Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development 英文参考文献.docVIP

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Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development 英文参考文献.doc

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Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development 英文参考文献

SelectionofReliableReferenceGenesforGene ExpressionStudiesUsingReal-TimePCRinTungTree duringSeedDevelopment XiaojiaoHan1,2,MengzhuLu1,3,YicunChen1,2,ZhiyongZhan2,QinqinCui2,YangdongWang1,2* 1State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People’s Republic of China, 2Research Institute of Subtropical Forestry, ChineseAcademyofForestry,Fuyang,People’sRepublicofChina,3ResearchInstituteofForestry,ChineseAcademyofForestry,Beijing,People’sRepublicofChina Abstract Quantitativereal-timePCR(RT-qPCR)hasbecomeanaccurateandwidelyusedtechniquetoanalyzeexpressionlevelsof selectedgenes.Itisverynecessarytoselectappropriatereferencegenesforgeneexpressionnormalization.Inthepresent study,weassessedtheexpressionstabilityof11referencegenesincludingeighttraditionalhousekeepinggenesandthree novelgenesindifferenttissues/organsanddevelopingseedsfromfourcultivarsoftungtree.All11referencegenesshowed awiderangeofCtvaluesinallsamples,indicatingthattheydifferentlyexpressed.Threesoftwares–geNorm,NormFinder andBestKeeper–wereusedtodeterminethestabilityofthesereferencesexceptforALB(2Salbumin),whichpresenteda littledivergence.TheresultsfromthethreesoftwaresshowedthatACT7(Actin7a),UBQ(Ubiquitin),GAPDH(glyceraldehyde- 3-phosphatedehydrogenase)andEF1a(elongationfactor1-a)werethemoststablereferencegenesacrossallofthetested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1b (elongationfactor1-beta),GAPDHandTEF1(transcriptionelongationfactor1)werethetopfourchoicesfordifferenttissues/ organswhereasLCR69didnotfavornormalizationofRT-qPCRinthesetissues/organs.Meanwhile,theexpressionprofilesof FAD2andFADXwererealizedusingstablereferencegenes.TherelativequantificationoftheFAD2andFADXgenesvaried accordingtotheinternalcontrolsandthenumberofinternalcontrols.Theresultsfurtherprovedtheimportanceofthe choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and qua