Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii / 中国中药杂志

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

The small subunit of geranyl diphosphate synthase: a tool to improve aroma and fl avour by metabolic engineering.

Monoterpenes represent a major class of volatile terpenes produced by plants and play important roles in pollination, defense and seed dispersal (Dudareva et al. 2006). Also, plant-derived terpenoids, including monoterpenes are used as natural fl avour and aroma compounds, and have benefi cial impact on humans as health promoting compounds (Wagner and Elmadfa 2003). All monoterpenes are derived from the common precursor, geranyl diphosphate (GPP, C10), which is synthesized by a head-to-tail condensation of one molecule each of isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP) in a reaction catalyzed by GPP synthase (GPPS, EC 2.5.1.1) (fi gure 1) (Ogura and Koyama 1998). GPPSs have been isolated and functionally characterized in a limited number of plant species and are known to exist as homodimeric and heterodimeric structures. Heterodimeric GPPSs have been described in peppermint (Mentha piperita), snapdragon (Antirrhinum majus), Clarkia (Clarkia breweri) and hop (Humulus lupulus), which produce large amounts of monoterpenes in specifi c organs (Burke et al. 1999; Tholl et al. 2004; Wang and Dixon 2009). Structurally, the heterodimeric GPPS constitutes a small http://www.ias.ac.in/jbiosci J. Biosci. 35(2), June 2010, 167–169, © Indian Academy of Sciences 167 Keywords. Geranyl diphosphate synthase; metabolic engineering; monoterpenes Figure 1. Overall effects of GPPS.SSU over-expression on the levels of monoterpenes, sesquiterpenes and GGPP derived products in tobacco plants. DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, DXP reductoisomerase; FPP, farnesyl diphosphate; FPPS, FPP synthase; GA-3P, glyceraldehyde-3-phosphate; GGPP, geranyl geranyl diphosphate; GGPPS, GGPP synthase; GPP, geranyl diphosphate; GPPS, GPP synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl- CoA; HMGR, HMG-CoA reductase; IPI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; MVA, mevalonic acid. Names of enzymes are boxed. Bigger and smaller font size of end products indicates increase and decrease in the content, respectively.