内蒙古医院团委与社会公益组织联合开展关爱血液

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Three vectors—pUC18, pPZP100, and pBin19 (15–17)—were digested with Eco RI and Hind III and a synthetic linker flanking by meganuclease I–Sce I including Kpn I, Not I, and Sma I (5′-AATTCATTACCCTGTTATCCCTACCCGGGCGGCCGCGGTACCATTACCCTGTTATCCCTAA-3′) and (5′-AGCTTTAGGGATAACAGGGTAATGGTACCGCGGCCGCCCGGGTAGGGATAACGGGTAATG-3′) were introduced, forming pUC18M, pPZP100M, and pBin19M, respectively. An intermediate vector was made by insertion of the crtI expression cassette excised from Hind III/Eco RI–digested pUCET4, originally derived from pYPIET4 (11), into pBluescriptKS with Hind III/Eco RI digestion, followed by insertion of psy expression cassette from Sac II–blunted/Kpn I–digested pGt1psyH (7) into the Kpn I/Xho I–blunted previous vector. Finally, crtI and psy expression cassettes were isolated with Kpn I/Not I digestion and inserted into Kpn I/Not I–digested pUC18M and designated as pBaal3. pBin19hpc was made by insertion of a Kpn I fragment originally from pCIB900 (18) containing aphIV selectable marker gene into pBaal3, followed by digestion of the I-Sce I fragment of the resulting plasmid and insertion into I-Sce I–digested pBin19M. pZPsC was obtained by insertion of the I-SceI fragment of pBaal3 bearing the psy and crtI genes into I-Sce I–digested pPZP100M. pZLcyH was constructed by digestion of pGt1LcyH with I-Sce I and insertion of the resulting fragment, carrying lcy and aphIV, into I-Sce I–restricted pPZP100M. The three vectors were separately electroporated into Agrobacterium tumefaciens LBA4404 (19) with corresponding antibiotic selection. Callus induction: Immature seeds of japonica rice cultivar TP 309 at milk stage were collected from greenhouse-grown plants, surface-sterilized in 70% ethanol (v/v) for 1 min, incubated in 6% calcium hypochloride for 1 hour on a shaker, and rinsed three to five times with sterile distilled water. Immature embryos were then isolated from the sterilized seeds and cultured onto NB medium [N6 salts and B5 vitamins, supplemented with 30 g/l maltose, 500 mg/l proline, 300 mg/l casein hydrolate, 500 mg/l glutamine, and 2 mg/l 2,4-D (pH 5.8)]. After 4 to 5 days, the coleoptiles were removed, and the swelled scutella were subcultured onto fresh NB medium for 3 to 5 days until inoculation of Agrobacterium. Transformation: 1-week-old precultured immature embryos were immersed in Agrobacterium tumefaciens LBA 4404 cell suspension as described (20). For co-transformation, LBA4404/pZPsC [optical density at 600 nm (OD₆₀₀) = 2.0] mixed with an equal volume of LBA4404/pZLcyH (OD₆₀₀ = 1.0) was used for inoculation after acetonsyrigone induction. The inoculated precultured embryos were co-cultivated onto NB medium supplemented with 200 mM acetonsyringone for 3 days, subcultured on recovery medium (NB with 250 mg/l cefotaxime) for 1 week and then transferred onto NB selection medium in the presence of 30 mg/l hygromycin and 250 mg/l cefotaxime for 4 to 6 weeks. Transgenic plants were regenerated from recovered resistant calli on NB medium supplemented with 0.5 mg/l NAA and 3 mg/l BAP in 4 weeks, rooted and transferred into the greenhouse.

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The crtI gene fused to the transit peptide sequence was kindly provided by N. Misawa (Kirin Co., Ltd., Japan). We thank W. Dong and P. Burkhardt for their valuable contributions, S. Klarer, K. Konja, and U. Schneider-Ziebert for skillful technical assistance, and R. Cassada for correcting the English version of the manuscript. Supported by the Rockefeller Foundation (1993–1996), the European Community Biotech Program (FAIR CT96, 1996–1999) (P.B.), the Swiss Federal Office for Education and Science (I.P.) and by the Swiss Federal Institute of Technology (1993–1996).