Use of Restriction Fragment Length Polymorphism Analysis to Differentiate Fungal Strains in Sunchang Meju

  • Jung, Jong-Hyun (Graduate School of Biotechnology, Institute of Life Science and Resources, KyungHee University) ;
  • Seo, Dong-Ho (Graduate School of Biotechnology, Institute of Life Science and Resources, KyungHee University) ;
  • Bhoo, Sung-Hee (Graduate School of Biotechnology, Institute of Life Science and Resources, KyungHee University) ;
  • Ha, Suk-Jin (Graduate School of Biotechnology, Institute of Life Science and Resources, KyungHee University) ;
  • Kim, Jong-Sang (Department of Animal Science and Biotechnology, Kyungpook National University) ;
  • Kim, Jeong-Hwan (Department of Food Science and Technology, Gyeongsang National University) ;
  • Kwon, Dae-Young (Korea Food Research Institute) ;
  • Cha, Jae-Ho (Department of Biological Science, Pusan National University) ;
  • Park, Cheon-Seok (Graduate School of Biotechnology, Institute of Life Science and Resources, KyungHee University)
  • 발행 : 2008.08.31

초록

Twenty-three fungal strains were isolated from meju that had originated from the Sunchang province, the famous location for making fermented soybean foods in Korea. The restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rDNA (ITS-RFLP) was applied to differentiate the isolated fungal strains. First, the ITS region by polymerase chain reaction (PCR) with specific primers was amplified and then cleaved the products with different restriction enzymes. Cleavage of the amplified fragments with the restriction enzymes AluI, HaeIII, HhaI, and TaqI revealed extensive polymorphisms. The ITS-RFLP results highly correlated with ITS sequence analysis. All of the 23 fungal strains were classified into 5 groups by ITS-RFLP analysis. Aspergillus oryzae was the major fungal strain isolated from Sunchang meju (12 out of 23), while Aspergillus fumigatus was the next most frequently isolated strain (7 out of 23). In contrast, it was found that Fusarium asiaticum, Aspergillus sydowii, and Arthrinium sp. were the minor fungal strains in meju.

키워드

참고문헌

  1. Park KY, Jung KO, Rhee SH, Choi YH. Antimutagenic effects of doenjang (Korean fermented soypaste) and its active compounds. Mutat. Res. 523-524: 43-53 (2003) https://doi.org/10.1016/S0027-5107(02)00320-2
  2. Kim YM, Lee BH, Park KJ, Lee BK. Fungal microflora on Korean home-made meju. Korean J. Mycol. 5: 7-12 (1977)
  3. Lee SS, Park DH, Sung CK, Yoo JY. Studies on the yellow fungal isolates (Aspergillus species) inhabiting at the cereals in Korea. Korean J. Mycol. 25: 35-45 (1997)
  4. de Souza-Motta CM, de Queiroz Cavalcanti MA, dos Santos Fernandes MJ, Lima DMM, Nascimento JP, Laranjeira D. Identification and characterization of filamentous fungi isolated from the sunflower (Helianthus annus L.) rhizosphere according to their capacity to hydrolyse inulin. Braz. J. Microbiol. 34: 273-280 (2003)
  5. Gardes M, Bruns TD. ITS-RFLP matching for identification of fungi. Method Mol. Biol. 50: 177-186 (1996)
  6. Bougoure JJ, Bougoure DS, Cairney JW, Dearnaley JD. ITS-RFLP and sequence analysis of endophytes from Acianthus, Caladenia, and Pterostylis (Orchidaceae) in southeastern Queensland. Mycol. Res. 109: 452-460 (2005) https://doi.org/10.1017/S095375620500225X
  7. Martinez-Culebras PV, Ramon D. An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine. Int. J. Food Microbiol. 113: 147-153 (2007) https://doi.org/10.1016/j.ijfoodmicro.2006.06.023
  8. Korabecna M, Liska V, Fajfrlik K. Primers ITS1, ITS2, and ITS4 detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi. Folia Microbiol. (Praha) 48: 233-238 (2003) https://doi.org/10.1007/BF02930961
  9. White T, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. pp. 315-322. In: PCR Protocols. A Guide to Methods and Applications. Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds). Academic Press, San Diego, CA, USA (1990)
  10. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882 (1997) https://doi.org/10.1093/nar/25.24.4876
  11. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425 (1987)
  12. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-789 (1985) https://doi.org/10.2307/2408678
  13. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5: 150-163 (2004) https://doi.org/10.1093/bib/5.2.150
  14. Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int. J. Syst. Bacteriol. 49: 329-337 (1999) https://doi.org/10.1099/00207713-49-1-329
  15. Rahman TJ, Marchant R, Banat IM. Distribution and molecular investigation of highly thermophilic bacteria associated with cool soil environments. Biochem. Soc. T. 32: 209-213 (2004) https://doi.org/10.1042/BST0320209
  16. Kobayashi T, Abe K, Asai K, Gomi K, Juvvadi PR, Kato M, Kitamoto K, Takeuchi M, Machida M. Genomics of Aspergillus oryzae. Biosci. Biotech. Bioch. 71: 646-670 (2007) https://doi.org/10.1271/bbb.60550
  17. Jiao W, Blunt JW, Cole AL, Munro MH. Fumagiringillin, a new fumagillin derivative from a strain of the fungus Aspergillus fumigatus. J. Nat. Prod. 67: 1434-1437 (2004) https://doi.org/10.1021/np049893p
  18. Lefkove B, Govindarajan B, Arbiser JL. Fumagillin: An antiinfective as a parent molecule for novel angiogenesis inhibitors. Expert Rev. Anti. Infe. 5: 573-579 (2007) https://doi.org/10.1586/14787210.5.4.573
  19. Guillamon JM, Sabate J, Barrio E, Cano J, Querol A. Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region. Arch. Microbiol. 169: 387-392 (1998) https://doi.org/10.1007/s002030050587
  20. de Llanos Frutos R, Fernandez-Espinar MT, Querol A. Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Anton. Leeuw. Int. J. G. 85: 175-185 (2004) https://doi.org/10.1023/B:ANTO.0000020154.56649.0f