研究报告

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

展开
  • 1山西师范大学生命科学学院, 临汾 041000;
    2中国医学科学院北京协和医学院药用植物研究所, 北京 100193

收稿日期: 2013-01-10

  修回日期: 2013-03-15

  网络出版日期: 2013-09-26

基金资助

973计划;国家自然科学基金

Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis of Gene Expression in Populus trichocarpa

Expand
  • 1College of Life Sciences, Shanxi Normal University, Linfen 041000, China;

    2Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China

Received date: 2013-01-10

  Revised date: 2013-03-15

  Online published: 2013-09-26

摘要

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8TUB6ubiquitinGAPDHactin18S rRNAEF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actinubiquitinEF1α18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。

本文引用格式

苏晓娟, 樊保国, 袁丽钗, 崔秀娜, 卢善发 . 实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J]. 植物学报, 2013 , 48(5) : 507 -518 . DOI: 10.3724/SP.J.1259.2013.00507

Abstract

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. We analyzed the expression patterns of 7 housekeeping genes, including TUA8, TUB6, ubiquitin, GAPDH, actin, 18S rRNA and EF1α, in various tissues of greenhouse-grown Populus trichocarpa and Zn-treated in vitro plantlets. The stability of housekeeping gene expression was analyzed with use of 3 software packages, including geNorm, Norm- Finder, and BestKeeper. The genes actin, ubiquitin, EF1α and 18S rRNA were suitable reference genes for efficient normalization of qRT-PCR data, whereas TUB6 and GAPDH were not suitable for analysis of greenhouse-grown plants and Zn-treated plantlets, respectively. These findings were confirmed by comparative profiling of 4 P. trichocarpa NAC genes. This study provides useful information for reference gene selection in qRT-PCR analysis of gene expression in P. trichocarpa. It is also helpful to elucidate the function of P. trichocarpa NAC genes.

参考文献

Andersen C L, Jensen J L, Orntoft T F (2004). 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, 5245-5250.
Bustin S A (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169-193.
Bustin S A (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29, 23-39.
Bustin S A, Benes V, Nolan T, Pfaffl M W (2005). Quantitative real-time RT-PCR–a perspective. J Mol Endocrinol 34, 597-601.
Basa B, Solti A, Sarvari E, Tamas L (2009). Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation. Funct Plant Biol 36, 1079-1087.
Boava L P, Laia M L, Jacob T R, Dabbas K M, Goncalves J F, Ferro J A, Ferro M I , Furtado E L (2010). Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR. BMC Res Notes 3, 43.
Carvalho K, de Campos M K, Pereira LF, Vieira L G (2010). Reference gene selection for real-time quantitative polymerase chain reaction normalization in‘‘Swingle’’ citrumelo under drought stress. Anal Biochem 402, 197-199.
Chen X, Truksa M, Shah S, Weselake RJ (2010). A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus. Anal Biochem 405, 138-140.
Dheda K, Huggett J F, Bustin S A, Johnson MA, Rook G, Zumla A (2004). Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112-119.
Han X, Lu M, Chen Y, et al (2012). Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. PLoS One 7, e43084.
Hochstrasser M (2000). Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol 2, E153-157.
Hong S Y, Seo P J, Yang M S, Xiang F N, Park C M (2008). Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8, 1-11.
Hu R, Qi G, Kong Y Z, Kong D J, G Q, Zhou G K (2010). Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa. BMC Plant Biol 10, 145.
Huis R, Hawkins S, Neutelings G (2010). Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol 10, 71.
Kim B R, Nam H Y, Kim S U, Kim S L, Chang Y J (2003). Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25, 1869-1872.
Lovdal T, Lillo C (2009). Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387, 238-242.
Lu S, Sun Y H, Chiang V L (2008). Stress-responsive microRNAs in Populus. Plant J 55, 131-151.
Luo H, Chen S, Wan H, Chen F, Gu C, Liu Z (2010). Candidate reference genes for gene expression studies in water lily. Anal biochem 404, 100-102.
Mafra V, Kubo K S, Alves-Ferreira M, Ribeiro-Alves M, Stuart R M, Boava L P, Rodrigues C M, Machado M A (2012). Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7, e31263.
Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines (2010). Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10, 4.
Maroufi A, Bockstaele E Y, Loose M D, (2010). Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11, 15-26.
Martin R C, Hollenbeck V G, Dombrowski J E (2008). Evaluation of reference genes for quantitative RT-PCR in Lolium perenne. Crop Sci 48, 1881-1887.
Mehdi Khanlou K, Van Bockstaele E (2012). A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.). Planta 236, 1381-1393.
Nolan T, Hands R E, Bustin S A (2006). Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559-1582.
Pettengill E A, Parmentier-Line C, Coleman G D (2012). Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies. BMC Res Notes 5, 366.
Pfaffl M W, Tichopad A, Prgomet C, Neuvians TP (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26, 509-515.
Ponton F, Chapuis M P, Pernice M, Sword G A, Simpson S J (2011). Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol 57, 840-850.
Regier N, Frey B (2010). Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar. BMC Mol Biol 11, 57.
Reid K E, Olsson N, Schlosser J, Peng F, Lund S T (2006). An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6, 27–37.
Santella L, Chun J T (2011). Actin, more than just a housekeeping protein at the scene of fertilization. Sci China Life Sci 54, 733-743.
Schmittgen T, 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.
Suzuki T, Higgins P J, Crawford D R (2000). Control selection for RNA quantitation. BioTechniques 29, 332-337.
Thellin O, Zorzi W, Lakaye B, Borman B D, Coumans B, Hennen G, Grisar T, Lgout A, Heinen E (1999). Housekeeping genes as internal standards: use and limits. J Biotechnol 75, 291-295.
Udvardi M K, Czechowski T, Scheible W R (2008). Eleven golden rules of quantitative RT-PCR. J Plant Cell 20, 1736-1737.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van RoyN, De Paepe A, Speleman F (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034.
VanGuilder H D, Vrana K E, Freeman W M (2008). Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 44, 619-626.
Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010). Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399, 257-261.
Warrington J A, Nair A, Mahadevappa M, Tsyganskaya M (2000). Comparison of human adult and fetal expression and identification of 535 housekeeping maintenance genes. Physiol Genomics 2, 143-147.
Xu M, Zhang B, Su X, et al (2011). Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem 408, 337-339.
Yang Y, Hou S, Cui G, et al (2010). Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Salvia miltiorrhiza. Mol Biol Rep 37, 507-513.
Zhou B, Cao C, Liu C X (2007). Advances in research on translation elongation factor 1 alpha. Lett Biotechnol 18, 281-284.
文章导航

/