RESUMO
Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein.
Assuntos
Proteínas de Bactérias/efeitos adversos , Endotoxinas/efeitos adversos , Inocuidade dos Alimentos , Proteínas Hemolisinas/efeitos adversos , Proteínas Mutantes/efeitos adversos , Testes de Toxicidade Aguda/métodos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Sequência de Aminoácidos , Animais , Aspartato Aminotransferases/sangue , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Contagem de Células Sanguíneas , Colesterol/sangue , Biologia Computacional , Creatinina/sangue , Endotoxinas/genética , Feminino , Proteínas Hemolisinas/genética , Insetos , Inseticidas , Dose Letal Mediana , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Tamanho do Órgão/efeitos dos fármacos , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Triglicerídeos/sangue , Ureia/sangueRESUMO
Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50 > 1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50 > 1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC > 1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Mutantes/genética , Plantas Geneticamente Modificadas/genética , Animais , Artemia/efeitos dos fármacos , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Endotoxinas/administração & dosagem , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/administração & dosagem , Humanos , Linfócitos/efeitos dos fármacos , Proteínas Mutantes/administração & dosagem , Controle Biológico de VetoresRESUMO
Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.
Assuntos
Proteínas de Helminto/genética , Nicotiana/genética , Nicotiana/parasitologia , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , DNA Complementar/genética , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Contagem de Ovos de Parasitas , Fenótipo , Doenças das Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas/parasitologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Nicotiana/ultraestrutura , Tylenchoidea/genética , Tylenchoidea/crescimento & desenvolvimento , Tylenchoidea/patogenicidadeRESUMO
Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.
