Combined transcriptome sequencing and prokaryotic expression to investigate the key enzyme in the 2-C-methylerythritol-4-phosphate pathway of Osmanthus fragrans

Abstract

Terpenoids are one of the main components of plant aromas. In the present study we investigated these compounds in Osmanthus fragrans Lour., which is a fragrant plant widely used for the production of essential oils. Quantitative real-time PCR (qRT-PCR) results of enzymes associated with the 2-C-methylerythritol-4-phosphate pathway confirmed that the TPS is a key enzyme for terpenoid synthesis in O. fragrans. In a series of experiments, we identified the TPS candidate genes in O. fragrans and revealed the underlying catalytic activity and subcellular localisation of the encoded proteins. Because there is no available O. fragrans reference genome, we sequenced and analysed its transcriptome and identified two putative TPS genes, OfTPS1 and OfTPS2. According to qRT-PCR analysis, both genes were most highly expressed at the full-bloom stage, suggesting that OfTPS1 and OfTPS2 contribute to O. fragrans terpenoid synthesis. To verify this hypothesis, we constructed prokaryotic expression vectors to obtain protein. In order to study the function of OfTPS1 and OfTPS2 in the synthesis of monoterpenes, the obtained proteins were reacted with geranyl pyrophosphate. As a result, two kinds of monoterpenes, (E)-β-ocimene and linalool, were detected from reaction products, respectively. In conclusion, OfTPS1 and OfTPS2 are both monoterpene synthases.