ABSTRACT
Some pest management programs employ PCR to identify cry1 genes from Bacillus thuringiensis to predict bacterial toxicity towards different insect pests. However, due to changes on the mode of action of the Cry proteins, new primers had to be designed to detect the new genes. Therefore, an 'in-silico' study of genetic sequences from five cry1 subclasses was carried out and characterized by molecular tools. The design of new primers allows for more precise selection of B. thuringiensis isolates, helping to better direct the programs employing biological control.
Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , DNA Primers/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Polymerase Chain Reaction/methods , Bacillus thuringiensis Toxins , Base Sequence , DNA, Bacterial/genetics , Genotype , Molecular Sequence Data , Sensitivity and Specificity , Sequence AlignmentABSTRACT
Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.
Subject(s)
Bacillus thuringiensis/physiology , Bacterial Proteins/metabolism , Endotoxins/metabolism , Gastrointestinal Tract/microbiology , Hemolysin Proteins/metabolism , Spodoptera/microbiology , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/toxicity , Endotoxins/toxicity , Hemolysin Proteins/toxicity , Larva/drug effects , Lethal Dose 50 , Protein BindingABSTRACT
Mozzarella cheese is traditionally prepared from bubaline (Bubalus bubalis) milk, but product adulteration occurs mainly by addition of or full substitution by bovine milk. The aim of this study was to show the usefulnes of molecular markers to identify the admixture of bovine milk to bubaline milk during the manufacturing process of mozzarella cheese. Samples of mozzarella cheese were produced by adding seven different concentrations of bovine milk: 0%, 1%, 2%, 5%, 8%, 12% and 100%. DNA extracted from somatic cells found in cheese were submitted to PCR-RFLP analysis of casein genes: alfa-s1-CN - CSN1S1 that encompasses 954 bp from exon VII to intron IX (Alu I and Hinf I), beta-CN - CSN2 including 495 bp of exon VII (Hae III and Hinf I), and k-CN - CSN3, encompassing 373 bp of exon IV (Alu I and Hind III). Our results indicate that Hae III-RFLP of CSN2 exon VII can be used as a molecular marker to detect the presence of bovine milk in "mozzarella" cheese.
Subject(s)
Animals , Cattle/genetics , Buffaloes/genetics , Caseins , Cheese/analysis , Milk , Polymorphism, Genetic , Polymorphism, Restriction Fragment LengthABSTRACT
The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 10(8) spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100% mortality and 16 presented an index between 70% and 90%. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1% probability for the cry1Ac and cry1E genes.
Subject(s)
Bacillus thuringiensis/genetics , Bacillus thuringiensis/isolation & purification , Insect Control/methods , Lepidoptera , AnimalsABSTRACT
A lagarta de Spodoptera frugiperda (J. E. Smith) é uma das principais pragas do milho e para seu controle o Bacillus thuringiensis se destaca por sua atividade entomopatogênica. Este trabalho objetivou a caracterização molecular de isolados de B. thuringiensis quanto à presença do gene cry1 e a avaliação da sua eficiência no controle de lagartas de S. frugiperda. Nas análises da PCR, foi utilizado o Gral-cry1 para confirmação da presença do gene cry1 nos 115 isolados. Uma suspensão de 3 x 10(8) esporos/ml banhou a dieta utilizada para alimentação de 30 lagartas por isolado, com três repetições. A identificação do tipo de genes cry1 dos diferentes isolados foi realizada para cinco sub-classes de genes e análises de regressão linear foram realizadas para verificar possíveis associações entre a presença de um gene cry individual e altos níveis de toxicidade. Ttodos os DNAs amplificados com os iniciadores Gral-cry1 apresentaram produto de amplificação com tamanho esperado. Quanto aos níveis de eficiência inseticida contra a lagarta-do-cartucho, 41 isolados apresentaram 100 por cento de mortalidade e 16 apresentaram índice entre 75 por cento e 90 por cento. O gene cry1Ab esteve presente em 80 isolados, cryB em 69 isolados, cry1Ac em todos os isolados, cryV e cry1E em 93 e 27 isolados, respectivamente. Os valores referentes ao efeito individual de cada gene na mortalidade de larvas foram significativos a 1 por cento de probabilidade, para os genes cry1Ac e cry1E.
The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 10(8) spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100 percent mortality and 16 presented an index between 70 percent and 90 percent. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1 percent probability for the cry1Ac and cry1E genes.
Subject(s)
Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis/isolation & purification , Insect Control/methods , LepidopteraABSTRACT
A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.
