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

Item Type Journal Article
Title A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability
Author Koo, Jachoon
Author Kim, Yumi
Author Kim, Jeongsik
Author Yeom, Miji
Author Lee, In Chul
Author Nam, Hong Gil
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 β-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.
Publication Plant and Cell Physiology
Volume 48
Issue 8
Pages 1121-1131
Date 08/01/2007
Journal Abbr Plant Cell Physiol
Language en
DOI 10.1093/pcp/pcm081
ISSN 0032-0781, 1471-9053
URL http://pcp.oxfordjournals.org/content/48/8/1121
Accessed 2012-10-19 09:47:06
Library Catalog pcp.oxfordjournals.org
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