Expressão heteróloga de novas toxinas Cry em diferentes sistemas para controle do bicudo-do-algodoeiro

• Ano de defesa: 2024
• Autor(a) principal: Santana, Julia Moura do Rosário
• Orientador(a): Grossi-de-Sa, Maria Fatima
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Stricto Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Saúde e Medicina
• País: Brasil
• Palavras-chave em Português: Proteína crystal; Bioensaio
• Palavras-chave em Inglês: Crystal protein; Bacillus thuringiensis; Bioassay; Cry23Aa/cry37Aa; Cry10Aa; Anthonomus grandis
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/3518

One of the main challenges in the production of cotton (Gossypium hirsutum) is the effective control of the damage caused by the cotton boll weevil (Anthonomus grandis, order Coleoptera), recognized as the main pest of the crop in Brazil. Currently, control of this insect pest depends on multiple applications of chemical insecticides during the crop cycle, which not only increases production costs but also has serious environmental impacts. Although genetically modified (GM) cotton plants expressing Cry toxins for insect resistance have been developed in different parts of the world, there are still no commercially available GM varieties to control cotton boll weevil. Therefore, this study aimed to validate molecules with potential entomotoxic effects against larvae of the A. grandis for their application in the development of GM cotton plants resistant to this insect pest. In this study, Cry toxins and their mutants were evaluated against larvae of A. grandis to determine their toxicity. Initially, were studied two new Bacillus thuringiensis toxins (Cry23Aa and Cry37Aa) that showed high efficacy in control of coleopteran insects. The genes encoding these toxins were expressed separately for in vitro toxicity assessments, both alone and in combination, against larvae of A. grandis, using bioassays with artificial diets containing the purified toxins. As a result, larvae fed exclusively with the Cry37Aa toxin showed no significant differences compared to the control group, while those exposed only to Cry23Aa had a reduction in survival of approximately 69%. However, the combination of Cry23Aa and Cry37Aa resulted in up to 100% larval mortality, revealing a powerful synergy between these two toxins. In addition, previous studies have demonstrated the efficacy of the Cry10Aa toxin against larvae of the A. grandis. However, its low solubility in the insect gut has been a challenge for its effective use to control coleopteran insects. To overcome this limitation, our study focused on developing mutants of the Cry10Aa toxin, based on the optimization of protein's isoelectric point, in order to improve entomotoxicity activity. The heterologous expression of these mutants was performed in two systems: Escherichia coli BL21 (DE3) pLysS and B. thuringiensis acrystalliferous. Although expression in E. coli showed low yields, the successful cloning of the cry10-mut2 and cry10-mut5 mutant genes into the pSVP27A expression vector enabled the efficient production of these molecules in B. thuringiensis acrystalliferous. Therefore, these advances represent a significant milestone in the development of sustainable strategies for controlling the cotton boll weevil through plant biotechnology. Furthermore, this study not only validates new biotechnological approaches using Cry toxins, but also stimulates safer and more efficient agricultural practices, contributing to sustainability and global food security.