苏云金芽胞杆菌cryⅡ基因的克隆和表达

Cloning and Expression of Cry I Gene of Bacillus thuringiensis

分离的苏云金芽胞杆菌(Bacillus thuringiensis)YBT-791对鳞翅目小菜蛾(Plutellaxylostella)有高毒力,将其质粒DNA提纯,经HindⅢ酶切后与杀虫晶体蛋白cryⅡ基因探针杂交,显示出分子量分别为5kb和9.4kb两条DNA阳性片段。把5kb的DNA阳性片段克隆到pUC18的HindⅢ位点上并转化大肠杆菌TG1,经酶切和杂交检测,证明斑点杂交阳性克隆子中含有5kb的cryⅡ片段。把这个含cryⅡ基因的5kb HindⅢ片段进行亚克隆,将4kb的BamHI-PstI酶切片段插入穿梭载体pXI61中,并用电脉冲法克隆于苏云金杆菌不产伴胞晶体的突变株中,得到产生单一cryⅡ基因编码的杀虫晶体蛋白的克隆菌株M-5。经电镜观察,该克隆菌株能形成出发菌YBT-791多种形态伴胞晶体中的一种方形伴胞晶体;经免疫双扩试验,它只能与CryⅡ晶体蛋白抗血清形成沉淀线;经SDS-PAGE电泳,克隆菌的伴胞晶体只含有一种65kDa的晶体蛋白。生物测定结果表明它既对鳞翅目小菜蛾(Plutella xylostella)有毒性,又对双翅目致倦库蚊(Culex quinquefasciatus)有毒性。

Isolated Bacillus thuringiensis strain YBT-791 is highly toxic to Plutella xy-lostella larvae. Hind I digested fragments from strain YBT-791 plasmid DNA were hybridized with Cry I DNA probe, which showed two positive bands of 5kb and 9. 4kb. The 5kb fragments were ligated into the Hind III site of vector pUCl8, and then transformed into E. coli TG1. Restriction and Southern blot analysis.showed that the positive-reacted clones of Dot blot analysis contained the 5kb Cry I gene fragment. By sub-cloning, a 4kb BamHI-PstI DNA fragment had been inserted into the poly-linker cloning site of shuttle vector pXI61 and transformed by electroporation into the B. thuringiensis acrystalliferous mutant Bti ?IPS ?78/11. The obtained strain M-5 with Cry I gene can express the pure Cry I insecticidal crystal protein. By electro-microscope, cubic crystals were seen formed by M-5 which could also be produced by YBT-791. Double-directed spread test showed antigen of M-5 could only react with Cry I protein antibody. SDS-PAGE showed the cubic crystals of M-5 contained pure protein of 65kDa. The result of bioassay showed M-5 was not only toxic to Plutella xylostella larvae, but also toxic to Culex quinquefasciatus larvae.