Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains
Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains
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DOI：
10.1007/s00299-013-1499-x
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发表时间：
2013-12-01
影响因子：
6.2
通讯作者：
Rombaldi, Cesar Valmor
中科院分区：
文献类型：
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作者：
Galli, Vanessa;Messias, Rafael da Silva;Rombaldi, Cesar Valmor
在玉米地方品种和多个谷物发育阶段中评估候选参考基因的稳定性，以通过RT-qPCR评估类胡萝卜素相关基因的表达，以应用于玉米生物强化。维生素A缺乏影响全球数百万儿童;因此，增加玉米谷物中维生素A前体的含量是令人感兴趣的。玉米籽粒中类胡萝卜素生物合成途径相关基因表达的研究为代谢工程方法提供了有用的信息。然而，使用实时定量聚合酶链反应（RT-qPCR）实验的可靠结果取决于使用适当的参考基因。在这项研究中，我们利用geNorm和Normalware软件，以确定最稳定表达的候选参考基因的样品从7个阶段的粮食发展和8个地方品种。geNorm分析结果表明，微管蛋白（TUB）和肌动蛋白（ACT）是所有实验条件中最合适的参考基因，而甘油醛-3-磷酸脱氢酶基因（GAPDH）的稳定性最差。用Normandy软件得到相同的结果。还通过geNorm确定了在每个实验条件下归一化基因表达数据所需的最小基因数量。当使用最不稳定的候选基因（GAPDH）而不是最稳定的基因对（ACT + TUB）作为内部对照时，八氢番茄红素合酶基因（PSY 1）（类胡萝卜素生物合成途径中的第一种酶）的表达被高估，因此突出了在进行RT-qPCR实验以获得准确结果之前验证参考基因的重要性。这项研究是第一次调查的稳定性基因作为参考基因，以标准化RT-qPCR数据从玉米地方品种在多个阶段的粮食发展。
The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.