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Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach

WW Wan-Qiu Wang
Wei-Hua Tang Wei-Hua Tang

Published: Aug 20, 2018 DOI: 10.21769/BioProtoc.2976 Views: 5774

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Abstract

Fusarium graminearum is a destructive phytopathogen and shows an impressive metabolic diversity. Gene deletion is an important and useful approach for gene function study. Here we present a protocol for generating gene deletion mutant by applying “split-marker” deletion strategy (Catlett et al., 2003) with PEG-mediated protoplast transformation (Yuan et al., 2008; Martín, 2015).

Keywords: Fusarium graminearum search Gene knock out search Split-marker recombination search

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
  2. Sterile centrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  3. PCR tubes (Corning, Axygen®, catalog number: PCR-02-C )
  4. 50 ml volume centrifuged tubes (Corning, Axygen®, catalog number: SCT-50ML-25-S )
  5. Scalpel (Fisher Scientific, catalog number: 08-920B )
  6. 15 ml volume centrifuge tube (Corning, Axygen®, catalog number: SCT-15ML-25-S )
  7. Petri dish
  8. Medical gauze (regular cotton yarn, Shanghai Honglong Medical Material Company)
  9. Niex nylon membrane
  10. Sterile toothpicks
  11. Fungal strains: F. graminearum PH-1(NRRL 31084)
  12. Primers (store at 4 °C for using within one month, store at -20 °C for using within six months)
    1. Primer HY-F
    2. Primer HY-R
    3. Primer YG-F
    4. Primer YG-R
    5. Primer Up-F
    6. Primer Up-R
    7. Primer ID-F
    8. Primer ID-R
    9. Primer ID-2F
    10. Primer ID-2R
  13. Ampicillin sodium Salt (Yeasen, catalog number: 60203ES60 )
  14. Hygromycin B (Sigma-Aldrich, Roche Diagnostics, catalog number: 10843555001 )
  15. Chloroform (Sinopharm Chemical Reagent, catalog number: 10006818 )
  16. Isopropyl alcohol (Sinopharm Chemical Reagent, catalog number: 80109218 )
  17. 75% ethanol (Sinopharm Chemical Reagent, catalog number: 80176961 )
  18. Sterile ddH2O
  19. AxyPrepTM DNA Gel Extraction kit (Corning, Axygen®, catalog number: AP-GX-250 )
  20. KOD FX PCR kit (TOYOBO, catalog number: KOD-101 )
  21. Agarose (Biowest, catalog number: 111860 )
  22. TAE buffer (Thermo Fisher Scientific, catalog number: B49 )
  23. Lysing enzymes (Sigma-Aldrich, catalog number: L1412 )
  24. Driselase (Sigma-Aldrich, catalog number: D9515 )
  25. Chitinase (Sigma-Aldrich, catalog number: C6137 )
  26. KCl (Sinopharm Chemical Reagent, catalog number: 10016318 )
  27. Ampicillin (Yeasen, catalog number: 60203ES10 )
  28. Yeast extract (Thermo Fisher Scientific, OxoidTM, catalog number: LP0021B )
  29. Casamino acids (Sigma-Aldrich, catalog number: 22090 )
  30. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  31. CaCO3 (Sinopharm Chemical Reagent, catalog number: 10005717 )
  32. Tris (AMRESCO, catalog number: 0826 )
  33. CaCl2 (Sinopharm Chemical Reagent, catalog number: 10005861 )
  34. PEG4000 (Sinopharm Chemical Reagent, catalog number: 30151626 )
  35. NaCl (Sinopharm Chemical Reagent, catalog number: 10019318 )
  36. EDTA (Sinopharm Chemical Reagent, catalog number: 10009617 )
  37. Peptone (Sinopharm Chemical Reagent, catalog number: 10014963 )
  38. D-glucose (Sinopharm Chemical Reagent, catalog number: 10010518 )
  39. V8 juice (Campbell Soup Company)
  40. CTAB (Sinopharm Chemical Reagent, catalog number: 30037416 )
  41. Plasmid pUCATPH (the sequence can be download from website: http://www.snapgene.com/resources/plasmid_files/yeast_plasmids/pUCATPH/)
  42. Genomic DNA preparation of F. graminearum in ddH2O
  43. 0.22 μm PES membrane (Merck, catalog number: GPWP14250 )
  44. YEPD liquid medium (see Recipes)
  45. Mycelium enzymolysis mixture (see Recipes)
  46. STC buffer (see Recipes)
  47. PTC (see Recipes)
  48. TB3 plate (see Recipes)
  49. Low-melting-temperature TB3 (see Recipes)
  50. V8 juice agar (see Recipes)
  51. 1.5x CTAB (see Recipes)
  52. Mung bean liquid medium (see Recipes)
  53. 1.2 M KCl (see Recipes)

Equipment

  1. Hemocytometer (0.10 mm, 1/400 mm2) (QIUJING, model: XB-K-25 )
  2. Flask (SiQi, model: SP250SJ )
  3. Electronic balance (OHAUS, model: AR1140 )
  4. Mold incubator (Yiheng, model: MJ-150-I )
  5. Micropipettes (Eppendorf)
  6. Gel electrophoresis chamber (Tanon, model: EPS300 )
  7. Gel apparatus (Helixx Mupid-exU) and image system (Tanon gel image system, model: 1600 )
  8. Microcentrifuge (Eppendorf, model: 5418 , catalog number: 022620304)
  9. Centrifuge (Beckman Coulter, model: Avanti® J-E )
  10. PCR system (Bio-Rad Laboratories, model: S1000 )
  11. Orbital shaker (Kylin-Bell Lab Instruments, model: TS-2 )
  12. Shaker (Boyn Industria, model: THZ-C-1 )
  13. Biological safety cabinet (ESCO Micro, model: FHC1200A )
  14. Microscope (Olympus, model: BX51 )
  15. Autoclave (SANYO, model: MLS-3781L-PC )
  16. Ultra-low Temperature Freezer (Thermo Fisher Scientific, model: TSE400V )
  17. Tube holder (LUOBENDE)
  18. Tissuelyser II (QIAGEN, model: 85300 )
  19. Growth chamber (JIANGNAN INSTRUMENT, model: RXZ-1000 )
  20. DuoFlow pH Monitor (Bio-Rad Laboratories, model: 760-2040 )

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial genetics > Mutagenesis

Molecular Biology > DNA > Mutagenesis

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