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

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 beta-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.

Carotenogenesis Is Regulated by 5′UTR-Mediated Translation of Phytoene Synthase Splice Variants

“…Expression was controlled by the 35Spro as above. As GUS enzyme activity reflects well GUS protein levels (Koo et al, 2007), the translation efficiency was estimated by determining the ratio of GUS activity and GUS mRNA expression in leaves of three independent transgenic events each (Fig. 5B).…”

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

“…The frequency of gus gene transcription fusion and gus gene expression in different plant tissues was higher [42]. Koo et al [43] revealed 13 % of gene trapping efficiency and Yamamoto et al [44] reported the same result for promoterless trapping vectors and 41 % of gene trapping efficiency for IRES-type trapping vector. We note that in our studies, however, enormous for promoter tagging experiments number of transgenic plants was resistant to PPT.…”

The Microbiome of Spodoptera littoralis: Development, Control and Adaptation to the Insect Host

“…For a gene to be selected as a reporter, it must be able to easily detect signals secreted by the expressed reporter gene in the cells [30]. The lux gene derived from bacteria, and luciferase from the firefly and click beetle (luc), are two other common reporter genes used in bioluminescence imaging.…”

“…pTRKH3 is a shuttle vector for E. coli, Enterococcus, Lactococcus, Streptococcus and Lactobacillus [60]. The vector has a copy number (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) in E. coli, and a somewhat higher copy number in Lactococcus and Streptococcus species [64]. It carries a gene for erythromycin resistance, which is expressed in E. coli and LAB.…”