Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Protoplast Isolation and Genetic Transformation of Polyporus brumalis

겨울우산버섯의 원형질체 분리와 유전자 형질전환

  • Ryu, Sun-Hwa (Division of Wood Chemistry & Microbiology, Korea Forest Research Institute (KFRI)) ;
  • Kim, Myung-Kil (Division of Wood Chemistry & Microbiology, Korea Forest Research Institute (KFRI))
  • 유선화 (국립산림과학원 임산공학부 화학미생물과) ;
  • 김명길 (국립산림과학원 임산공학부 화학미생물과)
  • Received : 2014.10.27
  • Accepted : 2014.11.22
  • Published : 2014.12.31
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Abstract

This experiment was undertaken to investigate proper conditions for protoplast isolation and genetic transformation of the white rot fungi, Polyporus brumalis. The protoplasts were formed from mycelia at a frequency of $1{\times}10^7/ml$ with 0.5% Usukizyme. The transformation vector (pHYgpt) was constructed using hygromycin resistance gene (hph) for the selectable maker. The yield was 100-160 transformants/${\mu}g$ DNA in a transformation mediated by 40% polyethylene glycol solution with aurintricarboxylic acid, heparin and supermidine. The genomic integration of the pHYgpt was confirmed by hph-specific PCR and the expected amplified band appeared only in the transformants. These results could be an efficient tool in gene engineering of the genus polyporus.

본 연구에서는 백색부후균인 겨울우산버섯의 원형질체 분리 조건과 유전자 형질전환 방법을 확립하였다. 겨울우산버섯 균사에 세포벽 분해효소로 0.5% Usukizyme을 처리하여 ml당 $1{\times}10^7/ml$개의 원형질체를 확보할 수 있었다. 형질전환체의 선별을 위해 hygromycin에 대한 저항성을 갖는 유전자(hph)를 선택표지로 이용하여 형질전환용 벡터(pHYgpt)를 제작하였다. 40% polyethylene glycol 용액과 aurintricarboxylic acid와 heparin, supermidine을 첨가하여 형질전환을 수행 한 결과 벡터 $1{\mu}g$ 당 100-160개의 수율로 형질전환체를 얻었다. 도입된 벡터는 hph 유전자의 PCR 증폭을 통해 형질전환체의 염색체내에 삽입되었음을 확인하였다. 이러한 결과는 polyporus 속의 유전자를 활용한 새로운 균주 개발에 유용한 기술이 될 것이다.

Keywords

References

  1. Barr, D.P. and Aust, S.D. 1994. Mechanisms white rot fungi use to degrade pollutants. Environ. Sci. Technol. 28, 78-87. https://doi.org/10.1021/es00051a724
  2. Becker, H.G. and Sinitsyn, A.P. 1993. Mn-peroxidase from Pleurotus ostreatus: the action on the lignin. Biotechnol. Lett. 15, 289-294. https://doi.org/10.1007/BF00128321
  3. Dickman, M.B. 1988. Whole cell transformation of the alfalfa fungal pathogen Colletorichum trifolii. Curr. Genet. 14, 241-246. https://doi.org/10.1007/BF00376744
  4. Hallick, R.B., Chelm, B.K., Gray, P.W., and Orozco, E.M. Jr. 1977. Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res. 4, 3055-3064. https://doi.org/10.1093/nar/4.9.3055
  5. Hatakka, A. 1994. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125-135. https://doi.org/10.1111/j.1574-6976.1994.tb00039.x
  6. Hwang, S.S., Choi, H.T., and Song, H.G. 2008. Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus. J. Microbiol. Biotechnol. 18, 767-772.
  7. Kim, Y.J., Kim, M., Song, H.G., and Choi, H.T. 2007. Genetic transformation of Irpex lacterus and Phlebia tremellosa to an antibiotic resistance. Kor. J. Microbiol. 43, 147-149.
  8. Kum, H., Lee, S., Ryu, S., and Choi, H.T. 2011. Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in Phlebia tremellosa. J. Microbiol. 49, 824-827. https://doi.org/10.1007/s12275-011-1230-y
  9. Lee, S.M., Lee, J.W., Koo, B.W., Kim, M.K., Choi, D.H., and Choi, I.G. 2007. Dibutyl phthalate biodegradation by the white rot fungus, Polyporus brumalis. Biotechnol. Bioeng. 97, 1516-1522. https://doi.org/10.1002/bit.21333
  10. Leem, Y., Kim, S.J., Ross, I.K., and Choi, H.T. 1999. Transformation and laccase mutant isolation in Coprinus congregatus by restriction enzyme-mediated integration. FEMS Microbiol. Lett. 172, 35-40. https://doi.org/10.1111/j.1574-6968.1999.tb13446.x
  11. Li, G., Li, R., Liu, Q., Wang, Q., Chen, M., and Li, B. 2006. A highly efficient polyethylene glycol-mediated transformation method for mushrooms. FEMS Microbiol. Lett. 256, 203-208. https://doi.org/10.1111/j.1574-6968.2006.00110.x
  12. Pasti-Grigsby, M.B., Paszczynski, A., Goszczynski, S., Crawford, D.L., and Crawford, R.L. 1992. Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58, 3605-3613.
  13. Peng, M., Singh, N.K., and Lemke, P.A. 1992. Recovery of recombinant plasmids from Pleurotus ostreatus transformants. Curr. Genet. 22, 53-59. https://doi.org/10.1007/BF00351742
  14. Pointing, S.B. 2001. Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol. 57, 20-33. https://doi.org/10.1007/s002530100745
  15. Ryu, S.H., Lee, A.Y., and Kim, M. 2008. Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis. J. Microbiol. 46, 62-69. https://doi.org/10.1007/s12275-007-0110-y
  16. Shin, E.H., Choi, H.T., and Song, H.G. 2007. Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus. J. Microbiol. Biotechnol. 17, 1147-1151.
  17. Yeo, S., Park, N., Song, H.G., and Choi, H.T. 2007. Generation of a transformant showing higher manganese peroxidase (MnP) activity by overexpression of MnP gene in Trametes versicolor. J. Microbiol. 45, 213-218.