Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development 英文参考文献.docVIP

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Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development 英文参考文献.doc

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Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development 英文参考文献

Kaczmarczyketal.BMCPlantBiology2012,12:184 /1471-2229/12/184 METHODOLOGY ARTICLE OpenAccess QuantitativeRT-PCRbasedplatformforrapid quantificationofthetranscriptsofhighly homologousmultigenefamiliesandtheir membersduringgraindevelopment AgnieszkaKaczmarczyk1,SteveBowra2,ZoltanElek3andEvaVincze1* Abstract Background:Cerealstorageproteinsrepresentoneofthemostimportantsourcesofproteinforfoodandfeed andtheyarecodedbymultigenefamilies.Theexpressionofthestorageproteingenesexhibitsatemporal fluctuationbutalsoaresponsetoenvironmentalstimuli.Analysisoftemporalgeneexpressioncombinedwith geneticvariationinlargemultigenefamilieswithhighhomologyamongtheallelesisverychallenging. Results:WedesignedarapidqRT-PCRsystemwiththeaimofcharacterisingthevariationintheexpressionof hordeingenesfamilies.AlltheknownD-,C-,B-,andγ-hordeinsequencescodingfulllengthopenreadingframes werecollectedfromcommonlyavailabledatabases.Phylogeneticanalysiswasperformedandthemembersofthe differenthordeinfamilieswereclassifiedintosubfamilies.Primersetsweredesignedtodiscriminatethegene expressionlevelofwholefamilies,subfamiliesorindividualmembers.Thespecificityoftheprimersetswas validatedbeforesuccessfullyapplyingthemtoacDNApopulationderivedfromdevelopinggrainsoffieldgrown Hordeumvulgarecv.Barke.Theresultsquantifythenumberofmolesoftranscriptcontributedtoaparticulargene familyanditssubgroups.Moreovertheresultsindicatethegenotypicspecificgeneexpression. Conclusions:QuantitativeRT-PCRwithSYBRGreenlabellingcanbeausefultechniquetofollowgeneexpression levelsoflargegenefamilieswithhighlyhomologuesmembers.Weshowedvariationinthetemporalexpressionof genescodingforbarleystorageproteins.TheresultsimplythatourrapidqRT-PCRsystemwassensitiveenoughto identifythepresenceofallelesandtheirexpressionprofiles.Itcanbeusedtocheckthetemporalfluctuationsin hordeinexpressionsortofinddifferencesintheirresponsetoenvironmentalstimuli.Themethodcouldbe extendedforcultivarrecognitionassomeofthesequencesfromthedatabaseoriginatedfromcv.Golde