• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• Korean Journal of Plant Tissue Culture
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• v.28 no.1
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• pp.47-52
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• 2001

Bet A and Bet B genes related to salt resistance were introduced into Lycium chinense through high efficiencies of plant regeneration. The explants were precultured on the shooting medium which is consisted of MS medium added 1mg/L kinetin and 0.05mg/L IBA for 2 days. After pre-culture, they were immersed in LB media containing Agrobacteria tumefaciens harboring Bet genes, and cultured on the same medium. Putative transformants could be selected after cocultivation of the explants with Agrobacteria on the shooting medium supplemented with 30mg/L kanamycin. The presence of both Bet A and Bet B genes from the transgenic plants were confirmed by PCR amplification with the gene specific primers and subsequent PCR-Southern blot with labeled Bet genes probe. The expression of Bet A and Bet B genes in the transgenic plants were observed by RT-PCR method.

• Journal of Plant Biotechnology
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• v.36 no.2
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• pp.144-148
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• 2009

The transfer of Mn-Superoxide Dismutase (SOD2) gene, complex gene (SA) of CuZnSOD and ascorbate peroxidase (APX), and NDP kinase 2 (NDPK2) gene into Korean 4 cultivars (cvs. Millenium White, Glory Blue, Glory Red, and Glory Purple) and 15 purebred lines of petunia was conducted using Agrobaterium-mediated technique. Two (Wongyo A2-16 and A2-36) of 15 purebred lines and one (cv. Glory Red) of 4 cultivars were effective for the transfer of SOD2 gene. The putative transgenic plants survived on the 2nd selection medium were 124. From PCR analysis, 118 (derived from 4 cultivars and 2 purebred lines) of 124 plants were confirmed to contain marker (npt II ) gene, while 58 of 118 plants did not have target genes. There were no plants with both npt II and SA genes. Twenty seven of 28 SOD2 transgenic plants were re-confirmed as transformants by Sothern analysis. SOD2 and NDPK2 genes were expressed in the transgenic petunias as the ratio of 77.8 to 100.0 % and 23.5%, respectively. T1 seeds were obtained from 36 acclimated transgenic plants (SOD2 34 plus NDPK2) in a glasshouse by self-pollination.

• The Plant Pathology Journal
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• v.15 no.6
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• pp.313-318
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• 1999

Cloning of the 5'untranslated region (5' UTR) and Nterminus of the glycoprotein precursor (G2G1) open reading frame of tomato spotted wilt virus has been problematic, possibly because of the toxicity of a signal peptide at the beginning of th G2G1 protein precursor. The toxicity of the signal peptide to bacterial growth and the reason for the expression of the peptide gene in Escherichia coli were investigated by cloning the 5' UTR and the signal peptide sequence separately. Cells transformed with the plasmid containing both the first 30 amino acids of the glycoprotein and the 5' UTR showed a severe growth inhibition whereas transformants harboring either the plasmid with the signal sequence or the 5'UTR alone did not show any ingibition. An E. coli promoter-like sequence was found in the 5'UTR and tis promoter acivity was confirmed with a promoter-less GUS gene cloned downstream of the 5'UTR. In the cloning of the Tomato spotted wilt virus (TSWV) glycoprotein G2G1 open reading frame all the recovered plasmids contained stop codons in the signal sequence region. However, clones containing no stop codon were recovered when the signal sequence and the 5'UTR were cloned separately.

• The Plant Pathology Journal
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• v.36 no.4
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• pp.378-383
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• 2020

The objective of this research was introduction of chit42 to tuber mustard plants through Agrobacteriummediated transformation against white mold caused by Sclerotinia sclerotiorum. The binary plasmid pGisPEC1 was used in this study. Polymerase chain reaction analysis detected the transgene in 27 transformants with a transformation efficiency of 6.9%. Southern blot test was used to assess the copy number of transgene in tuber mustard plants. One, two, two, and two chit42-related bands were observed in the transformed lines TMB4, TMB7, TMB12, and TMB18, respectively. Enzymatic tests showed a significant increase in the activity of endochitinase in protein isolated from leaf tissues of chit42 transgenic 75-day tuber mustard lines. The pathogenicity of three pathogen isolates was tested on the leaves of transformed plans. The results of current study showed that expression of the gene chit42 in tuber mustard plants markedly reduced infection radius on the leaves 7 days after inoculation with the fungus.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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• pp.434-439
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• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• Toxicological Research
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• v.17 no.1
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• pp.65-71
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• 2001

The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. A yeast-based steroid hormone receptor gene trascription assay was previously developed for the evaluation of chemicals with endocrine modulating activity. The yeast transformants used in this assay contain the human estrogen receptor along with the appropriate steroid response elements upstream of the $\beta$-galactosidase reporter gene. We tried to evaluate several natural and synthetic steroids of their potential to interact directly with the steroid receptor. Some putative endocrine disruptors, including nonylphenol, are weakly estrogenic. But the combined treatment oj these chemicals with di-(2-ethylhexyl)phthalate (DEHP) significantly increased the $\beta$-galactosidase activity in the yeast transformant. These results suggest that we also have to consider the synergistic effects of endocrine disruptors. In this study, we showed that yeast-based bioassay is a valuable tool for screening potential endocrine disruptors and quantitative determination of estrogenicity. And the possibility that the estrogen receptor binds multiple environmental chemicals adds another level of complexity to the interaction between the endocrine disruptors and the human hormone system.

• Toxicological Research
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• v.22 no.1
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• pp.15-22
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• 2006

Many of endocrine disrupting chemicals induce effects via interaction with hormone receptors and responsive elements in target cells. We investigated endocrine disrupting effects of some food additives and contaminants including BHA, BHT, ethoxyquin, propionic acid, sorbic acid, benzoic acid, CPM, aflatoxin B1, cadmium chloride, genistein, TCDD and PCBs in yeast transformants expressing human steroid hormone receptors along with steroid responsive elements. The response limit of genetically recombinant yeast to $17{\beta}$-estradiol, testosterone and progesterone was $1{\times}10^{-16},\;1{\times}10^{-12}\;and\;1{\times}10^{-13}M$, respectively. BHT induced weak transcriptional activity in estrogen sensitive yeast, while BHA and sorbic acid interacted weakly with androgen receptor/responsive element. CPM induced transcriptional activities in all types of yeasts sensitive to steroid hormones. Zearalenone and genistein induced high transcriptional activation in estrogen sensitive yeast with relative potencies almost $10^8$ folds lower than $17{\beta}$-estradiol. TCDD induced transcriptional activation weakly in estrogen- and progesterone- sensitive yeasts. This study elucidated that recombinant yeast is a sensitive and high-throughput system and can be used for the direct assessment on chemical interactions with steroid receptors and responsive elements. Also, the present study raises the requirement of evaluation on the endocrine disrupting effects of BHT, BHA, sorbic acid, CPM and TCDD for their transcription activity in yeast screening system though weak in intensity.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.491-496
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• 2000

A RAG25 gene regulating flowering time was introduced into Codonopsis lanceolata through high efficiencies (ca. 90%) of plant regeneration. The leaf explants were immersed in YEP media containing Agrobacterium tumefaciens (pGA 1209) harboring RAG25 gene, and cocultivated for 3 days. After cocultivation, they were cultured in shoot inducing media (SIM), N2B2 (NAA 2 mg/L, BA 2 mg/L and kanamycin 20 mg/L) and N2B4 (NAA 2 mg/L, BA 4 mg/L and kanamycin 20 mg/L), and the putative transformants were regenerated. The introduction of nptII and RAG25 gene into Codonopsis lanceolata was confirmed by 0.7 kb and 0.6 kb bands from polymerase chain reaction and reconfirmed by Southern hybridization using PCR product of RAG25 gene.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.361-366
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• 2005

Opium poppy (Papaver somniferum L.) is one of the most important medicinal plants, which is used as a sole commercial source of narcotic analgesic, morphine. The transformant of opium poppy we have established by infection of Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain MAFF03-01724 showed aberrant morphology and altered opium alkaloid composition. The major alkaloid produced by this transformant was thebaine (16.3%, opium dry weight) instead of morphine. It is likely that this 'thebaine poppy' phenotype was caused by the integration of T-DNA(s) into the poppy genome DNA, and their inserted loci are of great interest. To gain an insight into the mechanism of nonnarcotic thebaine accumulation for the further approach toward the creation of 'codeine poppy' which produces codeine as a major alkaloid, the genetical and morphological analyses on the transformant was carried out. Here we report the results of the detailed analysis on the T-DNA inserted loci of T0 transfromant and the correlation between opium alkaloid composition and segregated T-DNA integration pattern in the self-pollinated T1 transformants.