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建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法。通过体外重组构建了双T-DNA双元载体pDLBRBbarm。载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA。利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%。对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ。这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物。研究还利用位于2个不同载体上的nptⅡ基因与bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%~47.4%,低于使用双T-DNA转化的共转化频率。
A method for cultivating transgenic plants with no selectable marker using dual T-DNA vectors was established. The double T-DNA binary vector pDLBRBbarm was constructed by in vitro recombination. In the vector, the selectable marker nptII gene and another bar gene representing the foreign gene are located in two independent T-DNAs, respectively. In Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L.), the frequency of simultaneous integration of the nptII gene and the bar gene in the transformed plants was 59.2%. The results showed that two T-DNA isolates could be isolated in three T1 generation lines, of which about 19.5% of transgenic T1 generation plants Only the bar gene is present without the selection marker nptII. This result shows that the double T-DNA vector system can be effectively used for cultivating transgenic plants without selectable markers. In addition, co-transformation of tobacco with nptII gene and bar gene mediated by two different vectors on Agrobacterium tumefaciens was carried out. The frequency of co-transformation plants was 20.0% -47.4%, which was lower than that of co-transformation using double T-DNA transformation.