Characterization of the selective potential for the improvement of Cry toxins with nematicidal activity

• Autor(a) principal: Delgadinho, Mariana Isabel Neves
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/21412
• topic: Nemátodos; Toxinas

Cry proteins are naturally produced by Bacillus thuringiensis, a gram-positive spore-forming bacterium, which exhibit specific toxicity against a variety of insects and nematodes. The toxins produced are known to be highly efficient, completely biodegradable and harmless to humans and other vertebrates. All of these reasons make them an excellent alternative to the chemical pesticides currently used. The main goal of the present work was to characterize and evaluate the potential to improve a Cry protein (Cry21Fa1) against nematodes. In order to fulfil this aim, the protein was expressed in the Escherichia coli JM103 strain and given to Caenorhabditis elegans as food source. Several experiments were designed, such as: toxin efficiency tests, characterization of toxic components, bacterial fitness measurements, assessment of the bacterial genetic background impact, site-directed mutagenesis and toxin testing on pathogenic nematodes. The results of the present study demonstrated that Cry21Fa1 is a very efficient toxin in reducing the C. elegans growth rate and consequently providing a higher fitness to the bacteria expressing it. The affected worms have a slower development time, lay eggs later and the chances of survival were decreased. Additionally, with the mutagenesis procedure we were able to demonstrate that a specific region on Domain III of the protein is crucial for the toxin function, probability affecting the receptor binding affinities or structural integrity. This is the first mutation analysis performed on this Cry21 gene, hopefully contributing to a better understanding of their mode of action. However, further work of this kind will be needed in order to efficiently improve the toxin. Our ability to confer and control the function of proteins via genetic engineering has significantly improved our understanding of protein structure-function relationships and led to the creation of novel molecules of economic interest, in order to enhance human life. Given the increasing demand for eco-friendly products, the development of additional Cry products should be encouraged.