Cloning of a horn fly cDNA, Hi alpha E7, encoding an esterase whose transcript concentration is elevated in diazinon-resistant flies
2000
Guerrero, F.D.
Reverse transcriptase-polymerase chain reaction (PCR) was used to clone two esterase cDNAs from a diazinon-resistant field population of horn flies that expresses qualitative and quantitative differences in esterases compared with a susceptible population . The open reading frame from one of the esterase cDNAs, Hi(alpha)E7, exhibits substantial amino-acid identity to an esterase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that Hi(alpha)E7 mRNA was more abundant in the diazinon resistant population than the susceptible population. DNA copy number analysis did not reveal major differences in Hi(alpha)E7 gene copy number between the two populations. The full-length cDNA to Hi(alpha)E7 was cloned and sequenced, and found to contain all of the highly conserved elements associated with carboxyl/cholinesterases. The Hi(alpha)E7 homologs in diazinon-resistant strains of L. cuprina and Musca domestica have been shown to possess an amino-acid substitution conferring diazinon hydrolytic activity to the esterase enzyme. This amino-acid substitution was not found in diazinon-resistant horn flies examined by allele-specific PCR. Individual flies from the resistant field population were phenotyped as diazinon-resistant or diazinon-susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to Hi(alpha)E7 probe in ribonuclease protection assays. Hi(alpha)E7 transcript was expressed at five-fold higher level in resistant female individual flies than in susceptible female individuals.
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