• Journal of Microbiology and Biotechnology
• /
• 제18권2호
• /
• pp.207-218
• /
• 2008

The impacts of planted transgenic rice varieties on bacterial communities in paddy soils were monitored using both cultivation and molecular methods. The rice field plot consisted of eighteen subplots planted with two genetically modified (GM) rice and four non-GM rice plants in three replicates. Analysis with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were quite similar to each other in a given month, suggesting that there were no significant differences in bacterial communities between GM and non-GM rice soils. The bacterial community structures appeared to be generally stable with the seasons, as shown by a slight variation of microbial population levels and DGGE banding patterns over the year. Comparison analysis of 16S rDNA clone libraries constructed from soil bacterial DNA showed that there were no significant differences between GM and non-GM soil libraries but revealed seasonal differences of phyla distribution between August and December. The composition profile of phospholipid fatty acids (PLFA) between GM and non-GM soils also was not significantly different to each other. When soil DNAs were analyzed with PCR by using primers for the bar gene, which was introduced into GM rice, positive DNA bands were found in October and December soils. However, no bar gene sequence was detected in PCR analysis with DNAs extracted from both cultured and uncultured soil bacterial fractions. The result of this study suggested that, in spite of seasonal variations of bacterial communities and persistence of the bar gene, the bacterial communities of the experimental rice field were not significantly affected by cultivation of GM rice varieties.

• Journal of Microbiology and Biotechnology
• /
• 제17권6호
• /
• pp.905-912
• /
• 2007

A novel ${\beta}-glucosidase$ gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl ${\beta}-D-cellobioside$ (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. BglA included two conserved family 1 glycosyl hydrolase regions. When using $p-nitrophenyl-{\beta}-D-glucoside$ (pNPG) as the substrate, the maximum activity of the purified ${\beta}-glucosidase$ exhibited at pH 6.5 and $55^{\circ}C$, and was enhanced in the presence of $Mn^{2+}$. The $K_m\;and\;V_{max}$ values for the purified enzyme with pNPG were 0.16 mM and $19.10{\mu}mol/min$, respectively. The purified BglA enzyme hydrolyzed both pNPG and $p-nitrophenyl-{\beta}-D-fucoside$. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, BglA was able to convert the major ginsenoside $Rb_1$ into the pharmaceutically active minor ginsenoside Rd within 24 h.

• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• pp.193-199
• /
• 2006

Halotolerant spore-forming Gram-positive bacteria were isolated from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides. The different isolates were characterized genetically using an amplified ribosomal DNA restriction analysis (ARDRA), and phenotypically based on their colonial morphology, physiology, and nutritional requirements. Three different 16S rRNA gene-based genotypes were observed at a 100% similarity using the enzymes HinfI, MspI, and RsaI, and the phenotypic results also followed the ARDRA groupings. Selected strains, representing the different ARDRA groups, were analyzed by 16S rDNA sequencing, and members of the genera Halobaeillus, Virgibacillus, and Oceanobacillus were found. Two isolates showed low 16S rDNA sequence similarities with the closest related species of Halobacillus, indicating the presence of new species among the isolates. The majority of the strains isolated in this study seemed to belong to the species O. iheyensis and were compared using an AP-PCR to determine whether they had a clonal origin or not. Different patterns allowed the grouping of the strains according to Pearson's coefficient, and the resulting dendrogram revealed the formation of two main clusters, denoted as A and B. All the strains isolated from the soil were grouped into cluster A, whereas cluster B was exclusively composed of the strains associated with the B. portulacoides roots. This is the first report on the isolation and characterization of halotolerant spore-forming Gram-positive bacteria that coexist with B. portulacoides. As such, these new strains may be a potential source for the discovery of bioactive compounds with industrial value.

• 한국유기농업학회:학술대회논문집
• /
• 한국유기농학회 2009년도 하반기 학술대회
• /
• pp.302-302
• /
• 2009

유기농업에서 유기물은 양분의 공급, 토양의 이화학성 개선, 토양의 생물학적 건전성 유지 등 중요한 역할을 한다. 토양의 생물학적 건전성은 토양의 생태계적 기능을 지속적으로 유지시키는 토양미생물이 관여하고 있다. 따라서 본 연구는 유기물의 장기연용에 따른 밭토양 미생물의 다양성을 비교 분석하였다. 여러 가지 유기자원을 동일한 기준으로 매년 동일 장소에 처리하였다. 사용된 유기자원은 가축분퇴비, 채종유박인 유기질비료, 볏짚으로만 퇴비화한 볏짚퇴비와 겨울철 휴한기에 헤어리베치를 재배하여 이듬해 봄에 예취한 후 토양에 환원한 녹비처리구, NPK구, 가축분퇴비를 혼용처리한 NPK퇴비군, 양분을 전혀 시용하지 않은 무비구 등 총 7처리구였다. 각각의 처리구에서 토양(0-20 cm)을 채취하여 배양성 토양미생물은 희석평판법으로 해당 선백배지에 시료를 도말 하여 조사하였고 비배양성 미생물은 토양으로부터 genomic DNA를 추출하여 세균의 16S rDNA를 증폭시킨 후 denaturing gradient gel electrophoresis (DGGE)를 수행하여 분석하였다. 주요결과를 요약하면 밭토양에 서식하는 토양미생물의 균수는 처리별간의 차이를 보였으며 유기물처리구가 화학비료처리구보다 높았다. DGGE 분석을 통해 유기물 처리에 따른 군집의 다양성을 살펴본 결과 Fig. 1에서 보는바와 같이 Gel 상에서 다양한 위치의 밴드를 확인할 수 있었고 처리별로 특이 밴드가 있음을 확인할 수 있었다. Fig. 1에서 얻은 DGGE profile상의 밴드 강도와 수를 비교하여 Fig 2와 같은 dendrogram을 나타낼 수 있었다.

• 한국균학회지
• /
• 제43권4호
• /
• pp.267-271
• /
• 2015

제주도 토양에 서식하는 야생효모들의 분포 특성을 조사하기 위해, 동부지역 7곳, 서부지역 5곳에서 토양을 채취하여 효모를 분리한 후, 콜로니 PCR에 의해 증폭된 26S rDNA D1/D2 지역의 염기서열을 결정하여 BLAST를 이용하여 효모를 동정하였다. 동부지역에서 채집한 토양에서 12종에 속하는 20균주를, 서부지역에서 6종에 속하는 13균주를 분리하였다. 분리되는 효모 종은 강수량 차이를 보이는 지역에 따라서 현저한 차이가 있는 것을 알 수 있었다.

• 한국지하수토양환경학회지:지하수토양환경
• /
• 제14권4호
• /
• pp.10-14
• /
• 2009

본 논문은 지열시스템의 설치 시 사용되는 그라우트와 운영에 따른 수온변동이 유발할 수 있는 미생물학적 영향을 실내실험을 통해 살펴보았다. 시료는 지열히트펌프가 아직 가동되지 않는 관정(한방병원)과 지열 히트펌프가 가동 중인 곳(창업보육센터)에서 채취하였다. 실험에 사용한 그라우트는 볼클레이 벤토나이트로 나트륨(Na)계이며 Real-time PCR을 사용하여 각 시료에서 추출된 총 유전자 DNA 중 세균이 가지고 있는 16S rDNA를 정량함으로써 전체 세균의 양을 평가하였다. 실험 결과 지열히트펌프가 가동 중인 지하수에서 총 세균양이 가장 많았으며 벤토나이트를 주입하면서 세균수가 증가하는 경향을 보였다. 한편 그라우트 및 수온영향을 보다 명확하게 파악하기 위해서는 장기적인 현장모니터링이 요구된다.

• 한국토양비료학회지
• /
• 제44권5호
• /
• pp.836-840
• /
• 2011

Cellulase와 xylanase 분비능이 우수한 고온성 부숙촉진 세균을 분리하기 위하여 진주 인근지역의 새송이버섯 재배농장으로부터 새송이버섯 수확 후 배지를 수집하였다. 새송이버섯 수확 후 배지로부터 23종의 균주를 분리하였으며 이 중 cellulase와 xylanase을 동시에 분비하는 균주를 최종 선발하여 SJ21으로 명명하였다. Bacillus ID kit와 VITEK 2 system를 이용하여 분리균 SJ21의 생리적 생화학적 특성을 조사한 결과 분리균 SJ21은 B. lincheniformis와 유사한 특징을 나타내었으며 16S rDNA 염기서열 분석결과에서는 B. subtilis와 99%의 상동성을 나타내었다. 이와 같은 결과를 종합하여 분리균 SJ21은 Bacillus sp. SJ21 로 동정되었다. 분리균이 분비하는 cellulase와 xylanase 활성은 분리균이 증식함에 따라 대수증식기 중반부터 급격히 증가하였고 정지기에 진입하면 효소활성이 더 이상증가하지 않는 것으로 나타났으며 xylanase 활성은 대수증식기 초기부터 지속적으로 증가하여 대수증식기 중반에 최대활성을 나타내었다.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.65.2-65
• /
• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• 분석과학
• /
• 제21권2호
• /
• pp.129-134
• /
• 2008

순환전압전류법과 벗김전압 전류법을 사용한 폭발물(hexahydro-1,3,5-trinitro-1,3,5-triazine, RDX)의 흔적량 분석을 위하여 double-stranded ds calf thymus (DNA)와 카본 나노튜브 혼합 전극을 사용하였으며. 최적 분석 조건을 시험한 결과 0.2 V vs. Ag/AgCl 전위에서 봉우리 전류를 발견하였다, 이 전위를 사용하여 선형분석 농도 범위: 50-75 ug/의 순환전압전류법과, 5-80 ng/L의 벗김 전압 전류법에 도달하였으며, 10 ug/L의 농도에서15번 반복 측정한 상대 표준편차는 0.086% 이었다. 또한 300초의 벗김 분석 조건에서 0.65 ng/L ($2.92{\times}10^{-12}M$) (S/N=3)의 검출 한계에 도달 하였으며, 이 조건에서 폭약에 오염된 토양중의 RDX 흔적량을 분석 응용하였다.

• The Plant Pathology Journal
• /
• 제35권6호
• /
• pp.654-661
• /
• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.