实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

苏晓娟, 樊保国, 袁丽钗, 崔秀娜, 卢善发

doi:10.3724/SP.J.1259.2013.00507

植物学报 >

2013 , Vol. 48 >Issue 5: 507 - 518

DOI: https://doi.org/10.3724/SP.J.1259.2013.00507

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

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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

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

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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

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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TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 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actin、ubiquitin、EF1α和18S 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.

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