Abstract
Talinum paniculatum or Javanese ginseng in Indonesia is a plant widely used as a traditional medicine. The genus Talinum produces oleanane-type saponins, such as talinumoside I. The first aim of this study was to isolate the probable gene encoding β-amyrin synthase (bAS), a key enzyme involved in the cyclization of 2,3-oxidosqualene producing the backbone of the oleanane-type saponin β-amyrin and characterize the gene sequence and the predicted protein sequence using in silico approach. The second aim was to analyze the correlation between the TpbAS gene expression level and saponin production in various plant organs. Thus, TpbAS was isolated using degenerate primers and PCR 5'/3'-Rapid Amplification of cDNA Ends (RACE), then the gene sequence and the predicted protein were in silico analyzed using various programs. TpbAS expression level was analyzed using reverse transcriptase PCR (RT-PCR), and saponin content was measured using a spectrophotometer. The results showed that the full-length TpbAS gene consists of 2298 base pairs encoding for a 765-amino acid protein. From in silico study, the (GA)n sequence was identified in the 5'-untranslated regions and predicted to be a candidate of the gene expression modulator. In addition, functional RNA motifs and sites analysis predicted the presence of exon splicing enhancers and silencers within the coding sequence and miRNA target sites candidate. Amino acid sequence analysis showed DCTAE, QW, and WCYCR motifs that were conserved in all classes of oxidosqualene cyclase enzymes. Phylogenetic tree analysis showed that TpbAS is closely related to other plant oxidosqualene cyclase groups. Analysis of TpbAS expression and saponin content indicated that saponin is mainly synthesized and accumulated in the leaves. Taken together, these findings will assist in increasing the saponin content through a metabolic engineering approach.