Plant biotech & Risk Assessment
Okuzaki, Ayako , Toriyama, Kinya .
Repairing assay system of chromosomal mutated GUS gene in rice for improving the condition of oligonucleotide-directed gene targeting in rice.
Chimeraplasts, which consists of 68mer synthesized RNA-DNA oligonucleotides, are reported to cause site specific base change in chromosomal target genes in mammalian cells and also in some plants. In plants however, the efficiency of the oligonucleotide-directed gene targeting was estimated to be 10-4, which is low in comparison with mammalian cells, and the oligonucleotide-directed gene targeting was only demonstrated in acetolactate synthase gene, which confers chemically selectable phenotype. If oligonucleootide-directed gene targeting is able to convert various genes, it is thought to be useful for investigating gene function or producing functional crops. In this study, we established a chromosomal gene repair assay system in rice to compare the introduction condition of oligonucleotides in earlier stage. To facilitate the detection of the cells which having predicted gene conversion at the target site, the mutant GUS gene, which was connected to CaMV 35S promoter and CAT1 intron (35S:mGUS), was created and introduced into rice. Chimeraplasts for repairing mutant GUS gene were introduced into scutellum-derived calli containing 35S:mGUS by particle bombardment. GUS assay were carried out one week after bombardment. Some blue spots for GUS expression were detected with predicted gene conversion. Using this assay system, we are comparing an effective construct and amount of oligonucleotides. We are testing various kinds of oligonucleootides such as chimeraplasts, 68mer all-DNA analog of chimeraplasts and 35mer single stranded oligonucleotides, as well as co-bombardment of plasmid DNA containing genes for DNA repairing enzymes. Improvement of the condition of oligonucleotide-directed gene targeting is now in progress.
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1 - Graduate School of Agricultural Science, Tohoku University , 1-1, Tsutsumi-dori, Amamiya-mati,, Aoba-ku, Sendai, Miyagi, 981, Japan
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM