Dataset Information

Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.

ABSTRACT: Carrot, a biennial herb of the Apiaceae family, is among the most important vegetable crops in the world. In this study, nine candidate reference genes (GAPDH, ACTIN, eIF-4α, PP2A, SAND, TIP41, UBQ, EF-1α, and TUB) were cloned from carrot. Carrot plants were subjected to abiotic stresses (heat, cold, salt, and drought) and hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid). The expression profiles of the candidate reference genes were evaluated in three technical and biological replicates. Real-time qPCR data analyses were performed using three commonly used Excel-based applets namely, BestKeeper, geNorm, and NormFinder. ACTIN and TUB were the most stable genes identified among all sample groups, but individual analysis revealed changes in their expression profiles. GAPDH displayed the maximum stability for most of single stresses. To further validate the suitability of the reference genes identified in this study, the expression profile of DcDREB-A1 gene (homolog of AtDREB-A1 gene of Arabidophsis) was studied in carrot. The appropriate reference genes were selected that showed stable expression under the different experimental conditions.

SUBMITTER: Tian C

PROVIDER: S-EPMC4319972 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.

Tian Chang C Jiang Qian Q Wang Feng F Wang Guang-Long GL Xu Zhi-Sheng ZS Xiong Ai-Sheng AS

PloS one 20150206 2

Carrot, a biennial herb of the Apiaceae family, is among the most important vegetable crops in the world. In this study, nine candidate reference genes (GAPDH, ACTIN, eIF-4α, PP2A, SAND, TIP41, UBQ, EF-1α, and TUB) were cloned from carrot. Carrot plants were subjected to abiotic stresses (heat, cold, salt, and drought) and hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid). The expression profiles of the candidate reference genes were evaluated in three technical ...[more]

PMID: 25658122

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Project description:Background:Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress. Results:Here we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including ACT (Actin), ADP (ADP-ribosylation factor 1), CYP2 (Cyclophilin 2), EF-1α (Elongation factor 1-alpha), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), HSP90 (Heat shock protein 90), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), UBI (Ubiquitin), 18SrRNA-1 (guanine1575-N7-methyltransferase) and 18SrRNA-3 (adenine1779-N6-dimethyltransferase) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that ADP was the most suitable reference gene in salt stress while UBI showed peak stability under mannitol and ABA stress; EF-1α was the most appropriate reference gene for PEG, GA3, ethylene and heat stress; 18SrRNA-3 was the best choice for cold stress; and TUBα was the first stable gene across different tissues. Conclusions:Our main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research.

Project description:Background:Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized. Results:geNorm, NormFinder, BestKeeper, and ?Ct method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene-UNK), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession. Conclusions:Reference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.

Dataset Information

Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.

Publications

Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.

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