Bt基因表达载体的构建及对茶树遗传转化的研究

Studies on the Construction of Bt Gene Expression Vector and Its Transformation in Tea Plant

用限制性内切酶HindⅢ和BglⅡ从pGA4 71质粒中切取Bt基因并转入载体pCAMBIA2 30 1中 ,构建后的质粒含Bt基因、intron -GUS基因和NPTⅡ基因。将构建后的质粒转化大肠杆菌 ,通过三亲交配法分别导入农杆菌菌株LBA4 4 0 4、EHA10 5、pRi15834中。以茶树叶片、愈伤组织为转化的受体材料 ,用农杆菌介导法将其转入茶树中 ,获得了GUS瞬间表达。以潮霉素作为愈伤组织筛选的选择性试剂 ,效果明显优于卡那霉素 ,适宜浓度为 2 0 μ/ml;以卡那霉素作为茶树叶片材料的筛选试剂 ,50 μ/ml为宜。

The pGA471 plasmid Bt gene DNA was digested with HindⅢ and BglⅡand inserted into vector of pCAMBIA2301 The constructed plasmid with Bt gene, intron GUS gene and NPTⅡgene was transformed into Escherichia coli and introduced into Agrobacterium strains LBA4404, EHA105 and pRi15834 through triparental cross method The Bt gene was transformed into tea leaves and tea callus by Agrobacterium mediated method Transient expression of GUS gene was successful in both callus and leaves of tea plant Screening agent testing showed that hygromycin was a more effective screening agent than kanamycin for tea callus and its optimum concentration was 20 μ/ml Kanamycin was an effective screening reagent for tea leaf and its optimum cencentration was 50 μ/ml