• Journal of Life Science
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• v.14 no.4
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• pp.650-656
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• 2004

Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.71.2-71
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• 2003

Ralstonia solancearum infects many solanaceous plants, however race 3 infects only potato and tomato weakly. To identify genes responsible for race specificity of R. solanacearum, we mobilized genomic library of LSD2029 (race 3) into LSD341 (race 1) and inoculated 1,000 transconjugants into hot pepper. One transconjugant that did not induce wilt symptom in hot pepper was isolated. We found that a cosmid clone, pRSl, conferred avirulence to LSD341. By deletion and mutational analyses of pRSl, we found the 0.9-kb PstI/Hindlll fragment carries avirulence functions. We sequenced the fragment and identified one possible open reading frame, a rsal gene, possibly encoding 110 amino acids. The rsal was preceded with a plant-inducible promoter (PIP) box, indicating that the gene might be regulated by HrpB. Interestingly, the promoter region of the rsal homolog in the strain GM11000 (race 1) did not have the PIP box. Rsal did not show any significant homologies with proteins in the database, indicating th e protein is different from the previously reported avirulence proteins. When we mutated the rsal gene by marker-exchange in LSD2029, the mutant was less virulent in potato.

• KSBB Journal
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• v.6 no.3
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• pp.289-297
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• 1991

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7$\times$105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

• Journal of Photoscience
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• v.7 no.2
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• pp.53-58
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• 2000

We performed the cloning of a chalcone synthase (CHS) gene, the key enzyme in the anthocyanin biosynthesis, from the cDNA library constructed with grape suspension cells irradiated UV-B. The PCR fragment was used to cloning the CHS gene. One CHS cDNA clone containing an open reading frame and a partial stilbene synthase (STS)cDNA, the stilbene-type phytoalexin, were isolated. The CHS cDNA clone (VCHS) showed 87% sequence homology with VvCHS (V.vinifea) and 72.3% identity with VSTSY(V.vinifea). its amino acid sequences were longer than any other CHS genes as 454 residues. Two genes were weakly expressed in white light irradiated cells, but highly induced in UV-B irradiated condition during 32 hours. Interestingly, the STS was quickly and abundantly expressed from 2 hours when supplemented with jasmonic acid (JA) and the maximum expression was observed at 4 hours and then gradually decreased. But, the additional UV-B or white light quickly degraded the STS expression than only JA treated grape suspension cells. The CHS also was rapidly induced with JA and the synergistical effect was observed at the addigional light treatment of UV-B or white light. These results are indicated that CHS and STS have different response mechanisms against the environmental stresses.

• Journal of Photoscience
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• v.7 no.2
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• pp.73-78
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• 2000

To analyze molecular traits determining pigmentation between Pharbitis nill violet and white, Amplified Fragment Length Polymorphism(AFLP) and Differential Display Reverse Transcription(DDRT) experiments were carried out with either genomic DNAs or total RNAs isolated from both plants. Results of AFLP experiment in combination of 8 EcoRⅠ primers with 6 MseⅠ primers showed 41 violet-and 60 white-specific DNA bands. In the subsequent experiment, 22 violet-and 22 white-specific DNA fragments were amplified by PCR with DNAs eluted. The sizes of the fragments range from 200 to 600bp. DDRT using total RNA produced 19 violet-and 17 white-specific cDNA fragments, ranging from 200 to 600bp. The fragments obtained by both AFLP and DDRT had been cloned into pGEM T-easy vector, amplified and subjected to the nucleotide sequence analyses. As a result of Blast sequence analysis, most of them sequenced up to date showed no similarity to any Known gene, while few has similarity to known animal or plant genes. An AFLP clone V6, for example, has a strong sequence similarity to the human transcription factor LZIP-alpha mRNA and a DDRT clone W19 to Solanum tuberosum 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.50 no.4
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• pp.286-290
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• 2005

Ascorbate peroxidase (APX) plays a crucial role in the detoxification of hydrogen peroxide. APX activity is maintained significantly higher in the paraquat­treated leaves of the paraquat-tolerant Rehmannia glutinos. This study was conducted to understand structural and regulatory characteristics of APX gene in R. glutinosa. A putative APX cDNA clone (RgAPX1) was isolated from a leaf cDNA library using a partially sequenced expressed sequence tag clone. RgAPX1 is consisted of 1148 bp nucleotides and contains an open reading frame encoding a 250 amino acid-long polypeptide. Deduced RgAPX1 amino acid sequence shares higher sequence similarity to cytosolic APXs. RgAPX1. expression was higher in the leaf than in the flower and root. Southern blot result indicates the presence of one or two RgAPX1-related genes in R. glutinosa genome. RgAPX1 transcription was affected differentially by various stresses and phytohormone. The results indicate that RgAPXl is constitutively expressed in most tissues and its expression is modulated for the immediate and efficient detoxification of $H_2O_2$ under normal and stress conditions.

• The Korean Journal of Ecology
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• v.19 no.6
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• pp.507-517
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• 1996

To explain the horizontal expansion of a rhizomatous perennial, Convallaria keiskei(lily-of-the-valley), in a study site of Chunchon, Kangwon Province, Korea, ramet growth and clonal structure were studied. Remarkable growth stategies were clarified. First, the timing for the successive phenological events such as sprouting. flowering and rhizome growth for lily-of-the-valley was fitted to exploit early spring when the canopy of overstory was opened. Second, these events were supported by effective matter allocation pattern: for example, two-year investment for new rhizomes enabled the first year ramets to mature in six weeks after sprouting and to grow up to 85% of the leaf area of perennial ramets. Finally, the ramet population was increased by local disturbances such as freezing, herbivory and collection by human. The rule that a clone was supposed to produce one new thizome per year was broken by occasional disturbances. Then, up to 5rhizomes from latent bur could be redeveloped. Based on clonal structure, 80% or total clones have from 1 to 4 ramets. this means there have occurred minor disturbances. Therefore, in conclusion, the successful flourishing of lily-of-the-valley came from its effective frowth strategy to take advantage of site disturbance.

• Journal of Photoscience
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• v.5 no.3
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• pp.131-135
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• 1998

Using differential display reverse transcription technique, the present study attempted to isolate and characterize genes specifically expressed in cotyledons of Pharbitis nil Choisy cv. Violet during floral induction. A total of 107 bands specific to the inductive condition were initially obtained with 80 primer sets of 20 different arbitrary primers combined with 4 kinds of T12MN. In northern blot analysis with reamplified cDNAs as probes, three cDNAs were detected to be expressed specificcally in the induced cotyledon tissues, and designated PnFL-1, PnFL-2 and PnFL-3 , the size of which were 228 bp, 317 bp and 272 bp, respectively. A search for sequences similar to the decuced amino acid sequences was conducted using GenBank and EMBL database ; seequence encoded by PnFL-1 had 29% identity with the clone of Arabidopsis thaliana similiar to reverse trascriptase (Genbank Acc. N0.3047086), PnFL-2 shared 50% identity with hydroxiyproline-rich glycoprotein of Glycine max(GenBank Acc. No.347455), and PnFL-3 had 46% identity with TAMU 4. Thaliana genomic clone T23E16 (GenBank Acc. No.B67574). None of them was known gene in the plant system up to date, implying that the fragments may comprise parts of genes which are associated with the floral induction in Pharbitis nil.

• Journal of Life Science
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• v.18 no.11
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• pp.1499-1506
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• 2008

To examine antitumor activity of the edible plant Zanthoxylum schinifolium, the cytotoxic effect of various organic solvent extracts of its stems on human acute leukemia Jurkat T cells was investigated. Among these extracts such as methanol extract (SS-7), methylene chloride extract (SS-8), ethyl acetate extract (SS-9), n-butanol extract (SS-10), and residual fraction (SL-11), SS-8 exhibited the most cytotoxic activity against Jurkat T cells. The methylene chloride extract (SS-8) possessed the apoptogenic activity capable of inducing sub-G1 peak along with apoptotic DNA fragmentation in Jurkat T cells. Western blot analysis revealed that SS-8 induced apoptosis via mitochondrial cytochrome c release into cytoplasm, subsequent activation of caspase-9 and caspase-3, and cleavage of PARP, which could be blocked by overexpression of Bcl-xL. Jurkat T cell clone I2.1 $FADD^{-/-}$) and Jurkat T cell clone I9.2 (caspase-$8^{-/-}$ were as sensitive as was the wild-type Jurkat T cell clone A3 to the cytotoxic effect of SS-8, suggesting no contribution of Fas/FasL system to the SS-8-mediated apoptosis. The GC-MS analysis of SS-8 showed that it was composed of 16 ingredients including 9,12-octadecanoic acid (18.62%), 2,4-dihydro-5-methyl-4- (1-methylethylidene)- 2-(4-nitrophenyl)-3H- pyrazol-3-one (14.97%), hexadecanoic acid (14.23%), (z,z)-6,9-pentadecadien- 1-ol (13.73%), 5,6-dimethoxy-2-methyl benzofuran (10.95%), and 4-methoxy-2-methylcinnamic acid (5.38%). These results demonstrate that the methylene chloride extract of the stems of Z. schinifolium can induce apoptotic cell death in Jurkat T cells via intrinsic mitochondria-dependent caspase cascade regulated by Bcl-xL without involvement of the Fas/FasL system.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.77-85
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• 2009

Pseudomonas fluorescens strain Gpf01, isolated from ginseng rhizosphere showed antiviral activity against Cucumber mosaic virus, when tested in a local host of CMV, Chenopodium amaranticolor. Transposon mutant library of Gpf01 was prepared using pGS9::Tn5 and the mutant Gpf01-RS19 was found to loose antiviral production. We developed primers from the flanking region of Tn5 and found a cosmid clone pAV1123, harboring 1.2 kb antiviral compound producing (avcf01) locus. When a sub-clone pPH9, which carried 9.3 kb region of pAV1123, was introduced into antivirus deficient P. fluorescens wild type strain B16, it exhibited antiviral activity. Using Tn3-gus mutagenesis and complementation analysis, it was found that the genes related to antiviral activity production resided in a 9.3 kb HindIII-HindIII fragment of pAV1123, indicating that the plasmid carries an essential genes promoting antiviral activity.