Título Principal Toxinas recombinantes Cry2Aa e Cry11A de Bacillus thuringiensis expressas em células de inseto são tóxicas para larvas de Lepidoptera e Diptera

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Bacillus thuringiensis (Bt) is a Gram positive, tomopathogenic bacteria that produces crystalline inclusions called !-endotoxins or Cry proteins. These proteins are highly toxic to susceptible insects, not showing activity against others organisms. Some expression systems have been used to express crystal proteins with the aim to increase their toxicity for several insect orders. Baculoviruses are insect viruses that have been widely used as expression vectors of heterologous proteins in insect cells, mainly due to the presence of strong promoters that allow high levels of expression of the heterologous protein during the late phase of virus infection. In this work, cry genes (cry2Aa and cry11A) from B. thuringiensis subsp. Kursaki S447 and subsp. israelensis S1806, respectively, were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), by site-specific transposition, generating the recombinant viruses vAcCry2Aa and vAcCry11A. Transcription of the heterologous genes were confirmed using mRNA from recombinant viruses infectedinsect cells (72 h p.i) by RT-PCR . The heterologous Total extracts from Spodoptera frugiperda infected with the recombinant viruses (vAcCry2Aa and vAcCry11A) were analyzed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa and 70 kDa, respectively. Bioassays, using the heterologous proteins showed that Cry2Aa had toxicity to second instar Anticarsia gemmatalis larvae with a LC50 of 1.03 $g/mL and that Cry11A had toxicity to second instar Aedes aegypti larvae (Cry11A) with a LC50 of 53.3 ng/mL. Cuboid-shaped protein crystals (Cry2Aa) were observed only in vAcCry2Aa S. frugiperda infected-extracts by light and scanning electron microscopy. This work confirms the utility of the baculovirus expression system for the efficient expressionof Cry proteins.

ASSUNTO(S)

cry2aa cry11a, bacillus thoringiensis biologia molecular baculovírus

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