Molecular Cloning, Characterization and Functional Analysis of the Genes Encoding DXR and DXS, the Two Enzymes in the MEP Pathway, from Amomum villosum Lour.

Amomum villosum Lour. (officinal name Fructus Amomi) is one of the most well-known and authentic herbs in South China. Monoterpenes in its volatile oil are the main medicinal compounds. The condensation of pyruvate with D-glyceraldhyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (DXP) catalyzed by DXP synthase (DXS, EC: 2.2.1.7) and the conversion of DXP to 2-C-methyl-D-erythritol-4-phosphate (MEP), catalyzed by DXP reductoisomerase (DXR, EC:1.1.1.267), are the initial steps of the MEP pathway for isoprenoid biosynthesis. A DXR gene, AvDXR (GenBank accession no. FJ459894), and a DXS gene, AvDXS (FJ455512), were isolated from the leaves of Amomum villosum. The 1749-bp full-length cDNA of AvDXR contained a 1416-bp open reading frame (ORF) encoding a peptide of 472 amino acids, and the 2347-bp full-length cDNA of AvDXS contained a 2148-bp ORF encoding a peptide of 715 amino acids. The deduced amino acid sequences of the AvDXR and AvDXS proteins shared high homology with DXRs and DXSs from other plant species, respectively, and the AvDXS belonged to class 1 plant DXS. Chloroplast transit peptides were found in the N-terminal region of AvDXR and AvDXS. A proline-rich region, two highly conserved NADPH-binding domains and two substrate-binding domains consistent with the enzymatic functions of DXR were found in AvDXR. AvDXS contained a TPP-binding domain and a DRAG domain related to the catalysis of DXS. The functional color assay in Escherichia coli with pAC-BETA implied that AvDXR and AvDXS encoded functional proteins that manipulate the biosynthesis of isoprenoid precursors. Both AvDXR and AvDXS were expressed extensively in the leaves, stems, roots, pericarps and seeds of A. villosum. AvDXS expression was similar in all tissues investigated, whereas higher levels of AvDXR were observed in the fruits, the main part for the accumulation of volatile oil in A. villosum, which suggested that AvDXR might play a more influential role in monoterpene precursor biosynth- sis than AvDXS. The role of AvDXS in other isoprenoid biosynthesis, especially the primary isoprenoid biosynthesis remained to be investigated.