Avaliação da toxicidade de proteínas produzidas em sistema heterólogo sobre Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae)
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Data
2022-12-14
Autores
Brondi, Carolina Veluci
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Universidade Estadual Paulista (Unesp)
Resumo
O Brasil é o maior produtor mundial de cana-de-açúcar. Entre os fatores que prejudicam a produtividade do cultivo de cana-de-açúcar no País, destaca-se a broca-da-cana, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae). O manejo da broca-da-cana no Brasil é realizado por meio da utilização de produtos químicos e biológicos e, mais recentemente, pela adoção de plantas transgênicas com resistência à broca-da-cana. Considerando as limitações e questões negativas associadas aos tipos de métodos de controle citados, como a evolução da resistência de populações da broca-da-cana às plantas transgênicas atualmente disponíveis, o conhecimento sobre novas proteínas com atividade inseticida abre caminho para o desenvolvimento de novas plantas transgênicas com modos de ação distintos. O presente trabalho foi realizado para avaliação de duas proteínas com possível atividade inseticida sobre a broca-da-cana. A toxina Vip3Aa20 de Bacillus thuringiensis Berliner, 1915 foi escolhida por sua notável atividade inseticida sobre lepidópteros pragas. A outra proteína escolhida foi o peptídeo CfCBD derivado do parasitoide Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) que apresenta um domínio ligante em quitina comumente associado a toxinas com atividade inseticida. A toxina Vip3Aa20 foi produzida, purificada e avaliada em bioensaios de alimentação utilizando folhas de cana-de-açúcar e dieta artificial como substratos alimentares. Não foi possível observar mortalidade significativa da broca-da-cana ao ingerir Vip3Aa20 nas concentrações de 30, 60, 120, 240, 480 e 960 ng.cm-2. A ativação da toxina Vip3Aa20 pelo conteúdo do intestino da broca-da-cana foi incompleta. O peptídeo CfCBD não foi produzido e, por equívoco, um peptídeo não identificado de E. coli foi purificado e avaliado em bioensaios de alimentação utilizando folhas de cana-de-açúcar como substrato alimentar. Valores de mortalidade de 100% da broca-da-cana foram observados após ingestão desse peptídeo na concentração de 6.400 ng.cm-2. Porém, foi verificado que esse peptídeo não apresenta estabilidade e é completamente hidrolisado pelo conteúdo intestinal da broca-da-cana, fato que provavelmente está associado à alta concentração necessária para esse peptídeo causar mortalidade nas lagartas. Os resultados obtidos na presente dissertação auxiliam o desenvolvimento de toxinas proteicas para o controle da broca-da-cana.
Brazil is the largest producer of sugarcane in the world. The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is highlighted among the pests that reduce the productivity of sugarcane. In Brazil, sugarcane borer is controlled by the use of chemical and biological agents, and, more recently, by the adoption of transgenic plants resistant to the sugarcane borer. Considering the limitation and negative issues associated with each control tactic currently in use, such as the evolution of resistance in populations of sugarcane borer exposed to transgenic plants, the discovery of novel proteins with distinct modes of action for pest control would pave the way for the development of novel transgenic plants. The goal of the current work was to assess two proteins putatively lethal to the sugarcane borer. Vip3Aa20 derived from Bacillus thuringiensis Berliner, 1915 was selected given its remarkable insecticidal effectiveness against lepidopteran pests. The other selected protein was the CfCBD peptide derived from the parasitoid Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) which contains a chitin binding domain frequently associated with insecticidal activity over lepidopteran larvae. Vip3Aa20 toxin was produced, purified, and applied in bioassays using sugarcane leaves or artificial diet as feeding substrates. No mortality was observed for the sugarcane borer exposed to Vip3Aa20 at 30, 60, 120, 240, 480, and 960 ng.cm-2 . An incomplete activation of Vip3Aa20 by the midgut juice of the sugarcane borer was observed. A non-identified peptide from E. coli was erroneously purified instead of the CfCBD. This peptide was tested using sugarcane leaves as feeding substrate and the sugarcane borer experienced a 100% mortality rate after ingesting this peptide at a concentration of 6,400 ng.cm-2 . However, it was shown that this peptide lacks stability and was entirely digested by the midgut juice of the sugarcane borer, which is probably associated with the high concentration of this peptide required to result in larval mortality. The findings of the current dissertation aid in the development of proteins to be used for the control the sugarcane borer.
Brazil is the largest producer of sugarcane in the world. The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is highlighted among the pests that reduce the productivity of sugarcane. In Brazil, sugarcane borer is controlled by the use of chemical and biological agents, and, more recently, by the adoption of transgenic plants resistant to the sugarcane borer. Considering the limitation and negative issues associated with each control tactic currently in use, such as the evolution of resistance in populations of sugarcane borer exposed to transgenic plants, the discovery of novel proteins with distinct modes of action for pest control would pave the way for the development of novel transgenic plants. The goal of the current work was to assess two proteins putatively lethal to the sugarcane borer. Vip3Aa20 derived from Bacillus thuringiensis Berliner, 1915 was selected given its remarkable insecticidal effectiveness against lepidopteran pests. The other selected protein was the CfCBD peptide derived from the parasitoid Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) which contains a chitin binding domain frequently associated with insecticidal activity over lepidopteran larvae. Vip3Aa20 toxin was produced, purified, and applied in bioassays using sugarcane leaves or artificial diet as feeding substrates. No mortality was observed for the sugarcane borer exposed to Vip3Aa20 at 30, 60, 120, 240, 480, and 960 ng.cm-2 . An incomplete activation of Vip3Aa20 by the midgut juice of the sugarcane borer was observed. A non-identified peptide from E. coli was erroneously purified instead of the CfCBD. This peptide was tested using sugarcane leaves as feeding substrate and the sugarcane borer experienced a 100% mortality rate after ingesting this peptide at a concentration of 6,400 ng.cm-2 . However, it was shown that this peptide lacks stability and was entirely digested by the midgut juice of the sugarcane borer, which is probably associated with the high concentration of this peptide required to result in larval mortality. The findings of the current dissertation aid in the development of proteins to be used for the control the sugarcane borer.
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Palavras-chave
Biotecnologia, Cana-de-açúcar, Inseticidas, Insetos, Broca-da-cana-de-açúcar