大草蛉化学感受蛋白基因CpalCSP3组织表达谱及气味结合特性分析

昆虫化学感受蛋白(Chemosensory protein,CSP)具有结合和运载气味分子穿过感器淋巴液到达嗅觉受体的作用,但尚未见有关大草蛉(Chrysopa pallens)CSP功能的报道。本研究克隆得到大草蛉Cpal CSP3基因,利用RT-q PCR技术测定了其在成虫各组织的表达谱,发现Cpal CSP3基因主要在雌雄虫的触角和头部表达,暗示该基因参与了大草蛉的嗅觉及味觉感受。为探讨其嗅觉功能,利用原核表达系统和荧光竞争结合试验测定了Cpal CSP3蛋白与84种气味物质的结合能力。结果表明,Cpal CSP3蛋白与橙花叔醇、(+)￣雪松醇和β￣紫罗兰酮具有很高的结合能力,结合常数(Ki)为8.79~9.79μmol·L-1;与根皮苷、月桂醛、乙酸香叶酯和α￣蒎烯也有较高的结合能力,Ki为11.54~19.06μmol·L-1。分析认为,Cpal CSP3蛋白可能在大草蛉对这些气味的嗅觉感受中起作用,使得大草蛉能对害虫寄主植物有效定向进而搜寻到猎物。

Tissue Expression Profile and Ligand Binding Affinity of the Chemosensory Protein Gene CpalCSP3 in Chrysopa pallens(Rambur)

Insect chemosensory proteins(CSPs) play important roles in olfactory perception by transporting hydrophobic chemical volatiles across the sensillum lymph to olfactory receptors, but this role in Chrysopa pallens(Rambur) has not been addressed. In this paper, a CSP gene CpalCSP3 was cloned and the tissue expression profile of the CpalCSP3 gene in male and female adults was determined by real-time quantitative polymerase chain reaction. Results showed that the CpalCSP3 gene was highly expressed in the heads and antennae of both sexes, suggesting its involvement in olfaction and gustation. To further explore the olfactory role of the gene, recombinant CpalCSP3 protein was obtained by the Escherichia coli expression system, and the binding affinities of CpalCSP3 protein for 84 plant volatiles were measured using fluorescent competitive binding assay. Among the tested compounds, nerolidol, (+)-cedrol and beta-ionone had high binding affinities to CpalCSP3 protein, with K_i values ranging from 8.79 to 9.79 μmol·L^－1. In addition, phlorizin dihydrate, dodecyl aldehyde, geranyl acetate and (+/?)-alpha-pinene also showed high binding affinities, with Ki values from 11.54 to 19.06 μmol·L^－1. Together, these results suggest that CpalCSP3 protein may play a role in olfaction of these plant volatiles, which facilitate host-plant searching in adult C. pallens.