Culture independent analysis of bacterial community diversity associated with coastal sanddune plant

소개글

사구식물분리 미생물연구, PCR, DGGE, cloning.

목차

1. Introduction

2. Materials and Methods
2.1 Clone analysis
2.1.1 Sampling of rhizosphere soil and plants root soil
2.1.2 DNA extraction
2.1.3 PCR amplification and cloning of 16S rDNA
2.1.4. ARDRA analysis
2.1.5 Sequencing and phylogenetic analysis
2.2 DGGE analysis
2.2.1 Sampling of rhizosphere soil and plants root soil
2.2.2 DNA extraction
2.2.3 16S V3 region PCR
2.2.4 DGGE
2.2.5 Reamplification and Sequencing 16S rDNA
2.2.6 Phylogenetic analysis

3. Results
3.1 Clone analysis
3.1.1 Construction of 16S rDNA Clone Library
3.1.2 Whole community ARDRA patterns of the different soils and season.
3.1.3 Identification of ARDRA types
3.2 DGGE analysis
3.2.1 DGGE fingerprints and Phylogenetic analysis of 16S rDNA sequences.
3.2.2 Specific DGGE patterns of Lysobacter sp. relatives from the sand dune soil samples.
3.3 Comparison of ARDRA analysis and DGGE analysis

4. Discussion
영문 Abstract

5. References
국문 초록

본문내용

Abstract


Increasing dependence on molecular methods to analyze community structures have made molecular biological techniques including DNA extraction and purification from environmental samples very important part of ecological studies.
In this study, the bacterial community structure of sand dune rhizosphere soil and plant root was investigated by use of the rRNA based approach. Denaturing gradient gel electrophoresis (DGGE) with eubacterial primer (GC341F) and universal primer (518R) and cloning analysis of community 16S ribosomal DNAs amplified with universal bacterial primer sets were used to describe the geographical and seasonal succession of the bacterial community structure along the Tae-an area, Chungnam in 2003 and 2004. Root and rhizosphere soil samples were collected from 4 location and 9 plant species. The DGGE analyses revealed that all of the rhizosphere and root samples possessed an apparently common band, which was identified as that of Lysobacter sp. by sequencing. The genus Lysobacter is a member of the family Xanthomonadaceae, class Gammaproteobacteria.
DGGE analysis was carried out to compare diversity among various sets of samples. The analysis of bands from roots and rhizosphere of 9 plant species indicated that the bacterial composition was taxonomically diverse, and could be grouped in to 5 bacterial phyla. Members of Gammaproteobacteria were predominant.

참고 자료

Muyzer, G, C. Ellen and U, Andre G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reation-Amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59,695-700

Miller, M. R., C. J. Linton, D. Glancy, M. Hall, and H. Jalal. (1996). Application of 16S rRNA gene PCR to study bowel flora of preterm infants with or without necrotizing enterocolitis. J. Clin. Microbiol. 34, 2
506-2510.

Schabereiter-Gurtner, C., S. Maca, S. Rölleke, K. Nigl, J. Lukas, A. Hirschl, W. Lubitz, and T. Barisani-Asenbauer. (2001). 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting. Investig. Ophthalmol. Vis. Sci. 42, 1164-1171.

Temmermann, R., I. Scheirlinck, G. Huys, and J. Swings. (2003). Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 69:220-226.

Charlotte E. D., Katja E. H., Melanie J. W., Phil S. ,C., Michael H., Keith G. H., and David W. T. (2004). Use of 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis for Analysis of the Microfloras of Healing and Nonhealing Chronic Venous Leg Ulcers. J Clin Microbiol. 42(8), 3549–3557.

Muyzer, G., S. Hottentrager, A. T., and C. Wawer. (1996). Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA: a new approach to analyze the genetic diversity of mixed microbial communities. p.1-23. In A. D. L. Akermanns, J. D. van Elsas, and F. J. de Bruijn (ed.), Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Chun, J (1995). Computer-assisted classification and identification of

Atlas R.M., Sayler G., Burlage R.S., Bej A.K. (1992) Molecular approaches for environmental monitoring of microorganisms. Biotechniques.12, 706.

Head I.M., Saunders J.R., Pickup R.W. (1998) Microbial evolution, diversity, and ecology—A decade of ribosomal RNA analysis of uncultivated microorganisms. Microb. Ecol. 35, 1-21

Muyzer, G., S. Hottentrager, A. Teske, and C. Wawer. (1996) Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA-A new molecular approach to analyse the genetic diversity of mixed microbial communities, p.3.4.4:1–23. In A. Akkermans, et al. (ed.) Molecular microbial ecology manual. Kluwer Academic Publ., Nowell, MA.

Ferris M.J., Muyzer G., Ward D.M. (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62, 340-346

Torsvik V., Daae F.L., Sandaa R.A., Øvres L. (1998) Novel techniques for analysing microbial diversity in natural and perturbed environments. J. Biotech. 64, 53-62

Teske A., Wawer C., Muyzer G., Ramsing N.B. (1996) Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appl. Environ. Microbiol. 62, 1405-1415.

Ferris M.J., Ward D.M. (1997) Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 63, 1375-1381.

Øvreas L., Forney L., Daae F.L., Torsvik V. (1997) Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl. Environ. Microbiol. 63,3367-3373

Glick, B. R. (1995) The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41,109-117.

Jeon, J. S., S. S. Lee, H. Y. Kim, T. S. Ahn and H. G. Song. (2003). Plant growth promotion in soil by some Inoculated microorganisms. J. Microbiol. 41,271-276.

Lucy, M., E. Reed. and R. G. Bernard. (2004). Applications of free living plant growth-promoting rhizobacteria. Antonie Leeuwenhoek. 86,1-25

Mantelin, S. and B. Touraine. (2004). Plant growth-promoting bacteria and nitrate availability; impacts on root development and nitrate uptake. J. Exp. Bot. 55,27-34.

Singh, U. P., B. K. Sarma, and D. P. Singh. (2003). Effect of plant-promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cier arietinum). Curr. Microbiol. 46,131-140.

Park et al. (2005). Isolation and characterization of bacterial associated with two sand dune plants species, Calystegia soldanella and Elymus mollis. J. Microbiol. 43:219-227