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000829635.pdf: 1135736 bytes, checksum: 5f82225f1067c07c36424e25cc5c44f1 (MD5) / As proteínas Vip3Aa e Cry1Ia apresentam potencial no controle de lepidópteros-praga e surgem como alternativa promissora no manejo da resistência de pragas as proteínas Cry1A, que tem sido altamente utilizada na formulação de bioinseticidas comerciais à base de Bacillus thuringiensis (Bt) e em plantas transgênicas. Assim sendo, o presente trabalho teve como objetivo a clonagem e expressão das proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 em Escherichia coli, a fim de se analisar a correlação entre a união aos receptores por meio de análises de competição entre as diferentes toxinas Vip3Aa e a toxina Cry1Ia10, e a toxicidade a lepidópteros-praga, inferindo-se quais as combinações que poderiam ser utilizadas na produção de plantas transgênicas, contendo múltiplos genes, as quais vêm sendo empregadas para contornar a evolução da resistência dos insetos às toxinas Bt. Para tanto, os genes vip3Aa e cry1Ia10 foram clonados no vetor pET SUMO, expressos em E. coli e a toxicidade das proteínas foram testadas em bioensaios com lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalis e Heliothis virescens. As BBMVs (Brush Border Membrane Vesicles) foram preparadas a partir dos intestinos das três espécies e ensaios de competição homóloga e heteróloga foram realizados. As proteínas Vip3Aa42 e Vip3Aa43 apresentaram toxicidade para S. frugiperda e A. gemmatalis. Já a proteína Cry1Ia10 apresentou toxicidade apenas para A. gemmatalis e, as proteínas não se mostraram tóxicas para H. virescens. Os ensaios de ligação às BBMVs demonstraram que as proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 se unem aos receptores presentes no intestino médio de forma efetiva nas três espécies e que, portanto, houve correlação entre a toxicidade e a união aos receptores para as populações de S. frugiperda e A. gemmatalis, porém para H. virescens não houve relação entre a toxicidade e a união aos receptores. Sendo assim ... / Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through competition assays between the different Vip3Aa toxins and toxin Cry1Ia10, and toxicity to lepidopteran pests inferring up which combinations that could be used to produce transgenic plants containing multiple genes, which have been used to circumvent the development of insects resistance to Bt toxins. Therefore, vip3Aa and cry1Ia10 genes were cloned into the pET SUMO vector, expressed in E. coli and toxicity of proteins were tested in bioassays with neonate larvae of Spodoptera frugiperda, Anticarsia gemmatalis and Heliothis virescens. The BBMVs (Brush Border Membrane Vesicles) were prepared from the gut of the three species, and homologous and heterologous competition assays were performed. The Vip3Aa42 and Vip3Aa43 proteins were toxic to S. frugiperda and A. gemmatalis. Already Cry1Ia10 protein showed toxicity only for A. gemmatalis and proteins were not toxic to H. virescens. Binding assays to BBMVs demonstrated that Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins binding effectively to receptors present in the midgut in three species and, therefore, was correlation between toxicity and the binding to receptors for the populations of S. frugiperda and A. gemmatalis, but for H. virescens there was no relationship between toxicity and the binding to receptors. Thus, the combination of Cry1Ia10 and Vip3Aa42 or Vip3Aa43 proteins is indicated for the production of biological insecticidal, as well as for the production of transgenic plants to circumvent ...
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unesp.br:11449/123778 |
| Date | 06 February 2015 |
| Creators | Marucci, Suzana Cristina [UNESP] |
| Contributors | Universidade Estadual Paulista (UNESP), Desidério, Janete Apparecida [UNESP] |
| Publisher | Universidade Estadual Paulista (UNESP) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | iv, 67 p. : il. |
| Source | Aleph, reponame:Repositório Institucional da UNESP, instname:Universidade Estadual Paulista, instacron:UNESP |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | -1, -1 |
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