Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Abstract

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18S rRNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18S rRNA was found to be the most optimum reference gene under drought stress by the analyzing of geNorm and NormFinder software. Quantitative expression levels of P5CS using 18S rRNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5CS gene and proline content had a significantly positive relationship (R2=0.7763, P<0.05). This study established and validated 18S rRNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Keywords

• reference genes
• quantitative real-time PCR
• drought stress
• proline
• pyrroline-5-carboxylic acid synthetase
• Agropyron mongolicum