The HDR gene involved in the TIA pathway from Rauvolfia verticillata: Cloning, characterization and functional identification

Abstract

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR, EC 1.17.1.2) catalyzes the last reaction of the methylerythritol phosphate (MEP) pathway. The full-length cDNA sequence of HDR was cloned and characterized from terpenoid-indole-alkaloid-producing Rauvolfia verticillata. The new cDNA was named as RvHDR and submitted to GenBank® to be assigned with an accession number: EU034699. The full-length cDNA of RvHDR was 1679-bp containing a 1389-bp open reading frame (ORF) encoding a polypeptide of 462-amino acids with a calculated molecular mass of 52 kDa and an isoelectric point of 5.26. Comparative and bioinformatic analyses revealed that RvHDR had extensive homology with HDRs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic indicated that all HDRs could be divided into three groups and RvHDR belonged to plant HDRs family. RvHDR was found to be expressed in all tested tissues including roots, stems, leaves, flowers and fruits but at different levels. The highest expression level was found in flowers, and higher expression level in leaves and fruits; the expression level was low in roots and lowest in stems. Expression profiling analyses revealed that RvHDR expression was induced by exogenous elicitors including methyl jasmonate, acetyl salicylic acid, abscisic acid and UV, and showed the transcription levels were all up-regulated compared to the control. Finally, RvHDR was transformed into the E. coli HDR mutant strain MG1655 ara<>HDR, which was able to rescue the lethal phenotype of the E. coli HDR mutant. This confirmed that RvHDR had the typically function of HDR gene. The cloning, characterization and functional identification of RvHDR will be helpful to understand more about the function of HDR at the level of molecular genetics and help to unveil the biosynthetic mechanism of TIAs precursor and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata. Show more