RESUMO
Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.
Assuntos
Diamida/química , Proteínas de Insetos/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diamida/metabolismo , Diamida/farmacologia , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/efeitos dos fármacos , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/farmacologia , Mariposas/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
In this short review, we highlight three functional genomic technologies that have recently been contributing to the understanding of the molecular mechanisms underpinning insecticide resistance: the GAL4/UAS system, a molecular tool used to express genes of interest in a spatiotemporal controlled manner; the RNAi system, which is used to knock-down gene expression; and the most recently developed gene editing tool, CRISPR/Cas9, which can be used to knock-out and knock-in sequences of interest.