黄山栾树实时荧光定量PCR内参基因的筛选
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摘要
为了筛选可用于黄山栾树定量PCR的稳定内参基因,本研究选取看家基因EF1α,Actin,GAPDH,TUB6和18S rRNA作为候选内参基因,分析它们在黄山栾树的8个不同组织及一种黄叶品种‘金焰彩栾’的2个组织中的表达稳定性。结果表明,EF1α,Actin,GAPDH的CT值均集中在21～29之间,表明内参基因在不同组织中表达较稳定,它们之间的表达水平差异不明显。18S rRNA在个组织何总的表达丰度最高,CT值集中在15～20。geNorm分析结果表明基因GAPDH最稳定,NormFinder和BestKeeper计算得出最稳定的内参基因分别是18S r RNA和Actin,而TUB6在3个软件分析中均为最不稳定。因此,GAPDH,Actin和18S rRNA可作为研究基因在黄山栾树不同组织器官中表达的稳定内参。
In order to screen stable reference genes used for quantitative PCR of Koelreuteria bipinnata, 5 house keeping genes(EF1α, Actin, GAPDH, TUB6 and 18 S rRNA) were chosen as candidate reference genes and their expression stabilities in 8 different tissues of Koelreuteria bipinnata and 2 tissues of a yellow-leaf variety(Koelreuteria bipinnata var. integrifoliola 'Jinyan') were analyzed. Results showed that the CT values of EF1α, Actin,GAPDH were all between 21 to 29, indicating the expressions of these candidate genes were quite stable in different tissues. There was no significant difference in expression levels among them. 18 S rRNA had the highest expression in all tissues and CT values focused on 15～20. GAPDH was characterized as the most stable gene based on geNorm analysis, while 18 S r RNA and Actin were ranked in the first place according to NormFinder and BestKeeper. TUB6 was the most unstable one in 3 software analysis. Therefore, GAPDH, Actin and 18 S rRNA could serve as stable reference genes for studies on gene expression among different tissues in Koelreuteria bipinnata.
In order to screen stable reference genes used for quantitative PCR of Koelreuteria bipinnata, 5 house keeping genes(EF1α, Actin, GAPDH, TUB6 and 18 S rRNA) were chosen as candidate reference genes and their expression stabilities in 8 different tissues of Koelreuteria bipinnata and 2 tissues of a yellow-leaf variety(Koelreuteria bipinnata var. integrifoliola 'Jinyan') were analyzed. Results showed that the CT values of EF1α, Actin,GAPDH were all between 21 to 29, indicating the expressions of these candidate genes were quite stable in different tissues. There was no significant difference in expression levels among them. 18 S rRNA had the highest expression in all tissues and CT values focused on 15～20. GAPDH was characterized as the most stable gene based on geNorm analysis, while 18 S r RNA and Actin were ranked in the first place according to NormFinder and BestKeeper. TUB6 was the most unstable one in 3 software analysis. Therefore, GAPDH, Actin and 18 S rRNA could serve as stable reference genes for studies on gene expression among different tissues in Koelreuteria bipinnata.
引文
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Bustin S.A.,Benes V.,Garson J.A.,Hellemans J.,Huggett J.,Kubista M.,Mueller R.,Nolan T.,Pfaffl M.W.,Shipley G.L.,Vandesompele J.,and Wittwer C.T.,2009,The MIQEguidelines:minimum information for publication of quantitative real-time PCR experiments,Clin.Chem.,55(4):611-622
Brunner A.M.,Yakovlev I.A.,and Strauss S.H.,2004,Validating internal controls for quantitative plant gene expression studies,BMC Plant Biol.,4:14
Chang E.M.,Shi S.Q.,Liu J.F.,Cheng T.L.,Xue L.,Yang W.,Lan Q.,and Jiang Z.,2012,Selection of reference genes for quantitative gene expression studies in Platycladus orientalis(Cupressaceae)using Real-Time PCR,PLoS One,7(3):e33278
De Oliveira L.A.,Breton M.C.,and Bastolla F.M.,2012,Reference genes for the normalization of gene expression in Eucalyptus species,Plant Cell Physiol.,53(2):405-422
Galeano E.,Vasconcelos T.S.,and Ramiro D.A.,2014,Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak(Tectona grandis L.F.),BMC Research Notes,7(1):1-13
Galli V.,Borowski J.M.,and Perin E.C.,2015,Validation of reference genes for accurate normalization of gene expression for real time quantitative PCR in strawberry fruits using different cultivars and osmotic stresses,Gene,554(2):205-214
González-Aguilera K.L.,Saad C.F.,Chávez Montes R.A.,Alves-Ferreira M.,and de Folter S.,2016,Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genot ype MT-Rg1,Front.Plant Sci.,7:1386
Hu R.B.,Qi G.,Kong Y.Z.,Kong D.J.,and Zhou G.K.,2010,Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa,BMC Plant Biol.,10(1):145
Huang L.B.,Liang Z.H.,Dou Q.Q.,Dong X.Y.,Zhang M.,and Jiang Z.P.,2015,A new variety of ornamental Koelreuteria bipinnata var.Integrifoliola'Jinyan',Linye Kexue(Scientia Silvae Sinicae),51(5):165(黄利斌,梁珍海,窦全琴,董筱昀,张敏,蒋泽平,2015,观赏栾树新品种‘金焰彩栾’,林业科学,51(5):165)
Jian B.,Liu B.,and Bi Y.,2008,Validation of internal control for gene expression study in soybean by quantitative real-time PCR,BMC Mol.Biol.,9(9):59
Kanakachari M.,Solanke A.U.,and Prabhakaran N.,2016,Evaluation of suitable reference genes for normalization of Qpcr gene expression studies in Brinjal(Solanum melongena L.)during fruit developmental stages,Appl.Biochem.Biotechnol.,178(3):433-450
Karuppaiya P.,Yan X.X.,Liao W.,Wu J.,Chen F.,and Tang L.,2017,Correction:identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas-Abiodiesel plant,PLoS One,12(5):e0177039
Kozera B.,and Rapacz M.,2013,Reference genes in real-time PCR,J.Appl.Genet.,54(4):391-406
Li D.D.,Hu B.,Wang Q.,and Wu W.,2017,The research on reference genes in medicinal plant,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):903-910(李丹丹,胡博,王庆,吴卫,2017,药用植物内参基因研究进展,分子植物育种,15(3):903-910)
Libault M.,Thibivilliers S.,Bilgin D.D.,Radwan O.,and Benitez M.,2008,Identification of four soybean reference genes for gene expression normalization,Plant Genome,1(1):44-54
Liu W.Z.,Niu M.Y.,Li X.Y.,Lin E.P.,Huang H.H.,and Tong Z.K.,2016,The selection of reference genes for quantitative PCR in Betula luminifera,Liye Kexue(Scientia Silvae Sinicae),52(8):29-37(刘文哲,牛明月,李秀云,林二培,黄华宏,童再康,2016,光皮桦实时荧光定量PCR内参基因的筛选,林业科学,52(8):29-37)
Ma R.,Xu S.,Zhao Y.,Xia B.,and Wang R.,2016,Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea,Front.Plant Sci.,7(651):536
Mafra V.,Kubo K.S.,Alves-Ferreira M.,Ribeiro-Alves M.,Stuart R.M.,Boava L.P.,Rodrigues C.M.,and Machado M.A.,2012,Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions,PLoS One,7(2):e31263
Nolan T.,Hands R.E.,and Bustin S.A.,2006 Quantification of m R-NA using real-time RT-PCR,Nat.Protoc.,1(3):1559-1582
Pfaffl M.W.,Tichopad A.,Prgomet C.,and Neuvians T.P.,2004,Determination of stable housekeeping g enes,differentially regulated target genes and sample integrity:BestKeeper-Ex cel-based tool using pair-wise correlations,Biotechnol.Lett.,26(6):509-515
Schmittgen T.D.,and Zakrajsek B.A.,2000,Effect of experimental treatment on housekeeping gene expression:validation by real-time,quantitative RT-PCR,J.Biochem.Biophys.Methods,46:69-81
Stanton K.A.,Edger P.P.,Puzey J.R.,Kinser T.,Cheng P.,Vernon D.M.,Forsthoefel N.R.,and Cooley A.M.,2017,Awhole-transcriptome approach to evaluating reference genes for quantitative gene expression studies:a case study in mimulus,Genes Genomes Genet.,7(4):1085-1095
Udvardi M.K.,Czechowski T.,and Scheible W.R.,2008,Eleven golden rules of quantitative RT-PCR,Plant Cell,20(7):1736-1737
Vandesompele J.,De Preter K.,Pattyn F.,Poppe B.,Van Roy N.,De Paepe A.,and Speleman F.,2002,Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes,Genome Biol.,3(7):1-11
Xie B.B.,Wang X.D.,and Ye J.F.,2011,Research status and prospect on Koelreuteria integrifoliola Merr,Beifang Yuanyi(Northern Horticulture),(23):181-183(解冰冰,王小德,叶建丰,2011,黄山栾树的研究现状及展望,北方园艺,(23):181-183)
Yang X.X.,Lv Y.Z.,Dong X.Y.,Huang L.B.,and He K.Y.,2016,Photosynthetic characteristics of Koelreuteria bipinnata var.integrifoliola and its natural yellow mutant'Jinyan',Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University),40(4):74-80(杨小鑫,吕运舟,董筱昀,黄利斌,何开跃,2016,‘金焰彩栾’与黄山栾树光合特性比较,南京林业大学学报,40(4):74-80)
Yuan W.,Wan H.J.,and Yang Y.J.,2012,Characterization and selection of reference genes for real-time quantitative RTPCR of plants,Zhiwu Xuebao(Bulletin of Botany),47(4):427-436(袁伟,万红建,杨悦俭,2012,植物实时荧光定量PCR内参基因的特点及选择,植物学报,47(4):427-436)
Zhuang H.,Fu Y.,He W.,Wang L.,and Wei Y.,2015,Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments,Front.Plant Sci.,6:475
Bustin S.A.,Benes V.,Garson J.A.,Hellemans J.,Huggett J.,Kubista M.,Mueller R.,Nolan T.,Pfaffl M.W.,Shipley G.L.,Vandesompele J.,and Wittwer C.T.,2009,The MIQEguidelines:minimum information for publication of quantitative real-time PCR experiments,Clin.Chem.,55(4):611-622
Brunner A.M.,Yakovlev I.A.,and Strauss S.H.,2004,Validating internal controls for quantitative plant gene expression studies,BMC Plant Biol.,4:14
Chang E.M.,Shi S.Q.,Liu J.F.,Cheng T.L.,Xue L.,Yang W.,Lan Q.,and Jiang Z.,2012,Selection of reference genes for quantitative gene expression studies in Platycladus orientalis(Cupressaceae)using Real-Time PCR,PLoS One,7(3):e33278
De Oliveira L.A.,Breton M.C.,and Bastolla F.M.,2012,Reference genes for the normalization of gene expression in Eucalyptus species,Plant Cell Physiol.,53(2):405-422
Galeano E.,Vasconcelos T.S.,and Ramiro D.A.,2014,Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak(Tectona grandis L.F.),BMC Research Notes,7(1):1-13
Galli V.,Borowski J.M.,and Perin E.C.,2015,Validation of reference genes for accurate normalization of gene expression for real time quantitative PCR in strawberry fruits using different cultivars and osmotic stresses,Gene,554(2):205-214
González-Aguilera K.L.,Saad C.F.,Chávez Montes R.A.,Alves-Ferreira M.,and de Folter S.,2016,Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genot ype MT-Rg1,Front.Plant Sci.,7:1386
Hu R.B.,Qi G.,Kong Y.Z.,Kong D.J.,and Zhou G.K.,2010,Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa,BMC Plant Biol.,10(1):145
Huang L.B.,Liang Z.H.,Dou Q.Q.,Dong X.Y.,Zhang M.,and Jiang Z.P.,2015,A new variety of ornamental Koelreuteria bipinnata var.Integrifoliola'Jinyan',Linye Kexue(Scientia Silvae Sinicae),51(5):165(黄利斌,梁珍海,窦全琴,董筱昀,张敏,蒋泽平,2015,观赏栾树新品种‘金焰彩栾’,林业科学,51(5):165)
Jian B.,Liu B.,and Bi Y.,2008,Validation of internal control for gene expression study in soybean by quantitative real-time PCR,BMC Mol.Biol.,9(9):59
Kanakachari M.,Solanke A.U.,and Prabhakaran N.,2016,Evaluation of suitable reference genes for normalization of Qpcr gene expression studies in Brinjal(Solanum melongena L.)during fruit developmental stages,Appl.Biochem.Biotechnol.,178(3):433-450
Karuppaiya P.,Yan X.X.,Liao W.,Wu J.,Chen F.,and Tang L.,2017,Correction:identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas-Abiodiesel plant,PLoS One,12(5):e0177039
Kozera B.,and Rapacz M.,2013,Reference genes in real-time PCR,J.Appl.Genet.,54(4):391-406
Li D.D.,Hu B.,Wang Q.,and Wu W.,2017,The research on reference genes in medicinal plant,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):903-910(李丹丹,胡博,王庆,吴卫,2017,药用植物内参基因研究进展,分子植物育种,15(3):903-910)
Libault M.,Thibivilliers S.,Bilgin D.D.,Radwan O.,and Benitez M.,2008,Identification of four soybean reference genes for gene expression normalization,Plant Genome,1(1):44-54
Liu W.Z.,Niu M.Y.,Li X.Y.,Lin E.P.,Huang H.H.,and Tong Z.K.,2016,The selection of reference genes for quantitative PCR in Betula luminifera,Liye Kexue(Scientia Silvae Sinicae),52(8):29-37(刘文哲,牛明月,李秀云,林二培,黄华宏,童再康,2016,光皮桦实时荧光定量PCR内参基因的筛选,林业科学,52(8):29-37)
Ma R.,Xu S.,Zhao Y.,Xia B.,and Wang R.,2016,Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea,Front.Plant Sci.,7(651):536
Mafra V.,Kubo K.S.,Alves-Ferreira M.,Ribeiro-Alves M.,Stuart R.M.,Boava L.P.,Rodrigues C.M.,and Machado M.A.,2012,Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions,PLoS One,7(2):e31263
Nolan T.,Hands R.E.,and Bustin S.A.,2006 Quantification of m R-NA using real-time RT-PCR,Nat.Protoc.,1(3):1559-1582
Pfaffl M.W.,Tichopad A.,Prgomet C.,and Neuvians T.P.,2004,Determination of stable housekeeping g enes,differentially regulated target genes and sample integrity:BestKeeper-Ex cel-based tool using pair-wise correlations,Biotechnol.Lett.,26(6):509-515
Schmittgen T.D.,and Zakrajsek B.A.,2000,Effect of experimental treatment on housekeeping gene expression:validation by real-time,quantitative RT-PCR,J.Biochem.Biophys.Methods,46:69-81
Stanton K.A.,Edger P.P.,Puzey J.R.,Kinser T.,Cheng P.,Vernon D.M.,Forsthoefel N.R.,and Cooley A.M.,2017,Awhole-transcriptome approach to evaluating reference genes for quantitative gene expression studies:a case study in mimulus,Genes Genomes Genet.,7(4):1085-1095
Udvardi M.K.,Czechowski T.,and Scheible W.R.,2008,Eleven golden rules of quantitative RT-PCR,Plant Cell,20(7):1736-1737
Vandesompele J.,De Preter K.,Pattyn F.,Poppe B.,Van Roy N.,De Paepe A.,and Speleman F.,2002,Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes,Genome Biol.,3(7):1-11
Xie B.B.,Wang X.D.,and Ye J.F.,2011,Research status and prospect on Koelreuteria integrifoliola Merr,Beifang Yuanyi(Northern Horticulture),(23):181-183(解冰冰,王小德,叶建丰,2011,黄山栾树的研究现状及展望,北方园艺,(23):181-183)
Yang X.X.,Lv Y.Z.,Dong X.Y.,Huang L.B.,and He K.Y.,2016,Photosynthetic characteristics of Koelreuteria bipinnata var.integrifoliola and its natural yellow mutant'Jinyan',Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University),40(4):74-80(杨小鑫,吕运舟,董筱昀,黄利斌,何开跃,2016,‘金焰彩栾’与黄山栾树光合特性比较,南京林业大学学报,40(4):74-80)
Yuan W.,Wan H.J.,and Yang Y.J.,2012,Characterization and selection of reference genes for real-time quantitative RTPCR of plants,Zhiwu Xuebao(Bulletin of Botany),47(4):427-436(袁伟,万红建,杨悦俭,2012,植物实时荧光定量PCR内参基因的特点及选择,植物学报,47(4):427-436)
Zhuang H.,Fu Y.,He W.,Wang L.,and Wei Y.,2015,Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments,Front.Plant Sci.,6:475