Isolation of <i>Salvia miltiorrhiza</i> Kaurene Synthase-like (<i>KSL)</i> Gene Promoter and Its Regulation by Ethephon and Yeast Extract

Abstract

The presented study describes the regulation of the promoter region of the Salvia miltiorrhiza kaurene synthase-like gene (SmKSL) by ethylene and yeast extract. The isolated fragment is 897 bp and is composed of a promoter (763 bp), 5′UTR (109 bp), and a short CDS (25 bp). The initial in silico analysis revealed the presence of numerous putative cis-active sites for trans-factors responding to different stress conditions. However, this study examines the influence of ethylene and yeast extract on SmKSL gene expression and tanshinone biosynthesis regulation. The results of 72h RT-PCR indicate an antagonistic interaction between ethylene, provided as ethephon (0.05, 0.10, 0.25, and 0.50 mM), and yeast extract (0.5%) on SmKSL gene expression in callus cultures of S. miltiorrhiza. A similar antagonistic effect was observed on total tanshinone concentration for up to 60 days. Ethylene provided as ethephon (0.05, 0.10, 0.25, and 0.50 mM) is a weak inducer of total tanshinone biosynthesis, increasing them only up to the maximum value of 0.67 ± 0.04 mg g−1 DW (60-day induction with 0.50 mM ethephon). Among the tanshinones elicited by ethephon, cryptotanshinone (52.21%) dominates, followed by dihydrotanshinone (45.00%) and tanshinone IIA (3.79%). In contrast, the 0.5% yeast extract strongly increases the total tanshinone concentration up to a maximum value of 13.30 ± 1.09 mg g−1 DW, observed after 50 days of induction. Yeast extract and ethylene appear to activate different fragments of the tanshinone biosynthesis route; hence the primary tanshinones induced by yeast extract were cryptotanshinone (81.42%), followed by dihydrotanshinone (17.06%) and tanshinone IIA (1.52%).

Keywords

• callus culture
• promoter
• <i>cis</i>-active element
• tanshinone
• ethylene
• yeast extract