拟南芥吸收33P-PO4的能力和其在根茎中转移的测定

Improve Research Reproducibility A Bio-protocol resource

Peer-reviewed

Measurement of ³³P-PO₄ Absorption Capacity and Root-to-leaf Transfer in Arabidopsis

评审: Tie LiuMichael O. AduAnonymous reviewer(s)

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The authors used this protocol in:

Apr 2015

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Abstract

This method allows quantification of phosphate absorption capacity by Arabidopsis roots using very simple equipment, and can be scaled up or down.

Materials and Reagents

Plastic 12-well plates (Denmark, Nunc)
Note: One for the absorption step, one for the desorption step for each treatment (plant type or culture condition).
Plastic 20 ml vials for radioactivity measurement (Ratiolab GmbH, Dreieich)
Note: You will need one vial per plant, or 2 vials per plant if you want to quantify ³³P in both roots and leaves. The vials should be numbered from 1 to N before you start the experiment. They also should be placed in order in appropriate racks (PerkinElmer) adapted to the beta counter.
Tips
Young in vitro plantlets
MES hydrate (Sigma-Aldrich, catalog number: M8250 )
CaCl₂ (Sigma-Aldrich)
KH₂PO₄ (Sigma-Aldrich)
³³P-PO₄ 5 mCi/ml (40-158 Ci/mg, 1.48-5.84 TBq/mg, >99% isotopically pure, less than 0.5 μM Pi) (PerkinElmer)
Scintillation cocktail (PerkinElmer, Ultima Gold^TM)
MgSO₄
NH₄NO₃
KNO₃
NaH₂PO₄
KI
FeCl₂
MnSO₄
ZnSO₄
CuSO₄
CoCl₂
Na₂MoO₄
Thiamine
Pyridoxine
Nicotinic acid
Inositol
Sucrose
Agar
MS/10 medium (see Recipes)
Stock solution (see Recipes)
Incubation medium (see Recipes)
Desorption medium (see Recipes)

Equipment

Experiments should be performed on a bench or under a hood illuminated with white light (150 -180 μEm^-2s^-1) during the incubation step
Liquid scintillation counter (PerkinElmer, Packard Instrument Company, model: TRI-CARB )
Ice-containing large boxes for the desorption step (all 12-well plates will be placed horizontally on ice for 2 h)
Tweezers for handling the plantlets
If necessary, a razor to separate roots and aerial parts
Micropipets
Shield for protection against radiations (plexiglass)
Scanner or camera (Epson America, model: Perfection V850Pro or Canon, model: Powershot SX130 ), respectively but other devices from other manufacturers could suit perfectly

Software

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Thibaud, M. and Marin, E. (2016). Measurement of ³³P-PO₄ Absorption Capacity and Root-to-leaf Transfer in Arabidopsis. Bio-protocol 6(5): e1741. DOI: 10.21769/BioProtoc.1741.
Ayadi, A., David, P., Arrighi, J. F., Chiarenza, S., Thibaud, M. C., Nussaume, L. and Marin, E. (2015). Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. Plant Physiol 167(4): 1511-1526.