Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database.
Plant Mol Biol
; 54(4): 489-502, 2004 Mar.
Article
en En
| MEDLINE
| ID: mdl-15316285
ABSTRACT
We have developed a binary T-DNA vector, pGA2717, that contains the promoter-less beta-glucuronidase (gus) gene adjacent to the right border and the promoter-less green fluorescence protein (gfp) gene next to the left border of the T-DNA. Therefore, inserting T-DNA into a gene can result in the activation of either gus or gfp. A total of 12 169 T-DNA insertional lines of japonica rice were generated using this binary vector. Out of 3140 lines examined, 0.5% of their mature seeds and 2.0% of the 3-day-old etiolated seedlings were GFP-positive. However, GUS assays of the same materials resulted in the identification of 151 (4.8%) GUS-positive lines. Using DNA gel blot and reverse transcription (RT)-PCR analyses, we confirmed that the GFP-positive lines were a true indication of gene trapping. A fusion transcript was also obtained between gfp and the trapped gene. We isolated 990 genomic sequences flanking T-DNA from our analysis of 2099 transgenic plants. Among the insertions, 625 T-DNAs were integrated into genic regions; 361 were located in intergenic regions. These tagging lines will be valuable in trapping and studying various genes for their expression patterns, as well as providing a useful tool for genetic approaches.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN Bacteriano
/
Bases de Datos de Ácidos Nucleicos
/
Vectores Genéticos
Idioma:
En
Revista:
Plant Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BOTANICA
Año:
2004
Tipo del documento:
Article