Max-Planck Institut für Züchtungsforschung, D-5000 Köln 30, FRG.
EMBO J. 1987 Jun;6(6):1547-54. doi: 10.1002/j.1460-2075.1987.tb02399.x.
We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac big up tri, open), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 microg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed 25% as many calli resistant to 100 or 200 microg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac big up tri, open did not form any calli resistant to 200 microg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.
我们描述了一种表型测定法，旨在检测玉米调控元件 Ac 从可选择标记基因新霉素磷酸转移酶 II（NPT II）中的切除。构建了一个在烟草细胞中表达卡那霉素抗性的 NPT II 基因，该基因在未翻译的前导区含有一个独特的限制性内切酶位点。将 Ac 或有缺陷的 Ac 元件（Ac big up tri，open）插入该基因的前导区。通过瞬时 NPT II 表达测定，发现转座子插入使 NPT II 基因失活。将这三个质粒插入到根癌农杆菌 Ti 质粒载体的 T DNA 中，并转移到烟草原生质体中。用 100 或 200 μg/ml 卡那霉素选择转化的原生质体。被 Ac 中断的 NPT II 基因转化的原生质体形成的对 100 或 200 μg/ml 卡那霉素有抗性的愈伤组织比未被中断的 NPT II 基因转化的原生质体少 25%。在类似条件下，被 Ac big up tri，open 中断的 NPT II 基因转化的原生质体不能形成任何对 200 μg/ml 卡那霉素有抗性的愈伤组织。对被 Ac 中断的 NPT II 基因转化的七个卡那霉素抗性愈伤组织或植株进行 Southern blot 杂交分析表明，在所有情况下，Ac 都已切除，恢复了 NPT II 基因的结构。因此，该测定法可用于监测转座元件（如 Ac）的活性，并确定该元件参与转座活性的区域。
