Abstract
Normalisation of data, by choosing the appropriate reference genes, is fundamental for obtaining reliable results in quantitative real-time PCR (qPCR). This study evaluated the expression stability of 11 candidate reference genes with different varieties, developmental periods, tissues, and abiotic stresses by using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that ubiquitin-conjugating enzyme S (UBC) and ubiquitin-conjugating enzyme E2 (UBC E2) could be used as reference genes for different E. ulmoides varieties and tissues, UBC and histone H4 (HIS4) for different developmental periods, beta-tubulin (TUB) and UBC for cold treatment, ubiquitin extension protein (UBA80) and HIS4 for drought treatment, and ubiquitin-60S ribosomal protein L40 (UBA52) and UBC E2 for salinity treatment. UBC and UBC E2 for the group “Natural growth” and “Total”, UBA80 and UBC for the group “Abiotic stresses”. To validate the suitability of the selected reference genes in this study, mevalonate kinase (MK), phenylalanine ammonia-lyase (PAL), and 4-coumarate-CoA ligase (4CL) gene expression patterns were analysed. When the most unstable reference genes were used for normalisation, the expression patterns had significant biases compared with the optimum reference gene combinations. These results will be beneficial for more accurate quantification of gene expression levels in E. ulmoides.
Highlights
Gene expression analysis is an important part of molecular biology research
The results indicated that ubiquitin-conjugating enzyme S (UBC) and ubiquitin-conjugating enzyme E2 (UBC E2) were the most stable genes for different varieties and tissues, that UBC and HIS4 were the most stable genes for different development stages, that TUB and UBC were the most stable genes for cold treatment, that UBA80 and HIS4 were the most stable genes for drought treatment, and that UBA52 and UBC E2 were the most stable genes for salinity treatment
Gene expression pattern analysis in different experimental conditions is necessary for the functional analysis of genes[32]
Summary
Gene expression analysis is an important part of molecular biology research. Quantitative real time polymerase chain reaction (qRT-PCR) has become a popular technology for studying gene expression patterns[1,2]. As for the relative quantification, the qPCR data of target genes requires reference genes for calibration. To date there have been no systematic analyses of reference gene screening in E. ulmoides for different varieties, developmental periods, tissues, and abiotic stresses. Studies on the expression patterns of trans-polyisoprene rubber biosynthesis and CGA biosynthesis genes play very important roles in E. ulmoides research. Mevalonate kinase (MK) is a key enzyme-coding gene related to trans-polyisoprene biosynthesis[26]; phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL) are the key genes for the biosynthesis of CGA27. Their expression levels may be directly related to the contents of Eu-rubber and CGA
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