板栗实时定量PCR内参基因的筛选与验证
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摘要
实时荧光定量PCR技术具有高灵敏性和高特异性,被广泛应用于基因表达分析,选取合适的内参基因是利用该技术准确分析目的基因表达变化的前提。本文以板栗的7种不同组织器官(根、茎、叶、种子、花芽、雌花和雄花)为材料,采用qRT-PCR技术分析了板栗中6个候选内参基因(EF1α、TUA、TUB、UBQ、18S rRNA和Actin)的表达趋势,运用geNorm、NormFinder和BestKeeper 3种软件对候选内参基因的表达稳定性进行了综合分析,并利用目的基因CmSPL9对筛选出的内参基因进行验证。结果表明:Actin和EF1α在板栗不同组织器官中稳定性最佳, Actin、EF1α或Actin+EF1α可作为板栗的内参基因进行后续试验研究。
Quantitative RT-PCR(qRT-PCR) has been widely used in gene expression analysis because of its sensitivity, specificity, and reproducibility. Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results. In this study, seven different tissues and organs(roots, stems, leaves, seed, flower buds, female flowers, male flowers) of Castanea mollissima were used as materials. The expression trends of six candidate internal reference genes(EF1α, TUA, TUB, UBQ, 18 S rRNA, Actin) in chestnut were analyzed by RT-qPCR. The geNorm, NormFinder and BestKeeper software were used to analyze the expression stability of candidate internal reference genes. The selected internal reference gene was verified by the target gene CmSPL9. The results showed that Actin and EF1α had the most stability in different tissues and organs of C. mollissima. Actin, EF1α or Actin+EF1α can be used as reference test for C. mollissima internal reference genes.
Quantitative RT-PCR(qRT-PCR) has been widely used in gene expression analysis because of its sensitivity, specificity, and reproducibility. Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results. In this study, seven different tissues and organs(roots, stems, leaves, seed, flower buds, female flowers, male flowers) of Castanea mollissima were used as materials. The expression trends of six candidate internal reference genes(EF1α, TUA, TUB, UBQ, 18 S rRNA, Actin) in chestnut were analyzed by RT-qPCR. The geNorm, NormFinder and BestKeeper software were used to analyze the expression stability of candidate internal reference genes. The selected internal reference gene was verified by the target gene CmSPL9. The results showed that Actin and EF1α had the most stability in different tissues and organs of C. mollissima. Actin, EF1α or Actin+EF1α can be used as reference test for C. mollissima internal reference genes.
引文
Andersen CL,Jensen JL,?rntoft TF(2014).Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Res,64(15):5245-5250
Bustin SA,Benes V,Garson JA,et al(2009).The MIQEguidelines:minimum information for publication of quantitative real-time PCR experiments.Clin Chem,55(4):611-622
Chen LK,Lu D,Wang T,et al(2017).Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut(Castanea mollissima Blume)cotyledons.PLoS One,12(5):e0177792
Die JV,Román B,Nadal S,et al(2010).Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions.Planta,232:145-153
Fan LM,Wang C,Liu GS,et al(2014).Screening and validation of reference genes for real-time fluorescence quantitative PCR during coloring period in apple(Malus domestica).Plant Physiol J,50(12):1903-1911(in Chinese with English abstract)[樊连梅,王超,刘更森等(2014).苹果着色期实时定量PCR内参基因的筛选和验证.植物生理学报,50(12):1903-1911]
Giulietti A,Overbergh L,Valckx D(2010).An overview of real-time quantitative PCR:applications to quantify cytokine gene expression.Methods,25(4):386-401
Jiang TT,Gao YH,Tong ZK(2015).Selection of reference genes for quantitative real-time PCR in Lycoris.Acta Hortic Sin,42(6):1129-1138(in Chinese with English abstract)[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]
Li X,Pan XJ,Zhang WE,et al(2017).Stability evaluation of reference genes for quantitative real-time PCR analysis in walnut(Juglans spp.).Plant Physiol J,53(9):1795-1802(in Chinese with English abstract)[李雪,潘学军,张文娥等(2017).核桃内参基因实时荧光定量PCR表达稳定性评价.植物生理学报,53(9):1795-1802]
Liu WZ,Niu MY,Li XY,et al(2016).The selection of reference genes for quantitative PCR in Betula luminifera.Sci Silv Sin,52(8):29-37(in Chinese with English abstract)[刘文哲,牛明月,李秀云等(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]
Liu XT,Wang SL,Xue JQ,et al(2018).Selection of reference genes for quantitative real-time PCR in different tissue and organ of barbadoslily.Acta Hortic Sin,45(5):919-930(in Chinese with English abstract)[刘晓婷,王顺利,薛璟祺等(2018).朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选.园艺学报,45(5):919-930]
Lopez-Pardo R,Galarreta J,Ritter E(2013).Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress.Mol Breeding,31(1):39-45
Lu D,Wei W,Zhou W,et al(2017).Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut(Castanea mollissima Blume).Plant Cell Tiss Org Cult,130(3):601-616
Mafra V,Kubo KS,Alves-Ferreira M,et al(2012).Reference genes for accurate transcript normalization in Citrus genotypes under different experimental conditions.PLoSOne,7:e31263
Pang QQ,Li ZL,Luo SB,et al(2017).Selection and stability analysis of reference gene for qRT-PCR in eggplant under high temperature stress.Acta Hortic Sin,44(3):475-486(in Chinese with English abstract)[庞强强,李植良,罗少波等(2017).高温胁迫下茄子qRT-PCR内参基因筛选及稳定性分析.园艺学报,44(3):475-486]
Pfaffl MW,Tichopad A,Prgomet C,et al(2004).Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper Excel-based tool using pair-wise correlations.Biotechnol Lett,26(6):509-515
Ren R,Dai PH,Li M,et al(2016).Selection and stability evaluation of reference genes for real-time quantitative PCR in dove tree(Davidia involucrata).Plant Physiol J,52(10):1565-1575(in Chinese with English abstract)[任锐,戴鹏辉,李萌等(2016).珙桐实时定量PCR内参基因的筛选及稳定性评价.植物生理学报,52(10):1565-1575]
Su XJ,Fan BG,Yuan LC,et al(2013).Selection and validation of reference genes for quantitative RT-PCR analysis of gene expression in Populus trichocarpa.Chin Bull Bot,48(5):507-518(in Chinese with English abstract)[苏晓娟,樊保国,袁丽钗等(2013).实时荧光定量PCR分析中毛果杨内参基因的筛选和验证.植物学报,48(5):507-518]
Vandesompele J,De Preter K,Pattyn F,et al(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):research0034.1-research0034.11
Wang JX,Zhang LJ,Liao ZY,et al(2014).The selection of reference genes for real-time quantitative PCR normalization in black locust(Robinia pseudoacacia).Sci Silv Sin,50(9):167-172(in Chinese with English abstract)[王金星,张利军,廖资亿等(2014).刺槐实时定量PCR分析中内参基因的选择.林业科学,50(9):167-172]
Wang YJ,Chen YQ,Xue ZY,et al(2017).Selection and validation of reference genes for RT-qPCR normalization in lotus(Nelumbo nucifera)during petal coloration.J Nanjing Agr Univ,40(3):408-415(in Chinese with English abstract)[王彦杰,陈叶清,薛泽云等(2017).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.南京农业大学学报,40(3):408-415]
Wei DD,Zhang W,Wang CC,et al(2017).Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants.Plant Sci,257:74-83
Xia WX,Yu HY,Cao P,et al(2017).Identification of TIFYfamily genes and analysis of their expression profiles in response to phytohormone treatments and Melampsora larici-populina infection in poplar.Front Plant Sci,8(493):1-11
Yuan J,Meng J,Liang X,et al(2017).Organic molecules from biochar leacheates have a positive effect on rice seedling cold tolerance.Front Plant Sci,8(1624):1-13
Zhang YF,Zhao LJ,Zeng YL(2014).Selection and application of reference genes for gene expression studies.Plant Physiol J,50(8):1119-1125(in Chinese with English abstract)[张玉芳,赵丽娟,曾幼玲(2014).基因表达研究中内参基因的选择与应用.植物生理学报,50(8):1119-1125]
Zhou B,Cao C,Liu CX(2007).Advances in research on translation elongation factor 1 alpha.Lett Biotechnol,(2):281-284(in Chinese with English abstract)[周冰,曹诚,刘传暄(2007).翻译延伸因子1α的研究进展.生物技术通讯,(2):281-284]
Bustin SA,Benes V,Garson JA,et al(2009).The MIQEguidelines:minimum information for publication of quantitative real-time PCR experiments.Clin Chem,55(4):611-622
Chen LK,Lu D,Wang T,et al(2017).Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut(Castanea mollissima Blume)cotyledons.PLoS One,12(5):e0177792
Die JV,Román B,Nadal S,et al(2010).Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions.Planta,232:145-153
Fan LM,Wang C,Liu GS,et al(2014).Screening and validation of reference genes for real-time fluorescence quantitative PCR during coloring period in apple(Malus domestica).Plant Physiol J,50(12):1903-1911(in Chinese with English abstract)[樊连梅,王超,刘更森等(2014).苹果着色期实时定量PCR内参基因的筛选和验证.植物生理学报,50(12):1903-1911]
Giulietti A,Overbergh L,Valckx D(2010).An overview of real-time quantitative PCR:applications to quantify cytokine gene expression.Methods,25(4):386-401
Jiang TT,Gao YH,Tong ZK(2015).Selection of reference genes for quantitative real-time PCR in Lycoris.Acta Hortic Sin,42(6):1129-1138(in Chinese with English abstract)[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]
Li X,Pan XJ,Zhang WE,et al(2017).Stability evaluation of reference genes for quantitative real-time PCR analysis in walnut(Juglans spp.).Plant Physiol J,53(9):1795-1802(in Chinese with English abstract)[李雪,潘学军,张文娥等(2017).核桃内参基因实时荧光定量PCR表达稳定性评价.植物生理学报,53(9):1795-1802]
Liu WZ,Niu MY,Li XY,et al(2016).The selection of reference genes for quantitative PCR in Betula luminifera.Sci Silv Sin,52(8):29-37(in Chinese with English abstract)[刘文哲,牛明月,李秀云等(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]
Liu XT,Wang SL,Xue JQ,et al(2018).Selection of reference genes for quantitative real-time PCR in different tissue and organ of barbadoslily.Acta Hortic Sin,45(5):919-930(in Chinese with English abstract)[刘晓婷,王顺利,薛璟祺等(2018).朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选.园艺学报,45(5):919-930]
Lopez-Pardo R,Galarreta J,Ritter E(2013).Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress.Mol Breeding,31(1):39-45
Lu D,Wei W,Zhou W,et al(2017).Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut(Castanea mollissima Blume).Plant Cell Tiss Org Cult,130(3):601-616
Mafra V,Kubo KS,Alves-Ferreira M,et al(2012).Reference genes for accurate transcript normalization in Citrus genotypes under different experimental conditions.PLoSOne,7:e31263
Pang QQ,Li ZL,Luo SB,et al(2017).Selection and stability analysis of reference gene for qRT-PCR in eggplant under high temperature stress.Acta Hortic Sin,44(3):475-486(in Chinese with English abstract)[庞强强,李植良,罗少波等(2017).高温胁迫下茄子qRT-PCR内参基因筛选及稳定性分析.园艺学报,44(3):475-486]
Pfaffl MW,Tichopad A,Prgomet C,et al(2004).Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper Excel-based tool using pair-wise correlations.Biotechnol Lett,26(6):509-515
Ren R,Dai PH,Li M,et al(2016).Selection and stability evaluation of reference genes for real-time quantitative PCR in dove tree(Davidia involucrata).Plant Physiol J,52(10):1565-1575(in Chinese with English abstract)[任锐,戴鹏辉,李萌等(2016).珙桐实时定量PCR内参基因的筛选及稳定性评价.植物生理学报,52(10):1565-1575]
Su XJ,Fan BG,Yuan LC,et al(2013).Selection and validation of reference genes for quantitative RT-PCR analysis of gene expression in Populus trichocarpa.Chin Bull Bot,48(5):507-518(in Chinese with English abstract)[苏晓娟,樊保国,袁丽钗等(2013).实时荧光定量PCR分析中毛果杨内参基因的筛选和验证.植物学报,48(5):507-518]
Vandesompele J,De Preter K,Pattyn F,et al(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):research0034.1-research0034.11
Wang JX,Zhang LJ,Liao ZY,et al(2014).The selection of reference genes for real-time quantitative PCR normalization in black locust(Robinia pseudoacacia).Sci Silv Sin,50(9):167-172(in Chinese with English abstract)[王金星,张利军,廖资亿等(2014).刺槐实时定量PCR分析中内参基因的选择.林业科学,50(9):167-172]
Wang YJ,Chen YQ,Xue ZY,et al(2017).Selection and validation of reference genes for RT-qPCR normalization in lotus(Nelumbo nucifera)during petal coloration.J Nanjing Agr Univ,40(3):408-415(in Chinese with English abstract)[王彦杰,陈叶清,薛泽云等(2017).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.南京农业大学学报,40(3):408-415]
Wei DD,Zhang W,Wang CC,et al(2017).Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants.Plant Sci,257:74-83
Xia WX,Yu HY,Cao P,et al(2017).Identification of TIFYfamily genes and analysis of their expression profiles in response to phytohormone treatments and Melampsora larici-populina infection in poplar.Front Plant Sci,8(493):1-11
Yuan J,Meng J,Liang X,et al(2017).Organic molecules from biochar leacheates have a positive effect on rice seedling cold tolerance.Front Plant Sci,8(1624):1-13
Zhang YF,Zhao LJ,Zeng YL(2014).Selection and application of reference genes for gene expression studies.Plant Physiol J,50(8):1119-1125(in Chinese with English abstract)[张玉芳,赵丽娟,曾幼玲(2014).基因表达研究中内参基因的选择与应用.植物生理学报,50(8):1119-1125]
Zhou B,Cao C,Liu CX(2007).Advances in research on translation elongation factor 1 alpha.Lett Biotechnol,(2):281-284(in Chinese with English abstract)[周冰,曹诚,刘传暄(2007).翻译延伸因子1α的研究进展.生物技术通讯,(2):281-284]