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KD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,KD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。
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.
[1] 穆兰芳, 董双林, 修伟明. 无脊椎动物是怎样感受外界化学信号的?[J]. 自然杂志, 2004, 26(5): 305-309.
[2] Pelosi P, Zhou JJ, Ban LP, et al. Soluble proteins in insect chemical communication[J]. Cellular and Molecular Life Sciences CMLS, 2006, 63(14): 1658-1676.
[3] Pelosi P, Maida R.Odorant-binding proteins in insects[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1995, 111(3): 503-514.
[4] 任珍珍, 柳晓磊, 胡美英. 昆虫嗅觉相关蛋白的结构和功能[J]. 中国生物化学与分子生物学报, 2010, 26(6): 531-537.
[5] 王桂荣, 郭予元, 吴孔明. 昆虫触角气味结合蛋白的研究进展[J]. 昆虫学报, 2002, 45(1): 131-137.
[6] Scaloni A, Monti M, Angeli S, et al. Structural Analysis and Disulfide-Bridge Pairing of Two Odorant-Binding Proteins from Bombyx mori[J]. Biochemical and Biophysical Research Communications, 1999, 266(2): 386-391.
[7] L Ban, A Scaloni, A Brandazza, et al. Chemosensory proteins of Locusta migratoria[J]. Insect molecular biology, 2003, 12(2): 125-134.
[8] 李红亮, 张林雅, 倪翠侠, 等. 中华蜜蜂化学感受蛋白AcerCSP3的配基结合功能分析[J]. 昆虫学报, 2011, 54(3): 259-264.
[9] 孙红岩, 尹姣, 冯红林, 等. 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[J]. 昆虫学报, 2011, 54(4): 381-389.
[10] Gong ZJ, Zhou WW, Yu HZ, et al. Cloning, expression and functional analysis of a general odorant‐binding protein 2 gene of the rice striped stem borer, Chilo suppressalis (Walker)(Lepidoptera: Pyralidae)[J]. Insect Molecular Biology, 2009, 18(3): 405-417.
[11] Feng L, Prestwich GD.Expression and characterization of a lepidopteran general odorant binding protein[J]. Insect Biochemistry and Molecular Biology, 1997, 27(5): 405-412.
[12] ZHANG TT, WANG WX, GU SH, et al. Structure, Binding Characteristics, and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm, Helicoverpa armigera(Hübner)[J]. Journal of Integrative Agriculture, 2012, 11(3): 430-438.
[13] 李红亮, 张林雅, 庄树林, 等. 中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析[J]. 中国农业科学, 2013, 46(1): 154-161.
[14] 熊兴平. 茶尺蠖防治技术研究进展及展望[J]. 中国茶叶, 2003, 25(3): 15-17.
[15] 孙晓玲, 陈宗懋. 基于化学生态学构建茶园害虫无公害防治技术体系[J]. 茶叶科学, 2009, 29(2): 136-143.
[16] 范成平, 商志才, 郭明, 等. 荧光法研究加替沙星与牛乳铁蛋白的相互结合作用[J]. 浙江大学学报: 理学版, 2005, 32(1): 62-65.
[17] 黄安平, 韩宝瑜, 包小村. 茶刺蛾危害后茶树挥发性有机化合物释放变化[J]. 应用与环境生物学报, 2011, 17(6): 819-823.
[18] Zhu M, Li E, He H.Determination of volatile chemical constitutes in tea by simultaneous distillation extraction, vacuum hydrodistillation and thermal desorption[J]. Chromatographia, 2008, 68(7/8): 603-610.
[19] Maeda Y, Ueda T, Imoto T.Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone[J]. Protein Engineering, 1996, 9(1): 95-100.
[20] 李红亮, 聂文敏, 高其康, 等. 中华蜜蜂气味结合蛋白 ASP2 cDNA 的克隆及原核表达[J]. 中国农业科学, 2008, 41(3): 933-938.
[21] 郭萧, 王晓庆, 彭萍, 等. 茶树不同成熟度叶片对茶尺蠖发育适合度的影响[J]. 茶叶科学, 2012, 32(3): 229-235.
浙公网安备 33019902000101号 
