Gao Mengmeng, Liu Yaping, Ma Xiao, Shuai Qin, Gai Junyi, Li Yan
National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
PLoS One. 2017 Jan 3;12(1):e0168965. doi: 10.1371/journal.pone.0168965. eCollection 2017.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.
定量逆转录聚合酶链反应（qRT-PCR）被广泛用于分析相对基因表达水平，然而，qRT-PCR的准确性受到内参基因稳定性的极大影响，而内参基因的稳定性取决于组织和环境。因此，在特定组织和环境中选择最稳定的内参基因对于解读基因表达模式至关重要。铝（Al）、镉（Cd）和热胁迫是限制中国南方大豆（Glycine max）产量的三个重要非生物因素。为了鉴定在大豆对Al、Cd和热胁迫响应中用于通过qRT-PCR标准化靶基因表达水平的合适内参基因，我们使用BestKeeper、Delta Ct、geNorm、NormFinder和RefFinder这五种方法研究了十个常用管家基因在这三种非生物胁迫下大豆根和叶中的表达稳定性。我们发现，在Al胁迫下，TUA4是大豆根尖中最稳定的内参基因。在Cd胁迫下，Fbox和UKN2分别是根和叶中最稳定的内参基因，而在同时分析根和叶时，60S是最合适的内参基因。对于热胁迫，TUA4和UKN2分别是根和叶中最稳定的管家基因，并且UKN是同时分析根和叶的最佳内参基因。为了验证这些内参基因，我们分别定量了六个参与大豆对Al、Cd或热胁迫响应的靶基因的相对表达水平。使用最稳定和最不稳定内参基因时，这些靶基因的表达模式有所不同 , 这表明选择合适的内参基因对于基因表达研究至关重要。
