Dataset Information

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

ABSTRACT: Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4?A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24?h of cold and heat stresses (10 and 45?°C, respectively), and drought, including moderate (-0.5 to -0.7?MPa), severe (-1.1 to -1.8?MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4?A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.

SUBMITTER: Takamori LM

PROVIDER: S-EPMC5561021 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

Takamori Luciana Midori LM Pereira Alyne Valéria Carrion AVC Maia Souza Gustavo G Vieira Luiz Gonzaga Esteves LGE Ferreira Ribas Alessandra A

Scientific reports 20170817 1

Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 ...[more]

PMID: 28819216

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Project description:BackgroundNitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT-qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT-qPCR analysis.ResultsIn this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions.ConclusionThis study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Dataset Information

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

Publications

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

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