茶尺蠖信息素结合蛋白PBP2的基因克

发布时间：2018-01-29 04:01

本文关键词： 茶尺蠖信息素结合蛋白原核表达性信息素与茶树挥发物质结合特征　出处：《中国农业科学》2017年03期 　论文类型：期刊论文

【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、28.068和27.597μmol·L~(-1)。而性信息素成分之一的(Z,Z,Z)-3,6,9-十八碳三烯与EoblPBP2的解离常数KD仅为172.591μmol·L~(-1),仅小于苯甲醇的解离常数KD 230.880μmol·L~(-1),而远远大于上述5种气味物质。表明EoblPBP2重组蛋白具有茶尺蠖性信息素成分((Z,Z,Z)-3,6,9-十八碳三烯)和植物挥发物质广谱结合能力,而其与性信息素的亲和力弱于大部分测试植物挥发物。【结论】EoblPBP2能与包括性信息素成分在内的多种植物挥发物结合,可能是一种具备特殊复合功能的信息素结合蛋白,可用之进一步研究茶尺蠖的性信息素识别和传递途径。
[Abstract]:[objective] Ectropis obliqua is a major Lepidoptera pest in China. The larval gluttony often brings great loss to tea garden. Sex pheromone communication is common between male and female of Lepidoptera insects. However, the way of sex pheromone recognition of tea geometrid has been rarely reported. The purpose of this paper is to identify and study the binding protein gene in the process of sex pheromone recognition of tea geometrid. To provide theoretical basis for chemical communication of sex pheromone and transduction mechanism of olfactory information in tea geometrid. [methods] A tea geometrid was first identified and discovered from the data of male antennae transcriptome by RT-PCR technique. Xanthin-binding protein gene (. Pheromone-binding protein. The full-length cDNA sequence was cloned into Gen#en1# accession number KX421383). The expression vector was constructed by ligating the gene with the pET32a vector, and then the expression vector was transformed into E. coli BL21 (DE3), which was introduced into the pET32a/EoblPBP2 expression plasmid. The expression of the protein was induced by adding 1 mmol 路L ~ (-1) IPTG into the expression vector. The target protein was separated by nickel NTA agarose gel column and the supernatant buffer containing imidazole. The high concentration recombinant protein was purified with PBS buffer as dialysis solution. Finally, the recombinant protein was purified by fluorescence competition. N-phenyl-1-naphthylamine 1-NPN (N-phenyl-1-naphthylamine) was used as a fluorescent reporter to amplify the fluorescence signal. The relative fluorescence value was obtained by molecular fluorescence photometer scanning after adding ZZG ZZZZZZZHZZZZH6O6-18 carbotriene to the mixture system of recombinant protein and 1-NPN, respectively, and 10 kinds of odors were tested by molecular fluorescence photometer. The relative fluorescence value was obtained by molecular fluorescence spectrophotometer. The dissociation constants of each ligand were calculated to study the physiological function of recombinant protein, sex pheromone of tea geometrid and tea plant volatiles. [results] the total length of EoblPBP2 was 492bp. The protein encodes 163 amino acids with a relative molecular weight of 15.9 KD and a isoelectric point of 4.983, with 6 conserved cysteines. According to molecular phylogenetic tree analysis, it belongs to the PBPs family of insects. The relative fluorescence values of 1-NPN and geometrid EoblPBP2 recombinant protein at 420nm were decreased to below 50%, among them 尾 -ionone. Dibutyl phthalate, phenylacetaldehyde, decanal and trans -2-decenal had strong binding ability with EoblPBP2 recombinant protein, and the dissociation constants were 7.923, respectively. 14.830, 30.368, 28.068 and 27.597 渭 mol 路L ~ (-1), respectively. The dissociation constant KD of 9-18 carbotriene from EoblPBP2 is only 172.591 渭 mol 路L ~ (-1). The dissociation constant KD 230.880 渭 mol 路L ~ (-1) of benzyl alcohol is only smaller than that of KD230.880 渭 mol 路L ~ (-1). The results showed that the EoblPBP2 recombinant protein had the sex pheromone component of tea geometrid, ZZG ZZN ZN3, and the above five odors were much larger than those of the above five odors. 9- 18 carbotrienes) and plant volatile substances wide spectrum binding ability. But its affinity to sex pheromone is weaker than that of most tested plant volatiles. [conclusion] EoblPBP2 can bind to many plant volatiles including sex pheromones. It may be a kind of pheromone binding protein with special complex function, which can be used to further study the sex pheromone recognition and transmission pathway of tea geometrid.
【作者单位】：中国计量大学生命科学学院/浙江省生物计量及检验检疫技术重点实验室;杭州市农业科学研究院;
【基金】：国家自然科学基金(31272053,31372254) 浙江省自然科学基金(LY13C140004) 国家茶叶产业技术体系(CARS-23)
【分类号】：S435.711
【正文快照】： 【研究意义】昆虫在漫长的自然繁衍进程中,逐渐衍变出了复杂的信息感受机制来对外界环境作出响应,使其在取食、交配、产卵等多种行为中占据有利形势,从而能够生存下来并保持其自身种群的顺利繁衍[1],昆虫通过各种信息素来传递种间或种内信息,因此信息素对昆虫的集合、防御、觅

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