Internal reference genes for normalizing quantitative real-time PCR in different tissues of Gelsemium elegans or under low temperature, MeJA, and SA stresses

Abstract

Gelsemium elegans is a traditional Chinese medicinal plant, with indole alkaloids as its main active ingredient. The plant can be used as medicine, mainly for pain relief, anti-inflammation and anti-tumor. Exogenous stress, such as low temperature, methyl jasmonate (MeJA), and salicylic acid (SA), can affect the growth of G. elegans and the synthesis of secondary metabolites. Under these conditions, there are only a few reports on the selection and validation of internal reference genes in G. elegans. In this study, seven candidate internal reference genes that showed stable expression abundance in the transcriptome database of G. elegans were selected. The stability and reliability of these genes were analyzed in different G. elegans tissues and under low temperature, MeJA, and SA stresses. The results showed that CUL was the optimal reference gene for expression analysis in different G. elegans tissues and under cold stress, SA stress, followed by eEF-1α. Under MeJA stress, the best one was eEF-1α, and the next was CUL. Furthermore, the expression patterns of three different G. elegans genes, including GPPS, ERF, and 60S, were carried out to confirm that single reference gene (CUL or eEF-1α) or double reference genes (CUL + eEF-1α) can be selected as the best or perfect combination of reference genes for gene expression analysis in G. elegans. This study lays a foundation for the accurate normalization and quantification of gene expression in G. elegans.

Keywords

• gelsemium elegans
• quantitative real-time pcr
• reference gene
• methyl jasmonate
• salicylic acid
• cold stress