Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database

• Published in: Plant molecular biology Vol. 54; no. 4; pp. 489 - 502
• Main Authors: Ryu, C.H, You, J.H, Kang, H.G, Hur, J, Kim, Y.H, Han, M.J, An, K, Chung, B.C, Lee, C.H, An, G
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01.03.2004
• Subjects: Base Sequence; beta-glucuronidase; Binding Sites - genetics; Culture Techniques; Databases, Nucleic Acid; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Plant - chemistry; DNA, Plant - genetics; DNA, Plant - isolation & purification; enzyme activity; flanking sequence database; fluorescence; gene banks; Gene Expression; gene targeting; Genes; Genes, Plant - genetics; genetic techniques and protocols; Genetic Vectors - genetics; gfp gene; green fluorescent protein; Green Fluorescent Proteins; gusA gene; insertional mutagenesis; lines; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Molecular Sequence Data; Mutagenesis, Insertional; nucleotide sequences; Oryza - genetics; Oryza sativa; Plants, Genetically Modified; plasmid vectors; Plasmids - genetics; Polymerase Chain Reaction; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; reporter genes; rice; Seedlings; seeds; Sequence Analysis, DNA; Transcription, Genetic; transfer DNA; transgenes; Transgenic plants; Trapping
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/B:PLAN.0000038257.93381.05

We have developed a binary T-DNA vector, pGA2717, that contains the promoter-less β-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.