利用整合载体构建苏云金芽胞杆菌杀虫工程菌

Construction of Insecticidal Recombinant Bacillus thuringiensis Using An Integrative Vector

利用由苏云金芽胞杆菌整合子Tn4 4 30衍生出的整合载体pBMB F7E ,克隆了对鳞翅目夜蛾科昆虫有毒力的杀虫晶体蛋白基因cry1C ,获得了整合重组质粒pBMB FLC。用电脉冲法将该重组质粒转入对鳞翅目昆虫高毒力的野生菌株YBT80 3 1,在 4 6℃下 ,经过约 12 0代转接培养后 ,筛选出在染色体基因组上整合了cry1C基因的重组子 ,整合频率约为 3 4× 10 -5。Southernblotting验证了cry1C在菌株YBT80 3 1中于染色体的不同的位点整合。生物测定结果显示 ,重组子BMB80 3 Z对小菜蛾的毒力与出发菌株无明显差异 ,而对甜菜夜蛾则较出发菌株高。

Insecticidal crystal protein gene cry1C which is highly toxic to Spodoptera exigua was cloned into an integrative vector pBMB-F7E,which was derived from Bacillus thuringiensis transposon Tn4430.The recombinant integrative plasmid pBMB-FLC harboring the cry1C gene was gained and transformed into a wild-type Bt strain YBT803-1.A transformant BMB803-A was obtained,and grown at 46℃ for about 120 generations,From which three recombinants with cry1C gene integrating into the chromosome were achieved at a frequency of approximately 3.4×10 -5 .Southern blotting revealed that the integration occurred in different sites of the chromosome.The integrated cry1C were expressed effectively.The results of bioassays showed that the toxicity of recombinants BMB803-X and BMB803-Z against Plutella xylostella were similar to that of strain YBT803-1,and their toxicity to Spodoptera exigua were higher than that of strain YBT803-1.