• Applied Biological Chemistry
• /
• v.47 no.2
• /
• pp.182-186
• /
• 2004

Morphological observation of roots and molecular technique were used to investigate the symbiotic relationships between arbuscular mycorrhizal (AM) fungi and ginseng roots. Korean ginseng, Panax ginseng, was collected from 8 sites in Korea. Colonization pattern of AM fungi in ginseng roots was determined as an Arum type under light microscopes. Nested PCR using AM fungal specific primers was employed to amplify a partial region on 18s rDNA of AM fungi from the root extracted mixed DNA. The amplified DNA was cloned and analyzed by random fragment length polymorphism (RFLP) with restriction enzymes, AluI, HinfI and AsuC21. One from each RFLP pattern was selected for sequencing. A total 16 clones were sequenced and identified as 2 species of AM fungi; Paraglomus brasilianum and Glomus spurcum. Paramglomus brasilianum was found from most of the ginseng roots, in this syudy suggesting that this species of AM fungi could have specific relationship with the ginseng root. Possible roles of AM fungal species in ginseng roots are discussed.

• Journal of Microbiology
• /
• v.44 no.2
• /
• pp.155-161
• /
• 2006

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

• Korean Journal of Microbiology
• /
• v.37 no.2
• /
• pp.137-144
• /
• 2001

In order to investigate the diversity of bacterial community in the mud flat of Sunchon Bay, Chunnam province, diversity of amplified 16S rDNA was examined. Total DNA was extracted from sediment soils and 16S rDNAs were amplified using PCR primers based on the universally conserved sequences in bacteria. Clonal libraries were constructed and 111 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII. Clones were clustered based on restriction patterns using computer program, GelCompar II. One hundred different RFLP types were detected from 111 clones. The 20 clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the bacterial diversity in the clone libraries. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA databases and GenBank. All sequences showed between 77 and 96.8% similarity to the known 16s rRNA sequence from cultured organisms. The 20 clones sequenced fell into seven major lineages of the domain Bacteria: alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga) and Cyanobacteria (chloroplast). Among the clones, the Proteobacteria were dominant.

• The Plant Pathology Journal
• /
• v.15 no.1
• /
• pp.28-33
• /
• 1999

Phytophthora root rot of Chniese cabbage and spinach is reported for the first time in Korea. The diseases ocurred at Yangju, Seosan and Yeocheon in Korea from 1995 through 1998, mainly in lowland and submerged areas. Symptoms consisted of stunt, yellows, wilt and eventual death due to root rot. Fourteen isolates collected from naturally infected plants were all identified as P. drechsleri based on mycological characteristics. PCR-RFLP analysis of rDNA of the isolates confirmed the above result, since the restriction band patterns of the small subunit and internal transcribed spacers were identical to P. drechsleri and P. cryptogea, but distinct from closely related species of P. erythroseptica, P. cambivora, P. sojae and P. megasperma. The pathogen showed strong pathogenicity to Chinese cabbage, moderate to spinach, radish, cabbage and tomato, and weak or none to brown mustard, kale, chicory and pepper in pathogenicity tests.

• Journal of Animal Science and Technology
• /
• v.46 no.3
• /
• pp.295-300
• /
• 2004

The identification of the leptin gene in 1994 and it's adipocytes specific protein leptin hal provided the first physiological links to the regulatory system controlling body weight and fat deposits. The meat tastes is mainly determined by quantifY and quality of triglyceride stored in adipose tissue. This study was conducted to analyze genetic cbaracteristics of Hanwoo leptin gene and also to investigate the association of DNA marlcer with some economic meat traits for Hanwoo. The leptin hormone gene polymorphisms were identified by digestion with Kpn2 I and Msp I. Slaughter weight(SWI), slaughter peroentage(SP), longissimus muscle area(LMA), beef marbling score(MS) and back fat thickness(BF) were compared among three genotypes by P(R..RFlJ> and showed significant differences among genotypes. PCR-RFLP(Kpn2 I) were detected significant for SP, MS and BF. The allele was essociated with fatter carcasses and C allele with leaner carcasses.

• Parasites, Hosts and Diseases
• /
• v.40 no.1
• /
• pp.25-31
• /
• 2002

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involved the use of PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa 1. Unique RFCP of 17 strains in group II by Dde I. Taq I and Hae III were classified into: (1) four taxa that were identifiable at the species level. (2) a subgroup of 4 taxa and a pair of 2 taxi that were identical with each other. and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with those obtained by 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or a large number of environmental isolates of Acanthamoeba.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.4
• /
• pp.624-630
• /
• 2008

Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.

• Mycobiology
• /
• v.30 no.1
• /
• pp.13-17
• /
• 2002

The genomic DNAs were extracted from roots of Glycine max and Sorghum bicolor, and compared with those from spores of two arbuscular mycorrhizal(AM) fungi, Glomus mosseae and Scutellospora heterogama. The partial small subunit(SSU) of ribosomal RNA genes were synthesized and amplified by polymerase chain reaction with the fungal specific primers, AM1 and NS31. By the recent molecular techniques, the presence of another AM fungal DNA were confirmed in the roots of two plants, and three sequences of rDNA fragments amplified were identified to be close to those of G. caledonium, G. fasiculatum and G. proliferum. The two AM fungi, both, were found to colonize at the cortical layers of plant roots collected in the fields, together.

• Journal of Korean Society on Water Environment
• /
• v.22 no.4
• /
• pp.590-598
• /
• 2006

In-situ Air sparging (IAS) is a groundwater remediation technique, in which organic contaminants volatilize into air form the saturated to vadose zone. This study was carried out to evaluate the effect of sludge and soil microbial community structure on air sparging of Total Petroleum Hydrocarbons (TPH) contaminated groundwater soils. In the laboratory, diesel (10,000 mg TPH/kg) contaminated saturated soil. The Air was injected in intermittent (Q=1500 mL/min, 10 minute injection and 10 minute idle) modes. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for experiment with sludge soil samples that were closely related to Agrococcus, Flavobacterium, Thermoanaerobacter, Flexibacter and Shewanella, etc, in the clone library. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil the fate of microorganisms in natural microbial community.

• Journal of Korean Society on Water Environment
• /
• v.24 no.1
• /
• pp.19-29
• /
• 2008

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air ($34.9{\pm}2.7^{\circ}C$) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, ${\gamma}$-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.