Caracterização do gene vip3A e toxicidade da proteína Vip3Aa50 à lagarta-do-cartucho e à lagarta-da-soja

Abstract

Resumo -O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por "Western blot". Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL 50 de 20,3 e 79,6 ng cm -2 , respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A.Termos para indexação: Anticarsia gemmatalis, Spodoptera frugiperda, controle biológico, expressão heteróloga, proteína inseticida vegetativa. Characterization of the vip3A gene and toxicity of Vip3Aa50 protein to fall armyworm and velvetbean caterpillarAbstract -The objective of this work was to characterize the vip3A gene of Bacillus thuringiensis and to evaluate the toxicity of Vip3Aa50 protein to the fall armyworm (Spodoptera frugiperda) and velvetbean caterpillar (Anticarsia gemmatalis) larvae. The gene vip3A was amplified by specific PCR primers, generating a 2,370-bp fragment. This fragment was cloned into the pGEM-T Easy vector, and then it was sequenced, subcloned into the pET-28a (+)'s expression vector, and inserted into Escherichia coli BL21 (DE3) cells. The Vip3Aa50 protein expression was induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG), visualized in SDS-PAGE, and detected by Western blot. The toxicity bioassay showed a high activity of Vip3Aa50 protein against both velvetbean and fall armyworm neonate larvae, with LC 50 at 20.3 and 79.6 ng cm -2 respectively. The vip3Aa50 gene, is a new gene of vip3A class.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

“…Quantitative binding parameters were obtained by using 125 Ilabeled Vip3Aa and S. frugiperda BBMVs. This binding was found to be saturable, mostly irreversible, and differentially affected by the presence of divalent cations (89).…”

“…Vip3A proteins were also found to have lower affinities but higher numbers of binding sites than the Cry1A and Cry2A proteins. Interestingly, homologous competition showed that both the 62-kDa and the 20-kDa fragments of trypsin-activated 125 I-labeled Vip3Aa bound to BBMVs, and both fragments were displaced by the addition of nonlabeled Vip3Aa. In contrast, using biotin-labeled Vip3Aa, Liu et al (93) found that only the 62-kDa fragment was able to bind to H. armigera BBMVs and also that the 20-kDa fragment was found exclusively in the supernatant of the binding reaction mixture.…”

Relação entre toxicidade de proteínas Vip3Aa e sua capacidade de ligação a receptores intestinais de lepidópteros-praga

“…Como houve sobreposição dos intervalos de confiança (IC), as proteínas foram consideradas igualmente tóxicas para a espécie. Figueiredo et al (2013) realizaram ensaios com a proteína Vip3Aa50 e a mesma população de S. frugiperda e obtiveram CL 50 de 79,6 ng cm . Temse observado que a proteína Vip3Aa é altamente tóxica a S. frugiperda, com toxicidade superior à das proteínas Cry1.…”

Vegetative Insecticidal Protein (Vip): A Potential Contender From Bacillus thuringiensis for Efficient Management of Various Detrimental Agricultural Pests

“…Vip3 proteins are reported to be effective against black cutworm Agrotis ipsilon (Yu et al, 1997;Donovan et al, 2001), cotton boll worm; Helicoverpa armigera (Lee et al, 2003;Cai et al, 2006;Fang et al, 2007), Beet army worm Spodoptera exigua (Donovan et al, 2001;Cai et al, 2006), fall armyworm S. frugiperda (Yu et al, 1997;Sena et al, 2009;Figueiredo et al, 2013), tobacco budworm Heliothis virescens (Estruch et al, 1996;Gulzar and Wright, 2015), tobacco hornworm Manduca sexta (Lee et al, 2003), African cotton leafworm S. littoralis, European grapevine moth Lobesia botrana, Cabbage moth Mamestra brassicae (Palma et al, 2013;De Escudero et al, 2014), Mediterranean flour moth (Ephestia kuehniella) (Mesrati et al, 2005), cabbage looper Trichoplusia ni (Fang et al, 2007), Tomato leaf miner Tuta absoluta (Sellami et al, 2015), diamondback moth Plutella xylostella (Bhalla et al, 2005;Palma et al, 2013;Gulzar and Wright, 2015), locust bean moth Ectomyelois ceratoniae (Boukedi et al, 2015) and tobacco cutworm S. litura (Bhalla et al, 2005;Song et al, 2016).…”