Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China.
Plant Cell Rep. 2012 Jul;31(7):1245-54. doi: 10.1007/s00299-012-1245-9. Epub 2012 Mar 27.
Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. No report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus. This work identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abc1, Ubc1, Ubc2, Ubc4, Ubc5, eIF1, eIF2, eIF3, eIF4, EF1, EF2), and used the most suitable combination of reference genes to normalize the expression profiles of seven ROS-scavenging enzyme genes (AmSOD, AmAPX, AmGPX, AmCAT, AmGLR, AmPrx, and AmTrx). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of eIF1 and eIF3 was best for accurate normalization across all the treatments, confirmed by normalizing qPCR data with AmHsp90. In contrast, these data show that Tub1, Abc1, and EF1 are not suitable reference gene candidates. After being normalized against eIF1 and eIF3, the seven ROS-scavenging enzyme genes exhibited differentially up- or down-regulated expression patterns. AmSOD and AmGPX were up-regulated by all four treatments, indicating that they may participate in an anti-oxidative mechanism under abiotic stresses in A. mongolicus. AmCAT exhibited a much higher expression level than AmAPX, AmPrx, and AmGPX, suggesting a principle role in detoxifying excessive H₂O₂. AmSOD, AmGPX and AmAPX showing the most abundant transcripts under heat, AmCAT and AmGLR under drought, and AmPrx under salt, were observed. Expression patterns of the seven ROS-scavenging enzyme genes suggest different antioxidant protection roles of these genes under abiotic stresses. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.
沙冬青是中国西北荒漠地区特有的唯一常绿阔叶灌木，是植物非生物胁迫研究的有价值物种。迄今为止，尚无关于在沙冬青中进行 qPCR 严格归一化时选择参考基因的报道。本研究从 14 个候选参考基因（UBQ、Tub1、Tub2、Abc1、Ubc1、Ubc2、Ubc4、Ubc5、eIF1、eIF2、eIF3、eIF4、EF1 和 EF2）中鉴定了用于在非生物胁迫下归一化 qPCR 数据的可靠参考基因，并使用最合适的参考基因组合来归一化 7 种 ROS 清除酶基因（AmSOD、AmAPX、AmGPX、AmCAT、AmGLR、AmPrx 和 AmTrx）的表达谱。我们设置了一系列 22 个实验样本，涵盖了对照和不同时间点下的冷、干、盐和热胁迫。根据 geNorm 和 NormFinder，eIF1 和 eIF3 的组合最适合在所有处理中进行准确归一化，这通过用 AmHsp90 归一化 qPCR 数据得到了验证。相比之下，这些数据表明 Tub1、Abc1 和 EF1 不是合适的参考基因候选物。用 eIF1 和 eIF3 归一化后，7 种 ROS 清除酶基因表现出不同的上调或下调表达模式。AmSOD 和 AmGPX 被四种处理均上调，表明它们可能参与了沙冬青非生物胁迫下的抗氧化机制。AmCAT 的表达水平远高于 AmAPX、AmPrx 和 AmGPX，表明其在解毒过量 H₂O₂方面发挥着主要作用。在热胁迫下，AmSOD、AmGPX 和 AmAPX 的转录本最丰富，在干旱胁迫下 AmCAT 和 AmGLR 的转录本最丰富，在盐胁迫下 AmPrx 的转录本最丰富。7 种 ROS 清除酶基因的表达模式表明，这些基因在非生物胁迫下具有不同的抗氧化保护作用。这些结果对未来沙冬青基因表达和非生物胁迫耐受性的研究具有重要价值。
