Phenotypic assay for excision of the maize controlling element Ac in tobacco
Phenotypic assay for excision of the maize controlling element Ac in tobacco
复制标题
烟草中玉米控制元件 Ac 切除的表型测定
DOI：
10.1002/j.1460-2075.1987.tb02399.x
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Jozef Schell
中科院分区：
文献类型：
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作者：
Barbara Baker;George Coupland;Nina V Fedoroff;P. Starlinger;Jozef Schell
我们描述了一种表型分析，旨在检测玉米控制元件ac从一个可选择的标记基因新霉素磷酸转移酶II(NPT II)中删除。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。Ac或有缺陷的Ac元件(Ac)被插入到该基因的前导区。通过瞬时NPT II表达分析，转座子的插入使NPT II基因失活。将这3个质粒分别插入到根癌农杆菌T DNA载体中，转化烟草原生质体。转化的原生质体用100或200微克/毫升的卡那霉素进行筛选。经Ac中断的nptⅡ基因转化的原生质体产生的愈伤组织˜是未中断nptⅡ基因转化的原生质体的25%。在相同条件下，经Ac中断的NPT II基因转化的原生质体未形成对200µg/ml卡那霉素有抗性的愈伤组织。Southern杂交分析表明，在Ac中断的NPT II基因转化后获得的7个卡那霉素抗性愈伤组织或植株中，Ac都被切除，恢复了NPT II基因的结构。因此，该检测方法对于监测转座元件如Ac的活性和确定该元件参与转座活性的区域是有用的。
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△), 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 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac△ did not form any calli resistant to 200 µg/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.