Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, and , and Interaction Analysis with the Transcription Factor TwTGA1 from .

Miltiradiene synthase (MS) genes, and , are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in . However, the mechanism underlying the regulation of key genes in triptolide biosynthesis remains unclear. In this study, the promoters of (1496 bp) and (1862 bp) were isolated and analyzed. Some hormone-/stress-responsive elements and transcription factor (TF) binding sites were predicted in both promoters, which might be responsible for the regulation mechanism of . The β-glucuronidase (GUS) activity analysis in promoter deletion assays under normal and methyl jasmonate (MeJA) conditions showed that the sequence of -921 to -391 bp is the potential core region of the promoter. And the TGACG-motif, a MeJA-responsive element found in this core region, might be responsible for MeJA-mediated stress induction of GUS activity. Moreover, the TGACG-motif is also known as the TGA TF-binding site. Yeast one-hybrid and GUS transactivation assays confirmed the interaction between the promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in ), indicating that are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, and , as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.