茶尺蠖普通气味结合蛋白EoblGOBP2与茶树挥发物的结合功能研究

茶尺蠖是茶园中一种重要的鳞翅目害虫,主要以幼虫取食危害茶树叶片,给茶叶生产带来了严重威胁。茶尺蠖在寻找配偶、产卵地以及觅食活动中其嗅觉系统发挥了非常重要的作用。本研究以茶尺蠖Ectropis obliqua Prout嗅觉系统中的一个普通气味结合蛋白——EoblGOBP2为研究对象,在克隆得到的EoblGOBP2基因基础上,通过对EoblGOBP2进行原核表达、分离纯化后,利用1-NPN的荧光竞争结合法研究了体外重组EoblGOBP2与茶树挥发物的结合生理功能。Scatchard方程显示EoblGOBP2与1-NPN的解离常数K_D为2.310 μmol·L^-1。在荧光竞争结合实验中,7种待测配基均能使1-NPN的相对荧光强度降低到50%以下,表明与EoblGOBP2均有较强的结合能力,而其中邻苯二甲酸二丁酯结合能力最强,K_D值达到4.353 μmol·L^-1。本研究7种配基均为茶树叶片挥发物,表明EoblGOBP2在茶尺蠖嗅觉系统对茶树挥发物的信息识别结合功能中发挥了重要作用。

Molecular Binding Characterization with Tea Plant Volatiles of a General Odorant-binding Protein EoblGOBP2 in the Tea Geometrid,Ectropis oblique Prout (Lepidoptera: Geometridae)

As one main Lepidopteran pest in tea garden, the larvae of tea geometrid damages mainly the tea leaves, which severely reduce the yield of tea products. The olfactory system of tea geometrid plays a very important role in terms of seeking host and food. Therefore, a general odorant binding protein—EoblGOBP2, existing in the olfactory system of tea geometrid, the function binding with tea plant volatiles was studied in vitro here. Based on the cloned EoblGOBP2 gene by RT-PCR, the recombinant protein EoblGOBP2 was successfully expressed in an optimized prokaryotic prokaryotic expression system, then the binding characteristics of the purified EoblGOBP2 with 1-NPN was measured by fluorescence spectrometry. Scatchard plot analysis indicated that the dissociation constant between EoblGOBP2 and 1-NPN was 2.310 μmol·L^-1. In the competitive binding assays, to the seven candidate ligands, all of the relative fluorescence intensity of 1-NPN decreased more than 50%, which suggested that all of the tested ligands have strong binding capability with EoblGOBP2. The compound of dibutyl phthalate showed the maximum binding capability with EoblGOBP2, and their dissociation constant was 4.353 μmol·L^-1. As all of the candidate chemical ligands belong to the tea volatiles secondary metabolites, indicating that EoblGOBP2 plays an important role in the odor recognition process of olfactory identification in the olfactory system of tea geometrid.