中文摘要：

【目的】茶尺蠖（Ectropis obliqua）是中国茶区主要的鳞翅目害虫,其幼虫暴食性常给茶园带来巨大损失。鳞翅目昆虫雌雄个体间普遍存在着性信息素通讯环节,而茶尺蠖性信息素识别途径一直鲜有报道。论文旨在通过对茶尺蠖性信息素识别过程中的结合蛋白基因进行鉴定和功能研究,为茶尺蠖性信息素化学通讯和嗅觉信息识别传递机制提供理论依据。【方法】利用RT-PCR技术对从雄虫触角转录组数据中首次鉴定和发现的一个茶尺蠖信息素结合蛋白基因（pheromone-binding protein,PBP）2（EoblPBP2）进行全长cDNA序列克隆（Gen Bank登录号为KX421383）后,将该基因与pET32a载体连接构建表达载体。随后向导入pET32a/EoblPBP2表达质粒的大肠杆菌BL21（DE3）中对其表达载体加入终浓度1 mmol·L~（-1）的IPTG进行蛋白的诱导表达,进一步利用镍NTA琼脂糖凝胶柱和含有咪唑的上清缓冲液分离目的蛋白,以PBS缓冲液作为透析液纯化得到高浓度重组蛋白。最后通过荧光竞争法,利用N-苯基-1-萘胺（N-phenyl-1-naphthylamine,1-NPN）作为荧光报告子以放大荧光信号,随后向重组蛋白与1-NPN混合体系中分别加入（Z,Z,Z）-3,6,9-十八碳三烯以及10种测试气味后经过分子荧光光度计扫描得到相对荧光值,计算各配基的解离常数KD,研究重组蛋白与茶尺蠖性信息素和茶树挥发物的生理功能。【结果】EoblPBP2全长为492 bp,编码了163个氨基酸,蛋白相对分子质量为15.9kD,等电点为4.983,具有保守的6个半胱氨酸,根据分子进化树分析其应归属于昆虫PBPs家族。结合功能结果显示,10种测试气味均能将1-NPN与茶尺蠖EoblPBP2重组蛋白的混合体系于420 nm处的相对荧光值降至50%以下。其中β-紫罗兰酮、邻苯二甲酸二丁酯、苯乙醛、癸醛和反-2-癸烯醛等5种茶树挥发物质与EoblPBP2重组蛋白的结合能力较强,解离常数KD分别为7.923、14.830、30.368?

英文摘要：

[Objective] The larva of tea geometrid （Ectropis oblique） is a major Lepidoptera pest in tea plantation, which can cause enormous economic losses every year due to its violent foraging to tea leaves. Although pheromone communication is widely involved in the process of courtship of male moths towards females, the intrinsic pheromone cognitive mechanism has not been figured out yet in tea geometrid. This experiment aims to provide a theoretical basis of chemical communication and olfactory cognition and transportation for E. oblique, by means of the molecular characterization and functions for the binding protein involved in the process of sex pheromone cognition. [ Method] A pheromone binding protein gene, EoblPBP2, was amplified and cloned by RT-PCR （GenBank number KX421383）, and then subcloned into pET32a vector. Then the recombinant plasmid pET32a/EoblPBP2 was transformed into E. coli BL21 （DE3） and induced to express recombinant protein with IPTG at the concentration of 1 mmol＇L1. Furthermore, the protein was separated by Ni2＋-NTA agarose FF and bacterial supernatant buffer solution. The purified recombinant protein was purified by PBS buffer solution. Finally, for the investigation of molecular binding functions of EoblPBP2 with test ligands, the fluorescence competitive assay was applied to measure the binding profile of EobIPBP2 recombinant protein with tea geometrid sex pheromone and candidate tea leaves volatiles. With the help of the fluorescence reporter N-phenyl-1-naphthylamine （1-NPN）, the binding constants of EoblPBP2 protein binding with the candidate ligands, including a sex pheromone component （Z, Z, Z）-3, 6, 9-octadecatriene and 10 plant volatiles molecules, were measured and calculated. [Result] EoblPBP2 has 492 base pairs, encodes 163 amino acid residues with 6 conservative cysteines. The relative molecular mass is 15.9 kD and the isoelectric point is 4.983. The fluorescence experiment results indicated that there were 10 volatiles could quench relative fluorescence int