尖孢镰刀菌菌丝体的体内和体外葡萄糖的测定

Carmen Ruiz-Roldan; M. Isabel G. Roncero

doi:10.21769/BioProtoc.1868

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Peer-reviewed

Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum

尖孢镰刀菌菌丝体的体内和体外葡萄糖的测定

Carmen Ruiz-Roldan email

M. Isabel G. Roncero

发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1868 浏览次数: 10343

评审: Zhaohui LiuShahin S. AliSusheel KumarAnonymous reviewer(s)

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The authors used this protocol in:

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

Jan 2015

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Abstract

To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.

Keywords: Glucose determination (血糖测定)

Fusarium (镰刀菌)

Glucose utilization (葡萄糖的利用)

Materials and Reagents

Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15 )
2 ml Eppendorf tubes
Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
5 mm-diameter glass beads (Sigma-Aldrich, catalog number: 18406-500G )
Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and Δcon7-1 mutant strains
Sterile dH₂O
Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483 )
Glycerol (Merck, catalog number: 104092 )
Glucose (Coger SAS, catalog number: 24379.363 )
Na₂CO₃ (Sigma-Aldrich, catalog number: S2127 )
Glucose (GO) Assay Kit (Sigma-Aldrich, catalog number: GAGO-20 )
GO Assay includes:

Glucose Oxidase/Peroxidase Reagent (Sigma-Aldrich, catalog number: G3660 )
o-Dianisidine Reagent (Sigma-Aldrich, catalog number: D2679 )
Glucose Standard Solution (Sigma-Aldrich, catalog number: G3285 )

H₂SO₄, ACS reagent (Merck, catalog number: 108131.1000 )
MgSO₄·7H₂O (Merck, catalog number: 1058865.5000 )
KH₂PO₄ (Merck, catalog number: 104873.1000 )
KCl (Merck, catalog number: 104933.0500 )
NH₄NO₃ (Merck, catalog number: 101187.1000 )
FeSO₄ (Merck, catalog number: 103965.0500 )
ZnSO₄·7H₂O (Merck, catalog number: 108883 )
MnSO₄·H₂O (Merck, catalog number: 105941 )
0.1% benzoic acid
Extraction buffer (see Recipes)
Synthetic medium (SM) (see Recipes)
H₂SO₄(12 N) (see Recipes)

Equipment

Mini-BeadBeater -16 homogenizer (BioSpec Products)
Orbital incubator (Infors, Multitron Pro)
Sterile spatula (Fisher Scientific, catalog number: S50821 )
Microcentrifuge (Fisher Scientific, Eppendorf^TM MiniSpin plus^TM)
Hemocytometer (Thoma) (Marienfeld, catalog number: 06 407 10 )
Fluorimeter (TECAN, SpectraFluorPlus, model: F129005 )
Freeze-dryer (Virtis, model: BT4KZL-105 )
Water bath (Selecta, model: 6000138 )
Vortex (IKA, model: MS2 Mini shaker )

Procedure

English

中文翻译

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如何引用

Ruiz-Roldan, C. and Roncero, M. I. G. (2016). Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum. Bio-protocol 6(14): e1868. DOI: 10.21769/BioProtoc.1868.