Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.
A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.
Vontas
John G
JG
School of Biosciences, Cardiff University, Main College, Museum Avenue, Cardiff CF10 3TL, Wales, UK. vontas@imbb.forth.gr
Small
Graham J
GJ
Nikou
Dimitra C
DC
Ranson
Hilary
H
Hemingway
Janet
J
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006430:Hemiptera / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D007313:Insecta / Q000201:enzymology; D007305:Insecticide Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012275:Oryza / Q000469:parasitology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D005982:Glutathione Transferase