关键词: 梨木虱, 梨树, 化学感受蛋白, 原核表达, 植物挥发物, 荧光竞争结合

Abstract: 【Aim】 Cacopsylla chinensis is an important pest of pear trees in China. The objectives of this study are to analyze the binding properties of the chemosensory protein 8 of C. chinensis (CchiCSP8) to volatiles of pear trees, and to comprehensively study the 
olfactory function of CchiCSP8, so as to provide a preliminary basis for explaining the olfactory mechanism of C. chinensis locating pear trees in the future. 【Methods】 We cloned the full-length open reading frame (ORF) of CchiCSP8 by PCR, and performed homology 
alignment and phylogenetic analysis based on the deduced amino acid sequences of CchiCSP8 with CSPs from other hemipteran insects. We then constructed the prokaryotic expression vector pET30a(+)-CchiCSP8 and determined the binding affinities of the recombinant CchiCSP8 to 41 pear tree volatiles by in vitro expression of Escherichia coli, His-tag protein nickel beads purification technology and fluorescence competitive binding assay. CchiCSP8 was subsequently subjected to homologous modeling and molecular docking analysis. 【Results】 The full-length ORF sequence of CchiCSP8 was cloned and obtained. It is 426 bp 
in length, encoding 141 amino acids, with a signal peptide consisting of 21 amino acids at the N-terminus. The predicted molecular weight and isoelectric point of mature CchiCSP8 protein are 15.02 kD and 9.77, respectively. By sequence alignment with the CSPs of the same order insects, it was found that CchiCSP8 has four conserved cysteine sites and is of the closest evolutionary relationship with DcitCSP11 of Diaphorina citri. pET30a(+)-CchiCSP8 was successfully expressed in E. coli after induction by IPTG, and a clean 
CchiCSP8 protein was purified by nickel magnetic beads. The binding affinities of the recombinant CchiCSP8 analyzed by fluorescence competitive binding assay showed that CchiCSP8 had certain binding affinities to 18 volatiles of pear tree, including 4 alkenes, 6 alcohols, 4 aldehydes, 1 ketone, 1 ester and 2 aromatics ligands, with the Ki values of 14-30 μmol/L. The binding affinities of CchiCSP8 with ligands changed with the number of carbon atoms. The molecular docking results showed that the binding energy (-4.2-6.0 
kJ/mol) between CchiCSP8 and each ligand was not obviously different, being consistent with the results of the above fluorescence competitive binding assay. During the binding process between CchiCSP8 and different ligands, several common amino acid residues, including 5 non-polar residues (A38, A42, L93, L99 and V100) and 2 polar residues (Q107 and K46), play a most prominent role. 【Conclusion】 CchiCSP8 can bind 18 pear tree volatiles of different types, suggesting that it plays an important role in the process of identifying pear tree volatiles by C. chinensis. The results of this study lay a theoretical basis for clarifying the olfactory mechanism of C. chinensis in the process of locating pear trees, providing a new idea for behavioral regulation of C. chinensis based on disturbing the olfactory behavior of the pest.

Key words: Cacopsylla chinensis, pear tree, chemosensory protein, prokaryotic expression; plant volatiles, fluorescence competitive binding