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ISSN:0974-7435Volume10Issue20BioTechnologyAnIndianJournalFULLPAPERBTAIJ,10(20),2014[12331-12335]ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
ChenKe1*,JiangQi2,ZhuJing1,ZhangYuan11SchoolofLifeScienceandEngineering,SWUST,Mianyang,Sichuan621010,(CHINA)2AgricultureandForestryInstituteofPanzhihua,Panzhihua,Sichuan,617067,(CHINA)E-mail:583091910@qq.
comABSTRACTThehigh-qualitygenomicDNAwhichwassuitableforAFLPanalysiswasisolatedbyanimprovedCTABmethodfromJ.
curcasL.
SomekeyfactorsaffectingDNAextraction,restriction-ligasereaction,pre-amplificationandamplificationprocesseswerestudiedandanoptimizedAFLPreactionsystemofJ.
curcaswasestablished.
ReferringtotheAFLPreactionsystem,clearDNAelectrophoresisfragmentsofJ.
curcaswereobtainedandtheresultwaswellreproductive.
KEYWORDSJ.
curcasL;DNAextraction;AFLP.
12332ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
BTAIJ,10(20)2014INTRODUCTIONJ.
curcasL.
,alsoknownasXiaotongxi,Gaotong,etc,belongstotheEuphorbiaceae,isnativetotropicalAmerica,andisnowwidelydistributedintropicalandsubtropicalregionsacrosstheworldanddistributedinYunnan,Sichuan,Guangdong,GuangxiandHainan,etc,inChina[1].
stem,leafandbarkofJ.
curcasL.
arerichinmilk,containalotoftoxicprotein,ketonesandotherantivirus,anti-AIDS,anti-diabetesandanti-tumorsubstances,therefore,J.
curcasL.
isaresearchhotspotglobally,especiallyintheanalysisofchemicalcomposition,toxicityandpharmacologicalguards[2]-[4].
Inaddition,J.
curcasL.
hasahighoilcontentupto60%inseeds,itisahigh-qualityrenewableoilplantresource,andcanbedirectlyforbiodieselproduction.
J.
curcasL.
issignificantthankstoitsfastgrowth,droughtresistance,barrenresistanceandotheradvantagesinthegreeningofbarrenhills,waterandsoilconservation,returningfarmlandtoforest[1].
Therefore,J.
curcasL.
isFAO-listedasapreferredspeciesforrenewableenergyandreducingtheecologicalpoverty.
However,rarestudieshavefocusedonthegeneticevolutionevaluationofresources,geneticbreedingofJ.
curcasL.
TheJ.
curcasresourcehasbeenrestrictedinpromotionandcultivation,togetherwitheconomicandsocialbenefits.
Theamplifiedfragmentlengthpolymorphism(AFLP)hasbeenwidelyappliedsuchfieldsasbiologicaldiversity,phylogeny,linkagemapconstruction,genelocation,cultivaridentificationandmolecularbreedingowingtothelessDNAdosage,goodrepeatability,abundantpolymorphism,highefficiencyandreliabilityandthatthegenomesequenceinformationisnotknowninadvance[5][6].
Therefore,thepresentpaperisdesignedtocreateasetofmorereliableJ.
curcasL.
AFLPreactionsystemthroughcreatingandoptimizingtheAFLPreactionsystemofJ.
curcasL.
,andplaysagoodgroundforthegeneticdiversity,germplasmconservationandbreedingofJ.
curcasL.
MATERIALSANDMETHODSMateialsJ.
curcasL.
leaveswerecollectedfromJinshaRiverbasin,Panzhihua,packedinplasticbag,driedwithsilicagelparticlesandsavedinafridgeat-80Celsiusdegree.
DNAExtractionTheleafDNAwasextractedusingmodifiedCTABmethod.
0.
2gleavesweregroundintopowerinamortarwithliquidnitrogen.
Duringgrinding,addPVP-40,transferthepowdertoa2mLcentrifugaltube,add500ìL2*CTABpreheatedextractionbufferat65℃and10ìL10mmolL-1-ME,shakeevenly,batheat65℃for45-60min,slowlyshakeevery5minsothatthesolutionisadequatelymixed;coolthemixturedowntoroomtemperature,addchloroformandisoamylolinequalvolume(24:1,VV-1),gentlyputupsidedownforabout20min,centrifugeat4℃in8000rmin-1for6min,movethesupernatanttoanewcentrifugaltube;addchloroformandisoamylolinequalvolumeinthesupernatant,putupsidedownevenly,centrifugeat8000rmin-1for4min,andrecoverthesupernatant;addpre-cooledisoamylolin2/3volumeinthesupernatant,andsettletheDNA,pickuptheflocculentDNA;washtheDNAwith70%alcoholtriceanddrytheDNAatroomtemperature;dissolvetheDNAwithappropriateamountof0.
1*TEbuffer;purifytheDNAbyaddingRnasein1ìL:10ìL,andsavethepurifiedDNAat-20℃foruse.
DeterminethepurityandconcentrationofgenomicDNAbyelectrophoresisandSmartSpecTM3000nucleicacidproteinanalyzer.
TABLE1:AFLPreactionsystemofJ.
curcas酶切体系Digestionsystem连接体系Ligationsystem预扩增体系Pre-amplificationsystem2L10*NEBbuffer2L10*T4ligasebuffer2L10*PCRbuffer0.
2L100*BSA0.
5LEcoRIadaptor(10pmol/L)2LMg2+(25mmol/L)3UEcoRI5LMseIadaptor(10pmol/L)2LdNTPs(2.
5mmol/L)3UMseI1UT4DNALigase1UTaqpolymerse2LDNA(200ng/L)Digestionreactionsolution10L1Lligationtemplate2.
4LPrimerE-A00(10pmol/L)2.
4LPrimerM-C00(10pmol/L)AdddH2Oupto20LAddddH2Oupto20LAddddH2Oupto20LCreationandoptimizationofAFLPsystemJ.
curcasAFLPsystemiscreatedaccordingtothemethodsdescribedbyVosetal[7]andoptimizedaboutthekeyfactors.
SettingoftemplateDNAconcentrationDilutetheextractedleafDNAwith0.
1*TEupto100ng/Lastemplate,comparetheinfluenceofdifferenttemplateconcentrations(100,150,200,250and300ng/L)onAFLPreaction.
BTAIJ,10(20)2014ChenKeetal.
12333DigestionandligationDigestthetemplateDNAusingMseIandEcoRIenzyme,set4digestiontimes3,4,5and6h,anddigestiontemperature37℃,keepthetemperatureat70℃for20minbeforethereactionisstopped;andthenaddtheligationsolutionforovernightligationat16℃.
Testthedigestedproductsby1.
2%agarosegelelectrophoresis.
SeeTABLE1fordigestionandligationsystem.
PreamplificationDilutetheligatedproductsby10timesasthepre-amplificationtemplate.
SeetheTABLE1forpre-amplificationsystem.
ThePCRamplificationprocedureisdoneunderthereactionconditionsasbelow:predenaturateat94Cfor2min;denaturateat94Cfor3s,annealat56Cfor30sandextendat72Cfor1minintotally20cycles.
Afterthepreamplificationreactionisfinished,measure5Lpre-amplifiedproductsfor2%agarosegelelectrophoresistotestthepreamplificationeffect.
5Lreactionproducttodilutewith0.
1*TEbufferin1:50wasmeasuredSelectiveamplificationThekeyfactorsthataffecttheresultsofselectiveamplificationsystemwereoptimizedinthereactionsystem.
SeeTABLE3forgradientdesignofeachfactorinthereactionsystem.
Thebestselectiveamplificationssystemwasoptimizedthroughthesinglefactorandorthogonalexperiment.
WiththegradientPCRamplificationtechnique,thereactionconditionsareasfollows:predegenerateat94Cfor2min;denaturateat94Cfor30s,annealat65Cfor30sandextendat72Cfor1min;afterwards,reducetheannealingtemperatureby0.
7Cineachcycletill56Cafter13cycles;carryon24cycleswithoutanychangetotherestconditions.
TABLE2:AdapterandPrimersequencesofAFLPanalysisforJ.
curcasNameCodeSequenceEcoRIadapter5'-AATTGGTACGCAGTCTAC-3'3'-CCATGCGTCAGATGCTC-5'MseIadapter5'-TACTCAGGACTCAT-3'3'-GAGTCCTGAGTAGGAG-5'EcoRIPrimerE-A005'-GTAGACTGCGTACCAATTCA-3'MseIPrimerM-C005'-GATGAGTCCTGAGTAAC-3'EcoRI+3-ACTE-ACT5'-GTAGACTGCGTACCAATTCACT-3'MseI+3-CTTM-CTT5'-GATGAGTCCTGAGTAACCTT-3'ElectrophoreticanalysisofselectiveamplificationproductsTABLE3:ConcentrationgradientsdesignofdifferentingredientsforAFLPselectiveamplificationFactorUnitConcentrationingredientMg2+mmol/L1.
5,2,2.
5,3,3.
5,4dNTPmmol/L0.
1,0.
2,0.
3,0.
4,0.
5,0.
6TaqEU0.
5,0.
75,1,1.
25,1.
5,1.
75Primersmol/L0.
2,0.
3,0.
4,0.
5,0.
8,1.
0Testtheselectiveamplificationproductsfirstby2%agarosegelelectrophoresis,andthenmeasureandmix3Lproductswithequalvolumeof2*AFLPsamplingbuffer,denatureat95Cfor5min,conduct6%polyacrylamidegelelectrophoresisonModelDYCE-20Csequencer(BeijingLiuyiFactory)at50Wconstantpowerfor2h.
Afterelectrophoresis,conductthesilverstainingstep:fixwithfixingliquidcontaining20%methanoland10%aceticacidfor20min;washwithion-freewatertricefor5mineach;adequatelyoscillatefor45mininthestainingsolution(2gAgNO3,3mL37%formaldehyde,and2000mLultra-purewater);washthegelwithultra-purewaterfor9s;adequatelyoscillateinthedevelopingsolution(60gNa2CO3/2Lultra-purewater,added3mL37%formaldehyde,400L10g/Lsodiumthiosulfatesolutionpriortouse)tillallbandsappear;finallystopdevelopinthefixingsolution(containing20%methanoland10%aceticacid)andwashintheion-freewater;placetheglassplateverticallyovernightatroomtemperature,dryandscantheimagesandrecordthedata.
RESULTANDDISSCUSSDNAextractionofJ.
curcasAsshowninFigure1fortheelectrophoresisresultofCTAB-extractedDNA,DNAhasnoobviousdispersionphenomenonwithmainclearband,suggestingthatthegenomicDNAisperfect.
OD260/OD280wastestedbetween1.
7~1.
9inlinewiththeAFLPexperimentalrequirement.
12334ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
BTAIJ,10(20)2014AFLPconstructionofJ.
curcasL.
TemplateDNA:5DNAtemplatedosage100~300ngsetintheexperimentcangivetheclearofAFLPspectrum,suggestingthatthetemplateconcentrationrangedbetween100and300ngdoesnotaffecttheexperimentalresultsofJ.
curcasL.
AFLPsystem.
Figure1:AgarosegelelectrophporesisofgenomicDNAFigure2:RestrictionfigureofthegenomicDNAwithEcoRIandMseIon2.
0%agarosegelelectrophoresisDigestionandligation:thedigestionpatternofEcoRIandMseIrestrictionendonucleaseswasused.
ThedigestionresultsinFigure2showedthatthegenomicDNAcanbecompleteddigestedbyEcoRIandMseI.
Analysison5digestiontimeshowedthattheresultatdigestiontime4~5hisbest.
Butifthedigestiontimeistooshort,thedigestionwillbeincomplete;oncontrary,ifthedigestiontimeistoolong,thespecificityofbandwillbereduced.
Pre-amplification:pre-amplificationresultscannotonlyreflecttheligationresults,butalsodirectlyaffectstheresultsofselectiveamplification.
Figure3showsthepreamplifiedsignalisstrongconsistentlyamongallsamples.
Thissuggeststhatthepre-amplificationsystemhasabetterresultintheexperiment,thatcanprovideaidealtemplateforthelateselectivesubsequentamplificationandthatthepreviousoperationstepsandresultsareinlinewiththerequirement.
Figure3:PreamplificationFigure4:SelectiveamplificationCreationofselectiveamplificationsystem:Withcomparablestudiesofseveralkeyfactors,suchasMg2+,dNTP,Taqandprimerconcentrationsinsinglefactorandorthogonalexperiments,itmaybecreatedthat20Lselectiveamplificationsystemisabestcombination:2.
5mmol/LMg2+,0.
3mmol/LdNTP,1UTaq,0.
4mo/Lprimersconcentrations.
SeeFigure4forthe2%agarosegelelectrophoresisofselectiveamplificationproductsinthesystem.
Theselectiveamplificationproductsgiveaclearpolymorphismelectrophoresisspectrumthrough6%polyacrylamidegelelectrophoresis(seeFigure5),suggestingthatthereactionsystemissuitablefortheanalysisofJ.
curcasL.
AFLP.
Figure5:AFLPfingerprintsbyprimerE-ACT/M-CTTon6.
0%polyacrylamidegelelectrophoresisBTAIJ,10(20)2014ChenKeetal.
12335DISCUSSIONAsAFLPanalysishighlyrequirestheDNAtemplate,toextractthehigh-qualityandhigh-purityDNAiscriticaltotheAFLP'ssuccess.
Therefore,thepresentexperimentusedthemodifiedCTABmethodintermofthemoresubstancesandcomplexcomponentsinJ.
curcasL.
fortotalDNAextraction.
ThetestproofedthataddingasmallamountofPVP-40duringliquidnitrogengrindingprocesscangreatlyimprovetheDNAextractionquality;andthisresultwasagreedbyotherplantDNAextractionresearches[8][9].
PVP-40hastheabilitytostronglybindthepolyphenol,canpreventphenolicoxidationintoquininesafterbindingtoavoidthesolutionbrowningandhaveantioxidantproperties,andcanpreventDNAenzymetodegradetheDNA.
Inaddition,addingchloroform/isoamylalcoholextractiontimescanplayamarkedroleinremovingtheproteinandpolysaccharide,etcinDNA,however,itisalsomarkedthattheextractedDNAislost.
Therefore,itisrecommendedtoextractpreferablytwice.
TherearemanyaffectingfactorsandtheprocedureismorecomplexduringJ.
curcasL.
AFLPanalysis.
TheconcentrationisalsorequiredinadditiontothehighlyrequiredtemplateDNAquality.
IftheDNAconcentrationistoohigh,moreprimerordNTPswillbeconsumed,thesubstratewillbeamplifiedexcessively,andtheunstableillusionofamplificationresultoccurs[9].
Duringthepreamplificationandselectiveamplificationprocess,theMg2+concentrationnotonlyaffectstheTaqactivity,itcanalsocombinewithdNTPandDNAtemplateinthereactionsolution,affectthebindingefficiencybetweenprimerandtemplate,chaindecompositiontemperaturebetweentemplateandproductandproductspecificityandformtheprimerdimer[7].
Additionally,forthedNTPsastherawmaterialinPCRreaction,toohighconcentrationwillleadtoPRCmismatchandthenon-specificamplificationappears;toolowconcentrationwillaffectthesynthesisefficiency[10].
Thesamplesmustbeadequatelydenaturedpriortopolyacrylamidegelelectrophoresis,orotherwise,thebackgroundoftherunwillbedarken;toolongstainingtimewillleadtodarkergelplatebackgroundandaffectobservation.
Thedevelopingtimealsoservesasakeyfactorforthesilverstainingquality.
Ifthebanddevelopingtimeistoolong,thebandwillberelativelyblurred;ifthebanddevelopingtimeistooshort,itisdifficulttoobservethecolorandshadow;thedevelopingtemperaturemustalsobekeptatn12~15℃.
CONCLUSIONSThepresentstudycreatesandoptimizestheJ.
curcasL.
AFLPreactionsystem,provideagroundforthefoundingofJ.
curcasL.
molecularmarkertechnologicalplatformandresearchesonthegermplasmresourcediversity,andhasapracticalguidanceandbroadapplicationprospectsforJ.
curcasL.
breeding,molecularmarkerbreading,resourceutilizationandprotection.
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[J],FujianForestryScienceandTechnology,34(3),238-242(2007).
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Sirisomboon,P.
Kitchaiya,T.
Pholphoetal.
;PhysicalandmechanicalpropertiesofJatrophacurcasL.
fruits,nutsandkernels[J],BiosystemsEngineering,97,201-207(2007).
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Martiner-Herrera,P.
Siddhuraju,G.
Francis;Chemicalcomposition,toxic/antimetabolicconstituents,andeffectsofdifferenttreatmentsontheirlevels,infourprovenancesofJatrophacurcasL.
fromMexico[J],FoodChemistry,96,80-89(2006).
[4]FanJudi,YangSong,SongBaoanetal.
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pesticidesandpharmaceuticalandbiologicalactivity[J],Pesticides,45(5),298-301(2006).
[5]GuoYanyan,TangWenyu,XingShiyanetal.
;CreationandoptimizationofAFLPreactionsystemofGinkgobiloba[J],JiangxiAgriculturalUniversityJournal,28(3),377-381(2006).
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Tatikonda,S.
P.
Wani,S.
Kannanetal.
;AFLP-basedmolecularcharacterizationofanelitegermplasmcollectionofJatrophacurcasL.
,abiofuelplant[J],PlantScience,doi,10.
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(availableonline),(2009).
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;AFLP,AnewtechniqueforDNAfingerprinting[J],NeuAcidRes,23(21),4407-4414(1997).
[8]HuangWenkun,GuoJianying,WanFanghaoetal.
;CreationofeupatoriumadenophorumDNAextractionandAFLPreactionsystem,JournalofWuhanBotanicalResearch[J],24(6),498-504(2006).
[9]YangJuan,LiShan,CaoDongweietal.
;CreationandanalysisofdipteroniaAFLPreactionsystem,ActaBotanicaBoreali-OccidentaliaSinica[J],25(11),2173-2177(2005).
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ISSN:0974-7435Volume10Issue20BioTechnologyAnIndianJournalFULLPAPERBTAIJ,10(20),2014[12331-12335]ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
ChenKe1*,JiangQi2,ZhuJing1,ZhangYuan11SchoolofLifeScienceandEngineering,SWUST,Mianyang,Sichuan621010,(CHINA)2AgricultureandForestryInstituteofPanzhihua,Panzhihua,Sichuan,617067,(CHINA)E-mail:583091910@qq.
comABSTRACTThehigh-qualitygenomicDNAwhichwassuitableforAFLPanalysiswasisolatedbyanimprovedCTABmethodfromJ.
curcasL.
SomekeyfactorsaffectingDNAextraction,restriction-ligasereaction,pre-amplificationandamplificationprocesseswerestudiedandanoptimizedAFLPreactionsystemofJ.
curcaswasestablished.
ReferringtotheAFLPreactionsystem,clearDNAelectrophoresisfragmentsofJ.
curcaswereobtainedandtheresultwaswellreproductive.
KEYWORDSJ.
curcasL;DNAextraction;AFLP.
12332ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
BTAIJ,10(20)2014INTRODUCTIONJ.
curcasL.
,alsoknownasXiaotongxi,Gaotong,etc,belongstotheEuphorbiaceae,isnativetotropicalAmerica,andisnowwidelydistributedintropicalandsubtropicalregionsacrosstheworldanddistributedinYunnan,Sichuan,Guangdong,GuangxiandHainan,etc,inChina[1].
stem,leafandbarkofJ.
curcasL.
arerichinmilk,containalotoftoxicprotein,ketonesandotherantivirus,anti-AIDS,anti-diabetesandanti-tumorsubstances,therefore,J.
curcasL.
isaresearchhotspotglobally,especiallyintheanalysisofchemicalcomposition,toxicityandpharmacologicalguards[2]-[4].
Inaddition,J.
curcasL.
hasahighoilcontentupto60%inseeds,itisahigh-qualityrenewableoilplantresource,andcanbedirectlyforbiodieselproduction.
J.
curcasL.
issignificantthankstoitsfastgrowth,droughtresistance,barrenresistanceandotheradvantagesinthegreeningofbarrenhills,waterandsoilconservation,returningfarmlandtoforest[1].
Therefore,J.
curcasL.
isFAO-listedasapreferredspeciesforrenewableenergyandreducingtheecologicalpoverty.
However,rarestudieshavefocusedonthegeneticevolutionevaluationofresources,geneticbreedingofJ.
curcasL.
TheJ.
curcasresourcehasbeenrestrictedinpromotionandcultivation,togetherwitheconomicandsocialbenefits.
Theamplifiedfragmentlengthpolymorphism(AFLP)hasbeenwidelyappliedsuchfieldsasbiologicaldiversity,phylogeny,linkagemapconstruction,genelocation,cultivaridentificationandmolecularbreedingowingtothelessDNAdosage,goodrepeatability,abundantpolymorphism,highefficiencyandreliabilityandthatthegenomesequenceinformationisnotknowninadvance[5][6].
Therefore,thepresentpaperisdesignedtocreateasetofmorereliableJ.
curcasL.
AFLPreactionsystemthroughcreatingandoptimizingtheAFLPreactionsystemofJ.
curcasL.
,andplaysagoodgroundforthegeneticdiversity,germplasmconservationandbreedingofJ.
curcasL.
MATERIALSANDMETHODSMateialsJ.
curcasL.
leaveswerecollectedfromJinshaRiverbasin,Panzhihua,packedinplasticbag,driedwithsilicagelparticlesandsavedinafridgeat-80Celsiusdegree.
DNAExtractionTheleafDNAwasextractedusingmodifiedCTABmethod.
0.
2gleavesweregroundintopowerinamortarwithliquidnitrogen.
Duringgrinding,addPVP-40,transferthepowdertoa2mLcentrifugaltube,add500ìL2*CTABpreheatedextractionbufferat65℃and10ìL10mmolL-1-ME,shakeevenly,batheat65℃for45-60min,slowlyshakeevery5minsothatthesolutionisadequatelymixed;coolthemixturedowntoroomtemperature,addchloroformandisoamylolinequalvolume(24:1,VV-1),gentlyputupsidedownforabout20min,centrifugeat4℃in8000rmin-1for6min,movethesupernatanttoanewcentrifugaltube;addchloroformandisoamylolinequalvolumeinthesupernatant,putupsidedownevenly,centrifugeat8000rmin-1for4min,andrecoverthesupernatant;addpre-cooledisoamylolin2/3volumeinthesupernatant,andsettletheDNA,pickuptheflocculentDNA;washtheDNAwith70%alcoholtriceanddrytheDNAatroomtemperature;dissolvetheDNAwithappropriateamountof0.
1*TEbuffer;purifytheDNAbyaddingRnasein1ìL:10ìL,andsavethepurifiedDNAat-20℃foruse.
DeterminethepurityandconcentrationofgenomicDNAbyelectrophoresisandSmartSpecTM3000nucleicacidproteinanalyzer.
TABLE1:AFLPreactionsystemofJ.
curcas酶切体系Digestionsystem连接体系Ligationsystem预扩增体系Pre-amplificationsystem2L10*NEBbuffer2L10*T4ligasebuffer2L10*PCRbuffer0.
2L100*BSA0.
5LEcoRIadaptor(10pmol/L)2LMg2+(25mmol/L)3UEcoRI5LMseIadaptor(10pmol/L)2LdNTPs(2.
5mmol/L)3UMseI1UT4DNALigase1UTaqpolymerse2LDNA(200ng/L)Digestionreactionsolution10L1Lligationtemplate2.
4LPrimerE-A00(10pmol/L)2.
4LPrimerM-C00(10pmol/L)AdddH2Oupto20LAddddH2Oupto20LAddddH2Oupto20LCreationandoptimizationofAFLPsystemJ.
curcasAFLPsystemiscreatedaccordingtothemethodsdescribedbyVosetal[7]andoptimizedaboutthekeyfactors.
SettingoftemplateDNAconcentrationDilutetheextractedleafDNAwith0.
1*TEupto100ng/Lastemplate,comparetheinfluenceofdifferenttemplateconcentrations(100,150,200,250and300ng/L)onAFLPreaction.
BTAIJ,10(20)2014ChenKeetal.
12333DigestionandligationDigestthetemplateDNAusingMseIandEcoRIenzyme,set4digestiontimes3,4,5and6h,anddigestiontemperature37℃,keepthetemperatureat70℃for20minbeforethereactionisstopped;andthenaddtheligationsolutionforovernightligationat16℃.
Testthedigestedproductsby1.
2%agarosegelelectrophoresis.
SeeTABLE1fordigestionandligationsystem.
PreamplificationDilutetheligatedproductsby10timesasthepre-amplificationtemplate.
SeetheTABLE1forpre-amplificationsystem.
ThePCRamplificationprocedureisdoneunderthereactionconditionsasbelow:predenaturateat94Cfor2min;denaturateat94Cfor3s,annealat56Cfor30sandextendat72Cfor1minintotally20cycles.
Afterthepreamplificationreactionisfinished,measure5Lpre-amplifiedproductsfor2%agarosegelelectrophoresistotestthepreamplificationeffect.
5Lreactionproducttodilutewith0.
1*TEbufferin1:50wasmeasuredSelectiveamplificationThekeyfactorsthataffecttheresultsofselectiveamplificationsystemwereoptimizedinthereactionsystem.
SeeTABLE3forgradientdesignofeachfactorinthereactionsystem.
Thebestselectiveamplificationssystemwasoptimizedthroughthesinglefactorandorthogonalexperiment.
WiththegradientPCRamplificationtechnique,thereactionconditionsareasfollows:predegenerateat94Cfor2min;denaturateat94Cfor30s,annealat65Cfor30sandextendat72Cfor1min;afterwards,reducetheannealingtemperatureby0.
7Cineachcycletill56Cafter13cycles;carryon24cycleswithoutanychangetotherestconditions.
TABLE2:AdapterandPrimersequencesofAFLPanalysisforJ.
curcasNameCodeSequenceEcoRIadapter5'-AATTGGTACGCAGTCTAC-3'3'-CCATGCGTCAGATGCTC-5'MseIadapter5'-TACTCAGGACTCAT-3'3'-GAGTCCTGAGTAGGAG-5'EcoRIPrimerE-A005'-GTAGACTGCGTACCAATTCA-3'MseIPrimerM-C005'-GATGAGTCCTGAGTAAC-3'EcoRI+3-ACTE-ACT5'-GTAGACTGCGTACCAATTCACT-3'MseI+3-CTTM-CTT5'-GATGAGTCCTGAGTAACCTT-3'ElectrophoreticanalysisofselectiveamplificationproductsTABLE3:ConcentrationgradientsdesignofdifferentingredientsforAFLPselectiveamplificationFactorUnitConcentrationingredientMg2+mmol/L1.
5,2,2.
5,3,3.
5,4dNTPmmol/L0.
1,0.
2,0.
3,0.
4,0.
5,0.
6TaqEU0.
5,0.
75,1,1.
25,1.
5,1.
75Primersmol/L0.
2,0.
3,0.
4,0.
5,0.
8,1.
0Testtheselectiveamplificationproductsfirstby2%agarosegelelectrophoresis,andthenmeasureandmix3Lproductswithequalvolumeof2*AFLPsamplingbuffer,denatureat95Cfor5min,conduct6%polyacrylamidegelelectrophoresisonModelDYCE-20Csequencer(BeijingLiuyiFactory)at50Wconstantpowerfor2h.
Afterelectrophoresis,conductthesilverstainingstep:fixwithfixingliquidcontaining20%methanoland10%aceticacidfor20min;washwithion-freewatertricefor5mineach;adequatelyoscillatefor45mininthestainingsolution(2gAgNO3,3mL37%formaldehyde,and2000mLultra-purewater);washthegelwithultra-purewaterfor9s;adequatelyoscillateinthedevelopingsolution(60gNa2CO3/2Lultra-purewater,added3mL37%formaldehyde,400L10g/Lsodiumthiosulfatesolutionpriortouse)tillallbandsappear;finallystopdevelopinthefixingsolution(containing20%methanoland10%aceticacid)andwashintheion-freewater;placetheglassplateverticallyovernightatroomtemperature,dryandscantheimagesandrecordthedata.
RESULTANDDISSCUSSDNAextractionofJ.
curcasAsshowninFigure1fortheelectrophoresisresultofCTAB-extractedDNA,DNAhasnoobviousdispersionphenomenonwithmainclearband,suggestingthatthegenomicDNAisperfect.
OD260/OD280wastestedbetween1.
7~1.
9inlinewiththeAFLPexperimentalrequirement.
12334ConstructionandanalysisoftheAFLPreactionsystemofJ.
curcasL.
BTAIJ,10(20)2014AFLPconstructionofJ.
curcasL.
TemplateDNA:5DNAtemplatedosage100~300ngsetintheexperimentcangivetheclearofAFLPspectrum,suggestingthatthetemplateconcentrationrangedbetween100and300ngdoesnotaffecttheexperimentalresultsofJ.
curcasL.
AFLPsystem.
Figure1:AgarosegelelectrophporesisofgenomicDNAFigure2:RestrictionfigureofthegenomicDNAwithEcoRIandMseIon2.
0%agarosegelelectrophoresisDigestionandligation:thedigestionpatternofEcoRIandMseIrestrictionendonucleaseswasused.
ThedigestionresultsinFigure2showedthatthegenomicDNAcanbecompleteddigestedbyEcoRIandMseI.
Analysison5digestiontimeshowedthattheresultatdigestiontime4~5hisbest.
Butifthedigestiontimeistooshort,thedigestionwillbeincomplete;oncontrary,ifthedigestiontimeistoolong,thespecificityofbandwillbereduced.
Pre-amplification:pre-amplificationresultscannotonlyreflecttheligationresults,butalsodirectlyaffectstheresultsofselectiveamplification.
Figure3showsthepreamplifiedsignalisstrongconsistentlyamongallsamples.
Thissuggeststhatthepre-amplificationsystemhasabetterresultintheexperiment,thatcanprovideaidealtemplateforthelateselectivesubsequentamplificationandthatthepreviousoperationstepsandresultsareinlinewiththerequirement.
Figure3:PreamplificationFigure4:SelectiveamplificationCreationofselectiveamplificationsystem:Withcomparablestudiesofseveralkeyfactors,suchasMg2+,dNTP,Taqandprimerconcentrationsinsinglefactorandorthogonalexperiments,itmaybecreatedthat20Lselectiveamplificationsystemisabestcombination:2.
5mmol/LMg2+,0.
3mmol/LdNTP,1UTaq,0.
4mo/Lprimersconcentrations.
SeeFigure4forthe2%agarosegelelectrophoresisofselectiveamplificationproductsinthesystem.
Theselectiveamplificationproductsgiveaclearpolymorphismelectrophoresisspectrumthrough6%polyacrylamidegelelectrophoresis(seeFigure5),suggestingthatthereactionsystemissuitablefortheanalysisofJ.
curcasL.
AFLP.
Figure5:AFLPfingerprintsbyprimerE-ACT/M-CTTon6.
0%polyacrylamidegelelectrophoresisBTAIJ,10(20)2014ChenKeetal.
12335DISCUSSIONAsAFLPanalysishighlyrequirestheDNAtemplate,toextractthehigh-qualityandhigh-purityDNAiscriticaltotheAFLP'ssuccess.
Therefore,thepresentexperimentusedthemodifiedCTABmethodintermofthemoresubstancesandcomplexcomponentsinJ.
curcasL.
fortotalDNAextraction.
ThetestproofedthataddingasmallamountofPVP-40duringliquidnitrogengrindingprocesscangreatlyimprovetheDNAextractionquality;andthisresultwasagreedbyotherplantDNAextractionresearches[8][9].
PVP-40hastheabilitytostronglybindthepolyphenol,canpreventphenolicoxidationintoquininesafterbindingtoavoidthesolutionbrowningandhaveantioxidantproperties,andcanpreventDNAenzymetodegradetheDNA.
Inaddition,addingchloroform/isoamylalcoholextractiontimescanplayamarkedroleinremovingtheproteinandpolysaccharide,etcinDNA,however,itisalsomarkedthattheextractedDNAislost.
Therefore,itisrecommendedtoextractpreferablytwice.
TherearemanyaffectingfactorsandtheprocedureismorecomplexduringJ.
curcasL.
AFLPanalysis.
TheconcentrationisalsorequiredinadditiontothehighlyrequiredtemplateDNAquality.
IftheDNAconcentrationistoohigh,moreprimerordNTPswillbeconsumed,thesubstratewillbeamplifiedexcessively,andtheunstableillusionofamplificationresultoccurs[9].
Duringthepreamplificationandselectiveamplificationprocess,theMg2+concentrationnotonlyaffectstheTaqactivity,itcanalsocombinewithdNTPandDNAtemplateinthereactionsolution,affectthebindingefficiencybetweenprimerandtemplate,chaindecompositiontemperaturebetweentemplateandproductandproductspecificityandformtheprimerdimer[7].
Additionally,forthedNTPsastherawmaterialinPCRreaction,toohighconcentrationwillleadtoPRCmismatchandthenon-specificamplificationappears;toolowconcentrationwillaffectthesynthesisefficiency[10].
Thesamplesmustbeadequatelydenaturedpriortopolyacrylamidegelelectrophoresis,orotherwise,thebackgroundoftherunwillbedarken;toolongstainingtimewillleadtodarkergelplatebackgroundandaffectobservation.
Thedevelopingtimealsoservesasakeyfactorforthesilverstainingquality.
Ifthebanddevelopingtimeistoolong,thebandwillberelativelyblurred;ifthebanddevelopingtimeistooshort,itisdifficulttoobservethecolorandshadow;thedevelopingtemperaturemustalsobekeptatn12~15℃.
CONCLUSIONSThepresentstudycreatesandoptimizestheJ.
curcasL.
AFLPreactionsystem,provideagroundforthefoundingofJ.
curcasL.
molecularmarkertechnologicalplatformandresearchesonthegermplasmresourcediversity,andhasapracticalguidanceandbroadapplicationprospectsforJ.
curcasL.
breeding,molecularmarkerbreading,resourceutilizationandprotection.
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[J],FujianForestryScienceandTechnology,34(3),238-242(2007).
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Sirisomboon,P.
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Pholphoetal.
;PhysicalandmechanicalpropertiesofJatrophacurcasL.
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Martiner-Herrera,P.
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Francis;Chemicalcomposition,toxic/antimetabolicconstituents,andeffectsofdifferenttreatmentsontheirlevels,infourprovenancesofJatrophacurcasL.
fromMexico[J],FoodChemistry,96,80-89(2006).
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pesticidesandpharmaceuticalandbiologicalactivity[J],Pesticides,45(5),298-301(2006).
[5]GuoYanyan,TangWenyu,XingShiyanetal.
;CreationandoptimizationofAFLPreactionsystemofGinkgobiloba[J],JiangxiAgriculturalUniversityJournal,28(3),377-381(2006).
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[8]HuangWenkun,GuoJianying,WanFanghaoetal.
;CreationofeupatoriumadenophorumDNAextractionandAFLPreactionsystem,JournalofWuhanBotanicalResearch[J],24(6),498-504(2006).
[9]YangJuan,LiShan,CaoDongweietal.
;CreationandanalysisofdipteroniaAFLPreactionsystem,ActaBotanicaBoreali-OccidentaliaSinica[J],25(11),2173-2177(2005).
[10]ChenMin,TaoJing,MaChaodeetal.
;HolcocerushippophaecolusprimersscreeningandcreationofAFLPreactionsystem[J],JournalofBeijingForestryUniversity,30(4),116-120(2008).
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