Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii.

Yanbo Huo,Chuanshu Zhu,Xing Zhang,Jing Zhang,Bin Zhang,Ling Chen

doi:10.3390/plants10020418

Abstract

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

Highlights

Tripterygium wilfordii is a well-known traditional medicinal herb whose common name is Lei Gong Teng or thunder duke vine [1,2]
To distinguish the sequences of these two highly-homologous genes, the full-length coding sequence (CDS), gDNA, and 5 /3 -untranslated region (5 /3 -UTR) sequences of TwTPS27a and TwTPS27b were successfully obtained by polymerase chain reaction (PCR) amplification (Figure S2) and series of sequence alignment (Figures S3–S6)
After alignment analysis (Figure S7), it was determined that the 1496 bplength promoter sequence belongs to the TwTPS27a gene, whereas the 1862 bp-length promoter sequence belongs to the TwTPS27b gene, respectively

Summary

Introduction

Tripterygium wilfordii is a well-known traditional medicinal herb whose common name is Lei Gong Teng or thunder duke vine [1,2]. The identification of hundreds of effective compounds isolated from the roots of T wilfordii [3,4], such as diterpenoids, triterpenoids, sesquiterpenoids, and alkaloids, has stimulated the study of their pharmacological properties. Triptolide, an important bioactive diterpenoid in T. wilfordii, has a variety of pharmacological activities, such as antitumor, anticancer, immunosuppressive, and anti-inflammatory activities [5,6,7,8]. The biosynthetic pathway of triptolide has been widely studied. Less is known about the regulatory mechanism of key genes in triptolide biosynthesis in T. wilfordii, as compared with the progress of triptolide biosynthetic enzyme genes

Methods

Results

Discussion

Conclusion