运用PCR-RFLP技术区分赤潮藻

PCR-RFLPX张涛1,米铁柱1XX,卢青2,孙静1,赵丽媛1,袁建1,于志刚1(1.,2661000;2.,266033):(RestrictionFragmentLengthPolymorphism,RFLP)PCR-RFLP,,:(1)PCR918SrDNA(2)TaqÑPshBÑ,918SrDNARFLP,RFLP,9:;18SrDNA;PCR-RFLP:Q178.53:A:1672-5174(2009)03Ò-105-05,,DNA,,,[1-4]RFLP,PCR,DNAPCRRFLP,RFLP,DNA,PCRDNA,,,RFLP[5-6]PCR-RFLPPCR-RFLPScholin(SSU)rDNA[7],,58(SSU)rDNA,4:BsaAÑBsrÑHaeÓXbaÑRFLP[8]Adachi235.8SrDNA,4:AluÑRsaÑMvaÑNdeÑ,RFLP,23[9]RFLP,A.catenellaA.tamarenseITS,[10]NCBI18SrDNA,DNAstarMegalign,918SrDNAVectorNTISuite9,918SrDNARFLP,,,1Table1SpeciesandoriginsofthealgaeinthisstudySpeciesOriginsofsamplesGenbankAccessionnumberProrocentrummicansEF492511ProrocentrumdonghaienseDW521206ProrocentrumminiEF017804AlexandriumtamarenseCCMP1598AY831407KareniamikimotoiEF492505ThalassiosiranordenskioldiAY229884ThalassiosiraweissflogiiCCMP1010FJ600728ChaetoceroscurvisetusFJ536743ChaetocerosdebilisAY229896XXX:E-mail:[e-mail]:(2006AA09Z178);(30490232):2009-04-30;:2009-06-12:(1982-),,E-mail:[e-mail]3920095PERIODICALOFOCEANUNIVERSITYOFCHINA39(Sup.):105~108May,200911.111.2DNASinha[11],96%()31.3NCBI,1Megalign,,PrimerPremier5Megalign1.4PCRPCR:Mg2+(25mmol/L)4LL;10@PCR5LL;dNTP(TAGC2.5mmmol/L)4LL;1LL(30Lmol/L);Taq0.3LL;DNA1LL;milliQ50LLPCR:94e10min;94e40s,55e40s,72e90s;30;72e10minPCR,1%,130V,30min,2%15min,1.5PCR(1)VectorNTI,,(2)5LL,5LL,3LL,milliQ50LL65e90min(TaqÑ);37e90min(PshBÑ)(3)PCR22.1megalign,18SrDNA,,megalignPrimerPremier5,:18S(R):3.-AACCTGGTTGATCCTGCCAGT-5.18S(F):3.-FTGATCCTTCTGCAGGTTCACCTAC-5..2.218SrDNAPCR518SrDNA1,5,2000bp,PCR,,,118SrDNAPCRFig.118SrDNAPCRamplificationresultsof5speciesofPyrrophyta(¹Prorocentrummicans;ºProrocentrumdonghaiense;»Prorocentrummini;¼Alexandriumtamarense;½Kareniamikimotoi;¾¿Thenegativecontrol;ÀmarkerDL2000)2,,2000bp,Thalassiosiraweissflogii()218SrDNAPCRFig.218SrDNAPCRamplificationresultsof4speciesofBacillariophyta(¹Thalassiosiranordenskioldi;ºThalas-siosiraweissflogii;»Chaetoceroscurvisetus;¼Chaetocerosdebilis;½Thenegativecontrol;¾mark-erDL2000)2.3(1)3,4,,TaqÑ,3,4,(3,41),(3,42),,,,10620093Fig.3ComparisonofsimulativeandexperientalresultsofProrocentrumdonghaiense(1:;2:1:SimulativeresultofProrocentrumdonghaiense;2:ExperientalresultofProrocentrumdonghaiense)4Fig.4ComparisonofsimulativeandexperientalresultsofChaetoceroscurvisetus(1.;2.1.SimulativeresultofChaetoceroscurvisetus;2.ExperientalresultofChaetoceroscurvisetus)55TaqÑFig.5TaqÑMacrorestrictionMapof5speciesofPyrrophyta(¹Alexandriumtamarense;ºKare-niamikimoto;»Prorocentrummini;¼Prorocentrummicans;½Prorocentrumdonghaiense;¾markerDL200)(2)5TaqÑ5,5TaqÑ5,,5TaqÑ6PshBÑFig.6PshBÑMacrorestrictionMapofProrocentrumminiandProrocentrummicans(¹Prorocentrummini;ºProrocentrummicans;»markerDL2000)74TaqÑFig.7TaqÑMacrorestrictionMapof4speciesofPyrrophyta(¹markerDL2000;ºThenegativecontrol;»Chaetoceroscurvisetus;¼Chaetocerosdebilis;½Thalassiosiraweissflogii;¾Thalassiosiranor-denskioldi)PshBÑ6TaqÑ,,PshBÑ,6,PshBÑ4TaqÑ7,,107,:PCR-RFLP3NCBI18SrDNA,PrimerPremier5DNASTAR,9,,,18SrDNARFLP,:(1)18SrDNA,,,(2),18SrDNA,,VectorNTI,TaqÑPshBÑ,,,,,,,TaqÑ9RFLP,,PshBÑ:(1),,,(2)TaqÑPshBÑ,(3),(4),4918SrDNA,TaqÑ918SrDNA,TaqÑ,PshBÑ,9:[1]UsupG,LeawCP,AhmadA,etal.PhylogeneticrelationshipofAlexandriumtamiyavanichii(Dinophyceae)tootherAlexandriumspeciesbasedonribosomalRNAgenesequences[J].HarmfulAlgae,2002,1:59-68.[2],,,.-[J].,1999,21:106-112.[3],,,./(18SrRNA)[J].,2006,37:450-456.[4]ZechmanFW,ZimmerEA,TheriotEC.UseofribosomalDNAin-ternaltranscribedspacersforphylogeneticstudiesindiatoms[J].JournalofPhycology,1994,30:507-512.[5]AcinasS,Rodriguez-ValeraF.Spatialandtemporalvariationinma-rinebacterioplanktondiversityasshownbyRFLPfingerprintingofPCRamptified16SrDNA[J].Microbiology,1997,24:27-40.[6]RamJL,RamML,BaidounF.Authenticationofcannedtunaandbonitobysequenceandrestrictionsiteanalysisofpolymerasechainre-actionproductsofmitochondrialDNA[J].JournalofAgriculturalandFoodChemistry,1996,44:2460-2467.[7]ScholinCA,AndersonDM,SoginML.Twodistinctsmal-lsubunitribosomalRNAgenesintheNorthAmericantoxicdinoflagellateAlexandriumfundyense(Dinophyceae)[J].JournalofPhycology,1993,29:209-216.[8]ScholinCA,HerzogM,SoginM,etal.IdentificationofgroupandstrainspecificgeneticmarkersforgloballydistributedAlexandrium(Dinophyceae)RFLPanalysisofSSUrRNAgenes[J].JournalofPhycology,1994,30:744-754.[9]AdachiM,SakoY,IshidaY.Restrictionfragmentlengthpolymor-phismofribosomalDNAinternaltranscribedspacerand5.8SregionsinJapaneseAlexandriumspecies(Dinophyceae)[J].JournalofPhy-cology,1994,30:857-865.[10],.[J].,1999,30:45-51.[11]SinhaRP,DonatP.Asimpleandefficientmethodforthequantita-tiveanalysisofthyminedimersincyanobacteria,Phytoplanktonandmacroalgae[J].ActaProtozoologica,2001,40:187-195.(114)1082009CommunityStructureofMacrobenthosinSpringintheNorthYellowSeaQUFang-Yuan,YUZ-iShan,LIUWe-iXia,SUIJ-iXing,ZHANGZh-iNan(CollegeofMarineLifeSciences,OceanUniversityofChina,Qingdao266003,China)Abstract:ThecommunitystructureofmacrobenthosintheNorthYellowSeaispresentedinthispaper.Thestudymaterialswerecollectedfrom80stationslocatedin37b~40bNand121b~124bEfromApr.24thtoMay