Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR.
Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR.
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DOI：
10.1111/ppl.12206
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发表时间：
2014-11
影响因子：
6.4
通讯作者：
Hou-ling Wang;Jin-huan Chen;Qianqian Tian;Shu Wang;X. Xia;W. Yin
中科院分区：
文献类型：
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作者：
Hou-ling Wang;Jin-huan Chen;Qianqian Tian;Shu Wang;X. Xia;W. Yin
胡杨（Populus euphratica）是世界上最大的流沙沙漠中国境内唯一建立的乔木树种，对极端的沙漠环境具有良好的适应性，因此被广泛认为是木本植物非生物抗逆性研究的模式系统。然而，目前用于实时定量聚合酶链反应（qRT-PCR）的胡杨内参基因（RGs）鉴定较少。验证合适的RGs对基因表达规范化研究至关重要。本研究筛选了6种非生物胁迫处理（脱落酸、冷、脱水、干旱、短时间盐和长时间盐）下胡杨叶片16种内源RGs，每种处理有6个处理梯度。在计算PCR效率后，采用NormFinder、geNorm和BestKeeper三种不同的软件工具对qRT-PCR数据进行系统分析，并采用非加权无监督秩聚合法对输出结果进行合并。在ABA处理下，RPL17（核糖体蛋白L17）、低温处理下的EF1α（延伸因子-1 α）、脱水处理下的HIS（组蛋白超家族蛋白H3）、干旱和SS处理下的GIIα和LS处理下的TUB（微管蛋白）被选为最佳基因表达分析。60核糖体蛋白（60S核糖体蛋白）的表达变化最小。为了说明这些RGs的适用性，研究了不同RGs对PePYL1、PeSCOF-1和PeSCL7三个应激诱导基因的相对定量。使用qBasePlus软件计算的结果显示，与最不合适的RGs相比，推荐RGs归一化的表达谱更接近预期。本研究为胡杨基因表达表征提供了重要的RG应用指导。
Populus euphratica is the only arboreal species that is established in the world's largest shifting-sand desert in China and is well-adapted to the extreme desert environment, so it is widely considered a model system for researching into abiotic stress resistance of woody plants. However, few P. euphratica reference genes (RGs) have been identified for quantitative real-time polymerase chain reaction (qRT-PCR) until now. Validation of suitable RGs is essential for gene expression normalization research. In this study, we screened 16 endogenous candidate RGs in P. euphratica leaves in six abiotic stress treatments, including abscisic acid (ABA), cold, dehydration, drought, short-duration salt (SS) and long-duration salt (LS) treatments, each with 6 treatment gradients. After calculation of PCR efficiencies, three different software tools, NormFinder, geNorm and BestKeeper, were employed to analyze the qRT-PCR data systematically, and the outputs were merged by means of a non-weighted unsupervised rank aggregation method. The genes selected as optimal for gene expression analysis of the six treatments were RPL17 (ribosomal protein L17) in ABA, EF1α (elongation factor-1 alpha) in cold, HIS (histone superfamily protein H3) in dehydration, GIIα in drought and SS, and TUB (tubulin) in LS. The expression of 60S (the 60S ribosomal protein) varied the least during all treatments. To illustrate the suitability of these RGs, the relative quantifications of three stress-inducible genes, PePYL1, PeSCOF-1 and PeSCL7 were investigated with different RGs. The results, calculated using qBasePlus software, showed that compared with the least-appropriate RGs, the expression profiles normalized by the recommended RGs were closer to expectations. Our study provided an important RG application guideline for P. euphratica gene expression characterization.