The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification

Abstract

1-Hydroxy-2-methyl-butenyl 4-diphosphate reductase (HDR:EC: 1.17.1.2) is the last and key enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which provides the general precursors for taxol biosynthesis and is the important candidate gene for metabolic engineering of the taxol biosynthetic pathway. The gene encoding HDR from Taxus media (designated as TmHDR; GenBank accession number: EF541129) was cloned using RACE, characterized at the levels of bioinformatics and tissue expression pattern, and finally functionally identified with the genetic complementation. The full-length cDNA of TmHDR was 1987 bp containing an 1425-bp open reading frame (ORF) that encoded a polypeptide of 474 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 5.65. Comparative and bioinformatic analysis revealed that TmHDR showed extensive homology with HDRs from other plant species. The subcellular prediction showed that TmHDR owned a plastidial transit peptide of 47 amino acids at its N terminus, which directed TmHDR to plastid. The phylogenetic analysis revealed that TmHDR belonged to gymnosperm HDRs. The tissue expression pattern analysis indicated that TmHDR expressed in all tested tissues including cortices, stems, roots and leaves but at different levels. The highest expression level of TmHDR was found in leaves, followed by roots and cortices; and the expression of TmHDR was very low in stems. Finally, TmHDR was functionally expressed in lethal E. coli HDR mutant and could rescue the mutants. So, the genetic complementation assay demonstrated that TmHDR did encode the protein that had the typical activity of HDR proteins. In summary, the present study will be helpful to understand more about the role of HDR involved in the Taxol biosynthesis at the molecular level and provides an important candidate gene for metabolic engineering of the taxol biosynthesis in Taxus species plants. Key words: 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase (HDR), cloning, genetic complementation, Taxus media, tissue expression.