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(MVA) Pathway Genes and Triterpene Accumulation in Panax Ginseng

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(MVA) Pathway Genes and Triterpene Accumulation in Panax Ginseng J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 Online ISSN 2234-344X DOI 10.1007/s13765-014-4008-1 Print ISSN 1738-2203 ARTICLE Molecular Cloning and Characterization of Mevalonic acid (MVA) Pathway Genes and Triterpene Accumulation in Panax ginseng Yong-Kyoung Kim · Yeon Bok Kim · Jae Kwang Kim · Soo-Un Kim · Sang Un Park Received: 7 January 2014 / Accepted: 19 March 2014 / Published Online: 30 June 2014 © The Korean Society for Applied Biological Chemistry and Springer 2014 Abstract Panax ginseng Meyer is one of the most important Keywords gene expression · ginsenosides · mevalonic acid medicinal plants in Asia, and ginseng has attracted considerable pathway · Panax ginseng · triterpene attention worldwide. Triterpene saponins (ginsenosides) are the main bioactive compounds in P. ginseng. The isoprene units of triterpene are derived from the mevalonic acid (MVA) pathway. We cloned four genes involved in MVA pathway using rapid Introduction amplification of cDNA ends by polymerase chain reaction. Additionally, we investigated the transcript levels of 11 genes Panax ginseng C.A. Meyer is an important perennial herb plant involved in the terpenoid pathway in different organs and cell that belongs to the Araliaceae family. The major bioactive suspension cultures of P. g i n s e n g . The full-length cDNA sequences compounds of P. ginseng are the ginsenosides which possess were as follows: PgHMGS (1764 bp; 1407-bp ORF), PgHMGR numerous physiological and pharmacological effects (Sticher, (1992 bp; 1722-bp ORF), PgPMK (2170 bp; 1530-bp ORF), and 1998). These include exerting central nervous stimulating and PgMVD (1759 bp; 1263-bp ORF). The highest expression level of suppressing activity as well as anticancer activity (Kubo et al., all genes was found in fine roots. The total ginsenoside contents 1992; Shinkai et al., 1996; Yun, 1996; Iishi et al., 1997; Attele et in different organs were ranked in the following descending order: al., 2002; Dey et al., 2002). To date, more than 30 ginsenosides leaf > fine root > lateral root > red berry > main root > petiole > have been discovered. stem. Campesterol and stigmasterol were detected in all organs Isoprenoids are natural compounds that play a vital role in plant but at different concentrations. The total phytosterol content was metabolism (Bohlmann et al., 1998; Rodriguez-Concepcion and highest in fine root (147.8 µg/100 mg dry weight (DW)), and was Boronat, 2002). They constitute one of the largest structurally lowest in the stem (86.4 µg/100 mg DW). Four enzymes in the diverse groups of natural products, with over 30,000 known MVA pathway were cloned and characterized in P. ginseng. Such compounds (McGarvey and Croteau, 1995). In higher plants, they genes play important roles in terpenoid biosynthesis and may have are synthesized by 2 distinct biosynthetic pathways: the mevalonic applications in the metabolic engineering of ginsenoside production. acid (MVA) pathway and the 1-deoxyxylulose-5-phosphate or 2- C-methyl-D-erythritol-4-phosphate pathway. Isoprenoids are Y.-K. Kim · Y. B. Kim · S.-U. Park () synthesized by the head-to-tail condensation of isopentenyl Department of Crop Science, Chungnam National University, Daejeon diphosphate (IPP) and its isomer, dimethylallyl diphosphate 305-764, Republic of Korea E-mail: [email protected] (Rohmer et al., 1995; Lichtenthaler et al., 1997a; b). Ginsenosides are synthesized via the MVA pathway. The J. K. Kim enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Republic of Korea (HMGS) catalyzes the condensation of acetyl-CoA and acetoacetyl- CoA. The synthesis of MVA is catalyzed by 3-hydroxy-3- Y.-K. Kim · S. U. Kim methylglutaryl-CoA reductase (HMGR), which is a key regulatory Department of Agricultural Biotechnology and Research Institute for Agricultural Sciences, Seoul National University, Seoul 151-951, Republic enzyme, and the sequential action of MVA kinase, phospho- of Korea mevalonate kinase, and pyrophosphomevalonate decarboxylase 290 J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 Fig. 1 The ginsenoside biosynthetic pathway. AACT, acetyl-coenzyme A (CoA) acetyltransferase; β-AS, beta-amyrin synthase; CAS, cycloartenol synthase; DDS, dammarenediol-II synthase; FPS, farnesyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase; HMGS, HMG-CoA synthase; IDI, isopentenyl diphosphate isomerase; MVD, mevalonate diphosphate decarboxylase; MK, mevalonate kinase; MPK, mevalonate-5-phosphate kinase; SE, squalene epoxidase; SS, squalene synthase. converts MVA to IPP (Rohmer et al., 1995). Mevalonate diphosphate represent an attractive strategy to produce better quality medicinal decarboxylase (MVD) is an important enzyme in the MVA compounds. pathway, catalyzing the ATP-dependent decarboxylation of Recently, ginsenoside biosynthetic pathway genes from P. mevalonate 5-diphosphate to yield IPP. Ginsenoside is then ginseng (Chen et al., 2011) and panax quinquefolius (Sun et al., synthesized by the terpenoid biosynthetic pathway, involving 2010) were discovered by next generation sequencing. Huge enzymes such as farnesyl diphosphate synthase (FPS), squalene amount of genetic information was clarified using high throughput synthase (SS), squalene epoxidase (SE), dammarenediol-II synthase method. Therefore, cloning and molecular characterization of (DDS), and beta-amyrin synthase (β-AS) (Fig. 1). genes involved in ginsenoside biosynthesis are required. In the The studies of MVA pathway genes related to phytosetrol and present study, we cloned 4 genes, HMGS, HMGR, PMK, and terpenoid biosynthesis have been demonstrated in Nicotiana MVD involved in terpenoid biosynthesis. In addition, we tabacum (Schaller et al., 1995), Candida utilis (Shimada et al., investigated the transcript levels of 11 genes involved in the 1998), and Saccharomyces cerevisiae (Cordier et al., 1999). terpenoid pathway in different organs and cell suspension cultures Secondary metabolite production has been shown to be induced of P. ginseng. Finally, the ginsenoside and phytosterol contents in by various elicitors - including methyl jasmonate (MeJA) and P. ginseng were analyzed. yeast extract - in Panax species such as P. notoginseng (Hu and Zhong, 2007; 2008), P. quinquefolius (Ali et al., 2005), and P. ginseng (Lu et al., 2001; Hu et al., 2003). Triterpene biosynthetic Materials and Methods genes were upregulated by MeJA treatment in Medicago truncatula (Suzuki et al., 2002), and Glycyrrhiza glabra (Hayashi et al., Plant materials. P. gi n s e n g samples were collected from the 2003). Furthermore, MeJA has also been used to increase experimental farm of Chungnam National University. Different ginsenoside content in cell suspension cultures (Hu et al., 2003) organs were stored in sealed clear polyethylene plastic bags at and in root culture of P. ginseng (Han et al., 2006). Overproduction −80oC until further use and freeze-dried at −80oC for 72 h. of phytosterols and triterpenes by metabolic engineering may RNA isolation and cDNA synthesis. Total RNA was isolated J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 291 from different organs (red berry, leaf, petiole, stem, main root, Scattering Detector (Model 300s; SofTA, USA). The separation lateral root, and fine root) by using TRI-reagent (Molecular method was carried out according to Kim et al. (2009) using a Research Center, Inc., USA) and the RNeasy Plant Mini Kit ProntoSIL column (Bischoff Chromatography, Germany; 250× (Qiagen, USA) according to the manufacturers’ protocols. Total 4.6 mm) at a flow rate of 0.8 mL/min. The conditions were RNA of each organ was reverse transcribed using SuperScript II optimized by the solvent gradient system (Kim et al., 2009). First-Strand Synthesis Kit according to the manufacturer’s Identification and quantification of ginsenosides were performed instructions (Life technologies, USA). by comparing the retention times and peak areas with those of a Cloning of cDNAs encoding MVA pathway enzymes. The 5' ginsenoside standard or by direct addition of a ginsenoside and 3' rapid amplification of cDNA ends (RACE)-PCR was standard into the sample (spike test). Standard chemicals carried out using the GeneRacer Kit (Life technologies, USA) (ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rh1) with fine root RNA as the template and specific primers. The were purchased from Canfo Chemical Co., Ltd., China. All primer pairs (Supplementary Table 1) were based on conserved samples were run in triplicate. sequences from other known plant genes. All PCR products were Extraction and derivatization of triterpenes. Extraction of subcloned into the T-Blunt vector (Solgent, Korea) and sequenced triterpenes was performed according to the method of Du and Ahn (Solgent). (2002) with slight modifications. Triterpene components were Gene expression by real-time quantitative PCR. To investigate released from powdered samples (0.1 g) by the addition of 3 mL the expression pattern of MVA pathway genes from different of ethanol containing 0.1% ascorbic acid (w/v) and 0.05 mL of organs of P. ginseng, single-stranded cDNAs were synthesized 5α-colestane (10 µg/mL), mixed by vortexing for 20 s, and placed from total RNA by using the ReverTra Ace-α-Kit (Toyobo, in a water bath at 85°C for 5 min. After removal from the water Japan) and oligo (dT)20 primer according to the manufacturer’s bath, 120 µL of potassium hydroxide (80%) was added, and the
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