Selection and evaluation of reference genes by RT-qPCR analysis in aconitum vilmorinianum kom

Aconitum vilmorinianum Kom. is one of the most important traditional Chinese medicine. However, there are few reports on the molecular biology of A. vilmorinianum. Fluorescence quantitative real-time polymerase chain reaction (RTqPCR) is widely used detection method for target gene expression analysis, but the selection of the best reference gene is very important for the accuracy of the method. In this study, based on transcriptome data six candidate internal reference genes were selected for the first time and their expression stability in root, stem, leaf and flower of A. vilmorinianum was systematically evaluated by geNorm, Normfinder and BestKeeper. The results showed that MDH, GAPDH and 18S RNA could be used as reference genes in root; MDH and18S RNA can be used as reference genes in stem; MDH, GAPDH and β- TUB could be used as reference genes in leaf, and GAPDH, 18S RNA and ACT could be used as reference genes in flower. In addition, the applicability of the selected reference genes was verified by detecting two functional genes related to alkaloid metabolism, namely hydroxymethylglutaryl-CoA reductase (HMGCR) and phosphomevalonate kinase (PMK). The expression profiles of the HMGCR and PMK genes were similar after standardization to the stably expressed reference genes MDH, GADPH, 18S RNA and ACT. However, when UBQ was used as the reference gene, the expression profiles of HMGCR and PMK genes were different. These results provide useful information for obtaining reliable RT-qPCR standardized data in genetic research on A. vilmorinianum. (author)