长鬣蜥（Physignathus cocincinus）线粒体基因组全序列分析.pdf 6页

'中国科技论文在线http://www.paper.edu.cnThecompletemitogenomeandgeneorganizationofthe#Chinesewaterdragon(Physignathuscocincinus)**5YANLong,YANPengCollegeofLifeSciences,AnhuiNormalUniversity,AnhuiProvincialKeyLaboftheConservationandExploitationofBiologicalResources,Wuhu241000;10Abstract:ThecompletemitochondrialDNAsequenceoftheChinesewaterdragon(Physignathuscocincinus)wasdeterminedusingPCRandsequencedwithaprimerwalkingmethod.Thecompletemitochondrialgenomeis16,451bpinlength,whichcontains13protein-codinggenes,22tRNAgenes,2rRNAgenesandonecontrolregion(CR).ThegenearrangementandcompositionofP.cocincinuswassimilartomostothervertebrate,buttheorderofthetRNA-IleandtRNA-Glnwasswitchedand15lackoftheoriginofL-strandreplication(OL).Meanwhile,therewasonlyoneCRinP.cocincinusmitogenome,whereitwaslocatedbetweenthetRNA-ProandtRNA-Phegene.DuplicationofthecontrolregionwasnotfoundinP.cocincinus,whichwasoftenobservedinthemitogenomesofAgamidae.Keywords:Physignathuscocincinus;mitogenome;geneorganization200IntroductionThemitochondrionconstitutesanidealmodelforstudyingevolutionduetogenome[1]rearrangement.StructuralandfunctionalcharacterizationsofthemitochondrialgenomeshavebeenconsideredusefulforphylogeneticstudyinSquamata,suchasgenerearrangements,genetic25codechanges,simplificationoftRNAandrRNAstructures,peculiarmechanismsinreplication[2]-[3]-[4]andtranscription,andbasecompositionalbias.AgamidaeisoneofthelizardfamiliesinSquamata,about480membersofwhicharewidely[5]distributedinthetropicalandsubtropicalregionsofAfrica,AsiaandAustralia.TheChinesewaterdragonbelongstothekingdomAnimalia,phylumChordata,classReptilia,orderSquamata,30suborderLacertilia,infraorderIguania,familyAgamidae,subfamilyAgaminae,genusPhysignathusandspeciesPhysignathuscocincinus(Cuvier1829).ThegeneralcharacteristicsofP.cocincinusare:longsnout,moderatebodyinjuvenilesandrobustandcompressedinadults,smalldorsalscalesanduniform,femoralporesinmales(eightpairs),abrightgreendorsumchangeabletobrownish-green,awhiteventerwithbrightbluepatches,andasnout-ventlengthof250mmin[6]35adults.Forthepast10years,squamateshavebeenthefocusofmanystudiesusingphylogeniesto[7]addressquestionsabouttraitevolution.However,moreintegrateddatashouldbeconsideredto[8]-[9]useandaddresstheconflictsbetweentheresultsofmorphologicalandmolecularanalyses.However,thedataisstillnotenoughforanoverallanalysisinAgamidaetodate.Datafrom40GenBankindicatethatthereisnorecordofmitochondrialgenomeofgenusPhysignathusuntilFoundations:SpecializedResearchFundfortheDoctoralProgramofHigherEducationofChina(No.20133424120001)Briefauthorintroduction:YanLong,(1990-),male,postgraduatestudent.Researcharea:BiologicalConservationCorrespondanceauthor:YanPeng,(1976-),Male,AssociateProfessor.Researcharea:BiologicalConservation&Phylogeny.E-mail:mailyanpeng@126.com.E-mail:mailyanpeng@126.com-1- 中国科技论文在线http://www.paper.edu.cnJuly2016yet.SowesequencedthewholemitochondrialgenomeoftheChinesewaterdragon(Physignathuscocincinus)throwinglightonhelpinglaythegroundworkforacomprehensivesystematicstudyofsqumatesinthispaper.1MaterialsandMethods451.1SamplesandDNAExtractionThetissuesampleoftheChinesewaterdragonwasstoredat-85℃.TotalgenomicDNAwasextractedfromasmallquantityoftissuesfollowingtheproceduredescribedinaTIANampGenomicDNAKit(TIANGEN).IsolatedDNAwasstoredat-20℃tobeusedasatemplatefor50polymerasechainreactionanddilutedtotheappropriateconcentrationaccordingtothedemandforexperiments.Thegenomeshouldbeavoidedfreezingandthawingoverandoveraspossible.1.2PrimerDesign,PCRAmplificationandSequencingFirst,weemployedtheconventionalPCRmethodforamplifyingportionsequencesofprotein-codinggenesusingtheprimersdesignedforwhite-headedvipermtDNAfragments.The55partialprotein-codinggeneswereobtainedandthenwedesignedtaxon-specificprimerswithPrimerPremier5programtoamplifytheremainingmtDNAsequences(thelow-conservedregions)onaccountoftheabovesequencesknown.BecausethepitviperhasalongtandemrepeatswithinCRs,theseextremelyvariableregionswasreadilysequencedbyapplyingthelong-and-accuratepolymerasechainreaction(LA-PCR)technology.ThemtDNAsegmentswere60amplifiedusingEasyTaqDNApolymerase(Transgen)andLATaqDNApolymerase(Takara).WeamplifiedmitochondrialDNAfromtemplateDNAin30μlreactionsusingahot-startmethodinathermalcycler.PCRswereperformedundertheprofile:5mindenaturingstepat95℃followedby32cyclesofdenaturingfor40sat95℃,primerannealingfor40sat50-60℃,andelongationat72℃for50s,withafinalelongationfor10minat72℃.Moreover,weoptimized65PCRconditionsregardingtheannealingtemperatureofdifferentprimersandtheactualeffectofamplification.Atlast,PCRproductsweresubjectedto1%agarosegelelectrophoresisandthensenttoGenScript(Nanjing)Co.,Ltd.tobesequencedwithbidirectionalprimers(ABI3730).1.3SequenceAssembling,GeneIdentificationandSubmissionTheresultswereanalyzedbysoftwareChromas2.22andcorrectedmanually.Agreat70numberofshortfragmentswereassembledintoacompletenucleotidesequenceusingOfficeWord2007(MicrosoftCorporation).ThusafinalmtDNAsequenceof16,451bpwasgained.InthemtDNAsequence,13protein-codinggenesand2rRNAswereidentifiedreferredtothecorrespondinghomologuesfromotherlizardsappearingintheGenBanknucleotidesequencedatabaseusingBLAST.ForidentificationoftRNAgenes,wesearchedfortheregionsthatcan75formdistinctivecloverleafsecondarystructuresformitochondrialtRNAgenes.AsCRwassurroundedbytRNAgenes,theirrangesweretemporarilydefinedbytheboundariesofadjacent-2- 中国科技论文在线http://www.paper.edu.cn[10]tRNAgenes.ThemultiplesequencealignmentwascarriedoutwithClustalX1.8andtherestwaseasilyidentified.Werevisedandrecognizedthelocationsofallthegeneseventually.ThewholemitochondrialnucleotidesequencewasannotatedbySequin(Version13.05)andsubmitted80toGenBank(accessionnumberKM272197).2Results2.1GeneorganizationandBasecompositionThesizeoftheclosedcircularmtDNAmoleculeofP.cocincinusis16,451nucleotides.Therelativepositionandorientationofall37genesareexactlythesameasthoseinthetypicallizard[11]-[12]85organization.Justasfoundinotherlizards,thismitogenomecomprises13protein-codinggenes(PCGs),2rRNAgenes,22tRNAgenes,andacontrolregion(CR)playingaregulatoryroleintheprocedureofgenomereplicationandtranscription.Theheavystrandencodes28genes,yetthelightstrandcodesmerely9genes.ThegeneralstructurecharacteristicsofP.cocincinusmitochondrialgenomearepresentedinTable1.90TheoverallbasecompositionoftheH-strandisA:35.4%;C:28.4%;G:12.4%;T:23.8%.TherelativeorderofnucleotidecompositionisA>C>T>G,whichissimilartoothervertebrate[13]mitochondrialgenomes.Overlappingareasandspacercanbeobservedamongsomegenes.Theoverlapoccurs10timesandinvolvesatotalof32bp.Thespacerappears14timesandinvolvesaAsnCystotalof35bp.Thelongestspaceroccupies7bpwhichislocatedbetweentRNAandtRNA95genes.Table1LocationoffeaturesinthemtDNAofPhysignathuscocincinusNucleotideNo.CodonSpacer(+)/GeneSize(bp)Strand*FromToStartStopOverlap(-)phetRNA16565H12SrRNA66965900HValtRNA966103267H16SrRNA103325351503HLeu(UUR)tRNA25362608731HNADH126103584975ATGTAG-3HGlntRNA35823649696LIletRNA3656372671HMettRNA37273792661HNADH2379448281035ATTTAG-2HTrptRNA48274895692HAlatRNA4898496467LAsntRNA49655036727LCystRNA5044510259-1LTyrtRNA51025167661LCOI516967151547ATGAGA-3HSer(UCN)tRNA67136782704LAsptRNA6787685165HCOII68527538687ATGTCAHLystRNA7539760264H-3- 中国科技论文在线http://www.paper.edu.cnATPase876037758156CTGTAG-7HATPase677528431680ATGTA-1HCOIII84319215785ATGT-1HGlytRNA92159282682HNADH392859629345ATATAA-2HArgtRNA9628969164HNADH4L96929982291ATGTAA-7HNADH49976113471372ATGTHHistRNA1134811411641HSer(AGY)tRNA1141311472605HLeu(CUN)tRNA1147811549721HNADH511551133381788ATGTAA-5HNADH61333413828495ATGATG1LGlutRNA1383013897682LCytb13900150311132ATGTHThrtRNA1503215097661HProtRNA150991516365LCR15164164511288-Note:Theletter“H”denotesheavystrand,and“L”denoteslightstrand.2.2Protein-codinggenes(PCGs)Thetotallengthof13protein-codinggenes(PCGs)is11,288bp,whichoccupies68.6%ofP.100cocincinusmitogenome.Thelongestprotein-codinggeneisND5,andtheshortestisATP8.ND6istheonlyproteingeneencodedbythelightstrand,whereastherestofproteingenesarecodedontheheavystrand.Thesegeneshavenointrons,ofwhichcoincideslightlywithelsegenes(Tab.1).ThemitochondrialPCGsbeginwithanATGstartcodonlargely,whileATP8,ND2andND3respectivelyuseCTG,ATTandATAastranslationinitiationcodons.Stopcodonsinclude3AGA,1051AGA,1TCAand2TAA.Meanwhile,incompletestopcodonTisfoundin3genes(COIII,ND4,andCytb),whichmaybepresumablyconvertedintocompleteonesbyaddingapoly-adenylation[14]tailduringthetranscriptionprocess.2.3TransferRNAgenesandribosomalRNAgenesThe12SribosomalRNAgeneislocatedbetween66and965bp,whilethe16Sribosomal110RNAgeneislocatedbetween1033and2535bpinP.cocincinusmitogenome.MitochondrialgenomeoftheChinesewaterdragoncontains22tRNAgenes,whichcanmeettheneedsofallcodonsinproteintranslation.These22transferRNAgenesrangeinsizefrom59to73nucleotides.TheyallcanbefoldedintoacanonicalcloverleafsecondarystructurewiththeSer(AGY)exceptionoftRNA,whichlosesthe“DHU”arm.1152.4Non-codinggenes-ControlregionsThenon-codingregionisamostvariablepartofthemitochondrialgenome,whichincludesacontrolregion(CR)andafewspacers.Thespacersareonlyhaveseveralbasepairsinlengthandareusuallyinterspersedbetweenthecodinggenes.InP.cocincinusmitogenome,onlyonecontrolregionisfoundandlocatedbetweenthe-4- 中国科技论文在线http://www.paper.edu.cnProPhe120tRNAandtRNA.Itoccupysa1288-bplengthandtakepossessionof7.8%intheentiremitochondrialDNAsequence.Itcontainsa42-bpregion(5’-ACACATAAAATACTATTTTATTAGTGTATTAATATAATATTA-3’),whichrepeatedtwotimesinthecontrolregion.3DiscussionPro125FormostotherrepresentativesfromAgaminae,thepositionoftRNAislocatedatthe5’toPhe[15]-[16]thetRNAgeneingeneral.ForP.cocincinusmitogenome,however,theCRisinsertedProPhebetweenthetRNAandtRNAgene.ThisanomalyinthepositionoftRNA-Progenewassimilartootherreptiles.Itisspeculatedthatthearrangementisconcernedwiththecontrolregion.Controlregionsarerecognizedasthedomainswithhighsubstitutionalandvariableratesdueto130lackofbaseconstraintintheprotein-codinggenes.Intheacrodontlizards,itisrevealedthatduplicationofthecontrolregionhasprovedtobeadistinguishingfeatureoftheAgaminae,Amphibolurinae,ChamaeleonidaeandDraconinae.TheCRconsistsoftwopartscommonly,withThrProonepartexistingbetweenthetRNAgeneandtRNAgeneandanotherpartinsertingbetweenPhe[16]thetRNAand12SrRNAgene.Inthisstudy,however,thereisonlyoneCRlocatedbetweenProPhe135thetRNAandthetRNAinthemitogenomeofP.cocincinus.OnthegenusPhysignathus,duetothelackofmitogenomedata,whetheroneCRisageneralthingornotwerequirefurtherresearchtoconfirmit.Incommon,therearetwoderivedfeaturesintheacrodontlizards,suchasauniquetRNAgeneorderandthelackofrecognizableoriginofL-strandreplication(OL).IncontrastwiththeIleGln140typicalvertebratemitogenomes,theorderofthetRNAandtRNAareoftenswitchedinAgaminae,whichisalsoobservedinP.cocincinusmitogenome.Ontheotherhand,likeotheracrodontlizards,theOLwhichinvertebratesusuallylocatesinaclusteroffivetRNAgenes:TrpAlaAsnCysTyrtRNA-tRNA-tRNA-tRNA–tRNA(WANCY),isalsomissingintheP.cocincinusmitogenome.ThisphenomenonissimilartosomeonesinpreviouslysequencedmitogenomesofAsnCys145lizard.TheOLRregionbetweentRNAandtRNA,whichhasthepotentialtofoldintoastablestem-loopsecondarystructure,onlyhasa7-bplengthinP.cocincinus.Meanwhile,thephylogeneticpositionofthegenusPhysignathusiscontroversial.WeexpectedthecompletemitogenomesequenceoftheP.cocincinuscouldcontributetoabettersolutionofitsphylogeneticpositionwithinacrodontlizardsonabasisofanoverallpointofview.150AcknowledgementsThisworkwassupportedbytheSpecializedResearchFundfortheDoctoralProgramofHigherEducationofChina(No.20133424120001)andAnhuiProvincialKeyLaboratoryforConservationandExploitationofBiologicalResourcesinChina.155References[1]SankoffD,LeducG,AntoineN,PaquinB,LangBF,CedergrenR.Geneordercomparisonsforphylogeneticinference:Evolutionofthemitochondrialgenome.ProceedingsoftheNationalAcademyofSciences,1992,89(14):6575-6579.-5- 中国科技论文在线http://www.paper.edu.cn[2]KumazawaY.Mitochondrialgenomesfrommajorlizardfamiliessuggesttheirphylogeneticrelationshipsand160ancientradiations.Gene,2007,388(1):19-26.[3]AlbertEM,SanMauroD,García-ParísM,RüberL,ZardoyaR.Effectoftaxonsamplingonrecoveringthephylogenyofsquamatereptilesbasedoncompletemitochondrialgenomeandnucleargenesequencedata.Gene,2009,441(1):12-21.[4]KumazawaY,MiuraS,YamadaC,HashiguchiY.Generearrangementsingekkonidmitochondrialgenomes165withshuffling,loss,andreassignmentoftRNAgenes.BMCGenomics,2014,15:930.DOI:10.1186/1471-2164-15-930.[5]UetzP,FreedP,JiríHošek(eds.).TheReptileDatabase,http://www.reptile-database.org.Dec24,2016.[6]DasI.ThefieldguidetothereptilesofSouth-EastAsia.NewHollandPublishers,Chatswood,UK.2010.[7]PyronRA,BurbrinkFT,WiensJJ.AphylogenyandrevisedclassificationofSquamata,including4161species170oflizardsandsnakes.BMCEvolutionaryBiology,2013,13:93.DOI:10.1186/1471-2148-13-93.[8]ZhengY,WiensJJ.Combiningphylogenomicandsupermatrixapproaches,andatime-calibratedphylogenyforsquamatereptiles(lizardsandsnakes)basedon52genesand4162species.MolecularPhylogeneticsandEvolution,2016,94:537-547.[9]PoeS,Nieto-MontesDOA,Torres-CarvajalO,deQueirozK,VelascoJA,TruettB,GrayLN,RyanMJ,175KöhlerG,Ayala-VarelaF,Latella,I.APhylogenetic,Biogeographic,andTaxonomicstudyofallExtantSpeciesofAnolis(Squamata;Iguanidae).SystematicBiology.2017,DOI:10.1093/sysbio/syx029.[10]ThompsonJD,GibsonTJ,PlewniakF,JeanmouginF,HigginsDG.TheClustalXwindowsinterface:flexiblestrategiesformultiplesequencealignmentaidedbyqualityanalysistools.NucleicAcidsResearch,1997,25(24):4876-4882.180[11]AmerSA,KumazawaY.MitochondrialgenomeofPogonavitticepes(Reptilia;Agamidae):controlregionduplicationandtheoriginofAustralasianagamids.2005,Gene,346:249-256.[12]MaceyJR,LarsonA,AnanjevaNB,FangZ,PapenfussTJ.Twonovelgeneordersandtheroleoflight-strandreplicationinrearrangementofthevertebratemitochondrialgenome.MolecularBiologyandEvolution,1997,14(1):91-104.185[13]AsakawaS,KumazawaY,ArakiT,HimenoH,MiuraKI,WatanabeK.Strand-specificnucleotidecompositionbiasinechinodermandvertebratemitochondrialgenomes.JournalofMolecularEvolution,1991,32(6):511-520.[14]BooreJL.CompletemitochondrialgenomesequenceofthepolychaeteannelidPlatynereisdumerilii.MolecularBiologyandEvolution,2001,18:1413-1416.190[15]MaceyJR,SchulteJA,LarsonA.Evolutionandphylogeneticinformationcontentofmitochondrialgenomicstructuralfeaturesillustratedwithacrodontlizards.SystematicBiology,2000,49(2):257-277.[16]OkajimaY,KumazawaY.Mitochondrialgenomesofacrodontlizards:timingofgenerearrangementsandphylogeneticandbiogeographicimplications.BMCEvolutionaryBiology,2010,10:141.DOI:10.1186/1471-2148-10-141.195长鬣蜥（Physignathuscocincinus）线粒体基因组全序列分析晏龙，晏鹏200（安徽师范大学生命科学学院,安徽省重要生物资源保护与利用研究重点实验室,芜湖241000）摘要：基于PCR和引物步进法，本研究测定了长鬣蜥（Physignathuscocincinus）线粒体基因组的全序列。长鬣蜥线粒体基因组全长共16,451bp，包含了13个蛋白编码基因、22个tRNA基因、2个rRNA基因和1个控制区（CR）。其线粒体基因组的基因排列和组成方式与其它205脊椎动物类似，但tRNA-Ile和tRNA-Gln基因发生了换位，同时缺乏轻链复制起始区（OL）。结果显示长鬣蜥只有1个控制区，位于tRNA-Pro和tRNA-Phe基因之间。在其它鬣蜥科动物中广泛存在的控制区复制现象在长鬣蜥中未发现。关键词：长鬣蜥；线粒体基因组；基因构成中图分类号：Q959.6+2210-6-'