拟南芥幼苗中荧光素酶活性的测定

Mohan  TC; Gabriel  Castrillo; Antonio  Leyva

doi:10.21769/BioProtoc.1160

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Peer-reviewed

Determination of Luciferase Activity in Arabidopsis seedling

拟南芥幼苗中荧光素酶活性的测定

Mohan TC

GC Gabriel Castrillo

Antonio Leyva email

发布: 2014年06月20日第4卷第12期 DOI: 10.21769/BioProtoc.1160 浏览次数: 12628

评审: Ru Zhang

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参见作者原研究论文

The authors used this protocol in:

Cover of The Plant Cell, featuring study using the protocol.

Aug 2013

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Abstract

There are different direct and indirect methods available to study gene expression in plant systems. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the control of phosphate transporter PHT1;1 promoter. This assay was originally optimized for analyzing the repression of PHT1;1 promoter in response to arsenate As (V) which can be adapted to study the kinetics of transcriptional regulation of any gene in response to biotic or environmental stimuli measuring Luciferase activity in Arabidopsis thaliana.

Keywords: Luciferase (荧光素酶)

Gene expression (基因的表达)

Arabidopsis (拟南芥)

Materials and Reagents

Transgenic Arabidopsis seedlings expressing Luciferase fused to promoter of interest (i.e. PHT1;1:Luc in pGWB435 binary vector)
Note: Vector information can be find at: http://shimane-u.org/nakagawa/pgwb-tables/2.htm.
KH₂PO₄ (Sigma-Aldrich, catalog number: P5655 )
Sodium arsenate dibasic heptahydrate (Sigma-Aldrich, catalog number: A6756 )
30 mM arsenate (see Recipes)
0.36 mM D-Luciferin (Sigma-Aldrich, catalog number: L9504 ) (see Recipes)
Johnson media (see Recipes)

Equipment

Growth chamber [16-h-light/8-h-dark regime (24 °C/21 °C)]
White 96-well plates (Greiner Bio-One GmbH, catalog number: 655074 )
MicroAmp optical adhesive film (Life Technologies, Applied Biosystem^®, catalog number: 4311971 )
Luminometer (centro LB 960) (Berthold Technologies)

Software

Mikro Win 2000 software

Procedure

English

中文翻译

文章信息

版权信息

如何引用

TC, M., Castrillo, G. and Leyva, A. (2014). Determination of Luciferase Activity in Arabidopsis seedling. Bio-protocol 4(12): e1160. DOI: 10.21769/BioProtoc.1160.