• Title/Summary/Keyword: PCR 동정

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A Study on Microbial Community and Microbial Degradation of Diesel (디젤의 미생물 분해와 군집에 관한 연구)

  • Choi, Hee-Chol;Cho, Yoon-A;Choi, Sang-Il;Lee, Tae-Jin
    • Journal of Korean Society of Environmental Engineers
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    • v.32 no.5
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    • pp.509-516
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    • 2010
  • This study investigates characteristics of diesel degradation and variations of microbial community with the soil enrichment cultures. The cultures has yellow(YE-5) and transparent color's(WH-5) colony on solid plate medium. The bacillus type of YE-5 and WH-5 cultures showed diesel degradation at the rate of 99.07mg-Diesel/$L{\cdot}day$ and 57.82mg-Diesel/$L{\cdot}day$ in the presence of 1%(v/v) initial diesel concentration. Diesel degradation was 1.7 times faster than WH-5 culture. YE-5 or WH-5 culture could degrade a wide range of diesel compounds from $C_8$ to $C_24$. Microbial community analysis by PCR-DGGE technique shows that Psedomonas, Klebsiella, Escherichia and Stenotrophomonas as proteobacteria take role on the diesel degradation. uncultured Senotrophomonas sp. was only detected with YE-5 culture. It is concluded that proper combination of the microorganism should be present to stimulate the degradation of diesel and further studies are recommended for the effect of uncultured Senotrophomonas sp. or Escherichia hermannii on diesel degradation.

Isolation and Characterization of Actinomycete Strain BK185 Possessing Antifungal Activity against Ginseng Root Rot Pathogens (인삼 뿌리썩음병균에 항균활성이 있는 방선균 BK185의 분리 및 특성)

  • Kim, Byung-Yong;Bae, Mun-Hyung;Ahn, Jae-Hyung;Weon, Hang-Yeon;Kim, Sung-Il;Kim, Wan-Kyu;Oh, Dong-Chan;Song, Jaekyeong
    • The Korean Journal of Pesticide Science
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    • v.18 no.4
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    • pp.396-403
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    • 2014
  • Ginseng (Panax ginseng C. A. Meyer) is an economically valuable pharmaceutical crop in Korea. In order to find promising biocontrol agents for soil-borne fungal pathogens which infect ginseng roots, we have isolated actinomycete, BK185 from soil. The isolate was investigated for the antifungal activity against to ginseng rot pathogens prior to testing genetic and chemical properties. The strain was identified as Streptomyces sp. using phylogenetic analysis based on 16S rRNA gene sequence. The most closely related species was S. sporoclivatus and S. geldanamycininus with high similarities (>99%). The isolate, BK185 showed positive reaction for PCR detection targeting biosynthetic gene clusters of PKS (Type-I polyketide synthase) and NRPS (Non-ribosomal polypeptide synthetase) genes. Major metabolite from the BK185 was analyzed by The LC/MS and identified to geldamycin, which was known to contained broad antibacterial, antifungal or anticancer activities. The results provide evidences that the strain, BK185 can be promising biocontrol agent for ginseng organic farming.

Isolation and Characterization of Soil Bacteria Degrading a Fungicide Defenoconazole (살균제 디페노코나졸 분해 세균 분리 및 특성 분석)

  • Ahn, Jae-Hyung;Ro, Yu-Mi;Lee, Gwan-Hyeong;Park, InCheol;Kim, Wan-Gyu;Han, Byeong-Hak;You, Jaehong
    • The Korean Journal of Pesticide Science
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    • v.20 no.4
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    • pp.349-354
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    • 2016
  • Triazole fungicides occupy an important portion in the global fungicide market and are relatively persistent in soil compared to the other fungicides, suggesting possible adverse effects of the fungicides on human health and environment. In this study, we tried to isolate microorganisms from orchard soils, which can decompose the triazole fungicides, tebuconazole, fluquinconazole, and difenoconazole. Only difenoconazole was completely degraded in the enrichment culture, from which several difenoconazole-degrading bacteria were isolated. They showed the same rep-PCR pattern thus only one strain, C8-2, was further studied. The strain was identified as Sphingomonas sp. C8-2 based on its 16S rRNA gene sequence and decomposed 100 mg/L of difenoconazole in a minimum medium to an unknown metabolite with a molecular weight of 296 within 24 hours. The inhibition effect of the metabolite against representative soil microorganisms significantly decreased compared to that of difenoconazole thus the bacterial strain is expected to be used for the detoxification of difenoconazole in soil and crop.

A Paleogenetic Analysis of Human Skeletal Remains from the Myeongam-ri Site, Asan in Korea (아산 명암리 출토 인골의 고유전학적 연구)

  • Jee, Sang-Hyun;Kim, Yun-Ji;Chung, Yong-Jae;Seo, Min-Seok;Pak, Yang-Jin
    • Journal of Conservation Science
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    • v.23
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    • pp.81-93
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    • 2008
  • The analysis of ancient DNA (aDNA) in paleogenetics has become an increasingly important subject of archaeological, anthropological, biological as well as public interest. In this study, paleogenetic analyses were carried out on the human skeletal remains from a historical cemetery site in Myeongam-ri, Asan, Korea. Archaeological records show that this particular location had been used as a habitation or mortuary site as early as the Bronze Age and up until the Joseon Dynasty. Human remains of twenty individuals out of forty-nine tombs from the Goryeo to Joseon Dynasty were selected for the analysis of this study. In order to identify the genealogy of the population and traditional burial pattern of the cemetery, we conducted comparative analyses of the hyper variable regions (HVRs) in mitochondrial DNA (mtDNA) of each sample. A number of cautious steps were taken at all experimental stages in order to avoid erroneous recombination by the segmental and modern contaminations derived from the researchers. We sequenced segmental amplicons of HVRs andassigned relevant haplogroups according to the sequence polymorphism on the basis of the known mtDNA database. The result shows that diverse haplogroups were unexpectedly present in the small population group of the Myeongam-ri site. This diversity appears to be related to the geographical conditions and archaeological properties of the Myeongam-ri site.

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Isolation and characterization of lactic acid bacteria for use as silage additives (사일리지 제조를 위한 유산균 탐색 및 특성연구)

  • Ro, Yu-Mi;Lee, Gwan-Hyeong;Park, InCheol;Kim, Wan-Gyu;Han, Byeong-Hak;You, Jaehong;Ahn, Jae-Hyung
    • Korean Journal of Microbiology
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    • v.52 no.4
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    • pp.444-454
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    • 2016
  • Sixteen lactic acid bacterial strains were isolated from silage and cow dung samples, and characterized to identify their potential as silage additives. They were identified as the members of the genera Lactobacillus, Enterococcus, and Weissella, and clustered into nine groups based on the sequences of the genes for 16S rRNA, RNA polymerase alpha subunit, 60-kDa heat shock protein, and phenylalanyl-tRNA synthase alpha subunit. Among them, the three strains which were genetically similar to L. plantarum showed the fastest growth and pH decrease in MRS and rye extract media, the highest numbers of available carbohydrates, and the widest ranges of pH, temperature, and salinity for growth. In addition, they showed no amplified DNA products in the PCR examination targeting the genes for the production of biogenic amines, and the MRS media where they had been cultured showed relatively high inhibition effect against the growth of silage-spoiling microorganisms, including fungi, yeast, and clostridia. The results suggest that these strains are good candidates for silage additives. However, the rye extract media where the lactic acid bacteria had been cultured had no effect on or stimulated the growth of the silage-spoiling microorganisms, and the causes must be established for the practical use of the lactic acid bacteria as silage additives.

Identification and Molecular Characterization of Methionine Sulfoxide Reductase B Gene in Rice Blast Fungus, Magnaporthe oryzae (벼도열병균에서의 methionine sulfoxide reductase B 유전자의 분자적 특성)

  • Kim, Jeong-Hwan;Kim, Jin-Soo;Jeong, Mi-Yeon;Choi, Woo-Bong
    • Journal of Life Science
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    • v.19 no.3
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    • pp.343-348
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    • 2009
  • Magnaporthe oryzae, a major cause of rice blast, is one of the most destructive plant fungal pathogens. Secretion of reactive oxygen species (ROS) during the infection phase of plant pathogenic fungus plays a key role in the defense mechanism of a plant. ROS causes oxidative damage and functional modification to the proteins in a pathogenic fungus. Methionine, especially, is a major target of ROS, which oxidizes it to methionine sulfoxide. To survive from the attack of ROS, plant pathogenic fungus has antioxidative systems - one example would be methionine sulfoxide reductase B (MSRB), which reverses the oxidative alteration of methionine to methionine sulfoxide. In the present study, identification and molecular characterization of the MSRB gene in M. oryzae KJ201 were investigated. The MSRB gene was amplified by PCR from the M. oryzae KJ201 genomic DNA. The copy number of MSRB in the genome of M. oryzae KJ201 was identified by Southern blot analysis, which revealed that the gene exists as a single copy. To study the molecular function of an MSRB gene, the expression level of the MSRB gene was assayed with hydrogen peroxide treatment by Northern blot analysis and RT-PCR. The expression of the MSRB gene was increased by treatment of hydrogen peroxide, without significant correlation to hydrogen peroxide concentrations. These results indicate that the MSRB gene in M. oryzae KJ201 could contribute to protection against plant defense compounds such as ROS and offer a novel strategy for the control of rice blast.

Potential Probiotic Properties of Exopolysaccharide Producing Lactic Acid Bacteria Isolated from Fermented Soybean Product (장류유래 Exopolysaccharide 생성 유산균의 잠재적 Probiotic 특성)

  • Ahn, Yu-Jin;Choi, Hye-Sun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.5
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    • pp.749-755
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    • 2014
  • Exopolysaccharides (EPSs) have been widely used in the food industry as viscofying, stabilizing, and emulsifying agents as well as in the pharmaceutical industry for their immunomodulatory, anti-tumor, and anti-inflammatory effects. A total of 458 lactic acid bacteria (LAB) strains isolated from several kinds of soybean pastes were screened for the production of homo-EPS (HoPS). LAB isolates were primarily screened using thin layer chromatography (TLC) and further screened polymerase chain reaction (PCR) targeting genes involved in HoPS production. Six LAB isolates producing high amounts of HoPS were identified by TLC. Among these isolates, glucansucrase gene was amplified in two strains (JSA57, JSB22), whereas the fructansucrase gene was detected in three strains (JSA57, JSB22, JSB66). After isolating the strains, their morphological characteristics and 16S rDNA sequences were determined. Six species were identified as L. alimentarius HSB15, L. plantarum JSA22, L. pentosus JSA57, L. brevis JSB22, L. alimentarius JSB66, and L. parabrevis JSB89. To evaluate the potential probiotic properties of these LAB, their survival rates against a simulated intestinal environment were determined. After 2 hr of incubation in artificial gastric juice, survival rates of JSA57, JSB90, JSB22, and JSB66 were all greater than 50%. After 2 hr of incubation in bile juice, viable cell count of JSB22 was similar with initial vial cell counts. Growth of the six LAB was screened in arabino-oligosaccharide (AOS)-containing MRS broth. Results showed that growth of the isolates selectively increased after culture in AOS-containing media. Strain JSB22 (6 hr), JSB66 (6 hr), HSB15 (20 hr), and JSA22 (29 hr) showed maximum growth rate. Especially, JSB22 showed the highest growth rate. These results suggest that EPS-producing LAB isolated from Deonjang could be applied as synbiotics.

Antibiotic Resistance and Plasmid Profile of Vibrio parahaemolyticus Strains Isolated from Kyunggi-Incheon Coastal Area (경기인천 연안에서 분리된 장염비브리오균의 항생제 내성 및 플라스미드 보유 현황)

  • Han, A-Rheum;Yoon, Young-June;Kim, Jung-Wan
    • Korean Journal of Microbiology
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    • v.48 no.1
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    • pp.22-28
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    • 2012
  • Vibrio parahaemolyticus is one of the major agents responsible for food poisoning during summer in Korea, which is transmitted via seawater or seafoods. Recently, distribution of the bacteria in the marine environment has been increased due to global warming. Great concern also has been raised regarding public hygiene as well as marine culture by the emergence of pathogens with antibiotic resistance. Therefore, distribution of V. parahaemolyticus and antibiotic resistance of the isolates were monitored in 7 coastal areas of Kyonggi Province and Incheon by sampling seawater, fishes and clams monthly. V. parahaemolyticus was detected from 47.7% of 966 samples (seawater 61.9%, seafoods 41.8%) analyzed using $CHROMagar^{TM}$ and TCBS agar plates as well as multiplex PCR. Among 13 antibiotics tested, resistance to vancomycin and ampicillin was observed in 97.3% and 87.3% of the isolates, respectively, and the ratios of them resistant to cephalothin (48.8%) and rifampin (46.1%) were also high. The isolates were most highly sensitive to chloramphenicol (91.7%) and trimethoprim-sulfamethoxazole (91.8%). The ratio of sensitivity for other antibiotics was also high in the descending order of gentamycin (82.3%), tobramycin (74.8%), nalidixic acid (71.6%), tetracyclin (69.4%), cefotaxime (63.0%). About 69% of the isolates showed multiple drug resistance toward 3 antibiotics including vancomycin and ampicillin. Two of them exhibited resistance for 11 antibiotics used in this study. Plasmid profile analysis of the isolates with antibiotic resistance revealed that 55.1% of them retained plasmids of 24 different types. However, no clear inter-relationship between the resistance and the plasmid profile has been observed.

Characterization of Nitrile-hydrolyzing Enzymes Produced from Rhodococcus erythropolis (니트릴 분해효소 생산균인 Rhodococcus erythropolis의 발굴 및 효소 특성 연구)

  • Park Hyo-Jung;Park Ha-Joo;Uhm Ki-Nam;Kim Hyung-Kwoun
    • Microbiology and Biotechnology Letters
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    • v.34 no.3
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    • pp.204-210
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    • 2006
  • Ethyl (S)-4-chloro-3-hydroxybutyrate is a useful intermediate for the synthesis of Atorvastatin, a chiral drug to hypercholesterolemia. In this research, two 4-chloro-3-hydroxybutyro-nitrile-degrading strains were isolated from soil sample. They were identified as Rhodococcus erythropolis strains by 16S rRNA analysis. The nitrile-degrading enzyme(s) were suggested to be nitrile hydratase and amidase rather than nitrilase from the result of thin layer chromatography analysis. The corresponding genes were obtained by PCR cloning method. The predicted protein sequences had identities more than 96% with nitrile hydratase ${\alpha}-subunit$, nitrile hydratase ${\beta}-subunit$, and amidase of R. erythropolis. The 4-chloro-3-hydroxybutyronitrile-hydrolyzing activities in both strains were increased dramatically by ${\varepsilon}-caprolactam$ which was known as good inducer for nitrile hydratase. Both intact cells and cell-free extract could hydrolyze the nitrile compound. So, the intact cell and the enzymes could be used as potential biocatalyst for the production of 4-chloro-3-hydroxybutyric acid.

Isolation and Characterization of Eukaryotic Translation Initiation Factor 5A (eIF-5A) from Potato (감자로부터 Eukaryotic Translation Initiation Factor 5A (elF-5A) 유전자의 동정 및 발현 분석)

  • 인준교;신동호;최관삼;양덕춘
    • Korean Journal of Plant Tissue Culture
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    • v.28 no.5
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    • pp.283-287
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    • 2001
  • Differential display based on PCR was employed to identify genes expressed during tuber-developing stage of potato (Solanum tuberosum L. cv. Irish Cobbler). An eukaryotic initiation factor 5A (eIF-5A) clone isolated from a cDNA library constructed with developing micro-tuber using a probe of PCR fragment. We isolated three positive clones and ore of them contained open reading frame. This clone revealed high sequence similarity to tomato eIF 5A cDNA. At the DNA level, there is 94.8% identity with the tomato eIF-5A4, whereas at the protein level there is a high identity with 97.5%. The potato eIF 5A clone is 716 bp in length and contains a single open reading frame from 57 to 539 bp, a 56 bp 5'-untranslated region and a 177 bp 3'-untranslated region. The deduced protein composed of 160 amino acid residues, with a predicted molecular mass of 17.4 kD and an estimated pl of 5.5. The sequence of 12 (STSKTGKHGHAK) amino acids among eIF-5A proteins is perfectly conserved from yeast to human. That sequence in potato eIF-5A protein is also conserved at position 46 to 57 amino acid. This region embeds the post-translational modification site of the lysine residue (at the seventh K) to hypusine that is crucial to eIF-5A activity. The northern blot analysis of eIF5A has shown abundant expression, mainly in flower organs (stamen, ovary, petal, sepal), fruit and stolen.

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