Molecular Characterization and Functional Analysis of Triterpene Biosynthetic Genes in Panax ginseng and Platycodon grandiflorum

Abstract

The present work deals with the role of the genes, which are related to triterpene biosynthesis. Triterpene saponins (ginsenosides and platycosides) are main bioactive compounds in Panax ginseng and Platycodon grandiflorum. The ginsenoside content of berries and roots of three cultivars of Korean ginseng were investigated. For all cultivars, ginsenoside Re was the most abundant ginsenoside in roots and berries. However, berries produced more total ginsenosides and the berry ginsenoside profile differed from that of roots. The ginsenoside Re content of berries was 4-6 times more than that of roots. Averaged across all cultivars, the amounts of the five ginsenosides in berries was Re > Rc ≈ Rg1 ≈ Rb1 ≈ Rd. For roots, the amounts were Re > Rg1 > Rb1 > Rc >Rd. Roots of the Yunpoong cultivar had the greatest ginsenoside content, followed by roots of the Chunpoong cultivar and the Gumpoong cultivar. The total amount of ginsenosides (especially Rb1, Re, and Rg1) was greatest in the Yunpoong cultivar. Gene expression varied during flower and berry development. Total saponin levels were highest in the early berry stage and correlated well with that in the ginseng root. Therefore, berries may be of pharmaceutical interest and should be harvested at the early berry stage to ensure optimal ginsenoside levels. The isoprene units of the triterpene are derived from the mevalonic acid (MVA) pathway. This study provides a complete nucleotide sequence of the full-length cDNA involved in mevalonic acid biosynthesis. We had a full-length cDNA sequences as followed; PgHMGS (1764bp, 1407bp ORF), PgHMGR (1992bp, 1722bp ORF), PgPMK (2170bp, 1530bp ORF) and PgMVD (1759bp, 1263bp ORF) with partial poly A. HMGR belong to a multigene family and isoforms are key enzymes in the mevalonate biosynthetic pathway leading to production of a diverse array of isoprenoid compound. Homology searches in genome databases of other HMGRs allowed the identification of HMGR1. All genes were constitutively expressed in all tested tissues at different transcription level with the highest expression in fine roots. When cultures of hairy root were treated with 0.1 mM MeJA, MVA pathway genes were strongly expressed and the contents of ginsenoside reach the peak in 72 h. To elucidate the functions of mevalonate pathway genes in triterpene biosynthesis, Panax ginseng was transformed in hairy root. PCR analyses were used for the selection of hairy root lines. As a result of PCR determination, 6 lines of each genes (PgHMGR, PgMVD, PgFPS) were identified. Compare to the control hairy root, transgenic lines showed higher expression levels by qRT-PCR. Total ginsenosides were increased by transgenic lines to 1.4-1.84 fold in PgFPS lines. But, PgMVD overexpression line did not show large differences in ginsenoside levels. Phytosterols also showed increased in both lines. PgMVD and PgFPS overexpression lines showed high amount of total phytosterol than controls. In contrast, β-amyrin contents were slightly decreased in both lines. These results suggest that metabolic engineering of P. ginseng to enhanced production of phytosterols and triterpenoids by introducing the PgMVD and PgFPS genes was successfully achieved by Agrobacterium-mediated genetic transformation. But other genes data were not significance in this experiment. Platycodon grandiflorum is a medicinal plant widely used in northern China and Korea. This plant has been used as an expectorant and an antitussive for the treatment of coughs, colds, upper respiratory tract infections, sore throats, tonsillitis, and chest congestion. 5,760 expressed sequence tags (ESTs) were isolated from methyl jasmonate (MeJA)-treated hairy roots of P. grandiflorum, from which 811 contigs and 1,725 singletons were obtained, and 2,299 are passed the annotation. The unigenes were classified into the following 3 major functional categories according to the standard Gene Ontology (GO) classification: cellular component, molecular function, and biological process. The most abundant transcripts encoded fatty acid desaturase (FAD); numerous transcripts of secondary metabolite genes were also observed. We had isolated full-length cDNAs encoding BPgHMGS, BPgMK, and BPgMVD on the basis of the EST data. All the genes are expressed in every tissue of P. grandiflorum, but the levels are different. The contents of triterpene saponins and phytosterols are detected by HPLC and GC-MS, respectively. These EST data will provide important information of other secondary metabolite genes in P. grandiflorum. Transgenic hairy root lines were established using Agrobacterium rhizogenes. When transformed BPgMVD, hairy root produced total platycosides 1.15-1.9 fold more than the controls. Highest level of total platycosides (1.33 ± 0.05 mg/g DW) were detected in M22. This result reveals that BPgMVD are important enzyme in the production of saponin. However, no difference was found in the α-spinasterol and β-amyrin levels. And PgHMGR (Panax ginseng) overexpressed in P. grandiflorum, hairy root lines produced total platycosides 1.6-2.5 fold more than the controls. Highest level of total platycosides (1.60 ± 0.2 mg/g DW) were detected in H7 compared to the control hairy root lines. α-Spinasterol increased 1.1-1.6 fold compare to the control. H7 line highly accumulated α-spinasterol (1.78 ± 0.16 mg/g DW). However, β-amyrin contents were slightly decreased (0.3-0.8 fold). The result showed that overexpression of PgFPS in P. ginseng hairy root can lead to enhanced total ginsenosides production (up to 1.84 fold) and PgHMGR single gene transgenic hairy root in P. grandiflorum showed higher levels of total platycoside (up to 2.5 fold), which are the most effect single genes in this experiment. This result reveals that overexpression of mevalonate pathway genes showed slightly increased expression of each genes in hairy root of P. ginseng and P. grandiflorum containing triterpene saponin, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutical important compound. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the cytosolic isoprenoid biosynthesis pathway are examples of the metabolic engineering of isoprenoids biosynthesis in P. ginseng and P. grandiflorum.