气味结合蛋白MmedOBP19在中红侧沟茧蜂足部的表达及配体结合特征

摘要

[目的]明确气味结合蛋白基因MmedOBP19在中红侧沟茧蜂Microplitis mediator足部的表达定位及重组MmedOBP19的配体结合特征.[方法]利用扫描电镜观察化学感器在中红侧沟茧蜂足部的分布,通过原位杂交研究MmedOBP19在中红侧沟茧蜂雌蜂前足中的表达定位,并采用荧光竞争结合实验分析MmedOBP19重组蛋白与86种潜在配体的结合特征.[结果]扫描电镜观察发现,中红侧沟茧蜂的足部主要分布3种类型的感器,分别是毛形感器、刺形感器和锥形感器.原位杂交结果显示,MmedOBP19的表达主要集中在中红侧沟茧蜂前足跗节的感器下方.重组MmedOBP19与配体的结合力测定结果显示,MmedOBP19与非挥发性植物次生物质包括棉酚(Ki =2.32 ±0.17μmol/L)、硫酸黄连素(Ki=4.86±1.81 μmol/L)、槲皮素(Ki=4.93±0.21 μmol/L)、单宁(Ki=5.23±0.38 μmol/L)、芸香叶苷(Ki=5.91±0.26 μmol/L)和奎宁(Ki=6.05±0.51 μmol/L)等具有较强的结合能力,同时能够结合油酸(Ki=4.32±1.73 μmol/L)及亚油酸(Ki =3.55 ±0.44μmol/L),但与柠檬烯(Ki=12.65±2.55 μmol/L)的结合力较弱.[结论]推测中红侧沟茧蜂足部具有化学感受功能,MmedOBP19主要参与中红侧沟茧蜂足部味觉识别过程.%[Aim] This study aims to investigate the expression of the odor binding protein gene MmedOBP19 in the sensilla in legs of the braconid wasp Microplitis mediator and the ligand binding characteristics of recombinant MmedOBP19.[Methods] The ultrastructure of sensilla on the legs of female and male adults of M.mediator was observed by using scanning electron microscope.The in situ hybridization assays were conducted to explore the expression of MmedOBP19 in the forelegs of female adults of M.mediator.The fluorescence-based competitive binding assays were performed to measure the binding abilities of the recombinant MmedOBP19 to 86 candidate ligand compounds.[Results] There are three kinds of morphological sensilla on the legs of adult wasps,including sensilla trichodea,sensilla chaetica and sensilla basiconica,amongst which sensilla basiconica with two pores on the tip belong to taste sensilla.The in situ hybridization analysis showed that the expression of MmedOBP19 was concentrated at the base of the sensilla in tarsi of the foreleg.The results of fluorescence-based competitive binding assays revealed that the recombinant MmedOBP19 had strong affinities with non-volatile plant secondary metabolites,such as gossypol (Ki =2.32 ± 0.17 μmol/L),berberine sulphate (Ki =4.86±1.81 μmol/L),quercetin (Ki =4.93±0.21 μmol/L),tannin (Ki =5.23 ±0.38 μmol/L),rutin (Ki =5.91 ± 0.26 μmol/L) and quinine (Ki =6.05 ± 0.51 μmol/L).In addition,MmedOBP19 could bind to octadecenoic acid (Ki =4.32 ± 1.73 μmol/L) and linoleic acid (Ki =3.55±0.44 μmol/L),but had a weak binding ability with limonene (Ki =12.65 ± 2.55 μmol/L).[Conclusion] Legs of M.mediator wasps might be involved in chemical communication behavior,and MmedOBP19 in the legs of M.mediator wasps may play major roles in taste perception.