Abstract:

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 vectors pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker nptⅡ gene and the other a bar gene, 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 nptⅡ gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T1 lines from co-transformed T0 plants with nptⅡ and bar. PPT resistant and kanamycin sensitive plants were in approximate 19.5% of the T1 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.

利用双T -D N A 载体系统培育无选择标记转基因烟草
周红艳 陈松彪 李旭刚  肖桂芳 魏晓丽 朱 祯1

(1.中国科学院遗传与发育生物学研究所, 北京 100101; 2.北京师范大学细胞研究所, 北京 100875)
摘要: 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npt Ⅱ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TDNA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TDNA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 转化的共转频率。
关键词: 植物转化；无选择标记；双T-DNA 载体；烟草
通讯作者。 Tel(Fax): 010-64852890; E-mail: <zzhu@genetics.ac.cn>。