Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Plant Biol (Stuttg). 2022 Mar;24(2):367-379. doi: 10.1111/plb.13382. Epub 2022 Jan 6.
Increases in environmental temperature are directly linked to the issue of climate change and are known to significantly disrupt plant growth and development. Studies of gene expression in plants commonly include RT-qPCR but the reliability of the method depends on the use of suitable reference genes for data normalization. Despite this, no reference genes have been validated specifically for experiments in Arabidopsis thaliana employing treatments with elevated temperature. Here, ten genes were selected for expression stability analysis based on the screening of available literature and microarray data from temperature-treated A. thaliana. Expression levels of candidate reference genes were measured in 12-day-old seedlings, rosette leaves and flower buds of 5-week-old A. thaliana plants exposed to five different temperatures (22°C, 27°C, 32°C, 37°C and 42°C) and their expression stabilities were assessed using four statistical algorithms (BestKeeper, geNorm, NormFinder and comparative ΔC method). This study provides reliable reference genes for use in A. thaliana RT-qPCR expression analyses employing elevated temperature treatments, namely OGIO and PUX7 in seedlings, UBC21 and PUX7 in leaves, TIP41 and UBC21 in buds, and TIP41 and UBC21 in all three tissues combined. Orthologues of these genes can be of potential use in less studied plants, especially agricultural species heavily affected by climate change.
环境温度的升高与气候变化直接相关，已知其会显著干扰植物的生长和发育。植物基因表达的研究通常包括 RT-qPCR，但该方法的可靠性取决于用于数据归一化的合适参考基因的使用。尽管如此，在使用高温处理的拟南芥实验中，还没有专门针对该实验验证参考基因。在这里，根据可用的文献和温度处理的拟南芥微阵列数据筛选，选择了十个基因进行表达稳定性分析。在暴露于五种不同温度（22°C、27°C、32°C、37°C和 42°C）的 12 天大的幼苗、5 周大的拟南芥植株的莲座叶和花蕾中测量候选参考基因的表达水平，并使用四种统计算法（BestKeeper、geNorm、NormFinder 和比较 ΔC 方法）评估其表达稳定性。本研究为使用高温处理的拟南芥 RT-qPCR 表达分析提供了可靠的参考基因，即幼苗中的 OGIO 和 PUX7、叶片中的 UBC21 和 PUX7、花蕾中的 TIP41 和 UBC21 以及所有三种组织中的 TIP41 和 UBC21。这些基因的同源基因可能对研究较少的植物，特别是受气候变化严重影响的农业物种具有潜在用途。
