基于AlcR_alcA和Cre__省略_oxP系统的标记基因诱导删除体系_赵青

中国农业科学 2011,44(17):3491-3500 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2011.17.001

基于AlcR/alcA和Cre/loxP系统的标记基因诱导删除体系

赵 青

1,2

,郭仰东,谢 华,马荣才,姚 磊

1232

(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)

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。

关键词:Cre/loxP重组系统;AlcR/alcA诱导系统;删除;选择标记基因

Excision of Selectable Markers Based on Inducible

AlcR/alcA and Cre/loxP Systems

ZHAO Qing1,2, GUO Yang-dong1, XIE Hua2, MA Rong-cai3, YAO Lei2

(1College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193; 2Beijing Ago-Biotechnology Research

Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097; 3Beijing Municipal Bureau of Agriculture,

Beijing 100029)

Abstract: 【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 selectable markers can be removed based on AlcR/alcA inducible system and Cre/loxP 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, AlcR/alcA and Cre/loxP 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. After 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 AlcR/alcA and Cre/loxP systems is reliable, and has a bright future.

Key words: Cre/loxP recombination system; AlcR/alcA induce system; excision; selectable markers

0 引言

【研究意义】自1983年首次获得转基因烟草以

来,植物转基因技术飞速发展,转基因作物的种类、产量都迅速增加。然而,随着商业化转基因植物新品种的不断出现,人们对转基因植物的食用安全性和环

收稿日期:2011-01-07;接受日期:2011-03-16

基金项目: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)

联系方式:赵 青,Tel:13426211303;E-mail:zhaoqing404@http://www.wendangwang.com。通信作者姚 磊,Tel:13641376238;E-mail:yaolei@http://www.wendangwang.com

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