Isolation and characterization of a copalyl diphosphate synthase gene

Presented material concerns the promoter cloning and in-silico identification of cis-active elements localized in Salvia miltiorrhiza copalyl diphosphate synthase (CPS) gene. The plant is interesting in medical terms due to significant biological activity, dependent on terpenoid compounds known as tanshinones. As the metabolic pathways of tanshinones biosynthesis are not fully revealed, we decided to decipher the unknown aspect of the transcriptional regulation of CPS, the enzyme of crucial importance for tanshinone biosynthesis. We identified numerous cis-active motifs, together with homologous trans-factors potentially active in S. miltiorrhiza CPS gene regulation. Analysis of trans-factors genes co-expressed with A. thaliana CPS and their comparison with trans-factors identified by the in-silico searches of S. miltiorrhiza CPS promoter showed no common protein. To experimentally validate results of in-silico searches, the influence of auxin, gibberellin, salicylic acid and high-salt concentration on promoter activity was assessed: gus gene expression being measured 0, 12, 24, 48, 72 and 96 hours after hormonal treatment, or during a 96-hour exposure to 100mM NaCl.