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Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

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Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

    Screening of Transgenic Soybean

    Transformed by Means of Pollen-tube

    Using Kanamycin

    June2006JournalofNortheastAgficulturalUniversityV0I_l3.No.17l0

    ArticleID:l0068l04(2006)0l000704

    ScreeningofTransgenicSoybeanTransformedbyMeansof

    Pollen-tubeUsingKanamycin

    ZHANGShuzhen,XUPengfei,ZHANGDayong,LINShifeng,LIWenbin,

    HANYingpeng,YANGChuanping2

    (1.SoybeanResearchInstitute,NortheastAgriculturalUniversity,HarbinHeilongjiang150030,PRC;

    2.ForestryCollege,NortheastForestryUniversity,HarbinHeilongjiang150040,PRC) Abstract:KanamycinwasusedtoscreenT0seedsofthevarietyDongnong46transformedbymeansof

    pollen-tubemethod.Theresultsshowedthat400mg?L-kanamycincouldinhibitgrowthofnon-transgenicplants,

    and2positiveplantsweregottencombinedwithGusdyeingandPCRdetection.Itisprovedthatthismethodise

    conomicandeffectiveinpreliminaryscreeningthetransgenicplants. Keywords:soybean;pollen-tubemethod;kanamycin

    CLCnumber:S565.1Documentcode:A

    Thepollen——tubemethodhasbeenwidelyusedin

    soybeangenetransferforitsadvantageofsimplicity

    andavoidingtheobstacleofprocessofplantregener_

    ation,butthetestingofalargenumberofpotential

    transgenicseedsisveryarduousanddifficult.Zhou1

    screenedthesoybeanseedsbywayofGusdyeing combinedwithPCR.Althoughtheprocessisrelatively simple,itiscomplicatedandexpensive. Inthispaper,Kanamycinwasusedtopreliminar

    ilvscreenthetransgenicseedswithIImarker genegottenbywayofpollentube.

    Therapidgerminationmethodwasusedtoselect thetransgenicplantswhoseseedswereputontheMS mediaaddedwithappropriateconcentrationof Kanamycin,andthentheGusdyeingandPCRampli

    ficationwerecarriedouttofurthercorgitinthetrans——

    genicsoybean.

    Thepurposeofthisresearchistosimplifythese——

    lectingworkfromalargenumberofseedsbywayof pollentube,andthiscouldbeverysignificantinsoy

    beanwhichisacropdifficulttotransfer. 1MaterialsandMethods

    1.1Materials

    Soybeanseeds:non-transgenicseedsandtheTo seedsofthevarietyDongnong46transformedbyway ofthepollentubemethod.

    Plasmid:PGBI121S4ABCfconstructedbySandui GuoLabinChineseAcademyofAgriculturalSci——

    ences).includingBt,CPTI

    GUSand?P71?fselection

    (bi-insectresistancegene),

    markergene)(Fig.1).

    35SP

    Fig.1MapofplasmidPGBI121S4ABC

Receiveddate:200501-l2

    Foundationitem:HeilongjinagPostdoctoralFund Biography:ZHANGShuzhen(1972

    ),female,associateprofessor,Doctor,engagedintheresearchofsoybeandisease

    resistancebreeding.

    Email:dnzhshzh@163.com

    Correspondenceauthor

?

    8?JournalofNortheastAgriculturalUniversityV0l_l3 1.2Methods

    1.2.1Transformationofinsectresistantgeneby

    wayofpollen-tubemethod

    1.2.1.1TheextractionofplasmidDNA

    TheplasmidkeptinDH5ctwasextracted,elec

    trophoresedandenzymedigestedfortesting.Thecon

    centrationofplasmidwascalculatedwithelec——

    trophoresis.

    1.2.1.2Transfermationoff0reigngene Theflowerswhosecoronalswere1-2mmhigher thanthehighestcalycesortheopenedflowerswere selectedfortransfermationduringtheperiodofpro——

    fusefloweringI21.Thepetalswereremovedandstigma wascut,thentheplasmidDNAsolutionwasdripped intotheamplecavum.TheDNAsolutionjustim

    mergedtheovaryandformedaliquiddrop.Atagis hangedonthenextnodeofthestemwiththenotation ofdate.Checkingwasnecessarytopreventthenew budsfromcomingout.

    1.2.2TheinhibitionexperimentofKanamycinon

non-transgenicsoybeangrowth

    ThescreeningofappropriateKanamycinconcen——

    trationonnon-transgenicseedsofthevarietyDongnong 46[31wasdonefirst.Thenontransgenicseedsofthe

    varietyDonnong46weredisinfectdandputintoMS mediaaddedwith0,200,300,400,500mg.L(Kana

    mycinforgermination,respectively.Theirradication conditionwasobservedafter15days.Theexperiment wasrepeated3timesforassuringaccuracy.There were30seedstobetreatedpergroupandonecontrol wassetuppergrouptoconfirmtheappropriatecon——

    centration.

    1.2.3Theselectionoftransgenicsoybeanseedsby Kanamycin

    TheToseedsofthevarietyDongnong46trans——

    formedbymeansofpollen——tubeweregerminatedon

    solidMSmediumaddedwithappropriateKanamycin, andobservationwasdoneafter15days.

    1.2.4TheGusdyeingoftheplantIeavesscreened preliminarilybyKanamycin

    Theplantleavesscreenedpreliminarilyby Kanamycinwerecuttopiecesandputin0.5mL microcentrifugetubeaddedwithbufferfor30min. AfterthrowedoffbufferandaddedsomeXGlucbuffer

    inthetubeandplacedthemovernightat37oC, chlorophylloftheleaveswasfirstgottenridofby95% alcoholandthentheleaveswereputinto70%alcoho1. Atlast,thepositiveplantswerethenfound. 1.2.5PCRtesting

    1.2.5.1TheextractionofDNAinleaves

    TheDNAwasextractedbywayofSDS,referred to"MolecularCloningExperimentGuide'(.

    1.2.5.2PCRamplification

    a.Thesequenceoftwoprimersof?P?gene

    (synthesizedbyTakatak):

    SequenceofprimerI:5GATGGATTGCACGC AGGT3

    2.0L,Taqenzyme2U?L1.0L,primerI1.0

    L,primer111.0txL,ddH2O16.5L,templateDNA 1.0L,totalreactionvolume25IxL.

    c.PCRreactionprocedure:

    94oCdenaturalization.3min;94?denatural

    ization,1min;55oCannealing,1min;72oCelonga- tion,lmin;72oCelongation,5min;4oCkeeping warln,35cycles.

    1.2.5.3PCRproducttesting

    5-10LPCRamplificationproductwaselec

    trophoresedinl%gelofagarose,andthemarkerwas DL2000.Theelectrophoresisresultswereanalyzedby B10RADgelphotographsystem.

    1.2.5.4PCR-Southernhybridization

    ReferredtoDocument[4].

    2ResultsandAnalysis

    2.1Theextractionofplasmidandenzymediges- tiontest

    ThequalityofplasmidextractedfromDH5c~was good(Fig.2)(Misk-EcoT14IandPwastheplas

    mid).Andtheplasmidwasdigestedtotwofragments

    No.1JournalofNortheastAgnculturalUniversity?9?

PPPPM

    Fig.2TheplasmidinDH5ot

    19329bp

    7743bp

    4254bp

    (12.8kband4.7kb)(Fig.3).Theconcentrationofthe plasmidwasadjustedto100-300ng'Lusingsterile

    waterreferredtoZhout1.

    2.2TheinhibitionexperimentofKanamycinto non--transgenicvarietyDongnong46

    WiththeincreaseofKanamycinconcentration, theradiclegrowthofthevarietyDongnong46was 19329bp

    7743bp

    4254bp

    Fig.3PlasmiddigestedwithHindm

    inhibited(Tablel1.Therewasonly3-3%normalplants whentheKanamycinconcentrationwas300mg?IJI'.and thetaprootofotherseedswerebecomingblackanddie. WhentheKanamycinconcentrationwere400mg?L'

    and500mgL(,therewasnoseedgerminating.Sothe concentrationof400mg?L-wasthecriticalconcentra

    tionforscreening.

    Table1InhibitionofKmonradicleregenerationofthesoybeanDongnong46

    2.3PreliminarilyscreeningonT0seedsofthe varietyDongnong46usingKanamycin

    1200soybeanpodsweregottenwiththepollen——

    tubemethod,andoneseedfromeachpodwasputin

    totheMSmediumforgermination.12normal seedlingswereproducedafter15days.

2.4Gusdyeing

    ThreepositiveplantsweregottenthroughGus dyeingfrom12plantletsscreenedbyKanamycin. 2.5PCRamplificationandPCR-southernhy-- bridization

    Twopositiveplantswithaim——genefragmentfrom

    thethreeplantswereidentifiedthroughPCRamplifi——

    cationandPCRSouthernhybridization.Thenumber ofradicleandradiclelengthhadnosignificantdiffer——

    encecomparedwithcontrol,whichmeansthat400 mgL(Kanamycincouldinhibitthenontransgenic

    seedlingsandthemethodwasfeasibletoscreenthe transgenicoffspring.ThesewereshowninFig.4. 3ConclusiOIlSandDiscussion

    3.1Themechanismofscreeningresistantplants usingKanamycin

    Kanamycinbelongstoamidoindicanantibiotic. ItstoxicitymechanismiSthatitcancombinethe3OS sub——unitofribosomeofchloroplastandmitochondri——

    on,andthuspreventthetranslationprocessandinter

    ferethesynthesisofprotein.Thetransformedcells havegottentheresistancetoantibiotic,SOtheycan surviveonthemediumaddedwithsomeKanamycin andthenon——transgeniccellsmightdieorbeinhibited

?

    lO?JournalofNortheastAculturalUniversityVol_l3 300bp

    M

    MarkerofDL2000;Lane1,2-Positivetransformationplants;Lane3-Negativetransformatio

nplant

    Fig.4PCRamplificationandPCRs0utllernhybridizationofDongnong46 fornothavingtheresistancegene.

    Thetransformedplasmidinthisexperimenthad ?pflIgene,andthetransformedplantscouldhave resistancetoKanamycin.Buttheconcentrationof Kanamycincanvarywithdifferentplantspeciesand genotypes?

    TheappropriateconcentrationofKanamycinsho- uldbethatitcannotonlyinhibitthegrowthofnon

    transgeniccellsbutalsodonotaffectthegrowthof transgeniccells.Theappropriateconcentrationof Kanamycininthisexperimentforscreeningwas400 mg

    3.2

    L

    TheapplicationofKanamycininscreening thepositiveplantsfromtransgenicoffspringgot-. tenbypollen--tubemethod

    Thepollentubemethodisatechniquethattrans

    formforeignDNAthroughpollen——tubepassage.The

    principleofthismethodistomakeuseofthepassage, whichisformedwhenthepollentubepasstheovary afterthepollenhavegerminated.DNAcangothrough theformedpassageandbetransferredtothegastrula. ThismethodwasdevelopedbyZhouduringthe1980s.

    Itcanavoidtheobstacleoftheprocessofplantre

    generation,s0ithasbeenwidelyusedinsoybean transformationlr-s1.

    Butitisaveryarduousjobtoscreenthepositive

    plantsfromalargenumberofseeds.Kanamycinhas n0hatolivestock,humanandenvironment,andit isalsoverycheap.Soitiswidelyusedasascreening reagentintransgenicplantresearch.Kanamycinwas usedinthisresearchtoscreentheToseedsoftheva

    rietyDongnong46bymeansofpollen——tubemethod.

    Theresultsshowthattheappropriateconcentrationof Kanamycinforscreeningis400mg.L,and2positive

    plantsarefoundcombinedwithGusdyeingandPCR reactionfroml200seeds.Usingkanamycintoscreen thetransformedseedscouldbeeconomicandefficient. References

    1ZHOUSijun.Studyongenetransferofinsectresistanceandop

    timizationoftransformationsystems[D].Harbin:NortheastA

    culturalUniversity.2000(1):4549.

    2]LE1Bojun.Thestudyonthetransfer.fforeignDNAJ_Soybean

    Science,1991,lO(1):5863.

    [3]YUANYing,L1UDepu,WangYumin.Thestudyoninhibition andscreeningforsoybeangrowthusingkanamycinJ_Soybean

    Science,2003(4):6267.

    [4]HUANGPeitang.Molecularcloningexperimentguide[M]?Bei

    jing:SciencePress,1990:234239.

    [51ZhouGY,ZengYS,YangWX.Themolecularbasisofremotehy

    bridizationJJ.ActaGeneticaSinica,1980.7(2):119122.

    [6]HUZhanghua,HUANGRuizhi,L1UZhihong,eta1.Thetransgene DlantsofantisensePEPgottenbypollentube[J1.ggnculture

    ActaofZhejiang,1999,1l(2):99100.

    7]LE1Bojun,Q1NGuangchu,WANGShulin,eta1.Thevariationof G.maxforintroducingforeigngeneofsojae[J1.ChinaOil,1989 (3):11-14.

[8]XUXiangling,ZOULianpei,LIUWeihua,eta1.Thepreliminary

study0nchitinasegeneintroducingtosoybean[J1.SoybeanSci

ence,1999,l8f2):101108.

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