西南鸢尾花色变异实时定量PCR内参基因的筛选与验证
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摘要
为筛选适用于不同花色西南鸢尾花色合成途径相关基因表达分析的内参基因,本研究根据西南鸢尾及其白花变型白花西南鸢尾花蕾组织的转录组测序数据,筛选了6个常用内参基因(ɑ-TUB、β-TUB、AQP、ACT、GAPDH、UBQ),同时以百合18S做为对照内参基因,在西南鸢尾及其白花变型白花西南鸢尾的花蕾组织中,分别通过反转录PCR(RT-PCR)初筛和RT-qPCR检测表达量,并利用geNorm、NormFinder和BestKeeper软件对7个候选内参基因的稳定性进行评价。RT-PCR初筛结果显示,7个候选内参基因引物的特异性均较好,在6个样品间没有明显差异; RT-qPCR分析表明,7个候选内参基因的表达量存在一定差异,其中18S表达量最高,UBQ最低; geNorm、NormFinder和BestKeeper分析结果表明,ACT表现最稳定,最适合做为内参基因,β-TUB相对稳定性最低;以Actin为内参对西南鸢尾类黄酮/花青素和类胡萝卜素2类色素合成途径中部分相关基因的RT-qPCR分析结果与转录组测序结果相一致。本研究为鸢尾属植物花色素合成相关基因表达分析内参基因的筛选以及鸢尾属植物资源利用和花色育种研究提供了理论参考。
In order to screen the appropriate reference genes for real-time quantitative polymerase chain reaction( RTqPCR) analysis of the main pigments synthesis related genes in the blue/white flower buds of Iris bulleyana,in this study,6 traditional reference genes( ɑ-TUB、β-TUB、AQP、ACT、GAPDH、UBQ) were selected based on RNA-seq data of I. bulleyana Dykes and its white form-I. bulleyana Dykes f. alba Y. T. Zhao. lily 18 S was used as the control reference gene. The expression of all 7 candidate reference genes were examined by reverse transcriptional PCR( RT-PCR) and RT-qPCR,and the stability were analyzed by geNorm,NormFinder and BestKeeper programs. The RT-PCR results showed that the primers of the 7 candidate reference genes were all specifically amplified,and no significant difference was detected on the gene expression level among the 6 different samples. The RT-qPCR analysis showed a significant difference among the expression level of 7 reference genes,which displayed the highest level for 18S and the lowest level for UBQ. The analyses of geNorm,NormFinder and BestKeeper showed that Actin was the most stable reference gene andβ-TUB displayed the lowest stability,indicating Actin was suitable for reference gene. The RNA-seq data was exactly consistent with the RT-qPCR analyses of the partial relative genes in the two biosynthetic pathways of flavonoid/anthocyanin and carotenoid which using Actin as reference gene. This research gives an example of the reference gene screen for the relative genes expression analysis in the flavonoid/anthocyanin and carotenoid biosynthetic pathway of Iris,and provides a theoretical basis for plant resource utilization and flower color breeding.
In order to screen the appropriate reference genes for real-time quantitative polymerase chain reaction( RTqPCR) analysis of the main pigments synthesis related genes in the blue/white flower buds of Iris bulleyana,in this study,6 traditional reference genes( ɑ-TUB、β-TUB、AQP、ACT、GAPDH、UBQ) were selected based on RNA-seq data of I. bulleyana Dykes and its white form-I. bulleyana Dykes f. alba Y. T. Zhao. lily 18 S was used as the control reference gene. The expression of all 7 candidate reference genes were examined by reverse transcriptional PCR( RT-PCR) and RT-qPCR,and the stability were analyzed by geNorm,NormFinder and BestKeeper programs. The RT-PCR results showed that the primers of the 7 candidate reference genes were all specifically amplified,and no significant difference was detected on the gene expression level among the 6 different samples. The RT-qPCR analysis showed a significant difference among the expression level of 7 reference genes,which displayed the highest level for 18S and the lowest level for UBQ. The analyses of geNorm,NormFinder and BestKeeper showed that Actin was the most stable reference gene andβ-TUB displayed the lowest stability,indicating Actin was suitable for reference gene. The RNA-seq data was exactly consistent with the RT-qPCR analyses of the partial relative genes in the two biosynthetic pathways of flavonoid/anthocyanin and carotenoid which using Actin as reference gene. This research gives an example of the reference gene screen for the relative genes expression analysis in the flavonoid/anthocyanin and carotenoid biosynthetic pathway of Iris,and provides a theoretical basis for plant resource utilization and flower color breeding.
引文
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[3]Liu Q X,Qi X,Yan H D,Huang L K,Nie G,Zhang X Q.Reference gene selection for quantitative Real-time reversetranscriptase PCR in annual ryegrass(Lolium multiflorum)subjected to various abiotic stresses[J].Molecules,2018,23(1):172-184
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[8]杨丹,李清,王贵禧,马庆华,朱利泉.平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立[J].中国农业科学,2017,50(12):2399-2410
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[11]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738
[12]朱海生,陈敏氡,温庆放,蓝新隆,李永平,王彬,张前荣,吴卫东.丝瓜18S rRNA基因克隆及其作为内参基因的应用[J].核农学报,2016,30(1):35-41
[13]Hong S Y,Seo P J,Yang M S,Xiang F N,Park C M.Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR[J].BMC Plant Biology,2008,8(1):112-132
[14]Yi S J,Qian Y Q,Han L,Sun Z Y,Fan C M,Liu J X,Ju G S.Selection of reliable reference genes for gene expression studies in Rhododendron micranthum Turcz[J].Scientia Horticulturae,2004,99(2):128-133
[15]Volkov R A,Panchuk I I,Sch9ffl F.Heat-stress-dependency and developmental modulation of gene expression:The potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR[J].Journal of Experimental Botany,2003,54(391):2343-2349
[16]Gu C S,Liu L Q,Xu C,Zhao Y H,Zhu X D,Huang S Z.Reference gene selection for quantitative real-time RT-PCRnormalization in Irislactea var.chinensis roots under cadmium,lead,and salt stress conditions[J].The Scientific World Journal,2014,2014(2):532713
[17]任锐,戴鹏辉,李萌,刘志明,曹福祥.珙桐实时定量PCR内参基因的筛选及稳定性评价[J].植物生理学报,2016,52(10):1565-1575
[18]林萍,田昆,汪元超.云南野生鸢尾属植物种质资源及观赏应用[J].中国野生植物资源,2003,22(4):33-35
[19]中国科学院中国植物志编辑委员会.中国植物志[M].北京:科学出版社,1985
[20]罗建,费文群,兰小中.鸢尾属1新变型---白花宽柱鸢尾[J].西北植物学报,2016,36(5):1043-1045
[21]徐凌云,王俊丽,周宜君.喜盐鸢尾花色形成关键基因的克隆及表达分析[J].植物遗传资源学报,2017,18(2):340-348
[22]Wang H,Conchou L,Bessière J M,Cazals G,Schatz B,Imbert E.Flower color polymorphism in Iris lutescens(Iridaceae):Biochemical analyses in light of plant-insect interactions[J].Phytochemistry,2013,94(5):123-134
[23]肖月娥,俞新平,胡永红,杨庆华.西南鸢尾种子萌发特性初步研究[J].种子,2008,27(2):18-20
[24]王彦杰,叶清,薛泽云,周华,金奇江,徐迎春.荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证[J].南京农业大学学报,2017,40(3):408-415
[25]Chen D L,Pan X P,Xiao P,Farwell M A,Zhang B H.Evaluation and identification of reliable reference genes for pharmacogenomics,toxicogenomics,and small RNA expression analysis[J].Journal of Cellular Physiology,2011,226(10):2469-2477
[26]马璐琳,崔光芬,王祥宁,贾文杰,段青,杜文文,王继华.泸定百合过氧化氢酶(Ls-Cat1)基因的克隆及表达分析[J].核农学报,2017,31(9):1700-1707
[27]Tanaka Y,Ohmiya A.Seeing is believing:Engineering anthocyanin and carotenoid biosynthetic pathways[J].Current Opinion in Biotechnology,2008,19(2):190-197
[28]Tanaka Y,Sasaki N,Ohmiya A.Biosynthesis of plant pigments:Anthocyanins,betalains and carotenoids[J].The Plant Journal,2008,54(4):733-749
[29]Jeknic'Z,Jeknic'S,Jevremovic'S,Subotic'A,Chen T H.Alteration of flower color in Iris germanica L.Fire Bride through ectopic expression of phytoene synthase gene(crt B)from Pantoea agglomerans[J].Plant Cell Reports,2014,33(8):1307-1321
[30]Lou Q,Liu Y L,Qi Y Y,Jiao S Z,Tian F F,Jiang L,Wang Y J.Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth[J].Journal of Experimental Botany,2014,65(12):3157-3164
[31]Ahn J H,Kim J S,Kim S,Soh H Y,Shin H,Jang H,Ryu J H,Kim A,Yun K Y,Kim S,Kim K S,Choi D,Huh J H.De novo transcriptome analysis to identify Anthocyanin biosynthesis genes responsible for tissue-specific pigmentation in Zoysiagrass(Zoysia japonica Steud.)[J].PLoS One,2015,10(4):e0124497
[32]Ma P Y,Bian X F,Jia Z D,Guo X D,Xie Y Z.De novo sequencing and comprehensive analysis of the mutant transcriptome from purple sweet potato(Ipomoea batatas L.)[J].Gene,2016,575(3):641-649
[33]Yoshihara N,Imayama T,Fukuchi-Mizutani M,Okuhara H,Tanaka Y,Ino I,Yabuya T.c DNA cloning and characterization of UDP-glucose:Anthocyanidin 3-O-glucosyltransferase in Iris hollandica[J].Plant Science,2005,169(3):496-501
[34]Yoshihara N,Imayama T,Matsuo Y,Fukuchi-Mizutani M,Tanaka Y,Ino I,Yabuya T.Characterization of c DNA clones encoding anthocyanin 3-p-coumaroyltransferase from Iris hollandica[J].Plant Science,2006,171(5):632-639
[35]Hyun T K,Rim Y,Jang H J,Kim C H,Park J,Kumar R,Lee S,Kim B C,Bhak J,Nguyen-Quoc B,Kim S W,Lee S Y,Kim J Y.De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis[J].Plant Molecular Biology,2012,79(4/5):413-427
[36]Clotault J,Peltier D,Berruyer R,Thomas M,Briard M,Emmanuel Geoffriau E.Expression of carotenoid biosynthesis genes during carrot root development[J].Journal of Experimental Botany,2008,59(13):3563-3573
[37]Kishimoto S,Ohmiya A.Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum(Chrysanthemum morifolium)[J].Physiologia Plantarum,2006,128(3):436-447
[38]张金波,罗佳滨,张春斌,朱金玲,吕冬霞.荧光定量PCR技术原理及在分子诊断中的应用进展[J].中国优生与遗传杂志,2006,14(12):13-14
[39]贺婷停,宋婷,王超,张长斌,王海燕.短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选[J].生物技术通报,2016,32(11):99-106
[2]Kou X Y,Zhang L,Yang S Z,Li G H,Ye J L.Selection and validation of reference genes for quantitative RT-PCR analysis in peach fruit under different experimental conditions[J].Scientia Horticulturae,2017,225:195-203
[3]Liu Q X,Qi X,Yan H D,Huang L K,Nie G,Zhang X Q.Reference gene selection for quantitative Real-time reversetranscriptase PCR in annual ryegrass(Lolium multiflorum)subjected to various abiotic stresses[J].Molecules,2018,23(1):172-184
[4]Quackenbush J.Microarray data normalization and transformation[J].Nature Genetics,2002,32:496-501
[5]蒋婷婷,高燕会,童再康.石蒜属植物实时荧光定量PCR内参基因的选择[J].园艺学报,2015,42(6):1129-1138
[6]牙库甫江·阿西木,关波,张富春.植物基因表达转录分析中内参基因的选择与应用[J].生物技术通报,2011(7):7-11
[7]Zhang L,He L L,Fu Q T,Xu Z F.Selection of reliable reference genes for gene expression studies in the biofuel plant Jatropha curcas using real-time quantitative PCR[J].International Journal of Molecular Sciences,2013,14(12):24338-24354
[8]杨丹,李清,王贵禧,马庆华,朱利泉.平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立[J].中国农业科学,2017,50(12):2399-2410
[9]张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125
[10]孙美莲,王云生,杨冬青,韦朝领,高丽萍,夏涛,单育,骆洋.茶树实时荧光定量PCR分析中内参基因的选择[J].植物学报,2010,45(5):579-587
[11]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738
[12]朱海生,陈敏氡,温庆放,蓝新隆,李永平,王彬,张前荣,吴卫东.丝瓜18S rRNA基因克隆及其作为内参基因的应用[J].核农学报,2016,30(1):35-41
[13]Hong S Y,Seo P J,Yang M S,Xiang F N,Park C M.Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR[J].BMC Plant Biology,2008,8(1):112-132
[14]Yi S J,Qian Y Q,Han L,Sun Z Y,Fan C M,Liu J X,Ju G S.Selection of reliable reference genes for gene expression studies in Rhododendron micranthum Turcz[J].Scientia Horticulturae,2004,99(2):128-133
[15]Volkov R A,Panchuk I I,Sch9ffl F.Heat-stress-dependency and developmental modulation of gene expression:The potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR[J].Journal of Experimental Botany,2003,54(391):2343-2349
[16]Gu C S,Liu L Q,Xu C,Zhao Y H,Zhu X D,Huang S Z.Reference gene selection for quantitative real-time RT-PCRnormalization in Irislactea var.chinensis roots under cadmium,lead,and salt stress conditions[J].The Scientific World Journal,2014,2014(2):532713
[17]任锐,戴鹏辉,李萌,刘志明,曹福祥.珙桐实时定量PCR内参基因的筛选及稳定性评价[J].植物生理学报,2016,52(10):1565-1575
[18]林萍,田昆,汪元超.云南野生鸢尾属植物种质资源及观赏应用[J].中国野生植物资源,2003,22(4):33-35
[19]中国科学院中国植物志编辑委员会.中国植物志[M].北京:科学出版社,1985
[20]罗建,费文群,兰小中.鸢尾属1新变型---白花宽柱鸢尾[J].西北植物学报,2016,36(5):1043-1045
[21]徐凌云,王俊丽,周宜君.喜盐鸢尾花色形成关键基因的克隆及表达分析[J].植物遗传资源学报,2017,18(2):340-348
[22]Wang H,Conchou L,Bessière J M,Cazals G,Schatz B,Imbert E.Flower color polymorphism in Iris lutescens(Iridaceae):Biochemical analyses in light of plant-insect interactions[J].Phytochemistry,2013,94(5):123-134
[23]肖月娥,俞新平,胡永红,杨庆华.西南鸢尾种子萌发特性初步研究[J].种子,2008,27(2):18-20
[24]王彦杰,叶清,薛泽云,周华,金奇江,徐迎春.荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证[J].南京农业大学学报,2017,40(3):408-415
[25]Chen D L,Pan X P,Xiao P,Farwell M A,Zhang B H.Evaluation and identification of reliable reference genes for pharmacogenomics,toxicogenomics,and small RNA expression analysis[J].Journal of Cellular Physiology,2011,226(10):2469-2477
[26]马璐琳,崔光芬,王祥宁,贾文杰,段青,杜文文,王继华.泸定百合过氧化氢酶(Ls-Cat1)基因的克隆及表达分析[J].核农学报,2017,31(9):1700-1707
[27]Tanaka Y,Ohmiya A.Seeing is believing:Engineering anthocyanin and carotenoid biosynthetic pathways[J].Current Opinion in Biotechnology,2008,19(2):190-197
[28]Tanaka Y,Sasaki N,Ohmiya A.Biosynthesis of plant pigments:Anthocyanins,betalains and carotenoids[J].The Plant Journal,2008,54(4):733-749
[29]Jeknic'Z,Jeknic'S,Jevremovic'S,Subotic'A,Chen T H.Alteration of flower color in Iris germanica L.Fire Bride through ectopic expression of phytoene synthase gene(crt B)from Pantoea agglomerans[J].Plant Cell Reports,2014,33(8):1307-1321
[30]Lou Q,Liu Y L,Qi Y Y,Jiao S Z,Tian F F,Jiang L,Wang Y J.Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth[J].Journal of Experimental Botany,2014,65(12):3157-3164
[31]Ahn J H,Kim J S,Kim S,Soh H Y,Shin H,Jang H,Ryu J H,Kim A,Yun K Y,Kim S,Kim K S,Choi D,Huh J H.De novo transcriptome analysis to identify Anthocyanin biosynthesis genes responsible for tissue-specific pigmentation in Zoysiagrass(Zoysia japonica Steud.)[J].PLoS One,2015,10(4):e0124497
[32]Ma P Y,Bian X F,Jia Z D,Guo X D,Xie Y Z.De novo sequencing and comprehensive analysis of the mutant transcriptome from purple sweet potato(Ipomoea batatas L.)[J].Gene,2016,575(3):641-649
[33]Yoshihara N,Imayama T,Fukuchi-Mizutani M,Okuhara H,Tanaka Y,Ino I,Yabuya T.c DNA cloning and characterization of UDP-glucose:Anthocyanidin 3-O-glucosyltransferase in Iris hollandica[J].Plant Science,2005,169(3):496-501
[34]Yoshihara N,Imayama T,Matsuo Y,Fukuchi-Mizutani M,Tanaka Y,Ino I,Yabuya T.Characterization of c DNA clones encoding anthocyanin 3-p-coumaroyltransferase from Iris hollandica[J].Plant Science,2006,171(5):632-639
[35]Hyun T K,Rim Y,Jang H J,Kim C H,Park J,Kumar R,Lee S,Kim B C,Bhak J,Nguyen-Quoc B,Kim S W,Lee S Y,Kim J Y.De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis[J].Plant Molecular Biology,2012,79(4/5):413-427
[36]Clotault J,Peltier D,Berruyer R,Thomas M,Briard M,Emmanuel Geoffriau E.Expression of carotenoid biosynthesis genes during carrot root development[J].Journal of Experimental Botany,2008,59(13):3563-3573
[37]Kishimoto S,Ohmiya A.Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum(Chrysanthemum morifolium)[J].Physiologia Plantarum,2006,128(3):436-447
[38]张金波,罗佳滨,张春斌,朱金玲,吕冬霞.荧光定量PCR技术原理及在分子诊断中的应用进展[J].中国优生与遗传杂志,2006,14(12):13-14
[39]贺婷停,宋婷,王超,张长斌,王海燕.短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选[J].生物技术通报,2016,32(11):99-106
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