Abstract
Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions.
Highlights
Gentiana macrophylla Pall. is a well-known medicinal plant in the Gentianaceae family
The melting temperatures (Tm) of the PCR products ranged from 79.6◦C for Ubiquitinconjugating enzyme 10 (UBC10) to 84.9◦C for GAPC2 (Table 1)
cycle threshold (Ct) values ranged from 19.22 for UBC10 in stems to 32.9 for Exp1 at 4 L. Most of those values were between 22.0 and 28.0. These results indicated that none of the selected genes had stable expression among samples, thereby demonstrating how important it is to evaluate the suitability of reference gene(s) for normalizing expression under given experimental conditions when analyzing G. macrophylla
Summary
Gentiana macrophylla Pall. is a well-known medicinal plant in the Gentianaceae family. Is a well-known medicinal plant in the Gentianaceae family. Its dried roots, ‘Qinjiao,’ have been used in traditional Chinese medicine for over 2000 years, usually as an ingredient in numerous formulae. The biological and pharmacological effects of Qinjiao include stomachic, choleretic, and antihepatotoxic activities (Wang and Lou, 1987; Ji et al, 2002). Secoiridoids are its dominant active constituents, especially gentiopicrin (gentiopicroside). With the development of molecular biology tools, Gentiana is being used to study the molecular pathways of secondary metabolites and key related genes. Improving our understanding of gene expression patterns can provide insight into complex biological processes, such as signaling and metabolic pathways (Marino et al, 2008).
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