花椒窄吉丁化学感受蛋白AzanCSP3的cDNA克隆及原核表达  

cDNA Cloning and Prokaryotic Expression of Chemosensory Protein AzanCSP3 from the Agrilus zanthoxylumi

作　　者：杨平 巩雪芳 陈迪 郭莉 王延来 吕淑杰 谢寿安 YANG Ping;GONG Xue-Fang;CHEN Di;GUO Li;WANG Yan-Lai;LV Shu-Jie;XIE Shou-An(Forestry College,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学林学院

出　　处：《农业生物技术学报》2020年第2期302-312,共11页Journal of Agricultural Biotechnology

基　　金：国家公益性林业行业项目(201504324);2019年云南省林业科技推广示范项目(云[2019]TG01号)

摘　　要：花椒窄吉丁(Agrilus zanthoxylumi)是花椒(Zanthoxylum bungeanum)树上的重要蛀干害虫,深入了解其嗅觉感受机制,有助于研究化学感受蛋白(chemosensory proteins, CSPs)在其化学感受系统中的作用。基于本课题组转录组测序结果 ,本研究克隆获得花椒窄吉丁化学感受蛋白基因(A. zanthoxylumi chemosensory protein, Azan CSP3)的c DNA,利用在线工具(Expasy;Signal P4.1;Swiss model)对其进行理化特性分析;利用BLAST进行同源性对比,并用MEGA 10.0软件Neighbor-joining法构建进化树,进而分析其与鞘翅目其他11种昆虫化学感受蛋白的亲缘关系;将原核表达载体p ET28a(+)与克隆鉴定正确的重组质粒相连接,然后转入至大肠杆菌(Escherichia coli) BL21感受态细胞中,经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-thiogalactoside, IPTG)诱导后表达,用SDS-PAGE电泳分析目的融合蛋白的表达情况后,用Western blot检测所表达的蛋白是否为目标融合蛋白。结果表明,克隆获得花椒窄吉丁1条化学感受蛋白的c DNA序列,与转录组数据结果一致,其全长为846 bp,含有396 bp的完整开放阅读框,编码132个氨基酸,成熟蛋白分子质量为15.038 k D,等电点9.16,预测N末端含有28个氨基酸组成的信号肽;Azan CSP3的三维结构具有6个保守的α-螺旋,并形成1个疏水性的结合腔,具有化学感受蛋白家族的典型特征。氨基酸序列比对表示Azan CSP3与其他鞘翅目昆虫的CSP序列同源性较高,其中与苹果小吉丁(A. mali)Amal CSP4氨基酸序列同源性最高,为87.79%,系统发育结果表明,花椒窄吉丁Azan CSP3与苹果小吉丁Amal CSP4、白蜡窄吉丁(A. planipennis)的Apla CSP3以100%置信度聚为一支。Western blot检测结果表明,花椒窄吉丁Azan CSP3在大肠杆菌中成功表达。本研究明确了花椒窄吉丁化学感受蛋白Azan CSP3的结构特征,为深入研究花椒窄吉丁化学感受蛋白的功能特性、揭示花椒窄吉丁嗅觉识别机制提供了理论依据。Agrilus zanthoxylumi is an important trunk-boring pest on the Zanthoxylum bungeanum, which lays a foundation for understanding the mechanism of olfactory perception and studying the role of chemosensory proteins(CSPs) in its chemical sensory system. Based on the transcriptome sequencing results of the team,cloning of the c DNA of Azan CSP3;and the signal peptide, isoelectric point, molecular mass, and threedimensional structure of the signal peptide were predicted by using online tools(Expasy;Signal P4.1;Swiss model);the homology was compared by using BLAST, the phylogenetic tree was constructed using a neighbor-joining method with MEGA 10.0 software, and the affinity between Azan CSP3 and CSPs from other11 Coleoptera species was analyzed. The correct recombinant plasmid of prokaryotic expression vector p ET28 a(+) was ligated and cloned into Escherichia coli BL21 competent cells and expressed by isopropyl β-Dthiogalactoside(IPTG), the expression of the target protein was analyzed by SDS-PAGE, and using Western blot to detect whether the expressed protein was the target fusion protein. The results showed that the c DNA sequence of a chemosensory protein of A. zanthoxylumi was cloned and named as Azan CSP3. Consisted with the results of the transcriptome data, the full-length c DNA was 846 bp, contained a complete open reading frame of 396 bp, encoding 132 amino acids, the molecular weight of the mature protein was 15.038 k D, the isoelectric point was 9.16, and the signal peptide with 28 amino acids at the N-terminus was predicted. The three-dimensional structure of Azan CSP3 had 6 conserved α-helices and formed a hydrophobic binding cavity with typical characteristics of the chemosensory protein family. The amino acid sequence alignment indicated that Azan CSP3 had higher homology with other Coleoptera CSP sequences, and had the highest amino acid sequence homology with Amal CSP4, which was 87.79%, phylogenetic results indicated that Azan CSP3,Amal CSP4 and Apla CSP3 were clustered with 100% confidence. Wester

关 键 词：花椒窄吉丁 化学感受蛋白(CSPs) 分子克隆 原核表达 Western blot 

分 类 号：S763.38[农业科学—森林保护学]