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
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1868 浏览次数: 11108
评审: Zhaohui LiuShahin S. AliSusheel KumarAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
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, EppendorfTM MiniSpin plusTM)
- 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
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
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分类
生物化学 > 糖类 > 葡萄糖
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