稻瘟病菌中的甲壳素提取和含量测定

Xinyu Liu; Zhengguang  Zhang

doi:10.21769/BioProtoc.2164

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Peer-reviewed

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 email

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2164 浏览次数: 8319

评审: Zhaohui LiuAnna Дмитриевна KozhevnikovaAnonymous reviewer(s)

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Cover of Molecular Plant Microbe Interactions, featuring study using the protocol.

Jun 2016

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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)

Cell wall (细胞壁)

GlcNAc (GlcNAc)

Pathogenicity (致病性)

Magnaporthe oryzae (稻瘟病菌)

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

Microtube
Microtiter plates (Corning, Costar^®, catalog number: 42592 )
Miracloth (EMD Millipore, catalog number: 475855 )
Cells of Magnaporthe oryzae
Potassium hydroxide (KOH) (Sangon Biotech, catalog number: A610441 )
10x phosphate buffered saline (PBS, 1.35 M NaCl, 47 mM KCl, 100 mM Na₂HPO_4,20 mM NaH₂PO_4,pH = 7.4) (Beyotime, catalog number: ST476 )
Streptomyces plicatus chitinase (Sigma-Aldrich, catalog number: C6137 )
Sodium borate (Sangon Biotech, catalog number: A100390 )
GlcNAc (Sigma-Aldrich, catalog number: PHR1432-1G )
Sodium hydrogen (Na₂HPO₄) (Sangon Biotech, catalog number: A501727 )
Citric acid (Sangon Biotech, catalog number: A501702 )
p-dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: D2004 )
Hydrochloric acid (HCl)
Acetic acid (Sangon Biotech, catalog number: A501931 )
Biotin
Pyridoxin
Thiamine
Riboflavin
p-aminobenzoic acid
nicotinic acid
ZnSO₄·7H₂O
H₃BO₃
MnCl₂·4H₂O
FeSO₄·7H₂O
CoCl₂·6H₂O
CuSO₄·5H₂O
Na₂MnO₄·2H₂O
Na₄EDTA
NaNO₃
KCl
MgSO₄·7H₂O
KH₂PO₄
D-glucose
Peptone
Yeast extract
Casamino acid
Agar
McIlvaine’s buffer (see Recipes)
Ehrlich’s solution (see Recipes)
Vitamin solution (see Recipes)
Trace elements (see Recipes)
20x nitrate salts (see Recipes)
CM medium (see Recipes)

Equipment

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

Software

SPSS 2.0 (Chicago, IL, USA)

Procedure

English

中文翻译

文章信息

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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.