| Summary: | Miltiradiene synthase (MS) genes, <i>TwTPS27a</i> and <i>TwTPS27b</i>, are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in <i>Tripterygium wilfordii</i>. However, the mechanism underlying the regulation of key genes <i>TwTPS27a/b</i> in triptolide biosynthesis remains unclear. In this study, the promoters of <i>TwTPS27a</i> (1496 bp) and <i>TwTPS27b</i> (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 <i>TwTPS27a/b</i>. 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 <i>TwTPS27b</i> 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 <i>TwTPS27a/b</i> promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in <i>T. wilfordii</i>), indicating that <i>TwTPS27a/b</i> are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, <i>TwTPS27a</i> and <i>TwTPS27b</i>, as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.
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