Masson L, Mazza A, Gringorten L, Baines D, Aneliunas V, Brousseau R
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Mol Microbiol. 1994 Dec;14(5):851-60. doi: 10.1111/j.1365-2958.1994.tb01321.x.
The Bacillus thuringiensis crylA(a) and crylA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CrylA(c) is highly active against cultured Choristoneura fumiferana cells (CF-1) whereas CrylA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis revealed that the CrylA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258-358 and 450-510) were able to confer toxicity, although at lower levels, and one region (358-450) was present where progressive substitutions of crylA(a) with crylA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CrylA(c) with CrylE suggested that the N-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CrylA(b) but shows an 800-fold increase in toxicity to CF-1 cells relative to that shown by CrylA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450-620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.
通过在体内和体外针对培养的昆虫细胞或解离的中肠上皮细胞创建并测试杂交毒素，对苏云金芽孢杆菌crylA(a)和crylA(c)基因特异性区域进行了探究。毒素阈值剂量测定表明，CrylA(c)对培养的云杉卷叶蛾细胞（CF-1）具有高活性，而CrylA(a)则无毒。在活昆虫生物测定中，观察到了相反的毒性顺序。杂交分析表明，CrylA(c)毒性决定区域位于密码子258和510之间。该区域的两个较小亚区（残基258-358和450-510）能够赋予毒性，尽管水平较低，并且存在一个区域（358-450），用crylA(c)序列逐步取代crylA(a)对其没有影响。将CrylA(c)的非同源N端区域与CrylE交换表明，N端在特异性方面不起作用。一个杂交克隆MP80与CrylA(b)具有99.3%的同源性，但相对于CrylA(b)对CF-1细胞的毒性增加了800倍。对家蚕进行的活生物测定与使用来自同一种昆虫的解离中肠上皮细胞新开发的体外菌苔测定之间的直接比较显示，毒性存在显著差异。家蚕幼虫的毒性决定区域确定在密码子283和450之间，尽管450-620密码子区域可能对毒性有影响。一般来说，对昆虫几乎没有或没有中毒作用的天然或杂交毒素对上皮细胞非常活跃，这表明除毒素氨基酸序列外的其他因素在决定毒素特异性方面起着重要作用。
