中华蜜蜂信息素结合蛋白OBP10的基因克隆、原核表达和配基结合特性分析  

Molecular cloning, prokaryotic expression and lignand-binding characterization of a novel pheromone binding protein OBP10 in Apis cerana cerana(Hymenoptera: Apidae)

作　　者：吴帆[1] 黄君君[1] 谭静[1] 唐明珠[1] 李红亮[1] 

机构地区：[1]中国计量学院生命科学学院,浙江省生物计量及检疫检验重点实验室,杭州310018

出　　处：《昆虫学报》2016年第1期25-32,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31372254);中国博士后科学基金(2014M560488,2015T80624);杭州市科技计划项目(20140432B12)

摘　　要：【目的】气味结合蛋白在中华蜜蜂Apis cerana cerana嗅觉系统中起到重要作用,本实验拟研究中华蜜蜂一个新的信息素结合蛋白OBP10及其与蜜蜂信息素以及蜜源开花植物挥发物的结合特性。【方法】本实验通过RT-PCR扩增获得OBP10基因全长(Gen Bank登录号:KP717060.1),以p ET-30a构建原核表达载体,并以Ni2+琼脂糖柱进行重组蛋白表达和分离纯化,在N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)作为荧光报告子下利用荧光光谱法体外研究重组Acer OBP10与多种候选化学配基的结合特征。【结果】经多序列联配分析,发现Acer OBP10的多个同源基因均为信息素结合蛋白(pheromone binding proteins,PBPs)。配基结合特性分析显示,Acer OBP10对14种候选配基中的蜂王信息素成分对羟基苯甲酸甲酯(HOB)竞争力最大,相对荧光可降至6.06%,解离常数11.04μmol/L;进一步表明Acer OBP10属于一个新的中蜂PBPs。此外,Acer OBP10也能和包括工蜂信息素(香叶醇和橙花醇)、报警信息素(2-庚酮和乙酸异戊酯)等蜜蜂信息素以及蜜源开花植物挥发物之一的β-紫罗兰酮结合,表明Acer OBP10可能是一种以信息素结合为主的多功能结合蛋白。【结论】Acer OBP10是中蜂一个新的信息素结合蛋白,与此前我们鉴定的蜂王信息素结合蛋白Acer ASP1相比,Acer OBP10对蜜蜂信息素的结合谱更为广泛,这些结果将对进一步研究中华蜜蜂信息素识别和传递提供理论基础,对于深入了解中华蜜蜂嗅觉影响生理功能的行为机制具有重要意义。[ Aim ] Odorant-binding proteins （OBPs） play an important role in olfactory system of Chinese bee, Apis cerana cerana. In this study, we aim to research the binding characteristics of pheromones and some plant volatiles with a novel pheromone-binding protein OBP10 of A. cerana cerana. [ Methods ] We successfully cloned the gene AcerOBPIO from A. cerana cerana （ GenBank accession no. KP717060.1 ） by RT-PCR, constructed its prokaryotic expression vector pET30-AcerOBPIO, and conducted its expression in the optimized conditions in vitro. The recombinant protein with biochemical activities was purified by the method of Niz-agarose affinity chromatograph. Using 1-NPN as a fluorescence probe, thebinding capability of AcerOBP10 with diverse bee pheromones and plant volatiles was measured by competitive fluorescence assay. [ Results ] By analyzing the alignment of the deduced homologous proteins of other insect species, we speculated that AcerOBP10 is a novel PBP ofA. cerana cerana. The results of competitive fluorescence assay showed that eight out of 14 ligands were able to decrease the relative fluorescence intensity of 1-NPN by more than 50%. Among them, methyl 4-hydroxylbenzoate （ HOB）, one of queen pheromone components, showed the strongest capability to compete with 1-NPN, causing 93.94% reduction in the relative fluorescence intensity with the KD value of 11.04 txmol/L. AcerOBP10 was also found to have the capability of binding with worker bee pheromones （ geraniol and nerol） and alarm pheromones （heptanone and isoamyl acetate ）, suggesting that it plays a significant role in recognizing pheromones in the bee olfactory system. AcerOBP10 also had strong binding force with some plant volatiles, like 13-ionone. [ Conclusion] As a pheromone-binding protein, AcerOBP10 has a strong binding capability with diverse bee pheromones. Compared with another PBP （AcerASP1） , AcerOBP10 seems to have more extensive pheromone binding profiles. The results provide a theoretical basis to explore the molecular

关 键 词：中华蜜蜂 信息素结合蛋白 信息素 荧光竞争 结合特性 

分 类 号：Q966[生物学—昆虫学]