Molecular cloning and structural analysis of key enzymes in Tetrastigma hemsleyanum for resveratrol biosynthesis.

Abstract

Resveratrol (RES), a plant antitoxin, has antioxidant, anti-inflammatory, anti-cancer and cardiovascular protection effects. It has been reported that RES can be stably detected in a Chinese herbal medicinal plant Tetrastigma hemsleyanum. At present, the research of T. hemsleyanum mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites in T. hemsleyanum. In this experiment, four key enzymes (ThPAL/ThC4H/Th4CL/ThRS) involved in the RES biosynthesis pathway were cloned and obtained. They contained an open reading frame (ORF) of 2139\u202fbp, 1518\u202fbp, 1716\u202fbp and 1035\u202fbp, encoding 712, 505, 571 and 344 amino acids, separately. Various bioinformatics tools were used to analyze these deduced protein domains, secondary structures, three-dimensional (3D) structures and phylogenetic trees. Subsequently, quantitative primers were designed to conduct the tissue-specific expression. Quantitative results displayed that the four genes were expressed in all tested tissues, and their expression in root tubers was more stable. Moreover, the subcellular localization of the four genes was studied by constructed recombinant green fluorescent expression vectors. Herein, by digging out the key enzyme genes in the biosynthesis of RES in T. hemsleyanum, this experiment tried to reveal the expression patterns of these key enzyme genes. It also provided the basis for the research on the molecular level, which will help people further illuminate and clarify the biosynthesis and regulation mechanism of secondary metabolites in T. hemsleyanum.