利用Cre/lox定位重组系统获得无选择标记GFP转基因烟草  

Construction of Marker-free GFP Transgenic Tobacco by Cre/lox Site-specific Recombination System

作　　者：宋洪元[1] 任雪松[1] 司军[1] 李成琼[1] 宋明[1] 

机构地区：[1]西南大学园艺园林学院/重庆市蔬菜学重点实验室,重庆400715

出　　处：《中国农业科学》2008年第10期2973-2982,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30200185);重庆市教委应用基础项目(030208)

摘　　要：【目的】利用Cre/lox系统具有的特异重组特性,以绿色荧光蛋白GFP为目的基因,获得无选择标记的GFP转基因烟草。【方法】将Bar基因表达盒置于两个同向lox位点之间并与GFP基因表达盒融合后获得相应植物表达载体,转化烟草后获得抗除草剂Basta的GFP转基因植株。GFP转基因植株叶盘二次转化导入重组酶Cre基因后,实现与GFP基因连锁的Bar基因表达盒剔除,剔除Bar基因的植株开花后自交使重组酶Cre基因分离,获得无选择标记的GFP转基因烟草。【结果】将含Bar基因表达盒以及GFP基因表达盒的植物表达载体转化烟草Wisconsin38后获得了抗除草剂Basta以及GFP荧光表达的转基因植株。随机抽取5株转基因植株二次转化导入Cre基因,所获得的再生植株叶盘进行Basta的抗性检测,绝大多数单株对应叶盘在含8mg·L-1PPT(phosphinothricin)的筛选培养基上无法再生死亡,删除效率在76%～100%。对Bar基因删除后区域片段进行克隆测序分析显示,Bar基因表达盒已经被精确删除。Bar基因删除植株开花后自交,获得的自交后代进行NPTⅡ抗性检测,NPTⅡ敏感植株分子检测显示均只含有GFP基因。【结论】利用Cre/lox系统获得烟草无选择标记的转基因植物是稳定可行的,可广泛应用于其它无选择标记转基因植物的培育。[ Objective ] Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. [ Method ] A GFP gene was intoduced into the tobacco genome by using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation.The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene re-transformation. After removal of the Cre-NPTⅡ locus by genetic segregation through self-cross, plants were obtained that incorporated only the GFP transgene. [Result] Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently intoduced into five plants of them respectively by re-transformation. The leaf discs from regeneration plants were tested for the resistance to Basta on the medium with 8 mg·L^-1 PPT in it. The results showed few discs be able to regenerate normally, the excision at 76%-100% efficiency depended on individual re-transformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration any the submarginal nucleotides of the recombination sites. Bar gene excised plants were self-pollinated to allow segregation of the GFP gene from the Cre-NPTⅡ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPTⅡ locus in the Kan senstive plants were confirmed by PCR analysis. [Conclusion] Construction of the marker-free transgenic tobacco plants by Cre/lox site-specific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

关 键 词：Cre/lox定位重组系统 无选择标记转基因烟草 绿色荧光蛋白 转基因 

分 类 号：S572[农业科学—烟草工业] Q784[农业科学—作物学]