Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
권호 표지

Identification of Arbuscular Mycorrhizal Fungi Colonizing Panax ginseng Using 18S rDNA Sequence

18S rDNA를 이용한 인삼(Panax ginseng)의 내생균근 균의 동정

  • Eo, Ju-Kyeong (Department of Biology Education, Korea National University of Education) ;
  • Kim, Dong-Hun (Department of Biology Education, Korea National University of Education) ;
  • Jeong, Hyeon-Suk (Department of Biology Education, Korea National University of Education) ;
  • Eom, Ahn-Heum (Institute of Natural Science, Korea National University of Education)
  • 어주경 (한국교원대학교 생물교육과) ;
  • 김동훈 (한국교원대학교 생물교육과) ;
  • 정현숙 (한국교원대학교 생물교육과) ;
  • 엄안흠 (한국교원대학교 자연과학연구소)
  • Published : 2004.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

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.

아바스큘라 내생균근(arbuscular mycorrhizae, AM) 균은 대부분의 육상식물 뿌리와 공생하며, 식물의 성장에 도움을 주는 균이다. 인삼은 다년생 식물로서 뿌리를 약재로 사용하는 대표적인 약재 중 하나이다. 본 연구에서는 우리나라 8개 지역에서 인삼을 채집하여 AM 균을 염색을 통하여 형태적으로 관찰하고, 분자생물학적인 방법을 사용하여 뿌리내에 공생하는 다양한 AM 균을 확인하였다. 인삼의 뿌리에 감염되어 있는 AM 균을 염색하여 형태적으로 관찰한 결과 감염률이 낮고 흐리게 염색되었다. 또한 균사와 vesicle 이 발견되었고 이들은 Arum type 으로 판단되지만 arbuscule을 관찰하지 못했기 때문에 Arum-type으로 단정하기는 어려웠다. 식물 내에 공생하는 AM균들의 종류를 확인하기 위하여 채집된 인삼의 뿌리에서 내생균근 균의 DNA를 AM균에 특이적인 18s rDNA primer를 이용한 nested PCR을 수행하여 AM 균의 18S rDNA중 일부를 확보하였다. 증폭된 DNA는 클로닝을 통해서 개별 내생균근 균 종의 염기서열들로 분리하였고, 이들은 AluI, HinfI과 같은 제한 효소를 사용하여 RELP하였다. RELP 패턴에 따라 그룹을 나누어, 각 그룹에서 1개씩 염기서열 분석을 수행하였다. 염기서열은 기존의 서열과의 유사성과 계통 관계의 분석을 통하여 모두 AM균인 것으로 확인되었고, 다음 2개의 종과 가장 유사한 것으로 판단되었다; Paraglomus brasilianum, Glomus spurcum이 중 Paraglomus에 속하는 종인 P. brasil-ianum. 이 인삼의 뿌리에서 공통적으로 관찰되어 이들 균과 인삼과의 특이적 관계에 관하여 추측할 수 있었고, G. spurcum은 유사한 것으로 분석된 염기서열들이 계통도 상에서 특이한 분지를 형성하는 것으로 나타났는데, 이들 분지에 대해서는 확충된 염기서열들과 비교 분석과 같은 연구가 필요한 것으로 생각된다.

Keywords

References

  1. Abbas, J. D., Hetrick, B. A. D. and Jurgenson, J. E. (1996) Isolate specific detection of mycorrhizal fungi using genome specific primer pairs. Mycologia 88, 939-946 https://doi.org/10.2307/3761056
  2. Ausubel, F. M., Brent, R, Kingston, R E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1999) In Short protocols in molecular biology. John Wiley and Sons, Inc, New York
  3. Bonfante, P. and Perotto, S. (1995) Tansley Review No. 82: Strategies of arbuscular mycorrhizal fungi when infecting host plants. New Phytologist 130, 3-21 https://doi.org/10.1111/j.1469-8137.1995.tb01810.x
  4. Busse, M. D. and Ellis, J. R (1985) Vesicular-Arbuscular Mycorrhizal Glomus-Fasciculatum Influence on Soybean Glycine-Max Drought Tolerance in High Phosphorus Soil. Can. J. Bot. 63, 2290-2294 https://doi.org/10.1139/b85-327
  5. Dehne, H. W. and Backhaus, G. F. (1986) The use of vesiculararbuscular mycorrhizal fungi in plant production I. Inoculum production. Zeitschrift fiter Pflanzenkrankheiten Pflanzenschutz 93, 415-424
  6. Elmes, R. P. and Mosse, B. (1984) Vesicular-arbuscular endomycorrhizal inoculum production 2. Experiments with maize zea-mays and other hosts in nutrient flow culture. Can. J Bot. 62, 1531-1536 https://doi.org/10.1139/b84-203
  7. Gildon, A. and Tinker, P. B. (1983) Interactions of vesicular arbuscular mycorrhizal infections and heavy metals in plants 2. The effects of infection on uptake of copper. New Phytologist 95, 263-268 https://doi.org/10.1111/j.1469-8137.1983.tb03492.x
  8. Helgason, T., Daniell, T. J., Husband, R, Fitter, A. H. and Young, J. P. W. (1998) Ploughing up the wood-wide web? Nature 394, 431 https://doi.org/10.1038/28764
  9. Koske, R. and Gemma, J. N. (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486-505 https://doi.org/10.1016/S0953-7562(89)80195-9
  10. Lee, J. K. and Eom, A. H. (2003) Molecular identification of arbuscular mycorrhizal fungi colonizing plant roots. in press.
  11. Lee, J. K., Tae, M. S., Eom, A H. and Lee, S. S. (2003) Restriction analyses of PCR amplified partial SSU ribosomal DNA to distinguish arbuscular mycorrhizal fungi from other fungi colonizing plant roots. Mycobiology 31, 68-73 https://doi.org/10.4489/MYCO.2003.31.2.068
  12. Menge, J. A, Raski, D. J., Lider, L. A, Johnson, E. L. v., Jones, N. 0., Kissler, J. J. and Hemstreet, C. L. (1983) Interactions between mycorrhizal fungi soil fumigation and growth of grapes in California USA Am. J. Enology Viticulture 34, 117-121
  13. Merryweather, J. and Fitter, A (1998) The arbuscular mycorrhizal fungi of Hyacinthoides non-scripta: I. Diversity of fungal taxa. New Phytologist 138, 117-129 https://doi.org/10.1046/j.1469-8137.1998.00888.x
  14. Mosse, B. (1973) Plant Growth Responses to Vesicular Arbuscular Mycorrhiza Part 4 in Soil Given Additional Phosphate. New Phytologist 72, 127-136 https://doi.org/10.1111/j.1469-8137.1973.tb02017.x
  15. Pond, E. C., Menge, J. A. and Jarrell, W. M. (1984) Improved growth of tomato in salinized soil by vesicular arbuscular mycorrhizal fungi collected from saline soils. Mycologia 76, 74-84 https://doi.org/10.2307/3792838
  16. Redecker, D. (2000) Specific PCR primers to identify arbuscu1ar mycorrhizal fungi within colonized roots. Mycorrhiza 10, 73-80 https://doi.org/10.1007/s005720000061
  17. Redecker, D., ThierfeIder, H., Walker, C. and Werner, D. (1997) Restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA as a tool for species identification in different genera of the order glomales. Appl. Environ. MicrobioI. 63, 1756-1761
  18. Rosendahl, S. and Taylor, J. W. (1997) Development of multiple genetic markers for studies of genetic variation in arbuscular mycorrhizal fungi using AFLP. Mol. Ecol. 6, 821-829
  19. Safir, G. R., Boyer, J. S. and Gerdemann, J. W. (1971) Mycorrhizal enhancement of water transport in soy bean-D. Science 172, 581-582 https://doi.org/10.1126/science.172.3983.581
  20. Schenck, N. C. and Perez, Y. (1990) In Manual for the Identification of VA Mycorrhizal Fungi. Synergistic Publications, Gainesville, Florida. pp. 1-286
  21. Schuessler, A, Gehrig, H., Schwarzott, D. and Walker, C. (2001) Analysis of partial Glomales SSU rRNA gene sequences: implications ofr primer design and phylogeny. Mycol. Res. 105, 5-15 https://doi.org/10.1017/S0953756200003725
  22. Smith, F. A. and Smith, S. E. (1997) Tansley review No. 96 structural diversity in (vesicular)-arbuscular mycorrhizal symbioses. New Phytologist 137, 373-388 https://doi.org/10.1046/j.1469-8137.1997.00848.x
  23. Tae, M. S. (2002) In Molecular and morphological diversity of arbuscular mycorrhizal fungi in Mt. Work. Korea National University of Education, Chungbuk
  24. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL X: Improving the sensitivity of multiple sequence alignment though sequence weighting, position-specific gap penalties and wieght matrix choice. Nucleic Acid Res. 26, 179-182
  25. Trappe, J. M. (1981) In Advance in food producing systems for arid simiarid lands.: Mycorrhizae and productivity of arid and semiarid rangelands. pp. 581-599
  26. vanTuinen, D., Jacquot, E., Zhao, B., Gollotte, A and Gianinazzi, P. V. (1998) Characterization of root colonization profiles by a microcosm community of arbuscu1ar mycorrhizal fungi using 25S rDNA-targeted nested PCR. Molecular Ecology 7, 879-887. https://doi.org/10.1046/j.1365-294x.1998.00410.x
  27. Yeoman, M. M. and Yeoman, C. L. (1996) Tans1ey Review No.90. Manipulating scondary metabolism in cultured plant cells. New Phytologist 134, 553-569 https://doi.org/10.1111/j.1469-8137.1996.tb04921.x