Construction of A Plant Binary Expression Vector Containing Intron kanamycin Gene and Transformation in Nicotiana tabacum

Agrobacterium tumefaciens is a commonly used tool in plant genetic transformation. However, in selective medium,both cefotaxime and carbenicillin, antibiotics for eliminating Agrobacterium cells, showed plant hormone like effects on plant tissues,and can both influence the efficiency of Agrobacterium mediated transformation by reducing the regeneration efficiency of infected plants. In order to overcome this obstacle, a modified kanamycin resistant gene was designed. A plant intron was introduced into the N terminal part of the kanamycine coding sequence(Fig.4). In transgenic plants the intron is spliced efficiently, giving rise to kanamycin resistant activity. Due to the lack of a splicing apparatus, no kanamycin resistant activity was detected in agrobacteria containing this gene. The differentiation frequency of kanamycin resistant buds and the ratio of transformants with GUS activity in kanamycin resistant buds were analyzed in the presence of different concentration of kanamycin and carbenicillin. Interestingly, The differentiation frequency of kanamycin resistant buds was increased significantly when carbenicillin 500 mg/L was added in the selective culture medium(Table 1). However, the percentage of transformant with GUS activity was only 26.7% , much lower than that in medium containing no carbenicillin. The percentage of transformants with GUS activity was increased as the concentration of kanamycin increased from 50 mg/L to 200 mg/L(Table 2). From these results,it can be concluded that the intron kanamycin gene can be used as an appropriated gene in Agrobacterium mediated plant transformation.