Molecular cloning and characterization of strictosidine synthase, a key gene in biosynthesis of mitragynine from Mitragyna speciosa

Abstract

Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR from M. speciosa was cloned through reverse-transcription polymerase chain reaction (RT-PCR) and denoted as StrMs1. The clone is a full-length cDNA with a size of 1257 bp, which contains an open reading frame of 1056 bp starting from base pair 18 to 1076. Sequence analysis showed that StrMs1 has high homology with other STRs of TIA-producing plants. Nucleotide sequence of StrMs1 was deposited in GenBank with accession number ADK91432. The deduced amino acid sequence has 352 residues with a predicted molecular weight of 39 kDa and isoelectric point at pH 5.78. Southern blot performed showed that there is only one copy of StrMs1 present in the genome of M. speciosa. Expression pattern on different tissues tested using RT-PCR revealed that besides leaf, the expression was also detected in root, stem and flower. Expression profiles under plant defense signal using salicylic acid (SA) was investigated on leaf tissues and the results showed that the transcript of StrMs1 were detected before and after treatment with salicylic acid. Result obtained from phylogenetic analysis suggested that StrMs1 is the most evolved protein among other STRs. However, the 3-D prediction of StrMs1 showed that there are alpha helices and beta propeller structures, which remain conserved withother STRs.

Key word: Strictosidine synthase, Mitragyna speciosa, StrMs1, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), molecular evolution, protein prediction.