Park H W, Delécluse A, Federici B A
Department of Entomology and Interdepartmental Graduate Programs in, University of California-Riverside, Riverside, California 92521, USA.
J Invertebr Pathol. 2001 Jul;78(1):37-44. doi: 10.1006/jipa.2001.5038.
The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.
杀蚊细菌苏云金芽孢杆菌以色列亚种（Bti）产生四种主要的内毒素蛋白，即Cry4A、Cry4B、Cry11A和Cyt1A，其毒性与许多合成化学杀虫剂相当。Cry11B天然存在于苏云金芽孢杆菌杰加蒂森亚种中，是Cry11A的近亲，但对致倦库蚊的毒性约为Cry11A的10倍。为了确定向Bti中添加Cry11B是否会提高其毒性，我们在Bti中生产了这种蛋白。通过在cyt1A启动子和STAB - SD序列的控制下表达cry11B基因，获得了高水平的Cry11B合成。该构建体被克隆到穿梭载体pHT3101中，产生衍生质粒pPFT11Bs，然后通过电穿孔将其转化到无晶体（4Q7）和有晶体（IPS - 82）的Bti菌株中。在Bti 4Q7中合成的Cry11B产生的晶体比使用无STAB - SD的天然启动子时产生的晶体大约大50%。然而，与野生型构建体相比，使用该构建体每单位培养基产生的Cry11B较少，显然是因为后者构建体每单位培养基产生更多的细胞。尽管如此，用pPFT11Bs产生Cry11B的Bti IPS - 82菌株对致倦库蚊四龄幼虫的毒性是亲本IPS - 82菌株的两倍（LC(50)分别为1.4 ng/ml和3.3 ng/ml）。对于埃及伊蚊四龄幼虫，亲本Bti IPS - 82（LC(50) = 4.7 ng/ml）和产生Cry11B的Bti IPS - 82重组体（LC(50) = 3.5 ng/ml）在毒性上没有发现统计学上的显著差异。
