核桃(Juglans regia L.)MicroRNA实时荧光定量RT-PCR内参基因的筛选
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摘要
RT-qPCR是目前应用最广泛的基因表达分析方法,选择合适的内参基因对数据进行标准化至关重要。结合geNorm、Normfinder和RefFinder 3个软件分析了8个候选miRNA内参基因在核桃不同分化期花芽与叶芽、不同组织及不同品种中的表达稳定性。结果显示,jre-miR394a,jre-miR159a和jre-miR159c可作为不同分化期花芽中基因表达分析的内参;5.8S rRNA和jre-miRn3可作为不同分化期叶芽中基因表达分析的内参;5.8S rRNA,jre-miRn3、jre-miR159b-3p和jre-miR159c可作为不同组织中基因表达分析的内参;jre-miR159b-3p,jre-miR159c和jre-miR159a可作为不同品种中基因表达分析的内参。5.8S rRNA不适合作为不同分化期花芽和不同品种中基因表达分析的内参。本研究为核桃miRNA表达分析中的数据标准化提供数据支持和理论参考。
Reverse transcription quantitative real-time PCR(RT-qPCR) is the most widely used gene expression analysis method, and the selection of appropriate reference genes is crucial for data normalization. Eight candidate miRNA reference genes were screened to evaluate their expression stability in flower buds and leaf buds at different differentiation phase, different tissues and cultivars of walnut(Juglans regia L.) based on three softwares(geNorm, Normfinder and RefFinder). The results demonstrated that jre-miR394 a, jre-miR159 a and jre-miR159 c could be used as reference genes for gene expression analysis in flower buds at different stages of differentiation;5.8 S rRNA and jre-miRn3 could be used as reference genes for gene expression analysis in leaf buds at different stages of differentiation; 5.8 S r RNA, jre-miRn3, jre-miR159 b-3 p and jre-miR159 c could be used as reference genes for gene expression analysis in different tissues; jre-miR159 b-3 p, jre-miR159 c and jre-miR159 a could be used as reference genes for gene expression analysis in different cultivars. Moreover, 5.8 S rRNA was not a suitable reference gene for gene expression analysis in flower buds at different stages of differentiation and different cultivars. The study could provide data support and theoretical reference for data standardization in miRNA expression analysis of walnut.
Reverse transcription quantitative real-time PCR(RT-qPCR) is the most widely used gene expression analysis method, and the selection of appropriate reference genes is crucial for data normalization. Eight candidate miRNA reference genes were screened to evaluate their expression stability in flower buds and leaf buds at different differentiation phase, different tissues and cultivars of walnut(Juglans regia L.) based on three softwares(geNorm, Normfinder and RefFinder). The results demonstrated that jre-miR394 a, jre-miR159 a and jre-miR159 c could be used as reference genes for gene expression analysis in flower buds at different stages of differentiation;5.8 S rRNA and jre-miRn3 could be used as reference genes for gene expression analysis in leaf buds at different stages of differentiation; 5.8 S r RNA, jre-miRn3, jre-miR159 b-3 p and jre-miR159 c could be used as reference genes for gene expression analysis in different tissues; jre-miR159 b-3 p, jre-miR159 c and jre-miR159 a could be used as reference genes for gene expression analysis in different cultivars. Moreover, 5.8 S rRNA was not a suitable reference gene for gene expression analysis in flower buds at different stages of differentiation and different cultivars. The study could provide data support and theoretical reference for data standardization in miRNA expression analysis of walnut.
引文
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Bai S.L.,Saito T.,Ito A.,Tuan P.A.,Xu Y.,Teng Y.W.,and Moriguchi T.,2016,Small RNA and PARE sequencing in flower bud reveal the involvement of s RNAs in endodormancy release of Japanese pear(Pyrus pyrifolia'Kosui'),BMC Genomics,17:230
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Feng H.,Huang X.L.,Zhang Q.,Wei G.R.,Wang X.J.,and Kang Z.S.,2012,Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat,Plant Physiol.Biochem.,51:116-122
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Jiang T.T.,Gao Y.H.,and Tong Z.K.,2015,Selection of reference genes for quantitative real-time PCR in Lycoris,Yuanyi Xuebao(Acta Horticulturae Sinica),42(6):1129-1138(蒋婷婷,高燕会,童再康,2015,石蒜属植物实时荧光定量PCR内参基因的选择,园艺学报,42(6):1129-1138)
Kou S.J.,Wu X.M.,Liu Z.,Liu Y.L.,Xu Q.,and Guo W.W.,2012,Selection and validation of suitable reference genes for mi RNA expression normalization by quantitative RT-PCRin citrus somatic embryogenic and adult tissues,Plant Cell Rep.,31(12):2151-2163
Kulcheski F.R.,Marcelino-Guimaraes F.C.,Nepomuceno A.L.,Abdelnoor R.V.,and Margis R.,2010,The use of microR-NAs as reference genes for quantitative polymerase chain reaction in soybean,Anal.Biochem.,406(2):185-192
Li S.F.,Fan C.M.,Li Y.,Zhang J.H.,Sun J.S.,Chen Y.H.,Tian C.Y.,Su X.H.,Lu M.Z.,Liang C.Z.,and Hu Z.M.,2016,Effects of drought and salt-stresses on gene expression in Caragana korshinskii seedlings revealed by RNA-seq,BMCGenomics,17:200
Lin Y.L.,and Lai Z.X.,2013,Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis,Plant Physiol.Biochem.,66:20-25
Liu W.C.,Wang Q.,Zhou Y.G.,Deng Y.,Zhao L.D.,Wang X.C.,Jin J.,Dong Y.Y.,Wang N.,Wang F.W.,Chen H.,Li X.W.,and Li H.Y.,2016,Selection of reference genes for quantitative polymerase chain reaction of mi RNA and m RNA in soybean under drought stress,Xibei Nonglin Keji Daoxue Xuebao(Journal of Northwest A&F University(Natural Science Edition)),44(2):61-67(刘伟灿,王骐,周永刚,邓宇,赵利旦,王兴超,靳京,董园园,王南,王法微,2016,大豆干旱胁迫下mi RNA与m RNA荧光定量PCR内参基因的筛选,西北农林科技大学学报(自然科学版),44(2):61-67)
Ma X.R.,Li L.,Yang M.J.,Liao Z.P.,Wang W.T.,Liu J.Y.,Wu S.H.,and Li Y.Y.,2018,Selection of microRNA reference genes for real-time quantitative PCR in pear flower bud during dormant phase,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(14):4696-4704(马鑫瑞,李亮,杨梦洁,廖正萍,王文婷,刘靖媛,吴少华,李永裕,2018,梨花芽休眠进程中microRNA实时定量PCR内参基因的筛选,分子植物育种,16(14):4696-4704)
Niu K.J.,Shi Y.,and Ma H.L.,2017,Selection of candidate reference genes for gene expression analysis in Kentucky Bluegrass(Poa pratensis L.)under abiotic stress,Front.Plant Sci.,8:193
Nolan T.,Hands R.E.,and Bustin S.A.,2006,Quantification of m RNA using real-time RT-PCR,Nat.Protoc.,1(13):1559-1582
Qu D.,Yan F.,Meng R.,Jiang X.B.,Yang H.J.,Gao Z.Y.,Dong Y.H.,Yang Y.Z.,and Zhao Z.Y.,2016,Identification of microRNAs and their targets associated with fruit-bagging and subsequent sunlight re-exposure in the"Granny Smith"apple exocarp using high-throughput sequencing,Front.Plant Sci.,7(156):27
Sun Z.C.,Zhang L.S.,and Wang Z.J.,2017,Genome-wide analysis of mi RNAs in Carya cathayensis,BMC Plant Biol.,17:228
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 X.F.,Tian X.F.,Jiang C.J.,and Li Y.Y.,2015,Screening of microRNA reference genes for real-time fluorescence quantitative PCR under cold stress in Camellia sinensis,Chaye Kexue(Journal of Tea Science),35(6):596-604(谢小芳,添先凤,江昌俊,李叶云,2015,茶树低温胁迫下microRNA实时定量PCR内参基因的筛选,茶叶科学,35(6):596-604)
Yin D.M.,Zhao Z.Y.,Guo F.G.,Zhou G.Y.,Wang S.Y.,and Li J.,2017,Screening of reference genes for real-time quantitative PCR in Bergenia purpurascens,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(10):4256-4262(尹冬梅,赵振宇,郭凤根,周国雁,王仕玉,李娟,2017,岩白菜实时荧光定量PCR分析的内参基因的筛选,基因组学与应用生物学,36(10):4256-4262)
Bai S.L.,Saito T.,Ito A.,Tuan P.A.,Xu Y.,Teng Y.W.,and Moriguchi T.,2016,Small RNA and PARE sequencing in flower bud reveal the involvement of s RNAs in endodormancy release of Japanese pear(Pyrus pyrifolia'Kosui'),BMC Genomics,17:230
Bustin S.A.,Benes V.,Nolan T.,and Pfaffl M.W.,2005,Quantitative real-time RT-PCR--a perspective,J.Mol.Endocrinol.,34(3):597-601
Feng H.,Huang X.L.,Zhang Q.,Wei G.R.,Wang X.J.,and Kang Z.S.,2012,Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat,Plant Physiol.Biochem.,51:116-122
Gachon C.,Mingam A.,and Charrier B.,2004,Real-time PCR:what relevance to plant studies,J.Exp.Bot.,55(402):1445-1454
Jiang T.T.,Gao Y.H.,and Tong Z.K.,2015,Selection of reference genes for quantitative real-time PCR in Lycoris,Yuanyi Xuebao(Acta Horticulturae Sinica),42(6):1129-1138(蒋婷婷,高燕会,童再康,2015,石蒜属植物实时荧光定量PCR内参基因的选择,园艺学报,42(6):1129-1138)
Kou S.J.,Wu X.M.,Liu Z.,Liu Y.L.,Xu Q.,and Guo W.W.,2012,Selection and validation of suitable reference genes for mi RNA expression normalization by quantitative RT-PCRin citrus somatic embryogenic and adult tissues,Plant Cell Rep.,31(12):2151-2163
Kulcheski F.R.,Marcelino-Guimaraes F.C.,Nepomuceno A.L.,Abdelnoor R.V.,and Margis R.,2010,The use of microR-NAs as reference genes for quantitative polymerase chain reaction in soybean,Anal.Biochem.,406(2):185-192
Li S.F.,Fan C.M.,Li Y.,Zhang J.H.,Sun J.S.,Chen Y.H.,Tian C.Y.,Su X.H.,Lu M.Z.,Liang C.Z.,and Hu Z.M.,2016,Effects of drought and salt-stresses on gene expression in Caragana korshinskii seedlings revealed by RNA-seq,BMCGenomics,17:200
Lin Y.L.,and Lai Z.X.,2013,Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis,Plant Physiol.Biochem.,66:20-25
Liu W.C.,Wang Q.,Zhou Y.G.,Deng Y.,Zhao L.D.,Wang X.C.,Jin J.,Dong Y.Y.,Wang N.,Wang F.W.,Chen H.,Li X.W.,and Li H.Y.,2016,Selection of reference genes for quantitative polymerase chain reaction of mi RNA and m RNA in soybean under drought stress,Xibei Nonglin Keji Daoxue Xuebao(Journal of Northwest A&F University(Natural Science Edition)),44(2):61-67(刘伟灿,王骐,周永刚,邓宇,赵利旦,王兴超,靳京,董园园,王南,王法微,2016,大豆干旱胁迫下mi RNA与m RNA荧光定量PCR内参基因的筛选,西北农林科技大学学报(自然科学版),44(2):61-67)
Ma X.R.,Li L.,Yang M.J.,Liao Z.P.,Wang W.T.,Liu J.Y.,Wu S.H.,and Li Y.Y.,2018,Selection of microRNA reference genes for real-time quantitative PCR in pear flower bud during dormant phase,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(14):4696-4704(马鑫瑞,李亮,杨梦洁,廖正萍,王文婷,刘靖媛,吴少华,李永裕,2018,梨花芽休眠进程中microRNA实时定量PCR内参基因的筛选,分子植物育种,16(14):4696-4704)
Niu K.J.,Shi Y.,and Ma H.L.,2017,Selection of candidate reference genes for gene expression analysis in Kentucky Bluegrass(Poa pratensis L.)under abiotic stress,Front.Plant Sci.,8:193
Nolan T.,Hands R.E.,and Bustin S.A.,2006,Quantification of m RNA using real-time RT-PCR,Nat.Protoc.,1(13):1559-1582
Qu D.,Yan F.,Meng R.,Jiang X.B.,Yang H.J.,Gao Z.Y.,Dong Y.H.,Yang Y.Z.,and Zhao Z.Y.,2016,Identification of microRNAs and their targets associated with fruit-bagging and subsequent sunlight re-exposure in the"Granny Smith"apple exocarp using high-throughput sequencing,Front.Plant Sci.,7(156):27
Sun Z.C.,Zhang L.S.,and Wang Z.J.,2017,Genome-wide analysis of mi RNAs in Carya cathayensis,BMC Plant Biol.,17:228
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 X.F.,Tian X.F.,Jiang C.J.,and Li Y.Y.,2015,Screening of microRNA reference genes for real-time fluorescence quantitative PCR under cold stress in Camellia sinensis,Chaye Kexue(Journal of Tea Science),35(6):596-604(谢小芳,添先凤,江昌俊,李叶云,2015,茶树低温胁迫下microRNA实时定量PCR内参基因的筛选,茶叶科学,35(6):596-604)
Yin D.M.,Zhao Z.Y.,Guo F.G.,Zhou G.Y.,Wang S.Y.,and Li J.,2017,Screening of reference genes for real-time quantitative PCR in Bergenia purpurascens,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(10):4256-4262(尹冬梅,赵振宇,郭凤根,周国雁,王仕玉,李娟,2017,岩白菜实时荧光定量PCR分析的内参基因的筛选,基因组学与应用生物学,36(10):4256-4262)
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