MOSQUITO LARVICIDAL ACTIVITY OF ESCHERICHIA-COLI WITH COMBINATIONS OF GENES FROM BACILLUS-THURINGIENSIS SUBSP ISRAELENSIS
MOSQUITO LARVICIDAL ACTIVITY OF ESCHERICHIA-COLI WITH COMBINATIONS OF GENES FROM BACILLUS-THURINGIENSIS SUBSP ISRAELENSIS
复制标题
DOI：
10.1128/jb.177.10.2851-2857.1995
复制
发表时间：
1995-05-01
影响因子：
3.2
通讯作者：
ZARITSKY, A
中科院分区：
文献类型：
--
作者：
BENDOV, E;BOUSSIBA, S;ZARITSKY, A
分别编码134-和72-kDa蛋白质的基因cryIVA和cryIVD，以及苏云金芽孢杆菌苏云金亚种的调节性20-kDa多肽的基因。用大肠杆菌表达载体pT 7和pUHE以所有7种可能的组合克隆了以色列人（血清型H14）的菌株。含有cryIVA的四种组合（单独的cryIVA、与cryIVD的组合、与20-kDa-蛋白质基因的组合以及与两者的组合）在pUHE中显示出高水平的杀蚊幼虫活性。在重组大肠杆菌中，cryIVA和cryIVD的组合（有或没有20-kDa-蛋白基因）的毒性高于以往用δ-内毒素基因获得的毒性。杆菌这些克隆对三龄埃及伊蚊幼虫的50%致死浓度降低（即，毒性增加），诱导4小时后持续至约3 × 10（5）个细胞ml（-1）。诱导30分钟后获得的杀幼虫活性对于pT 7中的克隆较低，并且在另外3.5小时内降低。单独诱导cryIVD或20-kDa蛋白基因在pT 7或pUHE 20中均不产生杀幼虫活性。克隆在一起，这些基因在pT 7中有轻微毒性，但在pUHE 20中没有。在pT 7中诱导这种组合（cryIVD与20-kDa蛋白基因）5分钟产生约40%的最大死亡率，其迅速下降并在50分钟后完全消失。大肠杆菌，并部分稳定的20 kDa的调节蛋白。cryIVA和cryIVD的组合的杀幼虫活性比单独的cryIVA高7倍，这可能是因为多肽的交叉稳定性或它们的活性之间的协同作用。
The genes cryIVA and cryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide of Bacillus thuringiensis subsp. israelensis (serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquito larvicidal activity in pUHE. The toxicity of the combination of cryIVA and cryIVD, with or without the 20-kDa-protein gene, was higher than has ever been achieved with delta-endotoxin genes in recombinant E. coli. Fifty percent lethal concentrations against third instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 x 10(5) cells ml(-1) after 4 h of induction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of either cryIVD or the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVD with the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded in E. coli and partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination of cryIVA and cryIVD was sevenfold higher than that of cryIVA alone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities.