Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum

Cortleven, Anne; Remans, Tony; Brenner, Wolfram G.; Valcke, Roland

doi:10.1007/s11120-009-9470-y

Cited by 24 publications

(22 citation statements)

References 42 publications

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“…2; Table 2) were ranked poorly based on the two softwares in this study, indicating that these genes should be avoided as internal controls when analyzing gene expression levels in A. mongolicus under abiotic stresses, whereas tubulin (Cortleven et al 2009;de Almeida et al 2010), ATPbinding cassettes transporter gene (Libauh et al 2008) and elongation factor gene (Nicot et al 2005;Tong et al 2009), have been reported to be invariant genes in other studies. Fig.…”

Section: Discussionmentioning

confidence: 77%

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

et al. 2012

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Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. No report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus. This work identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abc1, Ubc1, Ubc2, Ubc4, Ubc5, eIF1, eIF2, eIF3, eIF4, EF1, EF2), and used the most suitable combination of reference genes to normalize the expression profiles of seven ROS-scavenging enzyme genes (AmSOD, AmAPX, AmGPX, AmCAT, AmGLR, AmPrx, and AmTrx). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of eIF1 and eIF3 was best for accurate normalization across all the treatments, confirmed by normalizing qPCR data with AmHsp90. In contrast, these data show that Tub1, Abc1, and EF1 are not suitable reference gene candidates. After being normalized against eIF1 and eIF3, the seven ROS-scavenging enzyme genes exhibited differentially up- or down-regulated expression patterns. AmSOD and AmGPX were up-regulated by all four treatments, indicating that they may participate in an anti-oxidative mechanism under abiotic stresses in A. mongolicus. AmCAT exhibited a much higher expression level than AmAPX, AmPrx, and AmGPX, suggesting a principle role in detoxifying excessive H₂O₂. AmSOD, AmGPX and AmAPX showing the most abundant transcripts under heat, AmCAT and AmGLR under drought, and AmPrx under salt, were observed. Expression patterns of the seven ROS-scavenging enzyme genes suggest different antioxidant protection roles of these genes under abiotic stresses. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.

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Section: Discussionmentioning

confidence: 77%

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

et al. 2012

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“…This is unsurprising since both genes demonstrate significant variation in many different organisms and experimental contexts (Dheda et al 2004;Nicot et al 2005;Jain et al 2006;Reid et al 2006). In contrast, Cortleven et al (2009) identified atubulin as one of the most stable reference genes in tobacco. However, this conclusion was based on a combined analysis of wildtype and transgenic plants, and may be specific to the lines tested.…”

Section: Discussionmentioning

confidence: 88%

“…These characteristics have encouraged the widespread use of N. tabacum in studies of plant stress responses (Kanneganti and Gupta 2008), gene expression (Delaney et al 2007), protein transport and localization (Delaney et al 2007), industrial protein production (Bock 2007), organ development (Volkov et al 2005;Ribarits et al 2007) and plastid evolution (Sheppard et al 2008). RT-PCR is commonly used to analyze gene expression in tobacco, and eight nuclear-encoded 'housekeeping' genes have been used as internal references in RT-PCR and Northern analysis of tobacco gene expression (Dean et al 2002;Volkov et al 2005;Kasukabe et al 2006;Ribarits et al 2007;Cortleven et al 2009). However, the relative stability of these and other potential reference genes in tobacco has not been studied in a range of experimental contexts, and this has constrained the use of real-time PCR in the tobacco system.…”

Section: Introductionmentioning

confidence: 99%

Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress

2010

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Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes. Primers targeting each gene (18S rRNA, EF-1alpha, Ntubc2, alpha- and beta-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses. L25 and EF-1alpha demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1alpha was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach. These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco.

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“…Experiments have shown the varied transcript level of the housekeeping genes under different environmental conditions (Jian et al, 2008;Maroufi et al, 2010). Several studies showed differential stabilities of different internal control genes which can be used for transcript level studies under any experimental condition (Exposito-Rodriguez et al, 2008;Cortleven et al, 2009;Artico et al, 2010). For more clarity, two or more reference genes can also be used for perfect interpretation of quantitative PCR data.…”

Section: Discussionmentioning

confidence: 99%

Identifications and Validations of Reference Genes for Gene Expression Data Normalization of Chenopodium album

Aman¹

2017

IJAB

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Quantitative real-time PCR (qRT-PCR) is considered as an important technique for gene expression and validation analysis for several high-throughput proteomics and genomics data. However, reliable qRT-PCR results are dependent on appropriate reference genes used for data normalization under specific experimental condition and such information is limited in case of plants whose genome is not sequenced yet. Therefore, we sequenced and evaluated stability of seven novel internal control genes (Act, Act α, β-tub, 18SrRNA, GAPDH, EF1α and Ubq) of Chenopodium album, an important edible plant with medicinal values and have been recently used in synthesis as well as analysis of several gold and silver nanoparticles. Statistical algorithms like geNorm and NormFinder were used and compared for the samples treated with different types of stresses including, high/low temperatures, drought, heavy metal and salt stress. β-tubulin was revealed as the most stable genes under heat, drought and metal stress. While, 18SrRNA has shown the stable expression only under cold and salt stresses. Our data support the use of the combinations of two stable internal control genes for normalization of the gene expression. We compared the relative expression of a stress induced Chloroplast small Heat Shock Protein (Cp-sHSP) of C. album to further support our results. Together with all novel internal controls genes, this study also provides a platform for future gene expression studies of C.album using validated housekeeping genes.

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Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum

Cited by 24 publications

References 42 publications

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress

Identifications and Validations of Reference Genes for Gene Expression Data Normalization of Chenopodium album

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