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Plant and Cell Physiology Advance Access originally published online on June 27, 2007
Plant and Cell Physiology 2007 48(8):1121-1131; doi:10.1093/pcp/pcm081
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability

Jachoon Koo1,3, Yumi Kim1, Jeongsik Kim1, Miji Yeom1, In Chul Lee1 and Hong Gil Nam1,2,*

1Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
2I-BIO Graduate School, Pohang University of Science and Technology, Pohang 790-784, Korea

*Corresponding author: E-mail, nam{at}postech.ac.kr; Fax, 82-54-279-5972.


  Abstract

A gene-trapping vector carrying a GUS/Luciferase dual reporter gene was developed to establish an efficient and convenient screening system for T-DNA-based gene trapping in plants. A key feature of this gene trap scheme is to place two different types of reporters, luciferase (Luc) and ß-glucuronidase (GUS), as a fusion protein within a trapped gene to probe the activity of the gene. Luc is then utilized as a non-invasive, vital and highly sensitive screening reporter to identify trapped lines, including direct screening of the trapped lines from the primary T-DNA mutant pools. GUS is utilized as a histochemical assay reporter to analyze detailed cellular expression patterns. Transgenic expression studies in Arabidopsis showed that this fusion reporter protein retains functional enzyme activity for both GUS and Luc. Using this system in Arabidopsis, we were able to identify 3,737 trapped lines from 26,900 individual T-DNA insertion lines. Sequence determination of the T-DNA insertion loci in the genome of 78 trapped lines identified GUS/Luc fusions with 27 annotated Arabidopsis genes which included a subset of transcription factors, protein kinases, regulatory proteins and metabolic enzymes. Of these, particular expression patterns of four tagged genes were further confirmed by analyzing putative promoter regions of the corresponding wild-type genes. Furthermore, the protein stability of the GUS/Luc fusion reporter was controlled by application of luciferase substrate (luciferin), overcoming the excessive stability problem of GUS that causes misrepresentation of the transcriptional activity of a promoter. These results demonstrate the utility of the GUS/Luc dual reporter system as a gene trap reporter for studying plant genome function and also as a convenient dual reporter system for study of gene expression.

Keywords: Arabidopsis - Dual reporter - Firefly luciferase - Gene trapping - GUS

Abbreviations: GFP, green fluorescent protein; GUS, ß-glucuronidase; HR, hypersensitive response; Luc, firefly luciferase; MU-GlcA, 4-methylumbelliferyl ß-d-glucuronide; PPT, phosphinothricin; UTR, untranslated region; Xgluc, 5-bromo-4-chloro-3-indolyl glucuronide

3Present address: Division of Science Education, Chonbuk National University, Jeonju 561-756, Jeonbuk, Korea.

(Received February 14, 2007; Accepted June 21, 2007)
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