Molecular ietfictodniiain and functional characterization of a stilbene synthase gene in Rheum offficnliiae

Stilbene synthase (STS) is a pivotal gene involved in the biosynthesis and regulation of stilbenes compounds in Rheum officinale Baill. However, the precise regulatory genes governing their biosynthesis remain elusive. A series of analyses was conducted herein, including cloning of Ro STS, subcellular localization, heterologous expression, enzyme reaction and product analysis, resveratrol content analysis, and Ro STS gene expression in hairy roots. The open reading frame (ORF) of the Ro STS gene was amplified, which is 1140 bp long and encodes a protein of 379 amino acids with a relative molecular mass of 41,640.25 Da. The Ro STS protein contains conserved residues (Cys 164, His 303, and Asn 336) and is closely related to the STS of Rheum palmatum L. and Rheum tanguticum Maxim. ex Balf. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that Ro STS had the highest expression in the roots and rhizomes of two-year-old plants. Moreover, it was significantly upregulated under methyl jasmonate (MeJA) and salicylic acid (SA) treatments. Subcellular localization analysis indicated that the Ro STS protein is localized in the nucleus and cell membrane. Ro STS was cloned into pGEX6p-1 and expressed in Escherichia coli BL21(DE3). The purified recombinant protein could catalyze the synthesis of resveratrol from specific substrates in vitro. The overexpression of Ro STS significantly increased the resveratrol content in the hairy roots of R. officinale. The qRT-PCR analysis also showed that the relative expression of Ro STS in transgenic hairy roots was significantly higher than that of the wild type. These results suggest that Ro STS might be involved in the stilbenes biosynthesis in R. officinale. This work may contribute to understanding the mechanism of resveratrol biosynthesis in R. officinale.