Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.
Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.
复制标题
DOI：
10.1371/journal.pone.0033278
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Jiang Z
中科院分区：
文献类型：
--
作者：
Chang E;Shi S;Liu J;Cheng T;Xue L;Yang X;Yang W;Lan Q;Jiang Z
侧柏是一种抗逆性强、适应性广、寿命长的树种，寿命可达数千年。为了探索这些特征背后的机制，通过 RNA-seq 结合数字基因表达 (DGE) 技术在转录组水平上研究了基因表达。因此，拥有一组可靠的内参基因，使用最准确、最灵敏的实时定量 PCR (qRT-PCR) 方法，在各种条件下标准化侧柏中的基因表达至关重要。在本研究中，我们从侧柏转录组数据中筛选出10个候选内参基因，并使用29个不同侧柏样品通过qRT-PCR检测其表达谱，这些样品采集自不同年龄、不同组织以及经过冷、热、盐度、聚乙二醇（PEG）和脱落酸（ABA）等不同处理的植物。使用三个分析软件包（geNorm、Bestkeeper 和 NormFinder）评估基因表达的稳定性。结果表明，泛素结合酶 E2 (UBC) 和 α-微管蛋白 (aTUB) 是所有发育阶段和所有胁迫条件下的最佳参考基因对。 ACT7 是不同组织和冷处理样品中最稳定的基因，而 UBQ 是 NaCl 和 ABA 处理样品中表达最稳定的参考基因。同时，单独或组合使用aTUB和UBC作为参考基因，以检测经过qRT-PCR各种处理的植物中NAC（AtNAC2的同源物）的表达水平。结果进一步证明了所选的两个内参基因的可靠性。我们的研究将有利于未来对侧柏和柏科其他成员应激/衰老反应的基因表达的研究。
Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.
登录
查看更多内容
影响因子：
5.3
作者：
Artico S;Nardeli SM;Brilhante O;Grossi-de-Sa MF;Alves-Ferreira M
通讯作者：
Alves-Ferreira M
影响因子：
2.7
作者：
Coker, JS;Davies, E
通讯作者：
Davies, E
影响因子：
6.5
作者：
Kanegae, H;Miyoshi, K;Takano, M
通讯作者：
Takano, M
影响因子：
10
作者：
Glare, EM;Divjak, M;Walters, EH
通讯作者：
Walters, EH
影响因子：
56.9
作者：
Kim, Jin Hee;Woo, Hye Ryun;Nam, Hong Gil
通讯作者：
Nam, Hong Gil