电脉冲穿孔法将苏云金杆菌δ-内毒素基因导入野生型芽孢杆菌

Transfer of Shuttle Vector Containing Bacillus thuringiensis Toxingene into Wild Type B. cereus-B. brevis and B. subtilis by Electroporation

利用电脉冲穿孔法将带有苏云金杆菌毒蛋白基因的穿梭质粒导入几株野生型芽孢杆菌中，它们是野生型的蜡状芽孢杆菌、短芽孢杆菌和枯草芽孢杆菌。通过观察在新霉素和氨苄青霉素平板上长出的抗性菌落数，计算出转化效率为１０＾１－１０＾４转化子／μｇＤＮＡ．从转化子中分离到的质粒ＤＮＡ大小及其用ＨｉｎｄⅢ酶切的片段与原始质粒ＤＮＡ相同，毒性测试表明重组转化子对烟青虫六天的致死率达９０－１００％。

Electroporation is one of the most effective methods for transferring DNA into cells. The technique is fast, simple and appears to be applicable to a wide rang of bacterial species praviously thought untransformable. We have successfully introduced shuttle vector containing B. thuringiensis Toxin gene into wild type B. cereus, B. brevis and .B. stibtilis by electroporation. The transformation efficiency was determined by counting the number of neomycin and ampicillin resistant colonies. The efficiencies for these bacteria were around 101 - 104 transformants per ug DNA. The structure of the transferred plasmid was unaffected by electroporation based on the size and restriction digestion pattern of the plasmid isolated from the transformants. Toxicity assays showed that the transformants gave a mortality of 90-100% against caterpillar of Heliothis assulta, indicating that the gene function was not changed by electropotation.