RESUMO
Cry9Aa, produced by Bacillus thuringiensis is reported to be not active against Spodoptera exigua (beet armyworm). In this study we have cloned a new cry9Aa5 gene encoding a protoxin with increased activity against S. exigua as compared to Cry9Aa1. When aligned to Cry9Aa1, four amino acid substitutions in domain I and one substitution in the C-terminal protein extension of Cry9Aa5 were identified. Toxicity of Cry9Aa5, produced in recombinant Escherichia coli was assessed and compared to the activity of Cry9Aa1, produced under the same conditions.
Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Controle Biológico de Vetores/métodos , Spodoptera/microbiologia , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectangular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Besouros/efeitos dos fármacos , Endotoxinas/toxicidade , Inseticidas/toxicidade , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Besouros/crescimento & desenvolvimento , Cristalização , Endotoxinas/genética , Escherichia coli/genética , Proteínas Hemolisinas , Corpos de Inclusão/metabolismo , Corpos de Inclusão/ultraestrutura , Inseticidas/química , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidadeRESUMO
We investigated the role of domain III of Bacillus thuringiensis delta-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic.
Assuntos
Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Endotoxinas/toxicidade , Lepidópteros/microbiologia , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas , Lepidópteros/efeitos dos fármacos , Dados de Sequência Molecular , Controle Biológico de Vetores , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidadeRESUMO
Twelve Cry1 and two Cry9 delta-endotoxins from Bacillus thuringiensis were tested for their activity against black cutworm ( Agrotis ipsilon). A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity against A. ipsilon. Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity.
