Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector  

利用双T-DNA载体系统培育无选择标记转基因烟草(英文)

作　　者：周红艳[1,2] 陈松彪[1] 李旭刚[1] 肖桂芳[1] 魏晓丽[1] 朱祯[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所 [2]北京师范大学细胞研究所,北京100875

出　　处：《Acta Botanica Sinica》2003年第9期1103-1108,共6页Acta Botanica Sinica（植物学报：英文版）

基　　金：国家高技术研究发展计划"863"项目(2001AA212041;2002AA212031);国家重点基础研究发展规划项目(G2001CB109001)。~~

摘　　要：We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法.通过体外重组构建了双T-DNA双元载体pDLBRBbarm.载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA.利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%.对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ.这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物.研究还利用位于2个不同载体上的nptⅡ基因与 bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%～47.4%,低于使用双T-DNA转化的共转化频率.

关 键 词：plant transformation MARKER-FREE double T-DNA vector TOBACCO 

分 类 号：S572.03[农业科学—烟草工业]