Molecular cloning of five chemosensory proteins from Conopomorpha sinensis eggs and gene expression after diflubenzuron treatment

Objective  To obtain the full-length sequences of CsinCSPs genes of five chemosensory proteins (CSP) from Conopomorpha sinensis, and analyze the effect of diflubenzuron on CsinCSP gene expression.

Method  Total RNA was extracted from C. sinensis eggs. The full-length cDNA of five CsinCSP genes were cloned by transcriptome sequencing results and rapid amplification of cDNA ends(RACE). The putative amino acid sequences of CsinCSPs were analyzed by bioinformatics software. The three-dimensional (3D) structure of CsinCSPs were predicted using the Phyre² online software. The binding capacities of CsinCSPs to pesticides were studied by AutoDock software. The changes of CsinCSP gene expressions after treating the C. sinensis eggs with diflubenzuron were analyzed by qRT-PCR.

Result  Full-length sequences of five CsinCSP genes were cloned from C. sinensis eggs. Sequence analysis results indicated these five genes encode proteins with typical characteristics of insect chemosensory proteins. The putative cysteines pattern of five CsinCSPs was C1-X₆-C2-X₁₈-C3-X₂-C4. The five CsinCSPs could be classified to clades with five α-helix and six α-helix. Based on analysis of model template, the 3D structures of five CsinCSPs were found to consist of α-helix, β-strand and loop, and were most similar to the 3D structure of Mamestra brassicae CSP2. The molecular docking results revealed that the five CsinCSPs had the highest binding affinities with two pyrethroids, while the lowest binding affinity with diflubenzuron. The expressions of CsinCSP3 and CsinCSP5 were up-regulated by more than 40 times at 6 and 24 h after diflubenzuron treatment at sublethal dose to C. sinensis eggs, and decreased at 48 h after treatment.

Conclusion  The chemosensory protein CsinCSPs may be involved in the detoxification process after pesticide treatment in C. sinensis eggs.