The optimal reference gene validation in Cyclocarya paliurus (Batal.) Iljinskaja under environmental stresses

Cyclocarya paliurus (Batal.) Iljinskaja is a special plant species grown at high elevation in southwestern China. The bark, leaves, and roots are used for many purposes including reduction of fever and enhancement of blood insulin level. However, its growth is usually limited by the fragile environment and climate variability. Previous studies attributed the environment-relevant yield reductions to stress induced gene expression changes. The accuracy and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR), a commonly accepted gene expression method, depends on the choice of appropriate reference genes, which are used for gene expression normalization. However, there are no reports at present about reference genes of C. paliurus under environmental stresses. Therefore, this study was conducted to identify reference genes for C. paliurus under environmental stresses. In this work, we selected seven common reference genes as our candidates: ACT2, ACTF, GAPDH, TUA, TUB, UBQ4, and 18SrRNA. The stability of these seven reference genes in C. paliurus was quantified by RT-qPCR and evaluated by geNorm and NormFinder algorithms. The results suggest that although there were variations of the stability of the internal reference genes, two of them were usually good enough for the normalization purpose for low temperature (4 °C), high salinity (NaCl), and drought stress (polyethylene glycol [PEG] treatment). However, these genes should not be used for high-temperature (42 °C) stress. This study provides insight for reference gene selection under experimental stresses in C. paliurus. It also sets the foundation of wide and accurate use of RT-qPCR under environmental stresses.