• 환경생물
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• 제17권3호
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• pp.279-284
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• 1999

토양으로부터 직접 추출한 DNA를 cross hybridization하는 방법을 통해서 서로 다른 토양 환경 간에 미생물 군집의 유전형적 유사성과 상대적 다양성을 비교하였다. 그 결과 소나무삼림토양이 다른 토양에 비해 상대적 다양성이 높은 것으로 밝혀졌으며, 경작지, 나지, 초지, 신갈삼림 순으로 다양성 정도를 나타내었다. 또한 유전형적 유사성의 정도에 따른 집괴 분석 결과 소나무삼림과 경작지 토양, 신갈나무삼림과 초지 토양 그리고 나지 등 세 부류로 구분되었다.

• 미생물학회지
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• 제35권1호
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• pp.72-77
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• 1999

BIOLOG GN microplate를 이용한 유일탄소원의 이용능 비교를 통한 대사적 유사성과 16S rDNA 의 PCR 증폭산물의 제한효소 지문 분석에 따른 유전적 유사성을 5종의 식생토양에 따른 토양미생물 군집을 대상으로 비교하였다. 16S rDNA를 증폭하여 제한효소 지문을 분석한 결과, 토양으로부터 직접 추출하여 증폭한 토양세균 군집의 16S rDNA의 유전적 유사도는 BIOLOG GN microplate를 이용한 대사적 구조와 일치하는 경향을 보았다. 그러나 배양된 종속영양세균 군집의 다양성과는 유전적 유사도가 매우 낮게 나타났다.

• Applied Biological Chemistry
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• 제39권5호
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• pp.403-408
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• 1996

토양에서 세균군집의 DNA를 추출하여 이사디 분해세균의 밀도와 군집변화를 tdfA 유전자와 Spa Probe를 이용하여 조사하였다. 이사디 분해균주인 Pseudomonas cepacia/pJP4을 토양에 여러 가지 밀도로 접종한 후 추출된 토양세균군집의 DNA를 Southern blot에서 분석한 결과, 본 실험에 사용된 DNA probe method에 의해 이 세균을 $10^5\;cells/g$ soil 수준까지 검출할 수 있는 것으로 나타났다. 이사디를 가해준 microcosm 토양에서 추출된 세균군집의 DNA를 분석한 실험에서는 Pseudemonas pickettii와 Sphingomonas Paucimobilis가 우점종으로 검출되었고, 사용된 두 가지의 DNA probes는 토양의 이사디 분해미생물에 대해 매우 높은 특이성을 가지고 있는 것으로 나타났다. 밭에 이사디를 장기 적으로 가해준 후 추출된 토양세균군집의 DNA를 분석 한 실험에서는 이사디를 최소한 10 ppm 이상 가해주어야 토양의 이사디 분해세균을 DNA probe method에 의해 검출할 수 있었고, tfdA 유전자는 실제의 밭토양에서도 높은 특이성을 나타냈으나 Spa probe는 일부의 토착세균에 비특이적으로 반응하는 것으로 나타났다. 토양에서 추출된 세균군집의 DNA를 분석하는 DNA probe method는 Southern blot과 함께 사용되었을 때 토양에 존재하는 이사디 분해미생물을 실험실 배지에 배양하지 않고 검출할 수 있었고,이 미생물들의 밀도, 군집변화, 유전적 변화 등을 효과적으로 분석할 수 있는 것으로 나타났다.

• Journal of Microbiology
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• 제34권3호
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• pp.229-235
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• 1996

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

• 한국지하수토양환경학회지:지하수토양환경
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• 제14권3호
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• pp.57-67
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• 2009

토양미생물은 토양 및 퇴적토 환경에서 생물학적 물질순환의 중요한 역할을 맡고 있다. 이러한 중요성으로 인해 토양미생물 생태 다양성과 군집구성이 토양 및 퇴적토 생태환경과 자연저감능력을 평가하는 지표로 유용하게 쓰일 수 있다. 보다 정확한 다양성과 군집구조 분석을 위해서는 다양한 토양 및 퇴적토 시료로부터 분석에 필요한 DNA수율과 순도를 확보해야 한다. 지금까지 토양 및 퇴적토에서 DNA추출하는 방법들에 대한 다양한 기초연구가 이루어졌지만, 실제 환경생태영향평가와 분해기능평가에 사용시 DNA추출방법 선정에 대한 지침 및 정보는 매우 부족하다. 이에 본 연구에서는 문헌조사를 통해서 다양한 방법들의 토양 및 퇴적토 내 미생물 DNA 추출 특성을 비교 분석하고, 현재 주로 사용되고 있는 DNA추출방법을 토양 및 퇴적토 환경평가나 오염지하수토양 자연저감능평가에 활용 할 경우 고려해야 할 기술적 사항에 대해 고찰하였다. 본 연구를 통해 하나의 특정 추출 방법으로는 미생물다양성, 군집구성 및 개체간 상호작용에 대한 정보를 얻기에는DNA양과 순도 차원에서 충분하지 않으며, 토양 및 퇴적토 미생물 군집분석의 목적에 따라 적합한 방법의 선택이 매우 중요함을 알 수 있었다.

• 한국지하수토양환경학회:학술대회논문집
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• 한국지하수토양환경학회 2005년도 총회 및 춘계학술발표회
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• pp.28-30
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• 2005

Because of high population diversity in soil microbial communities, it is difficult to accurately assess the capability of biodegradation of toxicant by microbes in soil and sediment. Identifying biodegradative microorganisms is an important step in designing and analyzing soil bioremediation. To remove non-important noise information, it is necessary to selectively enrich genomes of biodegradative microorganisms fromnon-biodegradative populations. For this purpose, a stable isotope probing (SIP) technique was applied in selectively harvesting the genomes of biphenyl-utilizing bacteria from soil microbial communities. Since many biphenyl-using microorganisms are responsible for aerobic PCB degradation In soil and sediments, biphenyl-utilizing bacteria were chosen as the target organisms. In soil microcosms, 13C-biphenyl was added as a selective carbon source for biphenyl users, According to $13C-CO_2$ analysis by GC-MS, 13C-biphenyl mineralization was detected after a 7-day of incubation. The heavy portion of DNA(13C-DNA) was separated from the light portion of DNA (12C-DNA) using equilibrium density gradient ultracentrifuge. Bacterial community structure in the 13C-DNAsample was analyzed by t-RFLP (terminal restriction fragment length polymorphism) method. The t-RFLP result demonstates that the use of SIP efficiently and selectively enriched the genomes of biphenyl degrading bacteria from non-degradative microbes. Furthermore, the bacterial diversity of biphenyl degrading populations was small enough for environmental genomes tools (metagenomics and DNA microarrays) to be used to detect functional (biphenyl degradation) genes from soil microbial communities, which may provide a significant progress in assessing microbial capability of PCB bioremediation in soil and groundwater.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.236-242
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• 2003

A culture-independent and -dependent survey of the bacterial community structure in the rhizosphere and soil samples from hot pepper plants was conducted using 16S rDNA clone library and FAME analyses. Out of the 78 clones sequenced, 56% belonged to Proteobacteria, 4% to high G+C Gram- positive group, 3% to Cytophyga-Flexibacter-Bacreroides, and 32% could not be grouped with any known taxonomic division. Among the 127 FAME isolates identified, 66% belonged to low G+C Gram-positive bacteria (Baciilus spp.) and 26% to high G+C Gram-positive bacteria. In a cluster analysis, the results for both methods were found to be strikingly dissimilar. The current study is the first comparative study of FAME and 165 rDNA clonal analyses performed on the same set of soil, rhizosphere soil, and root samples.

• Journal of Ginseng Research
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• 제40권2호
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• pp.176-184
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• 2016

Background: Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods: The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results: In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion: This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

• 미생물학회지
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• 제39권3호
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• pp.187-191
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• 2003

토양 시료와 Coli-spot 한천평판 배지를 이용하여 myxobacteria를 분리하였다. 용균 현상이 관찰되는 Coli-spot 한천평판에서 myxobacteria의 swarm및 자실체 형성 여부를 확인하고, 확인된 자실체를 분리하여 VY/2 한천평판 배지에서 순수배양을 실시하였다. 분리 균주의 동정을 위하여 myxobacteria표준 균주 및 토양에서 분리한 균주들의 16S리보좀 DNA를 중합효소 연쇄 반응을 통해 증폭시킨 다음, 제한효소(HaeIII, EcoRI 및 EcoRV)로 절단하여 RFLP 양상을 비교하였다. 그 결과, 토양에서 분리한 균주들이 Family I, II, III의 myxobacteria에 속하는 것을 확인하였다.

• 생명과학회지
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• 제15권6호
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• pp.851-856
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• 2005

Although valuable microbes have been isolated from the soil for the various productions of useful components, the microbes which can be cultivated in the laboratory are only $0.1-1\%$ of all microbes. To solve this problem, the study has recently been tried for making the valuable components from the environment by directly separating unculturable micrbial DNA in the soil. But it is known that humic acid originated from the soil interrupts various restriction enzymes and molecular biological process. Thus, in order to prevent these problems, this study modified the method separated soil DNA with phenol, CTAB and PEG. In order to compare the degree of purity for each DNA and the molecular biological application process, $A_{260}/A_{280}$ ratio, restriction enzymes, and PCR were performed. In case of DNA by the modified method, total yield of DNA was lower but $A_{260}/A_{280}$ ratio was higher than the previously reported methods. It was confirmed that the degree of purity is improved by the modified method. But it was not cut off by all kinds of tested restriction enzymes because of the operation of a very small amount of interrupting substances. When PCR was operated with each diluted DNA in different concentrations and GAPDH primer, the DNA by the modified method could be processed for PCR in the concentration of 100 times higher than by the previously reported separation method. Therefore, this experiment can find out the possibility of utilization for the unknown substances by effectively removing the harmful materials including humic acid and help establishing metagenomic DNA library from the soil DNA having the high degree of purity.