Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age
Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age
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DOI：
10.1007/s11738-012-0967-1
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发表时间：
2012-09-01
影响因子：
2.6
通讯作者：
Skorupa, Katarzyna
中科院分区：
文献类型：
--
作者：
Rapacz, Marcin;Stepien, Agata;Skorupa, Katarzyna
干旱是农业中最重要的非生物胁迫;因此，这一研究领域似乎是植物生物学中最重要的挑战之一。干旱条件下基因表达的数据是研究干旱响应机制和选择转基因方法的关键。定量RT-PCR是基因表达分析的一种有效方法，然而，获得正确的数据标准化需要具有稳定表达水平的内参基因。本论文对10个潜在的参考基因在大麦两个发育阶段中的表达稳定性进行了研究。结果表明，叶片生长本身对所研究基因表达的影响程度与干旱相似，并表明不同基因在苗期和抽穗期表达最稳定。因此，针对不同的应用选择了不同的参考基因组。例如，ADP-核糖基化因子1和泛素编码基因是最适合研究干旱诱导的基因表达在苗期的变化，而肌动蛋白和GAPDH基因是有用的抽穗期，和ADP-核糖基化因子1和HSP 90允许这两个阶段之间的比较。我们的数据证明了验证常用参考基因的必要性。结果表明，ADP的表达似乎是受本实验中研究的所有因素的影响最小。然而，当仅研究本工作中研究的因素中的一个因素的影响时，由于不同基因表达的稳定性较高，因此应考虑将其用作参考。
Drought is the most significant abiotic stress in agriculture; thus, this area of studies seems to be one of the most important challenges in plant biology. Data about gene expression under drought are crucial to study drought response mechanisms and to select the genes for a transgenic approach. Quantitative RT-PCR is a powerful method for gene expression analysis; however, obtaining proper data normalization requires internal reference genes with stable level of expression. In the present paper ten potential reference genes were examined in two developmental stages of barley for their expression stability during leaf growth and increasing drought level. The results indicated that leaf growth per se affects the expression of studied genes to the similar extent as the drought and showed that different genes were most stably expressed in the seedling and the heading stage. As a result, different sets of reference genes were selected for different applications. For instance, ADP-ribozylation factor 1 and ubiquitin encoding genes were most suitable to study drought-induced changes in gene expression at the seedling stage, whereas actin and GAPDH genes were useful during heading, and ADP-ribozylation factor 1 and HSP90 allowed for the comparison between these two stages. Our data proved the necessity for validation of commonly used reference genes. The results indicate that expression of ADP seems to be the least affected by all the factors studied in the present experiment. However, when the effect of only one factor among those investigated in this work will be studied, different genes should be considered to be used as the references due to the higher stability of their expression.