Jun-Gyo In

Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf.

The expressed sequence tags (ESTs) referenced in this report are the first transcriptomes in a leaf from a half-shade ginseng plant. A cDNA library was constructed from samples of the leaves of 4-year-old Panax ginseng plants, which were cultured in a field. The 2,896 P. ginseng cDNA clones represent 1,576 unique sequences, consisting of 1,167 singletons and 409 contig sequences. BLAST comparisons of the cDNAs in GenBanks non-redundant databases revealed that 2,579 of the 2,896 cDNAs (89.1%) exhibited a high degree of sequence homology to genes from other organisms. The majority of the identified transcripts were found to be genes related with energy, metabolism, subcellular localization, and protein synthesis and transport. The chlorophyll a/b-binding protein ESTs in the ginseng leaf samples manifested a substantially higher level of expression than was observed in other plant leaves. The ESTs involved in ginsenoside biosynthesis were also identified and discussed.

Isolation of Sesquiterpene Synthase Homolog from Panax ginseng C.A. Meyer

Sesquiterpenes are found naturally in plants and insects as defensive agents or pheromones. They are produced in the cytosolic acetate/mevalonate pathway for isoprenoid biosynthesis. The inducible sesquiterpene synthases (STS), which are responsible for the transformation of the precursor farnesyl diphosphate, appear to generate very few olefinic products that are converted to biologically active metabolites. In this study, we isolated the STS gene from Panax ginseng C.A. Meyer, designated PgSTS, and investigated the correlation between its expression and various abiotic stresses using real-time PCR. PgSTS cDNA was observed to be 1,883 nucleotides long with an open reading frame of 1,707 bp, encoding a protein of 568 amino acids. The molecular mass of the mature protein was determined to be 65.5 kDa, with a predicted isoelectric point of 5.98. A GenBank BlastX search revealed the deduced amino acid sequence of PgSTS to be homologous to STS from other plants, with the highest similarity to an STS from Lycopersicon hirsutum (55% identity, 51% similarity). Real-time PCR analysis showed that different abiotic stresses triggered significant induction of PgSTS expression at different time points.

Isolation of S-adenosyl-L-methionine synthetase gene from Panax ginseng C.A. meyer and analysis of its response to abiotic stresses.

A cDNA clone containing a S-adenosyl-L-methionine synthetase (SAMS) gene, named as PgSAM, was isolated from a commercial medicinal plant Panax ginseng. PgSAM is predicted to encode a precursor protein of 307 amino acid residues, and its sequence shares high homology with a number of other plant SAMS. PgSAM is expressed at different levels in various organs of ginseng. The expression of PgSAM in adventitious roots and hairy roots of P. ginseng were analyzed using reverse transcriptase (RT)-PCR and real-time PCR under various abiotic stresses. Salt, salicylic acid, abscisic acid and chilling stresses induced PgSAM significantly at different time points within 2–72 h post-treatment. This study revealed that PgSAM may help to protect the plants against various abiotic stresses.

A simple and rapid technique for the authentication of the ginseng cultivar, Yunpoong, using an SNP marker in a large sample of ginseng leaves

Yunpoong is an important Korean ginseng (Panax ginseng C. A. Meyer) cultivar, but no molecular marker has been available to identify Yunpoong from other cultivars. In this study, we developed a single nucleotide polymorphism (SNP) marker for Yunpoong based on analysis of expressed sequence tags (ESTs) in an exon region of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. This SNP marker had high specificity to authenticate Yunpoong in twelve different main ginseng cultivars. For application of the molecular marker, a rapid identification method was established based on the NaOH-Tris method and real-time polymerase chain reaction (PCR) in order to ensure more efficiency in the cultivar selection. The biggest feature of the NaOH-Tris method was that it made the extraction of DNA very simple and rapid in young leaf tissues. We only spent 1 min to extract DNA and directly used it to do PCR. In this report, the conventional DNA extraction method was used to develop molecular marker process, and the NaOH-Tris method was applied in screening large numbers of cultivars. Moreover, the greatest advantage of the real-time PCR compared with traditional PCR, is time saving and high efficiency. Thus, this strategy provides a rapid and reliable method for the specific identification of Yunpoong in a large number of samples.

Effects of Red Ginseng Extract on the Epididymal Sperm Motility of Mice Exposed to Ethanol

The protective effects of red ginseng extract and ginseng wine against ethanol-induced male reproductive toxicity were evaluated in male mice using computer-assisted sperm analysis. Mice were divided into 4 groups of 10 and fed plain saline, 6 g/kg per d of ethanol in saline, red ginseng extract plus ethanol, or a fermented preparation of red ginseng extract daily for 5 weeks. We found that the average seminal vesicle weight was significantly lower in the ethanol-treated group compared to the control group, while those of the ginseng-treated groups tended to be higher than the ethanol-treated group. We found a significant decrease in sperm motility and progressiveness in mice treated with ethanol for 5 weeks, while administration of ethanol plus red ginseng extract appeared to minimize the negative effects of ethanol toxicity on male fertility. Serum testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were insignificantly lower in the ethanol-treated group than in the control group.

Lactobacillus koreensis sp. nov., isolated from the traditional Korean food kimchi

A lactic acid bacterium, strain DCY50(T), isolated from the traditional Korean food kimchi, was studied to determine its taxonomic position. The strain was Gram-stain-positive, catalase-negative, facultatively anaerobic, rod-shaped and motile. The genomic DNA G+C content was 49 mol% and the peptidoglycan structure was of the A4α (l-Lys-d-Asp) type. Chemotaxonomic markers of the strain were consistent with its classification in the genus Lactobacillus. Comparisons of 16S rRNA and rpoA gene sequences showed that strain DCY50(T) was most closely related to the type strains of Lactobacillus parabrevis (98.4 and 91.6 % similarity, respectively, for the 16S rRNA and rpoA genes), L. hammesii (98.0 and 91.2 %), L. brevis (97.6 and 93.3 %) and L. senmaizukei (97.4 and 90.5 %). DNA-DNA relatedness of strain DCY50(T) to these type strains was below 36 %. According to the genotypic and phenotypic data, strain DCY50(T) could be differentiated from all known Lactobacillus species and should be classified in a novel species, for which the name Lactobacillus koreensis sp. nov. is proposed; the type strain is DCY50(T) ( = KCTC 13530(T)  = JCM 16448(T)).

Isolation and Characterization of Pathogenesis-Related Protein 5 (PgPR5) Gene from Panax ginseng

A pathogenesis-related protein (PgPR5) gene that isolated from the leaf of Panax ginseng was characterized. The ORF is 756 bp with a deduced amino acid sequence of 251 residues. The calculated molecular mass of the matured protein is approximately 27.5 kDa with a predicated isoelectric point of 7.80. A GenBank BlastX search revealed that the deduced amino acid of PgPR5 shares highest sequence similarity to PR5 of Actinidia deliciosa (80% identity, 87% similarity). PgPR5 has a C-terminal and N-terminal signal peptide, suggesting that it is a vacuolar secreted protein. The expression of PgPR5 under various environmental stresses was analyzed at different time points using real-time PCR. Our results reveal that PgPR5 is induced by salt stress, chilling stress, heavy metal, UV, and pathogen infection. These results suggest that the PgPR5 could play a role in the molecular defence response of ginseng to abiotic and pathogen attack. This is the first report of the isolation of PR5 gene from the P. ginseng.

Molecular identification of oriental medicinal plant Schizonepeta tenuifolia bunge (Hyung-Gae) by multiplex PCR

Schizonepeta tenuifolia (Korean name “Hyung-Gae”) is an oriental medicinal plant that is widely used in Korea, China and Japan. S. tenuifolia (Hyung-Gae) has many pharmacological activities and is mostly used for many medicinal preparations. The dried aerial part (spikes and stems) of three oriental medicinal plants, S. tenuifolia (Hyung-Gae), Agastache rugosa (Kwhak-Hyang) and Elsholtzia ciliata (Hyang-Yoo) belonging to the same family, mint family Labiaceae, have such similar shape and smell that it is difficult to differentiate between them. The trnL-F regions of chloroplast DNA of the three medicinal plants were sequenced and used as targets in multiplex PCR reaction to identify S. tenuifolia. After alignment of trnL-F sequences of the authenticated plant samples, one single nucleotide polymorphism (SNP) specific to S. tenuifolia was found. Based on this SNP, a new primer was designed that specifically amplifies the trnL-F region of S. tenuifolia. The established multiplex-PCR was proven to be effective in the differentiation of commercial S. tenuifolia samples from A. rugosa and E. ciliata. This rapid and accurate molecular method is highly promising for use in the food industry.

Microbacterium soli sp. nov., an α-glucosidase producing bacterium isolated from soil of a ginseng field

Five Gram-type-positive, aerobic, rod-shaped, non-motile strains of Microbacterium (DCY 17(T), Ms1, Ms2, Ms3 and Ms4) were isolated from soil from a ginseng field in Daejeon, South Korea. On the basis of 16S rRNA gene sequence similarity, these strains were shown to be related to Microbacterium esteraromaticum DSM 8609(T) (96.1 %), M. xylanilyticum DSM 16914(T) (96.0 %), M. aquimaris JS54-2(T) (95.6 %), M. insulae DS-66(T) (95.5 %), M. ketosireducens IFO 14548(T) (95.5 %) and M. arabinogalactanolyticum DSM 8611(T) (95.4 %). Chemotaxonomic data revealed that the type strain, DCY 17(T), possesses menaquinones MK-12, MK-11 and MK-13 and the predominant fatty acids C(15 : 0) anteiso (32.5 %), C(15 : 0) iso (27.5 %), C(16 : 0 ) iso (17.0 %), C(17 : 0) anteiso (13.2 %), C(17 : 0) iso (6.1 %) and C(14 : 0) iso (2.1 %). The DNA G+C content of strain DCY 17(T) is 70.2 mol% and those of strains Ms1 to Ms4 are in the range 68.9-73.5 mol%. The physiological and biochemical tests suggested that these strains represent a novel species. Based on these data, DCY 17(T) (=KCTC 19237(T) =LMG 24010(T)) is classified as the type strain of a novel Microbacterium species, for which the name Microbacterium soli sp. nov. is proposed.

Isolation and Characterization of Terpene Synthase Gene from Panax ginseng

Terpene synthase plays a key role in biosynthesis of triterpene saponins (ginsenosides) and is intermediate in the biosynthesis of a number of secondary metabolites. A terpene synthase (PgTPS) cDNA was isolated and characterized from the root of Panax ginseng C.A. Meyer. The deduced amino acid sequence of PgTPS showed a similarity with A. deliciosa (AAX16121) 61%, V. vinifera (AAS66357) 61%, L hirsutum (AAG41891) 55%, M truncatula (AAV36464) 52%. And the segment of a terpene synthase gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). We studied expression of terpene synthase under stressful conditions like chilling, salt, UV, and heavy metal stress treatment. Expression of PgTPS was increased gradually after exposure to stresses such as chilling, salt, and UV illumination. But its transcription seems to be reduced by cadmium and copper treatment.