatttattataattttttttatagttdq工ynt工vtahaf工m工ffmvataccaattataattggtggatttggaaattgattagtacctttaatattaggagctcctmp工m工ggfgnwlvplmlgapgatatagctttcccacgaataaataatataagattttgaatattacececeteattaactdmafprmnnmsfwmlppsltttattaatttctagaagaattgttgaaaatggagetggaacaggatgaacagtgtaccccll工sss工vengagtgwtvypccactatcatctaatattgctcacgetggaagttcagtagatttagcaattttctetttaplssn工ahagssvdla工fslcatttagctggaatttcttcaattctaggtgctattaattttattactacgattattaathlag工ss工lga工nf工tt工工natacgaattaatggtctttcatttgatcaaatacctttatttgtttgatccgtaggtattmr工nglsfdqmplfvwsvg工acagctttattattacttctatctctaceagtattagctggagcaattacaatattattatalllllslpvlaga工tmllaccgatcgbaatttaaatacatetttttttgateetgctggtggtggagatccaattctttdrnlntsffdpagggdp工ltatcaacatttattttgattttttggtcacccagaagtatatattttaattcttcctggayqhlfwffghpevy工l工lpgtttggtataatttctcaeattatttcacaagaaagaggaaaaaaggaaacttttggttgtfgm工sh工工sqesgkketpgcttaggtataatttatgctataatagcaattggattacttggatttgtagtatgagctcatlgm工yamma工gllgfvvwahcaeatatttacagttggtatagatattgatactcgagcttattttacttcggcaacaatahmftvgmd工dtrayftsatmatcattgccgtaecaacaggaattaaaatttteagatgattagctactttacatggaaca工工avptg工k工fswlatlhgtcaaattaattatagtccctcaactctttgaagtttaggatttgtatttttatttaetgtcq工nyspstlwslgfvflftvggaggattaaccggagtaattttagctaattcttcattgatgtagctttacatgatactggltgv工lanss工dvalhdttattatgtagtagctcattttcactatgttttatctataggagcagtatttgctattattyyvvahfhyvlsmgavfa工工gcaggatttattcactgatacccacttttaacaggattaaccctaaatccttacatattaagf工hwyplltgltlnpymlaaaattcaatttttaattatattcttaggagtaaatttaacatttttcccacaacattttk工qfl工mflgvnltffpqhfttaggattagcaggaataccacgacggtactcagattatccagatatttatatttcttgalglagmprrysdy.pd工y工swaatattatttcatctttaggatcatatatttcattattagcaattatacttttacttattn工工sslgsy工slla工mlll工attgtatgagagtcaataattaatcaacgaattattttattccctcttaacttaacctcc工vwesm工nqr工工lfplnltstcaattgaatgatatcaaaatctaccacctgctgaacattcttataatgaattaccaatts工ewyqnlppaehsynelp工ttaagaattttctaatatggcagactatatgtaatggattaaaccccatctataaaggls工c0x1rfig.2nucleotideandaminoacidsequencesofmtdnacox1geneofc.suppressalisulscusslonsinthisstudy.themitochondrialcox1geneofc.suppressaliswasamplifiedwithpcrmethod,sequencedandanalyzed.theresultsshowedthatcox1geneofsuppressal-scontained1531nucleotidesencodingaputativeproteinof510aminoacids.itsstartcodonwascga.anditsterminationcodonwascomposedofonlytbase.thestructureandcharacteristicsofcox1genesequenceofgsuppressaliswassimilartothatofotherlepidopteranmitochondriaicox1gene:thestadcodoncgamaybeuniquecharacteristicsofcox1geneoflepidoptera.themolecularphylogenetictreeconstructedonthebasisofcox1genewasgenerallysimilartotheclassicmorphologicalphylogenetictree,butalsoshowed59119271794723967299873591o74191274791475391675991876592o77192277792478392678992879593o7101932710793471139367119938712594o7131942713794471439467149948715595o71618510somedifferences:theadoxophyeshonmafoftortricoideashouldbedistantlyrelatedtootherspeciesinm0rph0ioqicalphylogenetictreeoflepidoptera,buttheresultsofthisstudyshowedthatthespeciesinpapilionoideaandnymphalidaewerethefarthestbranchinmolecularphylogenetictree.suchdifferencesmayberesultedfromthatinthisresearch.onlyagenewaschosentoconstructthemolecularphylogenetictreewhichcannotbefullyrepresentativeofalithegeneticjnformationaboutinsects.therefore.themolecularphyiogenetictreebasedonthecompletemitochondriaigenomeinformationmaybetterreflectphylogeneticrelationshipsbe-tweeninsects.inchina,csuppressalisisharmfuipesttorice.andawangai.rainetalcloning,sequencingandmolecularphylogeneticanalysisofthemitochondrialcytochromeoxidaseigeneofchi|osuppressalis6丌fig.3aphylogenetictreeoflepidopteransbasedontheami-noacidsequencesofmitochondrialcox1geneslotofmanpowerandmaterialresourcesarespenttocontroliteveryyear.themitochondrialgenomeencodessomekeyproteinsregulatingbiologicaloxidativerespiratorychain,andsomeproteinsrelatedtothedrugresistanceofinsect.ther