Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Abstract

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.

Keywords: ACT14; Actin 14 ANOVA, analysis of variance; Alpha-tubulin 10; CYP1; Cotton; Cydophilin 1; EF1A8; Eukaryotic translation initiation factor 5; GAPDH; Gene expression; GhHis3; GheIF5; Glyceraldehyde-3-phosphate dehydrogenase C subunit; Gossypium hirsutum; Histone H3; IQR; MS; Murashige and Skoog medium; PEG; PP2A; Polyethylene glycol; Protein phosphatase 2A catalytic subunit; Quantitative real time PCR; RT-PCR; Real time PCR; Reference gene; Reverse transcription-polymerase chain reaction; TUA10; The interquartile range; Translation elongation factor 1A-8; UBI1; UBQ7; Ubiquitin extension protein 7; Ubiquitin gene family; qRT-PCR.