Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).
Bottom Line: The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity.PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages.Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.
Affiliation: Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, AC, Av. Normalistas 800, Col. Colinas de la Normal, 44270 Guadalajara, Jalisco, México.Show MeSH
Mentions: The molecular basis for IVM susceptibility and resistance are not well understood in R. microplus ticks. This prompted us to search for novel molecular targets for IVM. Guerrero et al.  deposited a number of EST sequences with similarity to genes with possible roles in response to acaricide exposure or development of acaricide resistance. We focused on the EST TC2637 (GenBank accession no. CK184967), given that tentatively encodes for a chloride channel, a known target for IVM, and proceeded to further characterization. Based on the TC2637 sequence, we designed specific oligonucleotides GSP1GlyR and GSP2GlyR and used the 5′ RACE system to characterize the missing 5′ end of the GlyR sequence. Once we found the correct ORF and the deduced stop codon position, we designed PCR primers for the amplification of the respective full-length cDNA, which was cloned and sequenced (KJ476181). Sequence analysis showed that the flanked region has an ORF 1392 bp that encodes for a 464-amino acid polypeptide (Fig. 1), which contains features common to ligand-gated ion channels, such as a large amino-terminal extracellular domain, four transmembrane domains and a large intracellular loop (Fig. 2), with a predicted molecular mass of 52.85 kDa and a pI of 8.13. The deduced polypeptide appears to have no signal peptide.Figure 1.
Affiliation: Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, AC, Av. Normalistas 800, Col. Colinas de la Normal, 44270 Guadalajara, Jalisco, México.