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在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。
In plant transgenic plants, resistance screening of transformed cells is a common procedure and the resistance of the transformed cells is generally antibiotic or herbicide resistant. The deletion of the selectable marker gene conferring resistance to the transformed cells is a result of increased resistance of transgenic plants Biosecurity important measures. The FLP / frt site-specific recombination system from Saccharomyces cerevisiae can effectively delete the gene between the two sites frt. Through multi-step recombination, a FLP / frt site-specific recombination system that can be widely used in plants was established. The system includes the plant expression vector pCAMBIA1300-betA-frt-als-frt containing the frt site and the plant expression vector pCAMBIA1300-hsp-FLP-hpt containing the FLP recombinase gene initiated by the heat-inducible promoter hsp. The two transformations were transformed into tobacco plants by secondary transformation. After heat shock treatment, the expression of the FLP gene regulated by the heat-inducible promoter hsp catalyzed the recombination between the same-oriented frt sites on both sides of the selectable marker gene als The reaction effectively deleted the selectable marker gene als. The deletion of the selectable marker gene occurred in 41% of the heat-shocked secondary transformed plants, indicating that the system has good application value in obtaining transgenic plants without the selectable marker gene.