Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress
Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress
复制标题
DOI：
10.1016/j.ab.2009.01.024
复制
发表时间：
2009-04-15
影响因子：
2.9
通讯作者：
Lillo, Cathrine
中科院分区：
文献类型：
--
作者：
Lovdal, Trond;Lillo, Cathrine
我们检测了8个假定持续表达的基因--肌动蛋白(ACT)、β-微管蛋白、延伸因子1α(EF1)、甘油醛-3-磷酸脱氢酶(GAPDH)、磷酸甘油酸激酶(PGK)、核糖体蛋白L2(RPL2)、泛素(UBI)和蛋白磷酸酶2A催化亚基(PP2Acs)，以期作为番茄叶片基因表达正常化的参考。对候选参考基因在氮素饥饿、低温和次适光照条件下的表达稳定性进行了测试。为此目的，采用了geNorm算法，该算法在一大组候选参考文献之间进行相互交叉验证。被广泛使用的参考基因GAPDH和PGK在轻度胁迫中排名最高，在N和低温胁迫中排名较低。相反，EF1在N和冷胁迫中排名最高，但在轻度胁迫中排名较低。当将数据集作为一个整体来看时，新引用的RPL2和PP2Acs以及传统的引用ACT和UBI似乎可以稳定地表达。在所有实验条件下，没有一个基因表现出如此恒定的表达水平，以至于表现出优于其他候选基因的表现。因此，这些结果强调了使用多个精心选择的参考基因的几何平均值来使番茄的基因表达正常化的必要性。(C)2009 Elsevier Inc.保留所有权利。
We examined eight putative consistently expressed genes-actin (ACT), beta-tubulin, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ribosomal protein L2 (RPL2), ubiquitin (UBI), and a catalytic subunit of protein phosphatase 2A (PP2Acs)-for their potential as references for the normalization of gene expression in tomato leaves. Expression stability of candidate reference genes was tested during growth conditions of nitrogen (N) starvation, low temperature, and suboptimal light. The geNorm algorithm, using reciprocal cross-validation among a larger group of candidate references, was applied for this purpose. The widely used reference genes GAPDH and PGK were top ranked during light stress but poorly ranked during N and cold stress. In contrast, EF1 was top ranked during N and cold stress but poorly ranked during light stress. The novel references RPL2 and PP2Acs, as well as the traditional references ACT and UBI, appeared to be stably expressed when looking at the data set as a whole. No gene was identified that exhibited such a constant level of expression as to outperform the other candidates under all experimental conditions. Thus, the results highlight the need for normalizing gene expression in tomato using the geometric average of multiple carefully selected reference genes. (C) 2009 Elsevier Inc. All rights reserved.