Generation and characterization of enhancer trap lines in rice

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(1) Generation of transgenic rice lines having single copy (42 lines) or double copies (above 50 lines) of Ds-GUS sequences and 6 lines possesing 35S-Ac transposase genes were generated. Examination for DsGUS transposition frequencies by screening F2 plants derived from cross combinations between four DsGUS and six 35S-Ac TPase lines with PCR method showed average frequency was 6% (675 / 10, 524 plants). (2) The 35S-Ac TPase line which showed higest activity to traspose Ds-GUS element was crossed with 22 Ds-GUS lines. A part ofF2 progenies, 926 out of 3277 F2 plants, exhibited transposition and average frequency of Ds-GUS transposition was calculated 28%. (3) We next screened 540 F2 transposants for GUS expression by staining almost all tissues in different developmental stages ; leaf blade, leaf sheath, auricle, ligule, root, node, rachis, papillae, style, ovary, anther, filament, lodicle, palea, lemma, upper glume, lower glume etc. Between 10 to 20 % of the transposants examined so far revealed to show trapped enahancer-GUS activity in any tissue. (4) To detect original and transposed insertion sites of Ds-GUS elememt, we isolated flanking genome sequences of the insertion positions and sequenced. A part of the flanking sequences (about 100) isolated and sequenced could be mapped through homology searching against chromosome assigned genome sequences. Remaining clones will be soon mapped on the fully aligned genome sequences