Cloning and functional characterization of 2- C-methyl- d-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba

2- C-methyl- d-erythritol 4-phosphate cytidyltransferase gene was cloned from Ginkgo biloba and functionally characterized. Transient expression of this protein in the chloroplast of Arabidopsis was observed, confirming its subcellular localization. 2- C-methyl- d-erythritol 4-phosphate cytidyltransferase (MECT), the third enzyme of the 2- C-methyl- d-erythritol 4-phosphate (MEP) pathway, catalyzes formation of 4-(cytidine 5′-diphospho)-2- C-methyl- d-erythritol from MEP. GbMECT, presumably involved in ginkgolide biosynthesis, was cloned and characterized from Ginkgo biloba embryonic roots. The protein containing the N-terminal chloroplast transit peptide consisted of 327 amino acid residues. Complementation of GbMECT with Escherichia coli NMW33, ygbP ( EcMECT) knock-out mutant, rescued the mutant, confirming the function of the protein. Transcription levels of GbMECT remained generally constant in embryonic roots and leaves for 1 month. Full 88 N-terminal residues were necessary to deliver the protein into the chloroplast as shown by protein-targeting analysis with GFP as a reporter protein in Arabidopsis thaliana protoplasts.