‘砀山酥梨’实时荧光定量PCR内参基因的筛选
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摘要
对'砀山酥梨'不同组织器官及不同非生物胁迫条件下叶片中可以稳定表达的内参基因进行筛选,为后续荧光定量PCR试验提供参考基因。分别对生长45 d的'砀山酥梨'嫁接苗进行低温、高温和盐胁迫处理,采集不同处理时间的叶片;还分别采集'砀山酥梨'4个果实发育时期的果肉(分别为花后32、65、99、143d)、叶片、成熟果皮、花粉、花柱共26个样品。利用实时荧光定量PCR技术对4个传统内参基因:β微管蛋白基因(TUB)、多聚泛素酶基因(UBQ)、甘油醛-3-磷酸脱氢酶基因(GAPDH)和转录延伸因子基因(EF1α),以及通过转录组数据筛选出来的3个新内参基因Cytochrome b561(CyB)、WD-repeat protein(WDP)和mRNA capping enzyme(mRCE),共计7个内参基因在26个样品中的表达差异情况进行测定。结果表明:通过geNorm、NormFinder和Bestkeeper 3个软件对其表达稳定性进行分析,最终筛选出在不同组织及非生物胁迫条件下稳定表达的最优内参基因。UBQ在高温和盐胁迫处理的'砀山酥梨'叶片中表达最稳定,可作为优选内参基因;在低温胁迫处理下,TUB和WDP在'砀山酥梨'叶片中表达较稳定,可作优选内参基因;WDP在'砀山酥梨'不同组织器官中表达最稳定。不同内参基因在同一物种不同组织及胁迫处理条件下表达稳定性具有较大差异,在进行荧光定量PCR试验时,应根据具体的试验材料和方法选择最优内参基因。
The aim of this study is to detect the optimal reference genes for real-time fluorescence quantitative PCH analyses in different tissues of 'Dangshansuli' and abiotic stress-treated leaves. The abiotic stress-treated leaves were obtained from the grafted 'Dangshansuli' seedlings after 45 days growing which were grouped for heat, cold and salt treatments, respectively. The different tissues of 'Dangshansuli' including fleshes of four developmental stages(32 days after flower bloom, 65 days after flower bloom, 99 days after flower bloom, 143 days after flower bloom), leaf, pericarp,pollen and stigma, were collected from a 20-year-old tree. Seven candidate reference genes including Tubulin beta-2-chain(TUB),Poly-ubiquitin enzyme(UBQ), Glyceraldehyde-3-phosphate dehydrogenase(GAPDH), Elongation factor 1-α( EF1 α),Cytochrome b561(CyB), WD-repeat protein( WDP) and mRNA capping enzyme(mRCE) were selected to evaluate the expression stability by qRT-PCR among 26 samples. UBQ was the most stable reference gene in heat and salt treated leaves,TUB and WDP were the most suitable reference genes in the leaves under cold treatment. For different tissue samples, WDP was the most stable expressed reference gene. In this study, the results showed that the expression stability of different reference genes varies in different tissues and abiotic treated samples. According to specified materials and treatments, it is important to select suitable candidate reference genes for the qRT-PCR.
The aim of this study is to detect the optimal reference genes for real-time fluorescence quantitative PCH analyses in different tissues of 'Dangshansuli' and abiotic stress-treated leaves. The abiotic stress-treated leaves were obtained from the grafted 'Dangshansuli' seedlings after 45 days growing which were grouped for heat, cold and salt treatments, respectively. The different tissues of 'Dangshansuli' including fleshes of four developmental stages(32 days after flower bloom, 65 days after flower bloom, 99 days after flower bloom, 143 days after flower bloom), leaf, pericarp,pollen and stigma, were collected from a 20-year-old tree. Seven candidate reference genes including Tubulin beta-2-chain(TUB),Poly-ubiquitin enzyme(UBQ), Glyceraldehyde-3-phosphate dehydrogenase(GAPDH), Elongation factor 1-α( EF1 α),Cytochrome b561(CyB), WD-repeat protein( WDP) and mRNA capping enzyme(mRCE) were selected to evaluate the expression stability by qRT-PCR among 26 samples. UBQ was the most stable reference gene in heat and salt treated leaves,TUB and WDP were the most suitable reference genes in the leaves under cold treatment. For different tissue samples, WDP was the most stable expressed reference gene. In this study, the results showed that the expression stability of different reference genes varies in different tissues and abiotic treated samples. According to specified materials and treatments, it is important to select suitable candidate reference genes for the qRT-PCR.
引文
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[24]Libault M,Thibivilliers S.Bilgin D D.et al.Identification of four soybean reference genes for gene expression normalization.Plant Genome,2013,1(1):44-54.
[2]Vandesompele J,De P K.Pattyn F.et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biology 2002.3(7):1.
[3]Aswal A,Datta T K,Raghav S,et al.Development of a competitive quantitative PCR strategy for evaluating the expression stability of 18s rRNA during in vitro,maturation of buffalo(Bubalus bubalis)follicular oocytes.Reproduction in Domestic Animals,2007,42(2):195-201.
[4]Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):trends and problems.Journal of Molecular Endocrinology.2002,29(1):23-39.
[5]胡瑞波.范成明.傅永福.植物实时荧光定量PCR内参基因的选择.中国农业科技导报,2009,11(6):30-36.
[6]滕元文.柴明良,李秀根.梨属植物分类的历史回顾及新进展.果树学报,2004,21(3):252-257.
[7]张绍铃.梨学.北京:中国农业出版社,2013:70.
[8]田义轲,李节法,王彩虹,等.梨茎尖中PpKO基因表达量的实时荧光定量PCR分析.华北农学报,2012.27(3):62-66.
[9]闫洪波,葛文雅,程玉豆,等.鸭梨以GAPDH基因家族的克隆及其表达特征.西北农林科技大学学报(自然科学版),2012,40(9):181-186.
[10]Chen J,Li X,Wang D,et al.Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri.Scientia Horticulturae,2015,190:43-56.
[11]Andersen C L,Jensen J L,(?)rntoft T F.Normalization of real-time quantitative reverse transcription-PCH data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Research,2004,64(15):5245-5250.
[12]Pfaffl M W,Tichopad A,Prgomet C.et al.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations.Biotechnology Letters.2004,26(6):509-515.
[13]Wu T,Zhang R,Gu C,et al.Evaluation of candidate reference genes forreal time quantitative PCR normalization in pear fruit.African Journal of Agricultural Research,2012,7(25):3701-3704.
[14]Mafra V,Kuho K S,Alvesferreira M,et al.Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions.Plos One,2012,7(2):e31263.
[15]Luo X Y,Shi T,Sun H L,et al.Selection of suitable inner reference genes for normalisation of micro RNA expression response to abiotic stresses by RT-q PCR in leaves,flowers and young stems of peach.Scientia Horticulturae,2014,165:281-287.
[16]王梨嬛,潘永娟,杨莉,等.琯溪蜜柚'荧光定量PCR内参基因的筛选.果树学报,2013,30(1):48-54.
[17]赵晓,马会勤,陈尚武,等.葡萄果实发育后期半定量RT-PCR内参基因的优选.中国农业大学学报,2010,15(3):7-14.
[18]魏永赞,赖彪,胡福初,等.用于荔枝qPCR分析的内参基因克隆及稳定性分析.华南农业大学学报.2012.33(3):301-306.
[19]Xiu Y,Li H.Li X.et al.Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear.Acta Physiologiae Plantarum,2015,37(2):1-16.
[20]Imai T.Ubi B E,Saito T,et al.Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.Plos One,2014,9(1):e86492.
[21]Yan J W.Yuan F R.Long G Y,et al.Selection of reference genes for quantitative real-time RT-PCR analysis in citrus.Mol Biol Rep,2012,39(2):1831-1838.
[22]Farrioli P,Ciceri G P,Provero P,et al.A combined strategy of“in siliro"transrriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies.Plant Molecular Biology,2007.63(5):679-688.
[23]Czechowski T,Stitt M,Altmann T,et al.Genome-wide identification and testing of superior reference genes for transcript normalization in arahidopsis.Plant Physiology,2005,139(1):5-17.
[24]Libault M,Thibivilliers S.Bilgin D D.et al.Identification of four soybean reference genes for gene expression normalization.Plant Genome,2013,1(1):44-54.