Abstract
White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.
Highlights
Quantitative real-time reverse transcription PCR using cDNA as a template is a sensitive and powerful technique for measuring gene expression level [1]
Can be used to analyze the regulation of gene expression, monitor the expression pattern of mRNA, and quantitatively analyze the transcription level of genes, and to conduct spatial–temporal analysis of target genes in different tissues under various treatments; as such, Quantitative real-time reverse transcription PCR (qRT-PCR) has been widely applied in the field of molecular biology [2,3,4]
The quality of gene expression is affected by many experimental factors [5], and the relative quantitative method selected by researchers first needs to correct the expression amount of the target gene in the experimental process [6]
Summary
Quantitative real-time reverse transcription PCR (qRT-PCR) using cDNA as a template is a sensitive and powerful technique for measuring gene expression level [1]. Quantitative real-time PCR can be used to analyze the regulation of gene expression, monitor the expression pattern of mRNA, and quantitatively analyze the transcription level of genes, and to conduct spatial–temporal analysis of target genes in different tissues under various treatments; as such, qRT-PCR has been widely applied in the field of molecular biology [2,3,4]. White clover (Trifolium repens L.) is an important cool-season perennial legume forage which is widely cultivated in temperate grassland systems [11]. It has a high feed value and strong N fixation capacity in soil [12]. White clover is susceptible to drought, salt, and heat stress [13]
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