Made available in DSpace on 2014-08-20T14:31:29Z (GMT). No. of bitstreams: 1 dissertacao_ana_paula_de_lara.pdf: 4627911 bytes, checksum: 887ee69dd56b73509c1a603e3b25aebd (MD5) Previous issue date: 2013-03-08 / Bacillus thuringiensis (Bt) is a Gram-positive bacteria, ubiquitous, facultative anaerobic, and form spores. During sporulation produce a parasporal crystals inclusion. Within these inclusions there are δ-endotoxin proteins well known for its insecticides proprieties. Among them, the Cry (crystal) is wide employed for biological control of plagues. The δ-endotoxin has an advantage of been more specific than chemical insecticides, thus been consider more favorable for the environment. The aim of this study was to obtain the Cry 11Aa recombinant protein of Bacillus thuringiensis var. israelensis in Escherichia coli, active for use in biocontrol. Two expression E. coli strains were tested: BL 21 (DE3) C41 and BL 21 (DE3) Ril. The protein Cry 11Aa was expressed and secreted in a soluble form by the two strains. The expression was demonstrated by Western Blot using anti-histidin monoclonal antibody. The strain BL 21 (DE3) C41 express the protein Cry 11Aa ~3.6 times more than the strain Rill, and showed a biologic efficiency of 95% of mortality for Culex quinquefaciatus larvae. The data obtained in this study suggest that the protein recombinant Cry 11Aa expressed in E. coli has a potential to be used in biological control. / Bacillus thuringiensis (Bt) é uma bactéria Gram-positiva, de ocorrência ubíqua, anaeróbica facultativa, formadora de esporos. Produz cristais, como inclusões parasporal durante a esporulação. Estas inclusões contêm proteínas chamadas de δ-endotoxinas, que são bem conhecidas pelas suas propriedades inseticidas. Dentre elas as toxinas Cry (crystal) são largamente empregadas no controle biológico de pragas. As δ-endotoxinas têm a vantagem de serem mais específicos do que os inseticidas químicos sintéticos, portanto, são considerados como agentes de controles favoráveis ao meio ambiente. O objetivo deste estudo foi a obtenção da proteína Cry 11Aa recombinante de Bacillus thuringiensis var. israelensis em Escherichia coli, ativa, para utilização no controle biológico.Duas cepas de E. coli de expressão foram testadas: BL 21 (DE3) C41 e BL 21 (DE3) Ril. A proteína Cry 11Aa foi expressa e secretada na forma solúvel pelas duas cepas. A expressão foi demonstrada por Western blot utilizando-se anticorpo monoclonal anti-histidina. A cepa BL 21 (DE3) C41 expressou a proteína Cry 11Aa ~3.6 vezes mais que a cepa BL 21 (DE3) Ril, e apresentou, em teste biológico, uma eficácia de 95% de mortalidade sobre larvas de Culex quinquefaciatus. Com os dados obtidos neste trabalho podemos sugerir que a proteína recombinante Cry 11Aa expressa em E. coli é um potencial candidato para ser utilizado no controle biológico.

Bt é uma bactéria formadora da proteína Cry1Ac, tóxica a lepidópteros. Plantas geneticamente modificadas expressam essa toxina. O objetivo deste trabalho foi isolamento e identificação de bactérias do solo de algodão transgênico e convencional potencialmente biodegradadoras dessa proteína. Estudou-se a dinâmica de crescimento das bactérias em meios com a proteína Cry1Ac ou glucose, a biodegradação, os genes apr, npr e sub. Em solo de algodão convencional a contagem foi menor; para algodão transgênico foi maior. Seis bactérias foram identificadas. O crescimento da bactéria B. pumilus foi maior em meio com a proteína Cry1Ac que glucose e a bactéria G. rubripertincta em meio contendo glucose teve o crescimento maior do que com a proteína Cry1Ac. Foi verificada a biodegradação da proteína pelas bactérias Bp e Gr. Os genes apr e sub foram detectados em solo de planta transgênica e o gene npr não. / Bacillus thuringiensis is a bacterium that forms the Cry1Ac protein, toxic to lepidopteran. Genetically modify plants expressing this toxin. The objective of this work was the isolation and identification bacteria from soil cultivated with transgenic cotton and conventional with potential degradation of this protein. We studied the dynamics growth from bacteria in media with the Cry1Ac protein or glucose, the biodegradation genes apr, npr and sub. In soil of conventional cotton, count was lower, for Bt cotton were higher. Six strains were identified. The growth bacterium B. pumilus was higher in medium with the Cry1Ac protein than bacteria with glucose and G. rubripertincta in medium containing glucose had higher growth than the Cry1Ac protein. Were verified Cry1Ac protein degradation by the bacteria Bp and Gr. Genes apr and sub were detected in soil with transgenic plant and the gene npr were not.

No description available.

Orientador: Janete Apparecida Desidério / Banca: Ricardo Antonio Polanczyk / Banca: Janaína Fernandes Gonçalves / Resumo: A bactéria Bacillus thuringiensis é vista como uma das melhores opções no controle biológico devido à ação entomopatogênica e a especificidade de suas proteínas. As proteínas Vip3, que são secretadas durante o crescimento vegetativo do B. thuringiensis, atuam no controle de importantes lepidópteros praga. O objetivo deste trabalho foi caracterizar um gene vip3A de um isolado de B. thuringiensis, expressar a proteína e verificar sua toxicidade contra larvas de Anticarsia gemmatalis e. Spodoptera frugiperda Para tanto, o gene foi amplificado com iniciadores específicos por PCR, gerando um fragmento de 2370 pb. O fragmento foi clonado em vetor pGEM - T Easy para sequenciamento, subclonado em vetor de expressão pET-28a (+) e o conjunto inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína foi induzida por IPTG, a proteína Vip3Aa50 foi visualizada em SDS-PAGE e detectada por "Western blotting". Os ensaios de toxicidade revelaram alta virulência às larvas neonatas de A. gemmatalis e S. frugiperda, sendo as larvas da população de A. gemmatalis mais sensíveis. O isolado utilizado neste estudo é altamente promissor como fonte de gene vip3A para controle de ambas as pragas por meio da produção de plantas transgênicas como milho e soja. / Abstract: The bacterium Bacillus thuringiensis is seen as one the best options for the biological control due to entomopathogenic action and protein specificity. The Vip3 proteins, which are secreted during the vegetative growth of B. thuringiensis, act to the control the most important lepidopteran pests. The aim of this study was to characterize a vip3A gene from an isolate of B. thuringiensis, to induce the protein and evalute its toxicity against Anticarsia gemmatalis and Spodoptera frugiperda larvae. Therefore, the gene was amplified by specific PCR's primers, generating a 2370 pb fragment. The fragment was cloned into the pGEM-T Easy vector and subsequently, it was sequenced and subcloned into the pET-28a (+)'s expression vector and inserted in to the Escherichia coli BL21 (DE3) cells. The Vip3Aa50 protein expression was induced by IPTG, observed using SDS-PAGE and detected by Western blotting. The toxicity bioassay showed intense virulence for A. gemmatalis and S. frugiperda's neonate larvae. The results showed that A. gemmatalis larval population was more sensitive. The isolate used in this study is highly promising as vip3A source gene to control both plagues through transgenic plant production, such as, corn and soybean. / Mestre

Submitted by Mariana Dornelles Vargas (marianadv) on 2015-05-26T13:54:04Z No. of bitstreams: 1 analise_potencial.pdf: 1430284 bytes, checksum: 12436295c40ce78cf9c53abb0f1e16e3 (MD5) / Made available in DSpace on 2015-05-26T13:54:04Z (GMT). No. of bitstreams: 1 analise_potencial.pdf: 1430284 bytes, checksum: 12436295c40ce78cf9c53abb0f1e16e3 (MD5) Previous issue date: 2012-10-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A bactéria Bacillus thuringiensis exibe alta atividade tóxica específica para insetos devido à sintese de delta-endotoxinas, codificadas por genes cry. A presente pesquisa visou avaliar a atividade tóxica de cepas de Bacillus thuringiensis oriundas de regiões orizícolas do Estado do Rio Grande do Sul, como agente de controle de insetos-praga da cultura do arroz irrigado e milho Spodoptera frugiperda e Ostrinia nubilalis, a fim de selecionar cepas potenciais para o controle das mesmas e identificar se as atividades enzimáticas dos receptores alcalino fosfatase (ALP) e aminopeptidase (APN) podem ter um importante papel na resistência de B. thuringiensis. Neste trabalho foram utilizados testes fenotípicos e técnicas de PCR para a identificação das subclasses do gene cry1; perfil de proteínas para a observação da composição do complexo esporo-cristal, rep-PCR para a observação da variabilidade genética, DNA plasmidial, quantificação das enzimas ALP e APN para se observer perfil de resistência das cepas e a atividade tóxica frente aos insetos-praga. Os ensaios de quantificação de proteínas de ALP e APN apresentaram níveis reduzidos, sendo um biomarcador potencial para a resistência de toxinas Cry. Os resultados dos estudos de rep-PCR demonstraram um elevado grau de similaridade entre as regiões orizícolas, provavelmente associadas à especiação ecológica. Na caracterização do perfil protéico, os resultados revelaram diferenças entre as cepas em estudo, sendo algumas semelhantes àquelas utilizadas como padrão da análise (Bt. thuringiensis 4412; Bti IPS 82 e Bt. sorovar HD1). Nos ensaios do perfil plasmidial, as cepas formaram três padrões distintos. Para os dados de toxicidades avaliados pelos bioensaios com as lagartas de primeiro ínstar de O. nubilalis, todas as cepas testadas apresentaram mortalidade superior a 75%, com destaque à cepa Bt.1893-15 que causou 95%. Nos dados dos ensaios biológicos realizados contra as lagartas de S. frugiperda, a cepa Bt. 3420-11 destacou-se com mortalidade superior a 88%. / The bacterium Bacillus thuringiensis presents a high specific activity against insects due to delta-endotoxin syntheses, codified by cry genes. This study aimed to evaluate the toxic activity of strains of B. thuringiensis derived from rice fields of Rio Grande do Sul, as an agent of control of insect pest of rice and corn Spodoptera frugiperda and Ostrinia nubilalis, in order to select potential strains to control and identify whether the enzymatic activity of alkaline-phosphatase (ALP) and aminopeptidase (APN) receptors may play an important role in the resistance of B. thuringiensis. In this study we used phenotypic tests and PCR techniques to identify subclasses of gene cry1; proteins profile to observe the composition of the spore- crystal complex, rep-PCR for the observation of genetic variability, plasmid DNA, quantification of the enzymes ALP and APN to observe resistance profile of strains and bioassay capacity against insect pests. Assays for quantification of proteins ALP and APN showed reduced levels being a potential biomarker for resistance to Cry toxins. The results of studies of rep-PCR demonstrated a high degree of similarity between the rice regions, probably associated with ecological speciation. Regarding the protein profile characterization, the results revealed differences between the strains, some being similar to those used as standard analysis (Bt thuringiensis 4412, Bt IPS 82 and Bt HD1). In trials of plasmid profile, the strains formed three distinct patterns. In data and toxicity assessed by bioassays against first instar O. nubilalis larvae, all tested strains showed mortality exceeding 75%, highlighting the Bt strain 1893-15, which caused 95% mortality. In the data of the biological assays conducted against S. frugiperda larvae, the Bt strain 3420-11 stood out showing mortality greater than 88%.

Bibliography: leaves 120-145. Reports Bt crystal protein toxicity to a phthirapteran species. Although Bt strain WB3516 may produce other unidentified toxins effective against B. ovis, the results provide strong evidence that the [delta]-endotoxin crystal proteins of strain WB3516 significantly contribute to the lousicidal toxicity of this strain.

Lintroduction commerciale des cotonniers génétiquement modifiés (CGM), produisant les toxines insecticides de Bacillus thuringiensis (Bt), dans les pays en développement et notamment en Afrique, suscite des craintes de la part de la société civile au sujet de leur impact sur lenvironnement et léconomie des petits paysannats. Lobjectif de la thèse est de proposer, sur la base dune revue bibliographique et dune série détudes originales réalisées en Afrique du Sud et publiées par lauteur dans des revues scientifiques, un outil daide à la décision basé sur des critères techniques et scientifiques permettant de juger lintérêt de ladoption des variétés de cotonniers Bt en paysannat africain. Dans un premier temps nous présentons un état des lieux de la production cotonnière à léchelle mondiale ainsi que les modalités de la culture en Afrique. Cet inventaire permettra par la suite détablir un état des besoins nécessaire à lélaboration du cadre de décision. Dans un deuxième temps, limportance des cotonniers Bt dans lagriculture, leur efficacité agronomique et leurs effets sur lenvironnement sont discutés. Nous mettons en exergue le risque potentiel dune réduction de lefficacité de ces CGM sous linfluence de facteurs abiotiques tels que la sécheresse. Les recherches effectuées sur limpact environnemental des cotonniers Bt montrent que ceux-ci nont pas deffet direct sur la biodiversité. Cependant les pratiques agricoles mal adaptées en conjonction avec lusage de cette innovation peuvent entraîner des modifications de la diversité et de labondance de lentomofaune des agrosystèmes cotonniers. Concernant les flux de gènes et la probabilité de mélange non intentionnel de semences entre cultures Bt et non-Bt, huit sources potentielles de mélange ont été identifiées et leurs conséquences sont discutées. Limpact agroéconomique de lintroduction des cotonniers Bt est étudié dans un troisième temps. Les études entreprises en Afrique du Sud suggèrent que ces effets sont parfois discutables et la réussite de ladoption repose fortement sur la solidité économique de la filière, sur lorganisation de lencadrement technique, sur lefficacité du réseau de distribution dintrants agricoles et sur le niveau de rendement de la culture avant lintroduction de linnovation. La dernière partie de la thèse propose un cadre provisoire dévaluation de lopportunité dadoption des cotonniers Bt en petit paysannat africain. Ce cadre se fonde sur la formulation des problèmes à résoudre dans le paysannat et sur lévaluation des options techniques disponibles. Les conditions préalables à une introduction réussie des cultivars Bt sont identifiées et des mesures daccompagnement sont suggérées pour lusage durable de cette technologie.

Bacillus thuringiensis and .:scherichia coli were grown in submerged cultures in the presence of 1-014 labeled decanoic, palmitic and oleic acids and 2-C14 labeled malonic acid. The bacteria were harvested by centrifugation and fractionated by published methods. Cellular constituents were separated and identified by chromatographic techniques. Radioactivity was determined by liquid scintillation spectrometry and through the use of a radiochromatogram scanner.The bacteria incorporated all the fatty acids added to the medium but showed a preference for the longer chained compounds. The organisms incorporated 1-014 palmitic acid into mostly phospholipids and also into free fatty acids and neutral lipids. At least 90% of the incorporated palmitate remained as palmitic acid. Elongation to stearic acid was demonstrated by B. thuringiensis. The label from 1-014 palmitic acid was retained over several generations by both test organisms.Ball State UniversityMuncie, IN 47306

Hemocytes of the fifth instar European borer, Ostrinia nubilalis Hubner (Lepidoptera: Pyralidae) were examined in stained and unstained preparations. Prohemocytes, plasmatocytes, granular hemocyter, and oenocytoids, and spherule cells were found in this stage, The prohemocytes are characterized by a scant intensely basophilic cytoplasm. The highly pleomorphic plasiratocytes are characterized by a punctate nucleus and production of cytoplasmic extensions in vitro. Granular hemocytes are characterized by a small nucleus, accumulation of lipid droplets, and the production of extremely fine cytoplasmic extensions in living preparations. The oenocytoid is characterized by a small eccentric nucleus in a large amount of homogeneous basophilic cytoplasm. The spherule cell is characterized by large spherules within the cytoplasm which may tend to mask the nucleus.Treatment of larvae with per os doses of Bacillus thuringiensis did not affect the differential hemocyte count. However, there did appear to be an increase in the number of degenerating cells in Bacillus thuringiensis treated groups.Ball State UniversityMuncie, IN 47306

The insect pathogen Bacillus thuringiensis (Bt) holds great promise as an effective and friendly way for management of the pests with safety for nontarget animals and humans. However, high capital investment due to high production and formulation cost of commercial Bt preparations has caused prohibitive effect on companies. The present study mainly aimed at developing a low cost medium that supports the growth of different Bt strains and their specific bioinsecticidal &amp / #948 / -endotoxins (crystal proteins). A comparison was made between the representative members of three different subspecies of Bt to observe toxin yields in response to certain nutritional conditions. Three different Bt subspecies were Bt kurstaki (strain 81), Bt israelensis (strain HD500) and Bt tenebrionis (strain 3203), producing lepidoptera- and diptera-specific Cry1 and Cry2, diptera-specific Cry4Ba and Cry11Aa and coleoptera-specific Cry3Aa toxins, respectively. Studies were conducted to optimize glucose and inorganic phosphate concentrations in standard DSM medium for the production of these Bt-based biopesticides. General suppression of toxin yields in high glucose medium (10 g/L) thought the generality of carbon catabolite regulation for biosynthesis of different types of toxins. Inorganic phosphate (Pi) level was important for Cry4Ba, Cry11Aa and Cry3Aa biosynthesis while Cry1 and Cry2 production was not responsive to high Pi. Wastewater sludge, fruit residues and broiler litter were next tested as cheap raw materials for Bt-based biopesticide production in batch cultures. Broiler litter seemed to be a much better substrate among all since some degree of production of each toxin was observed at almost every stage of fermentation. The processing of broiler litter was found to significantly improve toxin yields. The medium prepared from processed broiler litter was successfully used to cultivate all Bt stains and obtain bioinsecticidal proteins in high yields which were comparable or higher than those that can be obtained on standard semi-synthetic media.