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Chitin Extraction and Content Measurement in Magnaporthe oryzae

稻瘟病菌中的甲壳素提取和含量测定

XL Xinyu Liu
Zhengguang Zhang Zhengguang Zhang

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2164 浏览次数: 9250

评审: Zhaohui LiuAnna Дмитриевна KozhevnikovaAnonymous reviewer(s)

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Abstract

Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example.

Keywords: Chitin (甲壳素) search Cell wall (细胞壁) search GlcNAc (GlcNAc) search Pathogenicity (致病性) search Magnaporthe oryzae (稻瘟病菌) search

Background

Chitin is an important component of the cell wall in fungal pathogens and is well known as a pathogen associated molecule pattern (PAMP). Determining the chitin content of a fungal species is important for studying fungal biology and host-pathogen interactions. The Morgan-Elson method based on colorimetric approach has been adapted in yeast to measure cellular chitin levels (Leloir and Cardini, 1953; Bulik et al., 2003; Baker et al., 2007). However, there is no specific protocol established for Magnaporthe oryzae, which is the causal agent of rice blast, the most important fungal disease in the world. Here, we describe a reliable and simple protocol which was modified from the Morgan-Elson method to test the chitin content of M. oryzae (Song et al., 2010).

Materials and Reagents

  1. Microtube
  2. Microtiter plates (Corning, Costar®, catalog number: 42592 )
  3. Miracloth (EMD Millipore, catalog number: 475855 )
  4. Cells of Magnaporthe oryzae
  5. Potassium hydroxide (KOH) (Sangon Biotech, catalog number: A610441 )
  6. 10x phosphate buffered saline (PBS, 1.35 M NaCl, 47 mM KCl, 100 mM Na2HPO4, 20 mM NaH2PO4, pH = 7.4) (Beyotime, catalog number: ST476 )
  7. Streptomyces plicatus chitinase (Sigma-Aldrich, catalog number: C6137 )
  8. Sodium borate (Sangon Biotech, catalog number: A100390 )
  9. GlcNAc (Sigma-Aldrich, catalog number: PHR1432-1G )
  10. Sodium hydrogen (Na2HPO4) (Sangon Biotech, catalog number: A501727 )
  11. Citric acid (Sangon Biotech, catalog number: A501702 )
  12. p-dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: D2004 )
  13. Hydrochloric acid (HCl)
  14. Acetic acid (Sangon Biotech, catalog number: A501931 )
  15. Biotin
  16. Pyridoxin
  17. Thiamine
  18. Riboflavin
  19. p-aminobenzoic acid
  20. nicotinic acid
  21. ZnSO4·7H2O
  22. H3BO3
  23. MnCl2·4H2O
  24. FeSO4·7H2O
  25. CoCl2·6H2O
  26. CuSO4·5H2O
  27. Na2MnO4·2H2O
  28. Na4EDTA
  29. NaNO3
  30. KCl
  31. MgSO4·7H2O
  32. KH2PO4
  33. D-glucose
  34. Peptone
  35. Yeast extract
  36. Casamino acid
  37. Agar
  38. McIlvaine’s buffer (see Recipes)
  39. Ehrlich’s solution (see Recipes)
  40. Vitamin solution (see Recipes)
  41. Trace elements (see Recipes)
  42. 20x nitrate salts (see Recipes)
  43. CM medium (see Recipes)

Equipment

  1. Freeze dryer (Marin Christ German)
  2. Vortex mixer
  3. Water bath (SHEL Lab, model: W6M-2 )
  4. Centrifuge (Eppendorf centrifuge) (Eppendorf, model: 5418 )
  5. Incubator shaker (Crystal Technology & Industries, model: IS-RSD3 )
  6. pH/ATU electrode (Sartorius, German)
  7. Microplate reader (Molecular Devices, model: VersaMax ELISA )
  8. PCR instrument (Takara Bio, model: TP600 )
  9. Eppendorf micropipette (1,000 μl, 100 μl, 10 μl)
  10. Dark glass bottle

Software

  1. SPSS 2.0 (Chicago, IL, USA)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Liu, X. and Zhang, Z. (2017). Chitin Extraction and Content Measurement in Magnaporthe oryzae. Bio-protocol 7(5): e2164. DOI: 10.21769/BioProtoc.2164.

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分类

微生物学 > 微生物生物化学 > 糖类

微生物学 > 微生物-宿主相互作用 > 真菌

生物化学 > 糖类 > 多糖

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