The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
권호 표지
DOI QR코드

DOI QR Code

Agrobacterium-Mediated Transformation of Flammulina velutipes with NaOH Treatment

NaOH처리에 의한 Agrobacterium이용 팽이균사체 형질전환

  • Shin, Dong-Il (Department of Biotechnology, Catholic University of Daegu, Kyungsan) ;
  • Park, Hee-Sung (Department of Biotechnology, Catholic University of Daegu, Kyungsan)
  • 신동일 (대구가톨릭대학교 생명공학과) ;
  • 박희성 (대구가톨릭대학교 생명공학과)
  • Received : 2011.09.20
  • Accepted : 2011.11.14
  • Published : 2011.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant($hyg^r$) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of $hyg^r$ gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.

팽이균사체의 형질전환을 위하여 Agrobacterium 세포를 사용하였다. 특히, Agrobacterium 세포의 감염단계 전에 약한 NaOH용액을 처리하였으며 이로써 균사체 세포들의 표면 상해 발생을 기대하였다. 그 결과, hygromycin 저항성 ($hyg^r$) 균사체는 NaOH 처리를 거친 경우에서만 출현하였다. 형질전환 균사체의 $hyg^r$ 유전자 도입은 PCR로 확인되었으며 또한 Southern blot hybridization과 western blotting 분석에 의하여 단일 유전자 copy의 삽입과 외래유전자의 발현을 확인할 수 있었다. 본 연구는 팽이균사체에 대한 효율적인 Agrobacterium 이용 형질전환수단을 보여주고 있다.

Keywords

References

  1. Bechtold, N. and Pelletier, G. 1998. In planta Agrobacteriummediated gene transfer of adult Arabidopsis thaliana plants by vacuum infiltration. Meth. Mol. Biol. 82:259-266.
  2. Cheng, M., Fry, J. E., Pang, S., Zhou, I., Hironaka, C., Duncan, D. R. I., Conner, T. W. L. and Wang, Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115:971-980. https://doi.org/10.1104/pp.115.3.971
  3. Cheng, M., Hu, T., Layton, J. L., Liu, C. N. and Fry, J. E. 2003. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell. Dev. Biol. Plant 39:595-604l. https://doi.org/10.1079/IVP2003471
  4. Enriquez-Obregon, G. A., Vazquez-Padron, R. I., Prieto-Samsonov, D. L. de la Riva, G. A. and Selman-Housein, G. 1998. Herbicideresistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium-mediated transformation. Planta 205:20-27.
  5. Flores Sols, J. L., Mlejnek, P., Studen, K. and Prochzka, S. 2003. Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L. Plant Soil Environ. 49:255-260. https://doi.org/10.1080/00380768.2003.10410005
  6. Floros, J. D., Wetzstein, H. Y. and Chinnan, M. S. 1987. Chemical (NaOH) peeling as viewed by scanning electron microscopy: pimiento peppers as a case study. J. Food Sci. 52:1312-1316. https://doi.org/10.1111/j.1365-2621.1987.tb14071.x
  7. Kim, S., Shin, D.-I. and Park, H.-S. 2007. Transient ${\beta}-glucuronidase$ expression in lily (Lilium longiflorum L.) pollen via woundingassisted Agrobacterium-mediated transformation. Biotech. Lett. 29:965-969. https://doi.org/10.1007/s10529-007-9326-5
  8. Kilaru, S. and Kes, U. 2005. Comparison of gpd genes and their protein products in basidiomycetes. Fungal Genet. Newslett. 52:18-23.
  9. Kuo, C. Y., Chou, S. Y., Hseu, R. S. and Huang, C. T. 2010. Heterologous expression of EGFP in enoki mushroom Flammulina velutipes. Bot. Studies. 51:303-309.
  10. Maehara T., Yoshida, M., Ito, Y., Tomita, S., Takabatake, K., Ichinose, H. and Kaneko, S. 2010. Development of a gene transfer system for the mycelia of Flammulina velutipes Fv-1 strain. Biosci. Biotechnol. Biochem. 74:1126-1128. https://doi.org/10.1271/bbb.100021
  11. Meyer, V. 2008. Genetic engineering of filamentous fungi - progress, obstacles and future trends. Biotech. Adv. 26:177-185. https://doi.org/10.1016/j.biotechadv.2007.12.001
  12. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  13. Olhoft, P. M. and Somers, D. A. 2001. L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep. 20:706-711. https://doi.org/10.1007/s002990100379
  14. Pan, S. Q., Charles, T., Jin, S., Wu, Z. L. and Nester, E. W. 1993. Preformed dimeric state of the sensor protein VirA is involved in plant-Agrobacterium signal transduction. Proc. Natl. Acad. Sci. USA 90: 9939-9943. https://doi.org/10.1073/pnas.90.21.9939
  15. Park, S.-Y., van Peer, A. F., Jang, K.-Y., Shin, P.-G., Park, Y., Yoo, Y.-B., Park, K.-M. and Kong, W.-S. 2010. Agrobacterium-mediated transformation using gill tissue of Flammulina velutipes. Kor. J. Mycol. 38:48-53. https://doi.org/10.4489/KJM.2010.38.1.048
  16. Sharma, K. K. and Kuhad, R. C. 2010. Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens. BMC Biotechnol. 10:67-74. https://doi.org/10.1186/1472-6750-10-67
  17. Shin, D.-I. and H.-S. Park. 2008. Expression of recombinant protein from Oenanthe javanic DC. leaf tissues as a biofactory. Kor. J. Biotechnol. Bioeng. 23:554-556.
  18. Singh, N. and Chawla, H. S. 1999. Use of silicon carbide fibers for Agrobacterium-mediated transformation in wheat. Curr. Sci. 76:1483-1485.
  19. Uze, M., Wunn, J., Pounti-Kaelas, J., Potrykus, I. and Sauter, C. 1997. Plasmolysis of precultured immature embryos improves Agrobacterium mediated gene transfer to rice (Oryza sativa L.). Plant Sci. 130:87-95. https://doi.org/10.1016/S0168-9452(97)00211-2
  20. Wasser, S. P. and Weis, A. L. 1999. Medicinal properties of substances occurring in higher basidiomycetes mushrooms: Current perspectives (review). Int. J. Med. Mushr. 1:31-62. https://doi.org/10.1615/IntJMedMushrooms.v1.i1.30