Abstract
Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs—geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. The results showed that α-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that α-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions.
Highlights
Drought, salt, and heavy metal stresses are major abiotic factors that contribute to the risk of environment and affect forestry productivity worldwide[1,2,3,4,5]; plants need to thrive in adverse circumstances[6]
Actin (ACT) and β-tubulin (β-TUB) have been used as reference genes for Quantitative real-time polymerase chain reaction (qRT-PCR) normalization in gene expression analysis in S. matsudana under salt and copper stresses[37,49]; a systematic study to validate reference genes has not been reported for S. matsudana under abiotic stresses
Our findings indicated that α-TUB2 and DnaJ either singly or in combination are suitable for normalization of gene expression in S. matsudana under different abiotic stresses
Summary
Salt, and heavy metal stresses are major abiotic factors that contribute to the risk of environment and affect forestry productivity worldwide[1,2,3,4,5]; plants need to thrive in adverse circumstances[6]. Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for gene expression analysis due to its high sensitivity, accuracy, specificity, and reproducibility[40,41,42]. Factors such as sample amount, RNA integrity, reverse transcription efficiency, and cDNA quality can significantly influence the reliability of the gene expression results[43,44,45]. To obtain accurate expression data, it is necessary to select suitable reference genes for each plant species and to verify their stability under the specific experimental conditions of interest. The results will provide suitable reference genes for qRT-PCR normalization for accurate gene expression analysis in S. matsudana under different stress conditions
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