梨木虱化学感受蛋白CchiCSP8与梨树挥发物的结合特性分析

【目的】梨木虱Cacopsylla chinensis是我国重要的梨树害虫。本研究通过分析梨木虱化学感受蛋白8(chemosensory protein 8, CchiCSP8)与梨树挥发物的结合特性，综合研究CchiCSP8的嗅觉功能，为今后阐释梨木虱定位梨树的嗅觉机理提供前期基础。【方法】采用PCR方法克隆CchiCSP8的全长开放阅读框序列，并对推导的氨基酸序列与其他半翅目昆虫CSPs进行同源比对和进化分析；构建pET30a(+)-CchiCSP8的原核表达载体，通过大肠杆菌Escherichia coli体外表达、组氨酸标签蛋白镍磁珠纯化技术和荧光竞争性结合实验，共测定了重组蛋白CchiCSP8与41种梨树挥发物的结合能力。随后对CchiCSP8进行同源建模和分子对接分析。【结果】克隆获得了CchiCSP8全长序列，开放阅读框全长426 bp，编码141个氨基酸，N端有一个包含21个氨基酸的信号肽。成熟的CchiCSP8蛋白的预测分子量和等电点分别为15.02 kD和9.77；与其他半翅目昆虫CSPs的序列比对发现，CchiCSP8具有4个保守的半胱氨酸位点，并与柑橘木虱Diaphorina citri化学感受蛋白DcitCSP11的进化关系最近；pET30a(+)-CchiCSP8在IPTG诱导下在大肠杆菌中成功表达，并通过镍磁珠纯化获得纯净的CchiCSP8蛋白；采用荧光竞争性结合实验分析重组蛋白CchiCSP8的结合特性，结果显示CchiCSP8对18种梨树植物挥发物具有不同程度的结合能力(Ki=14~30 μmol/L)，包括4种烯烃、6种醇类、4种醛类、1种酮、1种酯类和2种芳香类物质，CchiCSP8与配体的结合力表现出了随碳原子数不同而变化的特点；分子对接结果显示CchiCSP8和每个配体之间的结合能相差不大(-4.2~6.0 kJ/mol)，这与上述荧光竞争结合实验的结果一致。在CchiCSP8与不同配体结合过程中，存在多个共有氨基酸残基，其中5个非极性残基(A38， A42， L93， L99和V100)以及2个极性残基(Q107和K46)的作用最为突出。【结论】CchiCSP8能够结合18种不同类型的梨树挥发物，推测其在梨 木虱识别梨树挥发物的过程起到重要作用，本研究结果可为明晰梨木虱在定位梨树过程中的嗅觉机制提供理论基础，并为开发基于扰乱嗅觉行为的新型梨木虱行为调控技术提供新思路。

Binding properties of the chemosensory protein CchiCSP8 inCacopsylla chinensis(Hemiptera:Psyllidae) to pear tree volatiles

【Aim】 Cacopsylla chinensis is an important pest of pear trees in China. Theobjectives of this study are to analyze the binding properties of the chemosensory protein 8of 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 theolfactory mechanism of C. chinensis locating pear trees in the future. 【Methods】 Wecloned 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 CchiCSP8with CSPs from other hemipteran insects. We then constructed the prokaryotic expressionvector pET30a(+)-CchiCSP8 and determined the binding affinities of the recombinantCchiCSP8 to 41 pear tree volatiles by in vitro expression of Escherichia coli, His-tagprotein 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 atthe N-terminus. The predicted molecular weight and isoelectric point of mature CchiCSP8protein are 15.02 kD and 9.77, respectively. By sequence alignment with the CSPs of thesame order insects, it was found that CchiCSP8 has four conserved cysteine sites and is ofthe 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 therecombinant CchiCSP8 analyzed by fluorescence competitive binding assay showed thatCchiCSP8 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 of14-30 μmol/L. The binding affinities of CchiCSP8 with ligands changed with the number ofcarbon 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 withthe results of the above fluorescence competitive binding assay. During the binding processbetween 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 amost prominent role. 【Conclusion】 CchiCSP8 can bind 18 pear tree volatiles of differenttypes, suggesting that it plays an important role in the process of identifying pear treevolatiles by C. chinensis. The results of this study lay a theoretical basis for clarifyingthe olfactory mechanism of C. chinensis in the process of locating pear trees, providing anew idea for behavioral regulation of C. chinensis based on disturbing the olfactorybehavior of the pest.