Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators

Abstract

It has been shown that genes considered to be valid reference genes using semi-quantitative techniques (e.g. northern blot) appear to be less reliable when highly sensitive real-time PCR (qPCR) or microarrays are used. Therefore, the validation of expression stability of reference genes has become an important component of any study using such types of assay. No reference genes have been validated for expression studies of cucumber genes to date. Since the genome of this widely cultivated crop has been recently sequenced, the availability of suitable reference genes for expression analyses of the new cucumber genes is urgently required. For the purpose of normalization in studying expression of cucumber target genes, the stability of twelve reference genes in different cucumber tissues and under various stresses and growth regulators were determined in this study. These included commonly used cucumber reference genes, such as actin, EF, cyclophilin, ubiquitin and tubulin and the newly identified candidates for reference genes that encode clathrin adaptor complex subunit (CACS), F-box protein, PPA2 activator (tonoplast intrinsic protein, TIP41), mitosis protein (YSL8), protein phosphatase 2 (PDF2), helicase (HEL) and protein homolog of At4g33380. Analyses of quantitative real-time PCR data by three commonly used Excel-based applets, BestKeeper, geNorm and NormFinder, confirmed that expression stability of reference genes depends on the experimental parameters. In addition, they revealed that, except for EF, the most stable cucumber genes included mainly the new reference genes: CACS, F-box and TIP41, whereas the commonly used internal controls demonstrated various (actin, cyclophilin, ubiquitin) or much lower stability (tubulin). Hence, the authors of this study assume that the novel cucumber reference genes will enable better normalization and quantification of transcript levels in future expression studies on cucumber plants.