采用 GC-EI-MS分析拟南芥花粉管中热水提取物的糖组分

Marie Dumont; Arnaud Lehner; Corinne Loutelier-Bourhis; Jean-Claude Mollet; Patrice Lerouge

doi:10.21769/BioProtoc.1503

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Peer-reviewed

Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS

采用 GC-EI-MS分析拟南芥花粉管中热水提取物的糖组分

Marie Dumont

AL Arnaud Lehner

CL Corinne Loutelier-Bourhis

Jean-Claude Mollet

PL Patrice Lerouge email

发布: 2015年06月20日第5卷第12期 DOI: 10.21769/BioProtoc.1503 浏览次数: 8372

评审: Samik BhattacharyaFeng LiAnonymous reviewer(s)

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Cover of BMC Plant Biology, featuring study using the protocol.

Aug 2014

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Abstract

Extraction with hot water is the oldest and simplest method used to recover pectin from an alcohol insoluble residue extract, although this method has not been widely used for the cell wall analysis of pollen tube, a model used to study cell wall. This protocol described this method applied for pectin extraction from 6 h-old Arabidopsis pollen tubes followed by a sugar composition analysis by gas chromatography mass spectrometry.

Keywords: Pollen tube (花粉管)

Cell wall (细胞壁)

Monosaccharide composition (单糖组成)

GC-MS (气相色谱-质谱联用)

Materials and Reagents

Arabidopsis day-0 flowers according to Boavida and McCormick (2007)
Absolute ethanol (EtOH) for analysis (Merck Millipore, catalog number: 107017 )
Trifluoroacetic acid 12.93N (TFA) (Sigma-Aldrich, catalog number: T62200 )
Myo-inositol 2 mM (Sigma-Aldrich, catalog number: I5125 )
Methanolic-HCl 3N (Sigma-Aldrich, catalog number: 33051 Supelco )
Methanol (Thermo Fisher Scientific, catalog number: 10010280 )
Silylation reagent (HMDS:TMCS:Pyridine, 3:1:9 Sylon^TM HTP) (Sigma-Aldrich, catalog number: 33038 Supelco )
Cyclohexane (Acros Organics, catalog number: 279590010 )
Monosaccharides standards
Arabinose (Sigma-Aldrich, catalog number: A3256 )
Fucose (Sigma-Aldrich, catalog number: F8150 )
Galactose (Sigma-Aldrich, catalog number: G0750 )
Galacturonic acid (Sigma-Aldrich, catalog number: 857289 )
Glucose (Merck Millipore, catalog number: 8337.0250 )
Glucuronic acid (Sigma-Aldrich, catalog number: G8645 )
Mannose (Sigma-Aldrich, catalog number: M4625 )
Rhamnose (Sigma-Aldrich, catalog number: R3875 )
Xylose (Sigma-Aldrich, catalog number: X2126 )
Arabidopsis pollen germination medium (see Recipes)
Trifluoroacetic acid (2 N) (see Recipes)

Equipment

Inverted microscope Olympus CK2
Centrifuge Allegra^® X-15R Beckmann Coulter
Hot-water bath (70 °C and 90 °C)
40-ml glass potter homogenizer
Freeze-dryer
Incubator (22 °C, 80 °C and 110 °C)
Sample concentrator under air flow (Techne, catalog number: FSC400D )
GC-EI-MS instrument is composed of a Zebron Z5-MSi (30 m, 0.25 mm id, 0.25 µm film thickness, Phenomenex) capillary column for chromatographic separations, a Hewlett-Packard 6890 series gas chromatograph coupled to an Autospec mass spectrometer of EBE geometry (Micromass, Manchester, UK) equipped with a Opus 3.1 data system
1.5 ml Eppendorf tube
Vortex
Pair of tweezers
50-ml Falcon
Microliter syringe (10 µl, Hamilton)

Procedure

English

中文翻译

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如何引用

Dumont, M., Lehner, A., Loutelier-Bourhis, C., Mollet, J. and Lerouge, P. (2015). Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS. Bio-protocol 5(12): e1503. DOI: 10.21769/BioProtoc.1503.