Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database.
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.
Ryu
Choong-Hwan
CH
National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
You
Jung-Hwa
JH
Kang
Hong-Gyu
HG
Hur
Junghe
J
Kim
Young-Hea
YH
Han
Min-Jung
MJ
An
Kyungsook
K
Chung
Byoung-Chull
BC
Lee
Choon-Hwan
CH
An
Gynheung
G
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D030561:Databases, Nucleic Acid; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000235:genetics; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D018744:DNA, Plant; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins