Cloning,Sequence Analysis and Expression Characteristics of Carboxylesterase 2 Gene in Dermanyssus gallinae
[Objective]The aim of this study was to clone the CDS region sequence of the carboxylesterase 2(CarE2)gene in Dermanyssus gallinae and conduct bioinformatic analysis,as well as detect its expression characteristics in Dermanyssus gallinae,so as to explore the role of CarE2 gene in the formation and development of metabolic resistance in Dermanyssus gallinae.[Method]Fifty adult female Dermanyssus gallinae were selected,and the CDS region of CarE2 gene was amplified by PCR and sequenced.Multiple sequence alignment and phylogenetic tree construction were carried out for its coding amino acid sequence.The physical and chemical properties and structural functions of CarE2 protein were predicted by bioinformatics online software.Real-time quantitative PCR was used to detect the expression patterns of CarE2 gene in different developmental stages and different resistant strains of Dermanyssus gallinae,and to analyze the induction effect of cypermethrin on CarE2 gene in sensitive and resistant strains of Dermanyssus gallinae.[Result]The CDS region of CarE2 gene in Dermanyssus gallinae was 1 665 bp,encoding 554 amino acids.Multiple sequence alignment results showed that the amino acid sequence of CarE2 gene had highly conserved catalytic triad(Ser-Glu-His)and pentapeptide motifs(Gly-X-Ser-X-Gly).Phylogenetic tree analysis showed that CarE2 in Dermanyssus gallinae was closely related to Drosophila melanogaster,and was divided into epidermal esterase branch.Bioinformatics analysis results showed that CarE2 protein was a stable hydrophilic protein with signal peptide cleavage site,glycosylation site and multiple phosphorylation modification sites,but it did not have a transmembrane structure.The secondary structure of CarE2 protein was dominated by random coil,and the predicted tertiary structure was consistent with the secondary structure.The results of Real-time quantitative PCR showed that the expression of CarE2 gene was the highest in the nymph stage,which was significantly higher than that in other stages(P＜0.05),followed by in adult stage,and was relatively low in egg and larva stages.The relative expression of CarE2 gene in beta-cypermethrin resistant strain of Dermanyssus gallinae was 2.78 times that of sensitive strain.After treatment with beta-cypermethrin,the expression of CarE2 gene in sensitive strain was not significantly different compared with control group(P＞0.05),but the expression of CarE2 gene in beta-cypermethrin cypermethrin resistant strain was significantly higher than that of control group(P＜0.05).[Conclusion]In this study,the CDS region of CarE2 gene in Dermanyssus gallinae was cloned successfully,and it was expressed in all developmental stages of Dermanyssus gallinae,and overexpressed in beta-cypermethrin resistant strain.The expression of CarE2 gene was significantly up-regulated after treatment with cypermethrin,suggesting that CarE2 gene was involved in the metabolic resistance of beta-cypermethrin to carboxylesterase detoxification in Dermanyssus gallinae.
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