Isolation and Characterization of a Theta Glutathione S-transferase Gene from Panax ginseng Meyer

Abstract

Plants have versatile detoxification systems to encounter the phytotoxicity of the wide range of natural and synthetic compounds present in the environment. Glutathione S-transferase (GST) is an enzyme that detoxifies natural and exogenous toxic compounds by conjugation with glutathione (GSH). Recently, several roles of GST giving stress tolerance in plants have demonstrated, but little is known about the role of ginseng GSTs. Therefore, this work aimed to provide further information on the GST gene present in Panax ginseng genome as well as its expression and function. A GST cDNA (PgGST) was isolated from P. ginseng cDNA library, and it showed the amino acid sequence similarity with theta type of GSTs. PgGST in ginseng plant was induced by exposure to metals, plant hormone, heavy metals, and high light irradiance. To improve the resistance against environmental stresses, full-length cDNA of PgGST was introduced into Nicotiana tabacum. Overexpression of PgGST led to twofold increase in GST-specific activity compared to the non-transgenic plants, and the GST overexpressed plant showed resistance against herbicide phosphinothricin. The results suggested that the PgGST isolated from ginseng might have a role in the protection mechanism against toxic materials such as heavy metals and herbicides.

Keywords: Environmental stress; Glutathione-S-transferase; Panax ginseng; Phosphinothricin.

Fig. 1.. PgGST sequence analysis. (A) nucleotide and deduced amino acid sequence of a PgGST cDNA isolated from Panax ginseng. The deduced amino acid sequence is shown in single-letter code below the nucleotide sequence with the open reading frame from 10 to 763 bp. The position of nucleotides is shown on the right. Black box shows the transcription start codon and termination codon. The sequence has been deposited in GenBank as accession no. EU625298. (B) Expressions of PgGST in various organs of P. ginseng. Total RNAs were extracted from leaves (L), root (R), and stems (St). The actin gene of P. ginseng was used as a control.

Fig. 2.. Sequence homology and phylogenetic analysis of PgGST with other glutathione S-transferase (GST) proteins. (A) comparison of the putative amino acids sequence of PgGST with those of GST genes of theta class from other species with GenBank accession no. in parenthesis; Glycine max (AAG34813), Arabidopsis thaliana (AAM98138), Euphorbia esula (AAF64449), Oryza sativa (AAK98534), Canis familiaris (XP534751), Homo sapiens (AAV38754), Bos taurus (AAI11290), Mus musculus (NP598755), Rattus norvegicus (NP445745), Danio rerio (AAH56725), Anopheles gambiae (AAM61892), and Aedes aegypti (AAV68399). Hyphen was inserted within amino acid sequence to denote gap. Shadow box means well conserved residues, * represents conserved amino acid and, : represents very similar amino acid. Glutathione-binding residues are indicated by red boxes. (B) Phylogeny of the theta class GST protein family from Panax ginseng and other species. Phylogenetic analysis is based on the deduced amino acid sequences of GST genes from various species. Neighbor-joining method was used and the branch lengths are proportional to divergence, with the scale of 0.1 representing 10% changes.

Fig. 3.. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis for the expression of PgGST genes in Panax ginseng at various time points (h) post-treatment with various stresses including high light, UV, 100 mM NaCl, chilling (4℃), 10 mM H₂O₂, 11% mannitol (Man), 20% sucrose (Suc), 500 μM CuSO₄ (Cu), 500 μM CdSO4 (Cd), 5 mM salicylic acid (SA), 200 mM jasmonic acid (JA), or 0.1 mM abscisic acid (ABA). A RT-PCR employing actin specific primer was carried out to confirm equal RNA loading.

Fig. 4.. Genetic transformation of Nicotiana tabacum with ginseng glutathione S-transferase gene. Survived transgenic (T) calli compared with non-transgenic line (N) (A) on selection medium containing 100 mg/L kanamycin, and 500 mg/L cefotaxime produced resistant shoot (B), and transgenic tobacco explants acclimated in vitro (C).

Fig. 5.. Confirmation of transgenes in tobacco by polymerase chain reaction (PCR) and southern blot analysis. (A) PCR analysis of 35S cauliflower mosaic virus promoter (CaMV), glutathione S-transferase of Panax ginseng (PgGST), neomycin phosphotransferase II (NPTII), nopaline synthase terminator (NOS), and internal transcribed spacer (ITS) gene as control from non-transgenic (N) and two transgenic tobacco lines (T1 and T2) was conducted. (B) Southern blot hybridization of DNA prepared from transgenic tobacco plants. DNA (20 μg) was cut by restriction enzymes EcoRΙ or HindⅢ, separated on 1.3% agarose gel, transferred to a membrane and hybridized to a DIG-labeled PgGST-specific probe. (C) reverse transcriptase-PCR and (D) glutathione S-transferase (GST) activity of non-transgenic (N) and two transgenic lines (T1 and T2).

Fig. 6.. Leaves of non-transgenic (N) and transgenic (T and T2 line) tobacco plants treated with 0.1 M (A) or 1 M (B) phosphinothricin for 4 h.

Fig. 7.. Effect of phosphinothricin (PPT) on the chlorophyll content and PgGST expression in transgenic tobacco leaves (T2). Total chlorophyll content from leaf discs of wild-type and transgenic plants treated with 0.1, 1, 10, 100, or 1,000 mM of PPT for 4 h (A) and 24 h (B) was analyzed. Means (±SE) are for 10 replicates per treatment. Averages for treated samples were significantly different compared to the control at *p<0.01. PgGST expression by PPT (0.1, 1, 10, 100, or 1,000 mM) for 4 h was performed by reverse transcriptase polymerase chain reaction (C).