Abstract
Pleurotus ostreatus is widely cultivated worldwide, but the lack of an efficient transformation system regarding its use restricts its genetic research. The present study developed an improved and efficient Agrobacterium tumefaciens-mediated transformation method in P. ostreatus. Four parameters were optimized to obtain the most efficient transformation method. The strain LBA4404 was the most suitable for the transformation of P. ostreatus. A bacteria-to-protoplast ratio of 100:1, an acetosyringone (AS) concentration of 0.1 mM, and 18 h of co-culture showed the best transformation efficiency. The hygromycin B phosphotransferase gene (HPH) was used as the selective marker, and EGFP was used as the reporter gene in this study. Southern blot analysis combined with EGFP fluorescence assay showed positive results, and mitotic stability assay showed that more than 75% transformants were stable after five generations. These results showed that our transformation method is effective and stable and may facilitate future genetic studies in P. ostreatus.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AS:
-
Acetosyringone
- ATMT:
-
Agrobacterium tumefaciens-mediated transformation
- CM:
-
Complete medium
- GPD:
-
Glyceraldehyde-3-phosphate dehydrogenase
- LB:
-
Luria–Bertani
- MM:
-
Minimal medium
- PEG:
-
Polyethylene glycol
- WT:
-
Wild type
References
Ando A, Sumida Y, Negoro H, Suroto DA, Ogawa J, Sakuradani E, Shimizu S (2009) Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding. Appl Environ Microbiol 75:5529–5535. https://doi.org/10.1128/AEM.00648-09
Baldrian P (2003) Interactions of heavy metals with white-rot fungi. Enzyme Microb Technol 32:78–91. https://doi.org/10.1016/s0141-0229(02)00245-4
Chai R, Zhang G, Sun Q, Zhang MY, Zhao SJ, Qiu LY (2013) Liposome-mediated mycelial transformation of filamentous fungi. Fungal Biol 117:577–583. https://doi.org/10.1016/j.funbio.2013.06.008
Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66:4510–4513. https://doi.org/10.1128/aem.66.10.4510-4513.2000
Chen EC, Su YH, Kanagarajan S, Agrawal DC, Tsay HS (2009) AS-Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea. Mycol Res 113:290–297. https://doi.org/10.1016/j.mycres.2008.11.007
de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842. https://doi.org/10.1038/nbt0998-839
Ding Y, Liang S, Lei J, Chen L, Kothe E, Ma A (2011) Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus. Microbiol Res 166:314–322. https://doi.org/10.1016/j.micres.2010.07.001
Doerks T, Copley RR, Schultz J, Ponting CP, Bork P (2002) Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 12:47–56. https://doi.org/10.1101/gr.203201
Duarte RTD et al (2007) Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum. Lett Appl Microbiol 44:248–254
Haritash AK, Kaushik CP (2009) Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review. J Hazard Mater 169:1–15. https://doi.org/10.1016/j.jhazmat.2009.03.137
Honda Y, Matsuyama T, Irie T, Watanabe T, Kuwahara M (2000) Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus. Curr Genet 37:209–212. https://doi.org/10.1007/s002940050521
Irie T, Honda Y, Hirano T, Sato T, Enei H, Watanabe T, Kuwahara M (2001) Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals. Appl Microbiol Biotechnol 56:707–709. https://doi.org/10.1007/s002530100651
Jung-Hee C, Seung-Eun L, Chang WB, Jae-Soon C (2006) Agrobacterium mediated transformation of the winter mushroom Flammulina velutipes. Mycobiology 34:104. https://doi.org/10.4489/MYCO.2006.34.2.104
Kim S, Ha BS, Ro HS (2015) Current technologies and related issues for mushroom transformation. Mycobiology 43:1–8. https://doi.org/10.5941/myco.2015.43.1.1
Lavi I, Friesem D, Geresh S, Hadar Y, Schwartz B (2006) An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells. Cancer Lett 244:61–70. https://doi.org/10.1016/j.canlet.2005.12.007
Leclerque A, Wan H, Abschutz A, Chen S, Mitina GV, Zimmermann G, Schairer HU (2004) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana. Curr Genet 45:111–119. https://doi.org/10.1007/s00294-003-0468-2
Li G, Li RX, Liu QY, Wang Q, Chen M, Li BJ (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
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ., Ram AFJ (2008) Agrobacterium mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc 3:1671–1678. https://doi.org/10.1038/nprot.2008.154
Peng M, Singh NK, Lemke PA (1992) Recovery of recombinant plasmids from Pleurotus ostreatus transformants. Curr Genet 22:53
Sanchez C (2010) Cultivation of Pleurotus ostreatus and other edible mushrooms. Appl Microbiol Biotechnol 85:1321–1337. https://doi.org/10.1007/s00253-009-2343-7
Shao CW, Yin YP, Qi ZR, Li R, Song ZY, Li Y, Wang ZK (2015) Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi. Fungal Genet Biol 83:19–25. https://doi.org/10.1016/j.fgb.2015.08.002
Shi L, Fang X, Li M, Mu D, Ren A, Tan Q, Zhao M (2012) Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum. World J Microbiol Biotechnol 28:283–291. https://doi.org/10.1007/s11274-011-0818-z
Sun L, Cai H, Xu W, Hu Y, Gao Y, Lin Z (2001) Efficient transformation of the medicinal mushroom Ganoderma lucidum. Plant Mol Biol Rep 19:383–384
Sunagawa M, Magae Y (2002) Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment. FEMS Microbiol Lett 211:143–146. https://doi.org/10.1111/j.1574-6968.2002.tb11216.x
Wang HX, Gao JQ, Ng TB (2000) A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus. Biochem Biophys Res Commun 275:810–816. https://doi.org/10.1006/bbrc.2000.3373
Acknowledgements
This research was funded by the National Basic Research Program of China (2014CB138303), the China Agriculture Research System (CARS20), and the National Natural Science Foundation of China (31601803). Special thanks are given to Prof. Mingwen Zhao (College of life science in Nanjing Agricultural University, China) for his excellent technical instruction.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Lei, M., Wu, X., Zhang, J. et al. Establishment of an efficient transformation system for Pleurotus ostreatus . World J Microbiol Biotechnol 33, 214 (2017). https://doi.org/10.1007/s11274-017-2378-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-017-2378-3