Evaluation of the Use of the Polyubiquitin Genes, Ubi4 and Ubi10 as Reference Genes for Expression Studies in Brachypodium distachyon 英文参考文献.docVIP

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Evaluation of the Use of the Polyubiquitin Genes, Ubi4 and Ubi10 as Reference Genes for Expression Studies in Brachypodium distachyon 英文参考文献.doc

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Evaluation of the Use of the Polyubiquitin Genes, Ubi4 and Ubi10 as Reference Genes for Expression Studies in Brachypodium distachyon 英文参考文献

EvaluationoftheUseofthePolyubiquitinGenes,Ubi4 andUbi10asReferenceGenesforExpressionStudiesin Brachypodiumdistachyon JohnP.Chambers,AliBehpouri,AlisonBird,CarlK-Y.Ng* SchoolofBiologyandEnvironmentalScience,UniversityCollegeDublin,Belfield,Dublin,Ireland Abstract Background:Brachypodiumdistachyonisemergingasthemodelplantfortemperategrassresearchandthegenomeofthe community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformationforthegenerationofT-DNAinsertionallines.Recently,itwasreportedthatexpressionofthepolyubiquitin genes,Ubi4andUbi10arestableindifferenttissuesandgrowthhormone-treatedplantsamples,leadingtotheconclusion thatbothUbi4andUbi10aregoodreferencegenesfornormalizationofgeneexpressiondatausingreal-time,quantitative PCR(qPCR). PrincipalFindings:MiningoftheJointGenomeInstitute(JGI)8XBrachypodiumdistachyongenomeassemblyshowedthat Ubi4andUbi10shareahighlevelofsequenceidentity(89%),andinsilicoanalysesofthesequencesofUbi4(Bradi3g04730) and Ubi10 (Bradi1g32860) showed that the primers used previously exhibit multiple binding sites within the coding sequencesarisingfromthepresenceoftandemrepeatsofthecodingregions.Thiscanpotentiallyresultinover-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primersusedpreviouslyforamplificationofUbi4canbindtoUbi10andviceversa,resultingintheformationofnon-specific amplificationproducts. Conclusions:Theresultsfromthisstudyindicatethattheprimersusedpreviouslywerenotsufficientlyrobustandspecific. Additionally,theirusewouldresultinover-estimationofthesteady-stateexpressionlevelsofUbi4.Ourresultsquestionthe validityofusingthepreviouslyproposedprimersetsforqPCRamplificationofUbi4andUbi10.Wedemonstratethatprimers designedtotargetthe39-UTRsofUbi4andUbi10arebettersuitedforreal-timenormalizationofsteady-stateexpression levelsinBrachypodiumdistachyon. Citation:ChambersJP,BehpouriA,BirdA,NgCK-Y(2012)Eval