Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

Abstract

Persicaria odorata is an herbaceous plant with antifungal and antibacterial properties. The plant produces secondary metabolites, including phenols, sulphur-containing compounds and terpenoids, in response to biotic and abiotic stresses. Terpenoids in P. odorata are synthesized through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. 1-deoxy-D-xylulose-5phosphate isomerase (DXR) is a rate-limiting enzyme in the MEP pathway and may be regulated by microRNA (miRNA) miR398. There is a lack of evidence showing miR398 regulation in terpenoid biosynthesis in P. odorata through DXR-targeting. The study aimed to verify the stem-loop structure of miR398 and analyse its expression towards the target gene, DXR, in treated and control P. odorata. RNA was quantified and qualitatively analysed using a Nanodrop spectrophotometer and agarose gel electrophoresis. The stem loop of miR398 was verified using reverse transcriptase PCR (RT-PCR) and agarose gel electrophoresis. The expression of miR398 and DXR was compared between control and treated samples. Treated leaves were punctured with needles and left for 48h before harvest. Gene expressions were quantified and normalised using reference genes. The stem-loop structure of miR398 was confirmed, despite possible primer mismatches and unspecific binding. This step was essential before comparing and assessing the gene expression of miR398 and DXR. A decrease in abundance of miR398 whereas in increase in abundance was in the treated sample compared to the control sample indicating that miR398 negatively regulated DXR under stress conditions, suggesting an increase in terpenoid synthesis as a defence mechanism. DXR acts as a rate-limiting enzyme in the MEP metabolic pathway. Further studies are needed to quantify the effects of miR398 on terpenoid biosynthesis after wounding in P. odorata.