Gene trapping with firefly luciferase in Arabidopsis. Tagging of stress-responsive genes.
To monitor the expression of T-DNA-tagged plant genes in vivo, a collection of 20,261 transgenic lines of Arabidopsis (Columbia-0) were generated with the promoter trap vector pTluc, which carries a promoterless firefly luc (luciferase) reporter gene linked to the right T-DNA border. By detection of bioluminescence in 3-week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. To facilitate the identification of well-defined luciferase expression patterns, a pooled seed stock was established. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity. Segregation analysis of 215 promoter trap lines indicated that about 50% of plants contained single insertions, whereas 40% carried two and 10% carried three or more T-DNA tags. Sequencing the T-DNA insert junctions isolated from 17 luciferase-expressing lines identified T-DNA tags in 5'- and 3'-transcribed domains and translational gene fusions generated by T-DNA insertions in exons and introns of Arabidopsis genes. Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be similar to the luminescence patterns observed in the corresponding luciferase-tagged lines. Here, we describe the characterization of a transcriptional luc reporter gene fusion with the WBC-type ABC transporter gene At1g17840. Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar induction by salt, glucose, and ABA treatments and gibberellin-mediated down-regulation of ABA-induced expression. These results illustrate that luciferase gene traps are well suited for monitoring the expression of stress-responsive Arabidopsis genes in vivo.
Alvarado
Martha C
MC
Institute of Plant Biology, Biological Research Center, Temesvári krt. 62, 6726-Szeged, Hungary.
Zsigmond
Laura M
LM
Kovács
Izabella
I
Cséplö
Agnes
A
Koncz
Csaba
C
Szabados
László M
LM
D000818:Animals; D017360:Arabidopsis / Q000235:genetics*; D019677:Artificial Gene Fusion; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D017930:Genes, Reporter; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C036483:T-DNA; D008156:Luciferases