抗冻蛋白-GUS基因诱导性融合表达载体转化烟草植株中GUS活性测定

Determination of the GUS Activities in Transgenic Tobacco Plants Inducibly Expressing the Fusion Gene of Antifreeze Protein and GUS

把修饰合成的带有甘薯信号肽序列的抗冻蛋白成串基因与β-葡萄糖醛酸苷酶(GU S)报道基因融合克隆在大豆热休克启动区下游构建了温度诱导型双元载体pBF 04,三亲株配对法转化根癌农杆菌,叶盘法导入烟草,获得了转基因烟草植株.将10株转基因烟草放置在40℃诱导不同时间,利用荧光光度分析法测定水解液中GU S活性,结果诱导18、24、48小时的4株中GU S活性明显高于对照组和诱导6、12小时组,说明我们修饰合成的抗冻蛋白与GU S基因融合构建的载体在热休克启动区控制下可以诱导表达.经组织化学法测定这些阳性植株的叶片和茎段切片细胞间隙显示GU S活性,提示甘薯信号肽可能起作用.

To determine the expression of antifreeze protein （AFP） gene in transgenic tobacco plants, the temperature induced fusion expression vector, pBF04, was constructed. It encodes a signal peptide sequence of sweet potato and a modified synthetic flounder AFP with reporter GUS gene, under control of soybean heat shock promoter. Transgenic tobacco plants were obtained through tri-parental mating and leaf disk methods. Ten transgenic tobacco plants were induced at 40℃ for different periods ,then the GUS activities in the plant＇s extracts was determined with fluo- rescence spectrophotometer. The results show that GUS activities in 4 plantlets under inducing for 18, 24, and 48h were significantly higher than that of the control and other plantlets under inducing for 6, 12h. This suggests that the vectors with modified synthetic flounder AFP and reporter GUS genes were expressed under the control of heat shock promoter. In histochemical assay,the GUS activities were only observed in intercellular area of the stems and leaves from transgenic tobacco plantlets. This suggests that sweet potato＇s signal peptide genes might have effect.