橘小实蝇羧酸酯酶基因BdCAREB1的克隆及其表达模式解析  

Cloning and Expression Analysis of a Carboxylesterase Gene BdCAREB1 from Bactrocera dorsalis

作　　者：申光茂[1] 豆威[1] 王进军[1] 

机构地区：[1]西南大学植物保护学院昆虫学及害虫控制工程重庆市市级重点实验室,重庆400716

出　　处：《中国农业科学》2014年第10期1947-1955,共9页Scientia Agricultura Sinica

基　　金：重庆市自然科学基金重点项目(CSTC;2013jjB0176);中央高校基本科研业务费专项资金(XDJK2013A017)

摘　　要：【目的】在克隆获得橘小实蝇(Bactrocera dorsalis)1条羧酸酯酶基因全长序列的基础上,解析该基因在橘小实蝇不同发育阶段和组织以及经高效氯氰菊酯饲喂后的表达模式。同时结合之前完成的有关橘小实蝇羧酸酯酶生化特性以及增效剂处理研究结果,综合分析橘小实蝇羧酸酯酶对高效氯氰菊酯作用的应激反应,为明确羧酸酯酶对菊酯类杀虫剂的解毒代谢机制奠定基础。【方法】通过同源序列比对的方法,从橘小实蝇转录组数据中筛选出1条羧酸酯酶基因序列片段,利用cDNA末端快速扩增技术(RACE)扩增得到该基因的全长cDNA序列;应用生物信息学分析软件对该基因的开放阅读框、编码的氨基酸序列、分子量等信息进行预测,并基于最大似然法构建该基因与其他昆虫相关基因序列的系统发育树,阐明其分子生物学特征。此外,分别提取橘小实蝇不同发育阶段以及3龄幼虫的中肠、脂肪体、马氏管和气管等组织的RNA,在内参基因稳定性评估筛选的基础上,运用qPCR的方法解析该基因在不同发育阶段和组织间以及药剂处理后的表达模式。【结果】从橘小实蝇转录组数据库中筛选出1条长度为1 028 bp的羧酸酯酶基因片段,通过对5′端和3′端进行RACE扩增分别得到长度为1 073和625 bp的目的片段,经序列拼接和验证后,确定该基因全长为1 872 bp,完整开放阅读框1 710 bp,编码569个氨基酸,推测其蛋白质分子量为63.3 kD,理论等电点6.38。该基因经序列比对命名为BdCAREB1,GenBank登录号为KF539980。基于最大似然法构建的系统发育树显示,该基因编码的蛋白质与双翅目昆虫的羧酸酯酶具有高度的同源性。分析其在不同发育阶段的表达模式发现,BdCAREB1在3龄幼虫期的表达量最高,其次为成虫期,而在卵期的表达量最低。在橘小实蝇3龄幼虫不同组织间的定量分析结果表明,BdCAREB1在脂肪体中表达水平最高,[Objective]Based on the cloning of a carboxylesterase gene of Bactrocera dorsalis, the expressions of this gene among different developmental stages, tissues, and under insecticide stimulation were analyzed. Combined with previous data of enzyme activity assay and synergist analysis, the objectives of this study are to clarify the reaction of carboxylesterase when stimulated byβ-cypermethrin, and lay a foundation for the future study of the mechanism that how carboxylesterase works in thedetoxification of pyrethroid insecticides.[Method]From the transcriptome data of B. dorsalis, a partial sequence of carboxylesterase gene was screened out by using homologous blast, and the full sequence was cloned by using RACE technology. The ORF, deduced amino acid sequence, and molecular weight were predicted, and a phylogenetic tree with carboxylesterase genes from other insects was constructed by using maximum likelihood method to clarify its molecular characterization. RNA was extracted from different developmental stages and the midgut, Malpighian tubules, fat body, trachea of the 3rd instar larvae. Based on the reference evaluation, qPCR was used for the expression analysis of different developmental stages, tissues, and stimulated by insecticide.[Result]A partial sequence of 1 028 bp was found in the transcriptome data. The results of 5′and 3′amplification were 1 073 and 625 bp, respectively. After sequence assemble and verification, the full-length of this gene was 1 872 bp, contained a complete ORF of 1 710 bp, and encoding a protein of 569 amino acids with a predicted molecular mass of 63.3 kD. It was named as BdCAREB1 and submitted to GenBank with an accession number of KF539980. The phylogenetic tree of BdCAREB1 showed high homology with carboxylesterase of Diptera. The qPCR result of different developmental stages of the 3rd instar larvae showed BdCAREB1 was highly expressed in the 3rd instar larvae, followed by the adults, and it was the lowest in the eggs. Among different tissues, the BdCAREB1 was highly ex

关 键 词：橘小实蝇 羧酸酯酶 基因表达 脂肪体 解毒代谢 

分 类 号：S433[农业科学—农业昆虫与害虫防治]