Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition

Abstract

In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.

Molecular and biochemical properties of two P1B2‐ATPases, CsHMA3 and CsHMA4, from cucumber

“…Amplification reactions were run in a 96 well plate on an Lightcycler 480 (Roche) in a reaction containing 2× SYBR Green Mix B (A&A Biotechnology, Gdynia, Poland), as previously described (Warzybok & Migocka ) in the following conditions: 95 °C for 30 s, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and extension at 72 °C for 15 s. Lightcycler ProbeDesign Software 2.0 (Roche) was used to design primers that are highly specific to CsHMA3 and CsHMA4 sequences (Supporting Information http://onlinelibrary.wiley.com/doi/10.1111/pce.12447/suppinfo). The specificity of PCR products was verified by melting curve analysis followed by sequencing as previously described (Warzybok & Migocka ). Amplifications were normalized to clathrin adaptor complex subunit ( CACS ), and quantitation of gene expression was performed using the ΔΔ C T calculation, where C T is the threshold cycle.…”

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

“…A large number of research papers have been published on reference genes under different stresses or from different organs in plants (Warzybok and Migocka 2013; Lin et al 2014), and similar works in filamentous fungi are gradually being conducted (Zampieri et al 2014; Zhou et al 2011). It is striking that most of the applied reference genes for fungi were directly copied from the existing results in plants or animals, such as actin , tubulin and 18S rRNA (Fang and Bidochka 2006; Yan and Liou 2006).…”

Selection and evaluation of reference genes for expression analysis of Cassi

“…Based on the reference genes in other plants [9][10][11][12][13][14][15] and the result of pre-screening (the pre-screening was introduced in Discussion), we chose 10 candidate reference genes including ACT1, ACT2, CYP1, CYP2, EF1α2, UBQ1, UBQ2, α-TUB1, α-TUB2, and β-TUB2. Details of these genes were shown in Table 1.…”

“…6) Commonly, reference genes have been chosen from housekeeping genes. 7,8) For example, Actin (ACT), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Ubiquitin (UBQ), Tubulin (TUB), 18S rRNA (18S), translation elongation factor 1-α (EF1α), and cyclophilin (CYP1 and CYP2) are widely used in gene expression studies in Arabidopsis, Nicotiana tobacum, Oryza sativa, Cucumber Grown, Vitis amurensis, Triticum aestivum, and Glycine max, [9][10][11][12][13][14][15] etc. However, these genes have been shown to have variable expression levels across various plant species under different conditions.…”

“…However, these genes have been shown to have variable expression levels across various plant species under different conditions. 16) Therefore, many studies have focused on reference gene selection among the several candidate reference genes in specific plant species under different conditions such as Arabidopsis 9) , N. tobacum 10) , O. sativa 11) , C. Grown 12) , etc. Because universal reference genes expressed at a constant level are not available, the selection of appropriate reference genes in specific plant species under different conditions is fundamental for the normalization of target gene expression levels.…”