Cre/loxp系统的构建及无选择标记转基因苹果植株的获得

Constructed the Cre/loxp Recombination Inducible Excision System and Obtained Marker-free Transgenic Apple Plants

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。

Used the Cre/loxp to establish inducible expression systems with site-specific recombination ability,to eliminate the selectable marker genes in transgenic plants specifically. Firstly,a binary expression vector PBI121-Cre-GUS with the Cre/loxp system was constructed to eliminate a marker gene from transgenic plants conveniently by digesting three intermediate vector and p BI121. Secondly,transgenic Malus hupehensis var. pinyiensis were obtained by using Agrobacterium mediated leaf disc method. These transgenic plants were induced by heat shock. Subsequently,the elimination efficiency was determined by GUS staining and PCR amplification. Finally,the transgenic plants without selectable marker gene were obtained.