Characterization and functional analysis of a novel endogenous strong promoter discovered from Morinda officinalis

Iridoids represent the primary pharmacologically active constituents in Morinda officinalis, a traditional Chinese medicinal herb, and exhibit diverse pharmacological properties with broad applications in the pharmaceutical, chemical, and cosmetic industries. Geraniol synthase (GES), encoded by MoGES, catalyzes the conversion of geranyl diphosphate (GPP) to geraniol, representing a critical rate-limiting step in iridoid biosynthesis. Nevertheless, the regulatory mechanism of MoGES expression remains uncharacterized.

In this study, a 1598 bp promoter region of MoGES (designated as ProMoGES) was cloned from M. officinalis and systematically functionally characterized. Bioinformatics analysis revealed ProMoGES contains core elements (37 TATA-boxes, 40 CAAT-boxes) and cis-acting elements responsive to light, hormones, and abiotic stresses. Promoter truncation assays using a GUS reporter system demonstrated that all fragments drove GUS expression in both Nicotiana benthamiana and Arabidopsis thaliana, and the promoter activity exhibited a non‑monotonic trend with the P3 fragment showing peak activity as the fragment length decreased. The ProMoGES promoter exhibited stronger activity than the CaMV35S promoter in M. officinalis, driving 1.56-fold higher eGFP expression, which confirms its function as a potent endogenous promoter. ProMoGES-mediated MoGES expression was significantly induced by methyl jasmonate (MeJA), low temperature, and light, but suppressed by heat and salt stress. Furthermore, MoDREB6 functions as a negative regulator that suppresses MoGES expression by binding to the DRE/CRT element within the ProMoGES promoter.

The functional characterization of ProMoGES offers a valuable and potent endogenous promoter tool for future genetic engineering and metabolic manipulation in M. officinalis. This study also provides novel insights into the transcriptional regulatory mechanisms underlying iridoid biosynthesis.