Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions.
Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions.
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DOI：
10.1186/1471-2199-10-1
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发表时间：
2009-01-06
影响因子：
--
通讯作者：
Maia IG
中科院分区：
文献类型：
--
作者：
Barsalobres-Cavallari CF;Severino FE;Maluf MP;Maia IG
来自基因表达实验的定量数据通常通过参考基因或管家基因的转录水平进行规范化。使用它们的一个固有假设是，这些基因在生物体的不同发育阶段、不同细胞类型和不同环境条件下的表达是高度一致的。迄今为止，植物内参基因的鉴定很少受到重视，包括阿拉比卡咖啡在内的许多作物物种都没有确定合适的内参基因。本文报道的研究目的是比较一组潜在的内参基因在不同类型的咖啡组织/器官样本中的相对表达。我们还验证了所选内参基因在不同发育阶段和特定生物胁迫下的表达谱。采用实时荧光定量RT-PCR技术，检测了5个常用内参基因，即乙醇脱氢酶（adh）、14-3-3、多聚素（poly）、β-肌动蛋白（actin）和甘油醛-3-磷酸脱氢酶（gapdh）在5个组织/器官样本（根、茎、叶、花和果实）上的表达水平。除了这些常用的内部控制外，还测试了另外三个编码半胱氨酸蛋白酶（cys）、咖啡因合成酶（ccs）和60S核糖体蛋白L7 （rpl7）的基因。通过geNorm、NormFinder和BestKeeper程序验证了它们作为内参基因的稳定性和适宜性。结果显示，除gapdh外，在所研究的实验条件下，所有内参基因的表达水平均有显著变化。我们的数据表明，管家基因的表达在咖啡中并不完全稳定。根据我们的研究结果，gapdh、14-3-3和rpl7被发现是均匀表达的，因此足以用于标准化目的，在不同的组织/器官样本中显示出相同的转录水平。因此，Gapdh是测定阿拉比卡咖啡基因表达的推荐参考基因。它的使用将使这种重要的樱桃作物的组织/器官特异性基因表达研究更加准确和可靠。
Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), β-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.
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影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
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Ganesh, D.;Petitot, A. -S.;Fernandez, D.
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作者：
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作者：
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作者：
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Bustin, SA