苏云金杆菌.doc

摘要从苏云金杆菌( Bacillus thuringiensis ,简称 Bt菌) HD-1 中提取 Bt杀虫蛋白,作为抗原免疫家兔,得到高效价的抗血清。利用抗体-抗原-酶标抗体反应,建立了酶联免疫学测定方法( ELISA 法),可定量检测转 Bt杀虫蛋白基因植物组织内 Bt蛋白的含量。用建立的 ELISA 方法检测了转 Bt杀虫蛋白基因烟草( Bt烟草)的叶片和转 Bt杀虫蛋白基因棉花( Bt棉)的叶片、花器、铃和蕾等部位,并同生物测试法相比较,发现两种方法有很好的相关性。同生物测试法相比, ELISA 法具有检测周期短,操作简便的优点,并且可以精确定量。 Bt杀虫蛋白基因已被转入棉花、烟草、杨树和马铃薯等多种植物,其中一些已进入中试和大规模商业化种植阶段,在育种中, ELISA 法可提供简便易行的检测手段。用 ELISA 法检测 Bt棉不同时期不同器官杀虫蛋白含量,差异显著。叶和花瓣杀虫蛋白含量最高,铃和蕾次之。整个生长季中,杀虫蛋白的含量在盛花期较高,以后就开始下降。在生长的后期,各器官杀虫蛋白的含量都降至很低的水平,杀虫的效果也明显减弱。研究的结果对于棉铃虫的综合防治具有重要的指导意义。研究还发现,在种子萌发的初期,杀虫蛋白就开始表达,并随着时间的推移,表达量逐渐提高。而在未萌发的种子中,杀虫蛋白含量很低。对棉叶提取液进行超滤处理, ELISA 的检测结果显示,在胞浆中杀虫蛋白含量很低,而在膜系统处理液中含量较高,初步推测杀虫蛋白可能结合于膜系统上。关键词棉铃虫; Bt 棉; Bt 杀虫蛋白; ELISA ;生物测试 Abstract Extracting Bt insecticidal protein from Bacillus thuringiensis HD-1 and immunizing rabbits, we get antiserum with high rate. Using the reaction between antibody, antigen, and enzyme-marked antibody, ELISA method is established. Through determining leaves of Bt transgenic o and leaves, flowers, bolls and bracteal leaves of Bt transgenic cotton, it is found that ELISA method is relevant to bioassay. Compared with bioassay, ELISA method has the merits of spending shorter time, quantifying Bt insecticidal protein and easy operation. Bt insecticidal protein gene has been transferred into many kinds of plants, such as cotton, o, potato, and aspen. Some have reached the step of testing mercial planting. In the breeding of seeds, ELISA isa simple and applicable method. We find that Bt cotton has different Bt insecticidal protein content in an and growing period using ELISA method. Leaves and flower petals contain more Bt insecticidal protein than boll and bracteal leaves. In the late growing period, Bt insecticidal protein content of an decreases significantly. The result is very significant to the systemic prevention of bollworms. We also find that Bt insecticidal protein begins to express in the early stage of sprout of seeds. However, seeds that have not sprouted contain very little insecticidal prote