含双病原物诱导启动子无选择标记转基因烟草的获得

Acquirement of marker-free transgenic tobacco plants carrying two pathogen-inducible promoters

外源基因的引入造成转基因植物的食用安全性和环境安全性问题。此外，选择标记基因还可能使植株再生困难，及后继转基因工作不能再用相同的选择标记基因。若转基因植株在释放时其外源抗性选择标记基因得到剔除，则上述问题可得到解决。

Transgenic plants are controversial for their edible and environmental security. Marker-free transgenic plants can be produced by the construction and transformation of plant expression vectors carrying twin T-DNAs. The construction of plant expression vectors harboring twin T-DNAs and two pathogen-inducible promoters was previously reported. These vector plasmids were introduced into tobacco plants and the transgenic tobacco plants were obtained. In this paper we report that T1 transgenic tobacco seedlings were produced through classical genetics approach. Analysis of the seedlings demonstrated that some of them were marker-free lines. The segregation of exogenous genes in Tltransgenic tobacco seedlings was tested by using two methods. Firstly, the ability of resistance to kanamycin was analyzed in T1 transgenic tobacco seedlings from 14 transgenic plant lines. It was found that the segregation ratio of NPTII genes met well with Mendel＇ s law in 13 transgenic tobacco lines. So it was deduced that NPTII gene was as a single copy integrated into one of the homologous chromosomes. Then the NPTII genes and uidA genes of 130 T1 transgenic seedlings were detected from the above 13 tobacco lines. The results showed that uidA genes were only detected in 20.77% of the seedlings, NPTII genes were solely detected in 22.31% of the seedlings, but both exogenous genes were in 53.85% of the seedlings. The segregation ratio of the two genes was consistent with the law of independent assortment （9 ： 3 ： 3 ： 1）. These results suggested that the selective marker gene had no linkage with the reporter gene and they were segregated independently in the T1 transgenic tobacco plants. This method, as compared with traditional backcross, is confirmed a more easy and rapid way to eliminate the antibiotic resistance gene used as a selective marker in transgenic plants.