草地螟普通气味结合蛋白Ⅰ(Lsti-GOBP1)蛋白表达纯化及结合特性分析

Expression,purification and binding characteristics of general odorant binding protein Ⅰ(GOBP1) from the meadow moth,Loxostege sticticalia (Linnaeus) (Lepidoptera:Pyralididae)

气味结合蛋白在昆虫对寄主挥发性气味识别过程中有着重要的生理功能。本研究通过对草地螟Loxostege sticticalis L.普通气味结合蛋白Ⅰ(Lsti-GOBP1)进行克隆及原核表达的基础上,分离纯化得到体外重组的Lsti-GOBP1蛋白。并利用N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)作为荧光探针研究了Lsti-GOBP1蛋白与醇类、醛类、酯、烯等50种气味标样的结合特性,结果表明Lsti-GOBP1可与其中35种气味化合物结合,但只有1-己醇、1-庚醇、肉桂醛和莰烯4种气味标样能在20μmol/L浓度(探针与蛋白结合的饱和浓度值)下将1-NPN从Lsti-GOBP1中替换50%,其结合常数分别为8.997,7.283,7.289和9.814μmol/L。据此可以得出,Lsti-GOBP1蛋白的气味物质结合谱很广,同时具有较强的特异性,其中1-己醇、1-庚醇、肉桂醛、莰烯等化合物在草地螟识别寄主植物气味物质的过程中起重要作用。

Odorant-binding proteins （OBPs） are responsible for physiological function via perception of volatile odors. Here we reported the successful expression and purification of a general odorant binding protein Lsti-GOBP1 from Loxostege sticticalis L. Lsti-GOBP1 was expressed using Escherichia coli Rosetta （DE3） prokaryotic expression system,and then purified using Histrap HP column,and the protein functions of Lsti-GOBP1 were tested by fluorescent probe N-phenyl-1-naphthylamine （1-NPN） binding assays and competitive binding assays with 50 kinds of chemical molecules. The results showed that Lsti-GOBP1 had the capability of binding 35 odorants. However,only 1-hexanol,1-heptanol,cinnamaldehyde and camphene replaced 1-NPN from Lsti-GOBP1 by 50%,and their binding constants were 8.997,7.283,7.289 and 9.814 μmol/L,respectively. So,it can be speculated that Lsti-GOBP1 has a wide binding characteristics,but it has strong binding specificity to 1-hexanol,1-heptanol,cinnamaldehyde and camphene,which probably all play a critical role in the process of recognizing different odorants in L. sticticalis.