Skip to main content
SpringerLink
Log in

Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

  • Article
  • Published:
Science in China Series C: Life Sciences Aims and scope Submit manuscript

Abstract

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our experiments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experimental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR detection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Reyes-Ruiz J M, Barrera-Saldana H A. Proteins in a DNA world: Expression systems for their study. Rev Invest Clin, 2006, 58: 47–55 16789599, 1:CAS:528:DC%2BD28XmsVSlsr0%3D

    PubMed  CAS  Google Scholar 

  2. Singh M B, Bhalla P L. Recombinant expression systems for allergen vaccines. Inflamm Allergy Drug Targets, 2006, 5: 53–59 16613564, 10.2174/187152806775269312, 1:CAS:528:DC%2BD28XhtVOgsr8%3D

    Article  PubMed  CAS  Google Scholar 

  3. McNulty D E, Claffee B A, Huddleston M J, et al. Mis-translational errors associated with the rare arginine codon CGG in Escherichia coli. Protein Expr Purif, 2003, 27: 365–374 12597898, 10.1016/S1046-5928(02)00610-1, 1:CAS:528:DC%2BD3sXht1Gitrc%3D

    Article  PubMed  CAS  Google Scholar 

  4. Rai M, Padh H. Expression systems for production of heterologous proteins. Curr Sci, 2001, 80: 1121–1128 1:CAS:528:DC%2BD3MXksVyqs78%3D

    CAS  Google Scholar 

  5. Cho E J, Oh J Y, Chang H Y, et al. Production of exopolysaccharides by submerged mycelium culture of a mushroom Tremella fuciformis. J Biotechnol, 2006, 127: 129–140 16872706, 10.1016/j.jbiotec.2006.06.013, 1:CAS:528:DC%2BD28Xht1ShtL%2FO

    Article  PubMed  CAS  Google Scholar 

  6. Xie B G, Rao Y B, Zheng J G. Transformation of Tremella fuciformis by using ultrasonic mediation. Chin J Agric Biotechnol, 2005, 13: 42–45 1:CAS:528:DC%2BD2MXhtFSrurfL

    CAS  Google Scholar 

  7. Xie B G, Lu Q Q, Rao Y B, et al. Synthesis of human insulin gene and transformation into Tremella fuciformis. Acta Edulis Fungi, 2007, 14(2): 9–14

    Google Scholar 

  8. Taketo A. DNA transfection of Escherichia coli by electroporation. Biochim Biophys Acta, 1988, 949: 318–324 3280031, 1:CAS:528:DyaL1cXhvVWgu78%3D

    Article  PubMed  CAS  Google Scholar 

  9. Fiedler S, Wirth R. Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem, 1988, 170: 38–44 3133958, 10.1016/0003-2697(88)90086-3, 1:CAS:528:DyaL1cXhs1Cmurw%3D

    Article  PubMed  CAS  Google Scholar 

  10. Meilhoc E, Masson J M, Teissié J. High efficiency transformation of intact yeast cells by electric field pulses. Biotechnology (NY), 1991, 8: 223–227 10.1038/nbt0390-223

    Article  Google Scholar 

  11. Rohrer T L, Picataggio S K. Targeted integrative transformation of Candida tropicalis by electroporation. Appl Microbiol Biotechnol, 1992, 36: 650–654 1368068, 10.1007/BF00183243, 1:CAS:528:DyaK38XhsVOqsbg%3D

    Article  PubMed  CAS  Google Scholar 

  12. Minoru S, Toyomasa H. High-efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr Genet, 2003, 43: 206–211

    Google Scholar 

  13. Peng M, Lemke P A, Shaw J J. Improved conditions for protoplast formation and transformation of Pleurotus ostreatus. Appl Microbiol Biotechnol, 1993, 40: 101–106 10.1007/BF00170436, 1:CAS:528:DyaK2cXhs1Wkt78%3D

    Article  CAS  Google Scholar 

  14. van de Rhee M D, Graca P M A, Huizing H J, et al. Transformation of the cultivated mushroom, Agaricus bisporus to hygromycin B resistance. Mol Gen Genet, 1996, 250: 252–258 8602139

    PubMed  Google Scholar 

  15. Kuo C Y, Chou S Y, Huang C T. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Appl Microbiol Biotechnol, 2004, 65: 593–599 15168094, 10.1007/s00253-004-1635-1, 1:CAS:528:DC%2BD2cXns1eqsr0%3D

    Article  PubMed  CAS  Google Scholar 

  16. Borroso G, Perennes D, Labarere J. A mini-prep method for RFLP analysis and dsRNAs detection perfected in the cultivated fungus Agrocybe aegerita. In: Elliott T J, eds. Science and Cultivation of Edible Fungi. Rotterdam: A.A. Balkema Publisher, 1995. 87–94

    Google Scholar 

  17. Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning. 2nd Edition. New York: Cold Spring Harbor Laboratory Press, 1989. 456–535

    Google Scholar 

  18. Guo L Q, Yang F Y, Xiong S, et al. The transformation system for Volvariella volvacea by particle bombardment. Acta Hortic Sin, 2005, 32: 828–833 1:CAS:528:DC%2BD28XhtFKis7bM

    CAS  Google Scholar 

  19. Sunagawa M, Magae Y. Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment. FEMS Microbiol Lett, 2002, 211: 143–146 12076804, 10.1111/j.1574-6968.2002.tb11216.x, 1:CAS:528:DC%2BD38XksFyqtLY%3D

    Article  PubMed  CAS  Google Scholar 

  20. Wu S, Letchworth G J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques, 2004, 36: 152–154 14740498, 1:CAS:528:DC%2BD2cXlvFSjtg%3D%3D

    PubMed  CAS  Google Scholar 

  21. Zhu H, Wang T W, Sun S J, et al. Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis. Appl Microbiol Biotechnol, 2006, 72: 770–776 16501972, 10.1007/s00253-006-0322-9, 1:CAS:528:DC%2BD28XpvF2mur4%3D

    Article  PubMed  CAS  Google Scholar 

  22. Zhao S X, Lin J F, Guo L Q. Advances in the development of bio-safe selective markers for transgenic edible fungi. Acta Edulis Fungi, 2007, 14: 55–61

    Google Scholar 

  23. Mellon F M, Casselton L A. Transformation as a method increasing gene copy number and gene expression in the basidiomycete fungus Coprinus cinereus. Curr Genet, 1988, 14: 451–456 3224385, 10.1007/BF00521268, 1:CAS:528:DyaL1MXht12murc%3D

    Article  PubMed  CAS  Google Scholar 

  24. Nitta Y, Miyazaki Y, Nakamura M, et al. Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in Coriolus versicolor. Mycoscience, 2004, 45: 131–136 10.1007/s10267-003-0164-6, 1:CAS:528:DC%2BD2cXlsVCqtbk%3D

    Article  CAS  Google Scholar 

  25. Burns C, Gregory K E, Kirby M, et al. Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet Biol, 2005, 42: 191–199 15707840, 10.1016/j.fgb.2004.11.005, 1:CAS:528:DC%2BD2MXhtFOlsLc%3D

    Article  PubMed  CAS  Google Scholar 

  26. Yamazaki T, Hasebe T, Kajiwara S, et al. Structure and function of a pyrinidine/purine-biased sequence from the 5′-flanking region of the basidiomycete Lentinus edodes gene priA. Mol Genet Genomics, 2000, 263: 262–270 1:CAS:528:DC%2BD3cXivFSqs78%3D, 10.1007/s004380051167

    Article  CAS  Google Scholar 

  27. Irie T, Honda Y, Hirano T, et al. Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals. Appl Microbiol Biotechnol, 2001, 56: 707–709 11601618, 10.1007/s002530100651, 1:CAS:528:DC%2BD3MXnslGgsLo%3D

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Food Sciences, South China Agricultural University, Guangzhou, 510640, China

    LiQiong Guo, Yong Liu, ShuXian Zhao, ErXian Liu & JunFang Lin

  2. Institute of Biomass Research, South China Agricultural University, Guangzhou, 510640, China

    LiQiong Guo & JunFang Lin

Authors
  1. LiQiong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. ShuXian Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. ErXian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. JunFang Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to JunFang Lin.

Additional information

Contributed equally to this work

Supported by National High-tech Research and Development Program (863 Program) of China (Grant No. 2006AA10Z301), the National Natural Science Foundation of China (Grant Nos. 30371000 and 30671457) and the Natural Science Foundation of Guangdong Province, China (Grant No. 032239)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, L., Liu, Y., Zhao, S. et al. Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation. SCI CHINA SER C 51, 932–940 (2008). https://doi.org/10.1007/s11427-008-0121-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11427-008-0121-x

Keywords

Search

Navigation