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Master's Dissertation
DOI
https://doi.org/10.11606/D.59.2020.tde-17062021-190228
Document
Author
Full name
Ariadne Costa Sanches
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Polanczyk, Ricardo Antônio (President)
Desidério, Janete Apparecida
Pereira, Rodrigo Augusto Santinelo
Souza, Jackson Antonio Marcondes de
Title in Portuguese
Interação da toxina Cry1Ac de Bacillus thuringiensis às BBMVs de diferentes ínstares de Chrysodeixis includens (Lepidoptera: Noctuidae)
Keywords in Portuguese
Bacillus thuringiensis
Chrysodeixis includens
Competição homóloga
Ligand-Blotting
Abstract in Portuguese
Chrysodeixis includens é importante praga na cultura da soja, pois causa redução drástica da área foliar e, consequentemente grande redução da produtividade. Devido à intensa utilização de inseticidas químicos para o seu controle, populações frequentemente tem desenvolvido resistência. Desta forma, é necessário a utilização de outras formas de controle, como o controle microbiano com o uso da bactéria Bacillus thuringiensis (Bt) na forma de bioinseticida ou planta Bt. Dentre as toxinas ativas para lepidópteros praga, Cry1Ac se destaca com uma das mais promissoras. No entanto, há relatos de diferenças na suscetibilidade entre diferentes ínstares larvais da mesma espécie praga. Essa variação na susceptibilidade à toxina Cry1Ac pode estar relacionada a modificações nos receptores da toxina presentes nas BBMVs ("Brush Border Membrane Vesicles"), reduzindo a capacidade de ligação da toxina Cry ou a expressão destes receptores na membrana. Desta forma, os objetivos deste projeto foram avaliar a toxicidade da toxina Cry1Ac em diferentes ínstares de Chrysodeixis includens e correlacionar a susceptibilidade do inseto com a capacidade de ligação da toxina às suas BBMVs. A proteína Cry1Ac foi obtida a partir da expressão heteróloga em Escherichia coli e utilizada em bioensaios contra lagartas de 1º a 6º ínstar para estimar a CL50. A Cry1Ac foi ativada com tripsina, purificada e marcada com biotina. As BBMVs foram preparadas a partir dos intestinos de 2º a 6º ínstar de C includens para realizar os ensaios de competição e ligação. As lagartas de 6º ínstar foram 370 vezes menos suscetíveis que as lagartas de 1º ínstar. Houve maior competição pelos receptores nos ínstares tardios do que nos ínstares iniciais e diminuição da expressão dos receptores em ínstares tardios. Os resultados sugerem que a diminuição na suscetibilidade de C. includens à Cry1Ac pode estar relacionada com a quantidade dos receptores e a capacidade de ligação da proteína aos receptores.
Title in English
Interaction of Bacillus thuringiensis Cry1Ac toxin to BBMVs from different instars of Chrysodeixis includens (Lepidoptera: Noctuidae)
Keywords in English
Bacillus thuringiensis
Chrysodeixis includens
Homologous competition
Ligand-Blotting
Abstract in English
Chrysodeixis includens is an important pest in soybean crop, as it causes drastic reduction of leaf area and, consequently, great reduction of yield. Due to the intense use of chemical insecticides for their control, populations have often developed resistance. Thus, it is necessary to use other forms of control, such as microbial control with the use of Bacillus thuringiensis (Bt) bacteria in the form of bioinsecticide or Bt plant. Among the active toxins for pest lepidopterans, Cry1Ac stands out as one of the most promising. However, there are reports of differences in susceptibility between different larval instars of the same pest species. This variation in Cry1Ac toxin susceptibility may be related to changes in toxin receptors present in the Brush Border Membrane Vesicles (BBMVs), reducing Cry toxin binding capacity or membrane receptor expression. Thus, the objectives of this project were to evaluate the toxicity of Cry1Ac toxin in different Chrysodeixis includens instars and to correlate insect susceptibility with toxin binding capacity to its BBMVs. Cry1Ac protein was obtained from heterologous expression in Escherichia coli and used in 1st to 6th instar caterpillar bioassays to estimate the LC50. Cry1Ac was trypsin activated, purified and labeled with biotin. BBMVs were prepared from the 2nd to 6th instar intestines of C includens to perform the competition and binding assays. 6th instar larvae were 370 times less susceptible than 1st instar larvae. There was greater competition for receptors in late instars than in early instars and decreased receptor expression in late instars. The results suggest that the decrease in C. includens susceptibility to Cry1Ac may be related to the amount of receptors and the protein binding capacity to receptors.
 
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Publishing Date
2021-06-21
 
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