Abstract
We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.
Highlights
The isolation of specific members of gene families typically involves the use of degenerate primers designed in regions conserved across many members of the gene family of interest
Using the gene specific degenerate primers designed with CODEHOP software (Figures 1, 2, 3), fragments from the eight target genes were amplified from both genomic and cDNA
The amplicons ranged in size from 234–1277 bp (Figure 4) and were the same size for both gDNA and cDNA indicating that each degenerate primer pair was contained within one exon
Summary
The isolation of specific members of gene families typically involves the use of degenerate primers designed in regions conserved across many members of the gene family of interest. Clones are sequenced looking for the gene of interest This strategy is labour intensive, time consuming and often fails to isolate the target gene. We describe here a rapid, targeted approach which enables isolation of the gene of interest without cloning, using a degenerate PCR and sequencing strategy based on homologous amino acid motifs specific to each target gene. The pyrethroid, formamidine and macrocyclic lactone families of acaricides are among the most widely used acaricides for controlling cattle ticks at present; all of which are known or believed to target membraneassociated proteins, potentially including GPCR. We believe that R.microplus GPCR involved in the regulation of vital physiological functions offer valuable targets for new acaricides. We report the targeted isolation of eight putative G-protein coupled neurotransmitter receptors from the cattle tick, R. microplus
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