基于AlcR/alcA和Cre/loxP系统的标记基因诱导删除体系  

Excision of Selectable Markers Based on Inducible AlcR/alcA and Cre/loxP Systems

作　　者：赵青[1,2] 郭仰东[1] 谢华[2] 马荣才[3] 姚磊[2] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193 [2]北京市农林科学院农业生物技术研究中心,北京100097 [3]北京市农业局,北京100029

出　　处：《中国农业科学》2011年第17期3491-3500,共10页Scientia Agricultura Sinica

基　　金：国家"863"计划项目(2008AA10Z154;2009AA10Z104-2);北京市科技计划(Z07070501770704);北京市农林科学院科技创新能力建设专项(KJCX201102003);中央高校基本科研业务费专项资金(2009-2-06)

摘　　要：【目的】通过构建能够在植物生长发育阶段经乙醇诱导将选择标记基因删除的载体,消除选择标记基因带来的潜在安全隐患。【方法】利用AlcR/alcA诱导系统和Cre/loxP位点特异性重组系统删除选择标记基因。当外源诱导物乙醇存在时,激活下游Cre的表达。Cre重组酶识别2个同向loxP位点,剔除位点之间的DNA片段,包括选择标记基因、AlcR/alcA系统和Cre/loxP系统,而目的基因gus在诱导前后均为组成型表达。【结果】拟南芥转基因植株受乙醇诱导严格控制Cre表达的"开"和"关"。经诱导的转基因植株不能在选择培养基上继续生长。分子生物学检测显示,2个同向loxP位点间序列均已删除,表明标记基因删除效果明显。【结论】基于AlcR/alcA和Cre/loxP系统的标记基因诱导删除体系切实可行,有广泛的应用前景。[Objective] To eliminate the potential risk in safety raised by selectable markers, an ethanol inducible excision system of selectable markers was constructed, which can be used during plant growth and development. [ Method ] The sclectable markers can be removed based on A1cR/a1cA inducible system and Cre/1oxP site-specific recombination system. In the presence of the exogenous inducer, the activation of the downstream Cre gene was enabled. Cre recombinase identified and catalyzed excision of the intervening sequence between two directly oriented loxP sites, including selectable marker gene, A1cR/a1cA and Cre/1oxP system. The gene of interest, gus, was constitutively expressed before and after induction. [Result] The Arabidopsis thaliana transgenic plants were induced by ethanol. The results revealed that ethanol tightly control the ＂on＂ and ＂off＂ of the expression of Cre gene. Aider induction, the transgenic plants could not continuously grow on selective medium, which indicate the selectable marker was removed efficiently. The molecule analysis revealed the DNA fragment between two directly oriented loxP sites has been excised. [ Conclusion] The results demonstrate that the excision of selectable markers based on inducible A1cR/a1cA and Cre/1oxP systems is reliable, and has a bright future.

关 键 词：CRE/LOXP重组系统 AlcR/alcA诱导系统 删除 选择标记基因 

分 类 号：Q943.2[生物学—植物学]