• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.3
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• pp.220-226
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• 2014

Sixty-seven Vibrio parahaemolyticus isolates from surface seawater from the Wando area, on the southern coast of Korea, were analyzed for their susceptibility to 15 different antimicrobials and the presence of virulence genes. According to the disk diffusion susceptibility test, all of the strains studied were resistant to ampicillin and oxacillin, while decreasing percentages were resistant to vancomycin (64.2%), streptomycin (56.7%), amikacin (31.3%), kanamycin (22.3%), cephalothin (20.9%), erythromycin (10.4%), ciprofloxacin (4.5%), and tetracycline (3.0%). All of the strains were susceptible to five antimicrobials: chloramphenicol, gentamycin, nalidixic acid, sulfamethoxazole/trimethoprim, and trimethoprim. Fifty-nine isolates (88.1%) were resistant to three or more classes of antimicrobial and defined as multidrug resistant, and two strains were resistant to seven antimicrobial agents. The minimum inhibitory concentration (MIC) of the 67 V. parahaemolyticus isolates to ampicillin and oxacillin ranged from 512-2,048 and $64-512{\mu}g/mL$, respectively. All 67 isolates were also examined for the presence of the tdh and trh virulence genes using the polymerase chain reaction (PCR). However, no isolates possessed either tdh or trh. The VPA0477 (${\beta}$-lactamase) gene, present in all of the tested strains, was validated as a new specific marker gene in PCR assays for the accurate detection and identification of V. parahaemolyticus.

• Journal of Microbiology
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• v.44 no.6
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• pp.671-673
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• 2006

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminogly-coside phosphotransferase gene (aph) from Tn903, a lacZ' gene for $\alpha$-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DHS${\alpha}$ and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

• Journal of Animal Science and Technology
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• v.45 no.4
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• pp.659-666
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• 2003

Endoglucanase A from Clostridium thermocellum which is resistant to pancreatic proteinase was selected out of numbers cellulases then were expressed in lactobacilli. Recombinant lactobacilli expression vector, pSD1, harboring the endoglucanase gene from C. thermocellum under the control of its own promoter, was constructed. Both L. bulgaricus and L. plantarum were electrotransformed with pSD1. The endoglucanase activities of 0.120 and 0.144 U/ml were found in culture media of L. bulgaricus and L. plantarum containing pSD1, respectively. In vitro survival characteristics of the transformed lactobacilli were tested. Both L. bulgaricus and L. plantarum showed a similar resistance to low pH 3. Moreover, L. plantarum was bile-salt resistant in the presence of 0.3 and 1% oxgall. L. bulgaricus and L. plantarum showed a rather homogenous resistant pattern against the tested antibiotics. Both of the strains were resistant to amikacin, gentamicin, streptomycin, kanamycin, and colistin.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2002.11b
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• pp.30-30
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• 2002

꿩의비름 (Sedum erythrostichum)은 매우 우수한 지피식물이며 건조에 강한 대표적 식물로 바위정원 (rock garden)을 가꾸는데 있어서 중요한 수종으로 이용되며, 유럽등지에서는 지붕에 식재하기도 하며 최근에는 빌딩옥상녹화의 대표적 수종으로 식재되고 있다. 또한 한방에서는 경천이라 불리우기도 하는데 피부상처 치유 및 미백효과가 탁월하다고 알려져 있다. 본 연구에서는 Agrobacterium을 매개로한 꿩의비름의 형질전환 시스템을 개발하고 아울러 phosphinothricin-N-acetyltransferase (PAT) 유전자를 도입하여 제초제 저항 식물을 개발하고자 수행되었다. 꿩의비름 잎을 Agrobacterium에 담근후 0.5 mg/l NAA와 2 mg/1 BA가 첨가된 MS 배지에 3일간 공동배앙 하였다. 그 후 300 mg/1 cefotaxime이 첨가된 같은 배지에 옮겨 계대하면서 Agrobacterium을 제거하였다. 약 3주후에 잎 절편으로 부터 직접적으로 부정아가 형성되기 시작 하였는데 이 시기부터 잎 절편을 25 mg/1 kanamycin이 첨가된 선발배지에 옮겨 주었다. 이 결과 배양된 잎 절편 절편 중 3.75%에서 kanamycin에 저항하는 부정아를 얻을 수 있었다. 형질전환체는 X-gluc 반응, PCR, Southern, Nothern analysis를 통하여 확인하였다. 약 94%의 형질전환 식물체는 성공적으로 토양에 옮길 수 있었으며 약 3개월후에 꽃을 피웠다. 형질전환체는 제초제인 Basta ($^{(R)}$ phosphinothricine at 200 mg/1)를 살포하여 주었을 경우 생존함을 확인 하였다.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.430-435
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• 2013

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

• Korean Journal of Medicinal Crop Science
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• v.15 no.4
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• pp.285-290
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• 2007

This study was conducted to introduce phosphinothricin acetyl transferase (PAT) gene, resistant to basta which was non-selective herbicide, into balloon flower (Platycodon grandiflorum A. De. candolle). Seeds were germinated on MS medium, and 10-day-old immature cotyledon explants and 30-day-old leaf explants were cocultured with Agrobacterium tumefaciens strain MP 90 (pBinSyn) on 1/10 MS medium for 48 hours in the dark at $25^{\circ}C$. The cultures were transferred for selection of kanamycin-resistant shoots to the MS medium supplemented with 0.2 $mg/{\ell}$ NAA, 1.0 $mg/{\ell}$ BA, 3% sucrose, 100 $mg/{\ell}$ kanamycin, 500 $mg/{\ell}$ carbenicillin. Shoots were obtained from 10-day-old immature cotyledon explants after 4 weeks of culture. The shoots were subcultured twice every 4 weeks on the same medium for growth of transgenic shoots. Successful transformation was confirmed by histochemical GUS assay, PCR analysis, RT-PCR analysis, 10 $mg/{\ell}$ phosphinothricin treatment and 0.3% basta spray. The basta-resistant transgenic plants flowered normally.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.43-49
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• 2000

An efficient and reliable Agrobacterium transformation procedure based on TDZ (thidiazuron)-induced organogenesis was established and applied to six Brazilian eggp1ant varieties. Optimum transgenic plants recovery was achieved upon the study of the following parameters affecting transformation efficiency, using F-100 variety as a model: i) explant source; ii) pre-culture period; iii) physical state of the pre-culture medium and iv) coculture conditions. The highest frequency of kanamycin-resistant calli derived from leaf explants (5%) was obtained without a pre-culture period and co-cultivation for 24 h in liquid medium followed by five days on solid RM (regeneration medium). For cotyledon explants, best results were achieved upon a pre-culture of 24 h in liquid RM and a co-cultivation period of 24 h in liquid RM followed by three days in solid RM, resulting in a transformation Sequency of 22.7%. Kanamycin-resistant organogenic calli were also obtained from cultivars Emb, Preta Comprida, Round nose Shaded, Campineira and Florida Market. The expression pattern of an epidermis-specific promoter was studied using transformants expressing a chimaeric construct comprised by the promoter Atgrp-5 transcriptionally fused to the coding region of the gus gene. The expression pattern was similar to that previously observed in tobacco and Arabidopsis thaliana, with preferential expression at the epidermis and the stem phloem. These results support the idea that the Atgrp-5 promoter can be used to drive defense genes in these tissues, which are sites of pathogen interaction and spread, in programs for the genetic improvement of eggplant.

• KSBB Journal
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• v.16 no.5
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• pp.480-485
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• 2001

Genetic transformation in dandelion(Taraxacum mongolicum Hand). was studied. We used for transformation by Agrobacterium tumefaciens strian LBA4404 harboring a binary vector pBI121 carrying the CaMV 35S promoter-GUS gene fusion used as a reporter gene and NOS promoter-NPTII gene as a positive selection marker. To obtain transformed plants, leaf explants of dandelion were cocultured with Agrobacterium tumefaciens LBA4404 for 10 mins, then transferred to MS medium containing 1 $\mu$M IAA, 1$\mu$M BA, 100$\mu$g/ML carbenicillin and 50 $\mu$g/ML kanarmycin sulfate. After two weeks of subculture of the explants, Kanamycin-resistant shoots were formed on explants survived. When subjected to GUS histochemical assay, all of the regenerants showed the GUS-positive responses. Plantlets were be be transformed to soil for further growth.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.13-17
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• 2004

To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

• Journal of Plant Biotechnology
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• v.31 no.2
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• pp.175-178
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• 2004

Transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cell lines expressing a human histone H1.5 (referred to as hH1.5), which suppress collagen-induced rheumatoid arthritis, were developed under the oxidative stress-inducible peroxidase (SWPA2) promoter. Tobacco BY-2 cells were transformed by Agrobacterium-mediated method. The kanamycin-resistant calli were selected on the modified MS medium containing 150mg/L kanamycin and 300mg/L claforan. Transgenic cell lines were confirmed by PCR and northern blot analysis. Recombinant hH1.5 (rhH1.5) protein (42 kDa) was also detected by Western blot analysis, showing a different molecular weight of human hH1.5 (32 kDa). These results suggested that a hH1.5 gene was properly introduced in tobacco cultured cells under the control of SWPA2 promoter. The further characterization of rhH1.5 protein remains to be studied.