中华蜜蜂化学感受蛋白AcerCSP3的配基结合功能分析

Molecular binding characterization with chemical ligands of a chemosensory protein AcerCSP3 in the Chinese honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae)

为研究中华蜜蜂Apis cerana cerana化学感受蛋白AcerCSP3在化学感受系统中的生理功能,本实验通过对AcerCSP3进行原核表达、分离纯化后,利用荧光法研究了体外重组AcerCSP3与1-NPN以及候选化学配基的结合特征。Scatchard方程显示AcerCSP3与1-NPN的解离常数KD为8.29μmol/L,结合位点数约等于1。在候选配基竞争1-NPN与AcerCSP3结合的实验中,5种配基均能在200μmol/L浓度下使1-NPN的相对荧光强度下降至50%以下,其中β-紫罗兰酮甚至能使1-NPN的相对荧光强度下降至10%左右,表明候选配基均与AcerCSP3有较强的结合能力,而3,4-二甲基苯甲醛与中蜂AcerCSP3的结合能力最强,KD达到18.77μmol/L。本研究所用化学配基均为植物花与叶片的挥发性的次生代谢产物,表明AcerCSP3可能作为中蜂化学感受系统的一部分,在其搜寻某些植物花粉蜜源时作为气味分子运载体发挥一定的作用。

In order to clarify the physiological function of AcerCSP3, a chemosensory protein （CSP） in Apis cerana cerana, the recombinant protein AcerCSP3 was successfully expressed in a optimized prokaryotic expression system, and the binding properties of the purified AcerCSP3 with 1-NPN was then investigated. Scatchard plot analysis indicated that the dissociation constant between 1-NPN and AcerCSP3 was 8.29 μmol/L and the binding site number was about one. In the competitive binding assays, all the five candidate ligands at the concentration of 200 μmol/L reduced the relative fluorescence intensity of 1-NPN by more than 50%, with β-ionone causing 90% reduction in the relative fluorescence intensity, suggesting that all the tested ligands have strong binding capability with AcerCSP3. 3, 4-Dimethylbenzaldehyde had the strongest binding capability with a dissociation constant as high as 18.77 μmol/L. All the candidate chemical ligands are plant volatile secondary metabolites, indicating that AcerCSP3 may act as a carrier for odor molecules released from flower and nectar and play a role in searching for honey sources.