带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法，然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性，影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端，合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草，受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响，然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍，高达91.4％，然而具GUS活性的转化子占抗性芽的比例仅有26.7％，在200m/L的卡那霉素筛选下，比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。

Construction of A Plant Binary Expression Vector Containing Intron-ka-namycin 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.