Evaluation of housekeeping genes for normalizing RT-qPCR and analysis of the expression patterns of WRKY1 transcription factor and rhynchophylline biosynthetic-related genes in Uncaria rhynchophylla

Abstract

Background: Uncaria rhynchophylla（Miq.）Miq.ex Havil, a traditional medicinal herb, is enriched with a number of pharmacological active terpenoid indole alkaloids (TIAs). At present, a comprehensive selection and evaluation of the appropriate housekeeping genes for gene expression analysis, especially transcription factors and key enzyme genes involved in biosynthesis pathway of TIAs in U. rhynchophylla, have not been reported. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for gene expression level detection with its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on the selection of an optimal housekeeping gene for the accurate normalization of RT-qPCR results. Results: Ten candidate housekeeping genes, that are homologs of genes used in other plant species as common housekeeping genes, were used to evaluate their expression stability under three stress related experimental treatments (methyl jasmonate, ethylene and low temperature), using multiple stability analysis methodologies. The results showed that S-adenosylmethionine decarboxylase (SAM) had a higher expression stability than the other candidate housekeeping genes under the experimental conditions tested. Using SAM as a housekeeping gene, 14 genes of key TIA enzymes and a WRKY1 transcription factor had their expression profiles examined in the three experimental stress treatments that are known to affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was found to be similar that of tryptophan decarboxylase (TDC) and strictosidine- β-D-glucosidase (SGD). Conclusions: This research is first to report the stability of housekeeping gene in U. rhynchophylla and as such provides an important foundation for future gene expression analysis in U. rhynchophylla. WRKY1 expression indicated it is potentially capable of coordinating the expression of TDCand SGD, providing a possible means of enhancing alkaloid production in future with synthetic biology.