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利用Cre/loxp系统位点专一性重组的特点构建诱导表达的定位重组系统,特异性地敲除转基因植物的选择标记基因。通过多重酶切、连接及测序后成功构建植物表达载体p BI121-Cre-GUS。利用农杆菌介导的叶盘法将此目标基因转入平邑甜茶中,通过抗性筛选和PCR鉴定获得转基因阳性植株。对阳性植株进行热激诱导后通过PCR及GUS染色进行检测转基因植株的删除效果,最终获得了无选择标记基因的转基因苹果植株。
The specific recombination site of Cre / loxp system was used to construct the inducible expression system of recombination and localization, specifically knocking out the selectable marker gene of transgenic plants. The plant expression vector pBI121-Cre-GUS was successfully constructed by multiple enzyme digestion, ligation and sequencing. The Agrobacterium-mediated leaf disc method was used to transfer the target gene into Pingmai sweet tea, and the transgenic plants were obtained through resistance screening and PCR identification. After the positive plants were induced by heat shock, the deletion effect of the transgenic plants was detected by PCR and GUS staining. Finally, the transgenic apple plants without the selectable marker gene were obtained.