Abstract
Tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine in mitragynine biosynthesis via the shikimate pathway. Using the rapid amplification of cDNA ends (RACE) technique, the gene encoding TDC from Mitragyna speciosa was cloned (designated as MsTDC). The MsTDC cDNA contained an open reading frame (ORF) of 1,521 base pairs (bp) encoding 506 amino acid residues. It had a pyridoxal-phosphate (PLP)-binding site at the amino acid position 313–334 residues. The MsTDC showed homology of 68–76 % to the TDC of other plants. Heterologous expression in Escherichia coli afforded the soluble proteins as an apparent band of 57 kDa as judged by SDS-PAGE. Expression of the MsTDC in M. speciosa hairy roots under the 35S promoter was performed by insertion of MsTDC into pCAMBIA1300-gfp. The transgenic hairy root lines were detected by fluorescence microscopy and showed an increased accumulation of tryptamine.
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Acknowledgments
Authors thank for the Thailand Research Fund (TRF), the Office of the Higher Education Commission (HEC), Prince of Songkla University (PSU) and Faculty of Pharmaceutical Sciences (Grant No. RMU5380015). Grants from the PSU Graduate School and Songklanakarin Research Abroad to T.C. are also acknowledged. We thank Prof. Dr. Johan Memelink (Sylvius Laboratory, Leiden University, The Netherlands) for the lab facilities and kindly provide the pTH2, pRT101 and pMOG463 plasmids. We also thank Dr.Brian Hodgson and Mr. Joseph Materson for assistance with the English.
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Communicated by J.-H. Liu.
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Charoonratana, T., Wungsintaweekul, J., Keawpradub, N. et al. Molecular cloning and expression of tryptophan decarboxylase from Mitragyna speciosa . Acta Physiol Plant 35, 2611–2621 (2013). https://doi.org/10.1007/s11738-013-1296-8
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DOI: https://doi.org/10.1007/s11738-013-1296-8