Laboratory of Cell Biology, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, China.
PLoS One. 2013;8(1):e53196. doi: 10.1371/journal.pone.0053196. Epub 2013 Jan 2.
Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.
实时荧光定量聚合酶链反应（qPCR）是一种敏感的基因表达分析技术，其依赖于用于数据归一化的参考基因的稳定性。中间锦鸡儿是一种具有强抗旱性、固沙能力和高饲料价值的本地沙漠灌木，广泛分布在中国西部和西北部的沙漠地区，但尚未对适合 qPCR 数据归一化的参考基因进行鉴定。在这项研究中，分析了中间锦鸡儿在不同非生物（渗透、盐、冷和热）胁迫下的 10 个候选参考基因，在两个不同的植物器官（根和叶）中。使用 geNorm、NormFinder 和 BestKeeper 算法评估这些基因的表达稳定性。最佳排名的参考基因因不同的样本集而异，但 UNK2、PP2A 和 SAND 在所有测试样本中最稳定。UNK2 和 SAND 适合归一化盐处理根的基因表达数据，而 UNK2、SAND 和 EF-1α 的组合适合盐处理叶的基因表达数据。UNK1、UNK2 和 PP2A 适合 PEG（渗透）处理的根，而 TIP41 和 PP2A 的组合最适合 PEG 处理的叶。SAND、PP2A 和 TIP41 在热处理叶中表现出最稳定的表达。在冷处理叶中，SAND 和 EF-1α 表达最稳定。为了进一步验证本研究中鉴定的参考基因的适用性，还同时研究了 DREB1 和 DREB2（AtDREB1 和 AtDREB2 的同源物）的表达水平。本研究是首次对中间锦鸡儿在不同非生物胁迫条件下进行 qPCR 的优秀参考基因进行系统分析，将有助于未来对中间锦鸡儿和豆科锦鸡儿属其他物种基因表达的研究。
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