摘要
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.
出处
《园艺学报》
CAS
CSCD
北大核心
2016年第2期205-217,共13页
Acta Horticulturae Sinica
基金
国家高技术研究发展计划(‘863’)项目(2008AA10Z157)
北京市农林科学院科技创新能力建设专项(KJCX20140202)
国家自然科学青年基金项目(31201606)
科技创新能力建设专项(KJCX20140110)
北京市科委重大项目(D131100000113001)
关键词
平邑甜茶
烟草
Cre/loxp重组删除系统
无选择标记基因
Malus hupehensis var.pinyiensis
tobacco
Cre/loxp recombination excision system
marker-free selectable gene