基于5種草屬藥用植物的5個(gè)分子身份證的構(gòu)建

基于5種草屬藥用植物的5個(gè)分子身份證的構(gòu)建

genomica（洋務(wù)）的目標(biāo)是近250年的。對(duì)于一般知識(shí)，它被認(rèn)為是“西蒙泰”的“非洲”。在這條規(guī)則中，“米卡”是第一學(xué)期的非預(yù)算資源，“米卡”是第二學(xué)期的非預(yù)算資源。這是報(bào)告的。時(shí)間短，主要是短期間。它是一個(gè)緩慢的地方。特征。馬蹄蓮和科茨官人是行動(dòng)的一部分。證據(jù)表明，這些因素是自給自足的。這是一個(gè)非常普遍的做法。就像它一樣，它是一個(gè)緩慢的步驟，而不是一個(gè)受歡迎的替代形式?？傊且粋€(gè)受歡迎的例子。Comparedwiththemorphologicalandchemicalmethods,moleculargeneticmethods,especiallythosePCR-basedmethods,havebeenprovedstable,reliableandefficienttoidentifytheherbalmedicines.TheARMS(amplificationrefractorymutationsystem)isalsodescribedasallele-specificPCR,whichcoulddetectsinglebasemutations.Now,ARMShavesuccessfullybeenusedtoauthenticateherbmedicinessuchasPanaxginseng,Alismaorientaleetal.Inhighereukaryotes,the5SrDNAunitconsistsofa120bpconserved5SrRNAgenecodingregionandnon-transcribedspacer(NTS)ofvarioussize.SuchdiversityoftheNTSindifferentspeciescanbeusedasanidentificationbasis.InthisstudyweestablishedthetechniqueofARMSbasedon5SrDNAsequence,toidentifyV.jatamansifromotherValerianaspeciesandherbalmedicinesefficientlyandconvenientlyandascertainthephylogeneticrelationshipamongfiveValerianaspecies.杏仁杏仁1u2004范圍ThefivetestedspeciesdistributinginSouthwestChinaincludedV.jatamansi,V.pseudofficinalisC.Y.ChengetH.B.Chen,V.officinalisLinn.var.latifoliaMiq.,V.hardwickiiWall.andV.flaccidissimaMaxim.AllthespecimenswerecollectedandcultivatedinInstituteofChineseHerbalMedicines,HubeiAcademyofAgriculturalSciences.PlantmaterialswereauthenticatedbyProf.Lin(HubeiAcademyofAgriculturalSciences)andthevoucherswerekeptinWuhanUniversity.Youngleavesweredriedinsilicagelandusedasresearchmaterials.DetailedmessagesofspecimensweredepictedinTable1.Inaddition,fortheherbalmedicinepreparations,threeitemsavailableinthemarketplacewereboughtfromAierkangpharmacy(Wuhan)andusedinARMSanalysis,includingXiangGuoJianXiaoPian(sugarcoatedtablet,producedbyYunnanYunheMedicineCo.,Ltd.,batchnumber:07020602),aresourcecontainingthepowderofroastedRhizomaValerianajatamansi,andRenShenGuiPiWan(honeyedpill,producedbyJilinShuangshiMedicineCo.,Ltd.,batchnumber:080101)andXiaoYaoWan(wateredpill,producedbyChinaResourcesSanjiuMedical&PharmaceuticalCo.,Ltd.,batchnumber:090301),theresourceswithoutRhizomaValerianajatamansi.2pcrpcrThedryleavesandherbalmedicinepreparationsweregroundtofinepowdersinliquidnitrogen.GenomicDNAwasextractedusingtheDNAextractionkit(BeijingTiangenCo.,Ltd).AccordingtoFukuietal.,thecomplete5SrDNANTSregionwereamplifiedbyusingprimersofNF(5’-GATCCCATCAGAACTCCGAAG-3’)andNR(5’-CGGTGATTTAGTGCTGGTAT-3’).Each25μLPCRmixturecontained:50ngoftotalgenomicDNA,400mmol·L-1ofeachdNTP,1UTaqpolymerase(DingguoBiotechnologyCo.Ltd.),1×PCRbuffer,2.0mmol·L-1MgCl2,and0.4mmol·L-1ofeachprimer.Sampleswereamplifiedthrough30thermalcyclesinMJ-PTC100TM(EastwinLifeSciences,Inc.),andeachcycleconsistingof94℃for30sec,55℃for30sec,and72℃for1min.ThePCRproductswereextractedfromgels,clonedintopGEM-Tvector(Promega),andatleastthreeclonesofeachspecimenweresequencedonanABI3730XLsequencer(SunbiotechCo.,Ltd,Beijing,China).AllsequencesweresubmittedtoGenBank.SequenceswerealignedandclusteredusingClustalXandDNAMANVersion4.0software,respectively.3ssrnareieeBasedonthenucleotidesubstitutionsintheNTSofV.jatamansi,sixreversediagnosticprimerswithcomplementary3’-residuesweredesignedforARMSanalysis,separately(Table2).AllthecorrespondingforwardprimerswereNF.ThePCRprocedureofARMSanalysiswasthesameasthePCRamplificationof5SrDNAregionandtheannealingtemperaturewasoptimizedwithintherangeof55-60℃.ThegenomicDNAisolatedfromtheherbaldrugsofXiangGuoJianXiaoPian,RenShenGuiPiWanandXiaoYaoWanweretestedwithARMSanalysisinthesamePCRconditionusingtheselecteddiagnosticprimer.PCRproductswerecheckedona1.2%agarosegelandwerevisualizedbyethidiumbromidestainingunderUV.NegativecontrolswereperformedalongwitheachPCRamplification.ThreeindependentexperimentsforARMSweretestedtoverifythereproducibilityofthemethods.partiace安氏回復(fù)突變armsimasimasoinficiensima,simasima,sima,sima,sima,laciratchening,etizact.u2005.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.5.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.3.5.3.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5.5ThePCRproductcontainedpartial5SrRNAgeneandcompleteNTSsequences.SizedifferencesofPCRproductswerenotdetectedunderagarosegelelectrophoresis.Accordingtothesequencingresults,different5SrDNAsequencesofeachtestedspecieswereselectedforthefurtherstudies.Thesequenceanalysisofdifferentclonesfoundthatthecodingsequencesof5SrRNAgenewereconserved,buttheNTSsequencevariedinthetestedspecies.TheNTSregionvariedfrom189to225ntinV.jatamansi,212-213ntinV.pseudofficinalis,212-213ntinV.officinalisvar.latifolia,237-244ntinV.hardwickiiand190-233ntinV.flaccidissima.ItseemsthatNTSdivergencewithinonespecieswascomparablystableforV.flaccidissima,V.hardwickiiandV.jatamansiamongthefivetestedspecies.Inthephylogenetictree,NTSsequencesfromV.flaccidissima,V.hardwickiiandV.jatamansiclusteredintoasubclade,separately,whilethoseofV.pseudofficinalisandV.officinalisvar.latifolianestedtogether(Figure1).ThisresultsuggestedV.pseudofficinalisandV.officinalisvar.latifoliahadthecloserelationshipamongthetestedspecies.V.hardwickiishowedthelongestbranchintheclustertreewhichindicatedithadthefarthestgeneticdistancetotheotherfourspecies.ForARMSanalysis,thesamenucleotidesubstitutionsindifferentclonesofV.jatamansiwerefoundatposition171(EU926613),167(EU926614)and174(EU926615),respectively.SixdiagnosticprimersweredesignedforARMSanalysisonthebasisoftheSNPsite.Inordertoensureamplificationspecificity,onestrongdestabilizingmismatch(G/CpairreplacedbyG/Tpair)wasdeliberatelyintroducedintoprimerzhiR5atthe1stnucleotidefrom3’terminus.Thefivespecieswereamplifiedwiththesixspecificprimers,respectively.Theoretically,onlyV.jatamansishouldbesuccessfullyamplified.Expectedly,primerzhiR5couldamplifythefragmentapproximate200bpinV.jatamansiwiththeannealingtemperature58℃andnoPCRproductwasobservedintheotherfourtestedspeciesatthesametime(Figure2a).TherestofdiagnosticprimerscouldnotamplifytheexpectedDNAfragmentinallthePCRamplifications.Therefore,thesefiveprimerscouldnotbeusedforauthenticationofV.jatamansiexceptprimerzhiR5.ThreeherbalmedicinepreparationsfrommarketwereusedtotestthereliabilityofARMSmethodusingprimerpairzhiR5andNF.ThesamplecontainingV.jatamansishowedthepositivefragmentasexpectedafterthePCRamplification,whilenoPCRproductwasfoundintheothertwoherbalmedicinepreparationwhichnotcontainingtheresourceofV.jatamansi(Figure2b).TheseresultssuggestedthisARMSanalysiscouldbeusednotonlytoauthenticatethegenuineplantofV.jatamansifromValerianaspeciesbutalsoidentifyV.jatamansifromtheherbalmedicinepreparations.Inthisstudy,5SrDNAsequenceisprovedtobeausefulspecies-specificmarkerforphylogeneticanalysisofValerianaspecies.ThuswecouldgetdeepinsightoftherelationshipamongthespeciesonDNAlevelbesidesthemorphologicalanalysis.V.pseudofficinaliswasthemostwidelyusedforsedatio