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1、BreakthroughTechnologiesAVersatileSetofLigation-IndependentCloningVectorsforFunctionalStudiesinPlants1[C][W][OA]223BertDeRybel,WillyvandenBerg,AnnemarieS.Lokerse,Che-YangLiao,HildavanMourik,BarbaraMo¨ller,CristinaI.Llavata-Peris,andDolfWeijers*WageningenUniversity,LaboratoryofBiochemistry
2、,6703HAWageningen,TheNetherlandsWithplantmolecularbiologyintheomicsera,thereisaneedforsimplecloningstrategiesthatallowhighthroughputtosystematicallystudytheexpressionandfunctionoflargenumbersofgenes.Suchstrategieswouldfacilitatetheanalysisofgene(sub)familiesand/orsetsofcoexpressedgeneside
3、ntifiedbytranscriptomics.Here,weprovideasetof34ligation-independentcloning(LIC)binaryvectorsforexpressionanalysis,proteinlocalizationstudies,andmisexpressionthatwillbemadefreelyavailable.ThissetofplantLICvectorsoffersafastalternativetostandardcloningstrategiesinvolvingligaseorrecombination
4、enzymetechnology.Wedemonstratetheuseofthisstrategyandournewvectorsbyanalyzingtheexpressiondomainsofgenesbelongingtotwosubcladesofthebasichelix-loop-helixtranscriptionfactorfamily.WeshowthatneithertheclosesthomologsofTARGETOFMONOPTEROS7(TMO7/ATBS1)northemembersoftheATBS1INTERACTINGFACTORsu
5、bcladeofputativeTMO7interactorsareexpressedintheembryoandthatthereisverylimitedcoexpressionintheprimaryrootmeristem.Thissuggeststhatthesebasichelix-loop-helixtranscriptionfactorsaremostlikelynotinvolvedinTMO7-dependentrootmeristeminitiation.Whole-genomeanalysisisbecomingastandardanal-such
6、asBACrecombineering(Zhouetal.,2011),whileysistoolinreversegeneticsplantresearch.Further-allowingprecisioncloning,haveacleardisadvantagemore,thereisoftentheneedtostudylargegeneinthattheyintroducenotonlyageneofinterest,butafamiliesinArabidopsis(Arabidopsisthaliana)duetocompletegenomicregion
7、.Anemergingsingle-stepredundancy.Fortheseandotherreasons,thereisanmethodthatisverysuitableformoderatehigh-increasingneedinplantresearchforfastcloningstrat-throughputcloningisligation-independentcloningegies.Besidesspeed,thesemethodshavetobecharac-(LIC;LiandElledge,2
