Mycobiology

The Korean Society of Mycology (한국균학회)
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A Rapid and Effective Colony PCR Procedure for Screening Transformants in Several Common Mushrooms

  • Wang, Yuanyuan (College of Food Science and Technology, Huazhong Agricultural University) ;
  • Xu, Danyun (College of Food Science and Technology, Huazhong Agricultural University) ;
  • Liu, Dongmei (College of Food Science and Technology, Huazhong Agricultural University) ;
  • Sun, Xueyan (College of Food Science and Technology, Huazhong Agricultural University) ;
  • Chen, Yue (College of Food Science and Technology, Huazhong Agricultural University) ;
  • Zheng, Lisheng (College of Plant Science and Technology, Huazhong Agricultural University) ;
  • Chen, Liguo (College of Plant Science and Technology, Huazhong Agricultural University) ;
  • Ma, Aimin (College of Food Science and Technology, Huazhong Agricultural University)
  • Received : 2018.12.31
  • Accepted : 2019.06.02
  • Published : 2019.09.30
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Abstract

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue ($1-10{\mu}g$) to $20{\mu}l$ 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at $34^{\circ}C$ for 15 min. Finally, $2{\mu}l$ of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.

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References

  1. Mueller GM, Schmit JP, Leacock PR, et al. Global diversity and distribution of macrofungi. Biodivers Conserv. 2007;16:37-48. https://doi.org/10.1007/s10531-006-9108-8
  2. Li H, Wu S, Ma X, et al. The genome sequences of 90 mushrooms. Sci Rep. 2018;8:9982. https://doi.org/10.1038/s41598-018-28303-2
  3. Walti MA, Villalba C, Buser RM, et al. Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs. Eukaryot Cell. 2006;5:732-744. https://doi.org/10.1128/EC.5.4.732-744.2006
  4. Sato M, Kurahashi A, Nishibori K, et al. Development of a transformation system for the edible mushroom Grifola frondosa: demonstrating heterologous gene expression and RNAi-mediated gene silencing. Mycoscience. 2015;56:364-372. https://doi.org/10.1016/j.myc.2014.11.004
  5. Hamer L, Adachi K, Montenegro-Chamorro MV, et al. Gene discovery and gene function assignment in filamentous fungi. Proc Natl Acad Sci USA. 2001;98:5110-5115. https://doi.org/10.1073/pnas.091094198
  6. Salame TM, Knop D, Tal D, et al. Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus. Appl Environ Microbiol. 2012;78:5341-5352. https://doi.org/10.1128/AEM.01234-12
  7. van Burik JAH, Schreckhise RW, White TC, et al. Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Med Mycol. 1998;36:299-303. https://doi.org/10.1080/02681219880000471
  8. Walch G, Knapp M, Rainer G, et al. Colony-PCR is a rapid method for DNA amplification of hyphomycetes. J Fungi. 2016;2:2-10. https://doi.org/10.3390/jof2010002
  9. Pancher M, Ceol M, Corneo PE, et al. Fungal endophytic communities in Grapevines (Vitis vinifera L.) respond to crop management. Appl Environ Microbiol. 2012;78:4308-4317. https://doi.org/10.1128/AEM.07655-11
  10. Zeijl C, Kamp E, Punt PJ, et al. An improved colony-PCR method for filamentous fungi for amplification of PCR-fragments of several kilobases. J Biotechnol. 1997;59:221-224. https://doi.org/10.1016/S0168-1656(97)00170-3
  11. Alshahni MM, Makimura K, Yamada T, et al. Direct colony PCR of several medically important fungi using Ampdirect(R) plus. Jpn J Infect Dis. 2009;62:164-167.
  12. Packeiser H, Lim C, Balagurunathan B, et al. An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. Appl Biochem Biotechnol. 2013;169:695-700. https://doi.org/10.1007/s12010-012-0043-8
  13. Izumitsu K, Hatoh K, Sumita T, et al. Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience. 2012;53:396-401. https://doi.org/10.1007/S10267-012-0182-3
  14. Lin X, Yang F, Zhou Y, et al. Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants. Yeast. 2012;29:467-474. https://doi.org/10.1002/yea.2926
  15. Ding Y, Liang S, Lei J, et al. Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus. Microbiol Res. 2011;166:314-322. https://doi.org/10.1016/j.micres.2010.07.001
  16. Zhu H, Liu D, Wang Y, et al. Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin. Biotechnol Lett. 2017;39:1167-1173. https://doi.org/10.1007/s10529-017-2343-0
  17. Liu D, Zhu H, Chen Y, et al. Agrobacterium tumefaciens-mediated transformation of the king tuber medicinal mushroom Lentinus tuber-regium (Agaricomycetes). Int J Med Mushrooms. 2018;20:791-796. https://doi.org/10.1615/IntJMedMushrooms.2018026991
  18. Bokulich NA, Mills DA. Improved selection of internal transcribed spacer-specific primers enables quantitative, ultra-high-throughput profiling of fungal communities. Appl Environ Microbiol. 2013;79:2519-2526. https://doi.org/10.1128/AEM.03870-12
  19. Bellemain E, Carlsen T, Brochmann C, et al. ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiol. 2010;10:1-9. https://doi.org/10.1186/1471-2180-10-1
  20. Wang YN, Liu XY, Zheng RY. Umbelopsis changbaiensis sp. nov. from China and the typification of Mortierella vinacea. Mycol Progress. 2014;13:657-669. https://doi.org/10.1007/s11557-013-0948-9

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