中国农业科学 ›› 2014, Vol. 47 ›› Issue (16): 3184-3194.doi: 10.3864/j.issn.0578-1752.2014.16.007

• 植物保护 • 上一篇    下一篇

橘小实蝇谷氨酸脱羧酶的生化及分子特性

 魏冬, 王涛, 豆威, 王进军   

  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
  • 收稿日期:2014-02-19 出版日期:2014-08-18 发布日期:2014-03-28
  • 通讯作者: 王进军,Tel:023-68250255;E-mail:jjwang7008@yahoo.com E-mail:jjwang7008@yahoo.com
  • 作者简介:魏冬,Tel:023-68250653;E-mail:dong_wei1988@yahoo.com。王涛,Tel:023-68250653;E-mail:wangtao244152302@163.com。魏冬与王涛为同等贡献作者
  • 基金资助:

    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(XDJK2013A017)、公益性行业(农业)科研专项(201203038)

Biochemical and Molecular Characteristics of Glutamic Decarboxylase from Bactrocera dorsalis

 WEI  Dong, WANG  Tao, DOU  Wei, WANG  Jin-Jun   

  1. College of Plant Protection, Southwest University/Key Laboratory of Entomology and Pest Control Engineering, Chongqing 400716
  • Received:2014-02-19 Online:2014-08-18 Published:2014-03-28

摘要: 【目的】在测定橘小实蝇(Bactrocera dorsalis)γ-氨基丁酸(GABA)含量和谷氨酸脱羧酶(GAD)活性的基础上,克隆获得橘小实蝇GAD基因(BdGAD1)全长序列,进一步解析GABA、GAD以及BdGAD1在橘小实蝇各发育阶段、成虫不同体段以及经阿维菌素刺激后的表达模式,分析橘小实蝇GAD对阿维菌素作用的应激反应,为系统解析GABA介导的阿维菌素抗药性机制提供基础数据。【方法】采用高效液相色谱法测定橘小实蝇体内GABA含量,分析阿维菌素刺激对GABA含量的剂量和时间效应;以谷氨酸为底物,采用微量滴度酶标板法测定橘小实蝇经阿维菌素刺激后GAD活力的变化;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GAD基因片段,利用cDNA末端快速扩增技术(RACE)获得该基因的全长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,明确其系统进化关系。此外,分别提取橘小实蝇各发育阶段和成虫不同体段的RNA,以表达稳定的α-Tubulin为内参基因,应用qPCR技术,解析BdGAD1在橘小实蝇各发育阶段(卵、幼虫、蛹和成虫)和成虫不同体段(头、胸、腹)以及经阿维菌素刺激后的表达模式。【结果】经阿维菌素刺激后,橘小实蝇体内GABA含量升高,且与阿维菌素剂量及处理时间呈正相关,暗示橘小实蝇可能通过调节GABA含量以抵御阿维菌素的毒害。同时,橘小实蝇体内GAD的比活力也随药剂剂量增加而升高。通过RACE扩增,获得了橘小实蝇BdGAD1的cDNA全序列,长度1 755 bp,开放阅读框1 197 bp,编码398个氨基酸,GenBank登录号为KC763804。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冈比亚按蚊的GAD亲缘关系最近,序列一致性高达97%。qPCR分析结果表明,BdGAD1在幼虫期表达量最高,不同体段间相比较发现该基因在成虫腹部的表达量最高。经阿维菌素刺激后,BdGAD1表达水平上调。【结论】BdGAD1的表达具有发育阶段和体段特异性。阿维菌素能够刺激橘小实蝇体内BdGAD1表达水平上升,进而引起GAD活力增加促使虫体合成产生大量的GABA,这可能是橘小实蝇抵御阿维菌素毒害甚至产生抗药性的原因。

关键词: 橘小实蝇 , 谷氨酸脱羧酶 , &gamma, -氨基丁酸 , 阿维菌素 , 抗药性

Abstract: 【Objective】 The study aimed to determine the γ-aminobutyric acid (GABA) content and glutamic acid decarboxylase (GAD) specific activity of Bactrocera dorsalis, and clone the complete sequence of a GAD gene (BdGAD1). The changes of GABA content, GAD specific activity, and expression of BdGAD1 in different developmental stages and body tagmata of adults after avermectin stimuli provided basic data of the resistance mechanism of avermectin mediated by GABA. 【Method】 The content of GABA in B. dorsalis was determined through the method of high performance liquid chromatography, and the dose and time effects of avermectin stimuli on GABA content were determined. The change of specific activity of GAD in B. dorsalis was determined via the microplate method with the substrate of glutamate. According to the screened GAD gene sequence fragment from the transcriptome data of B. dorsalis, the complete sequence of cDNA was amplified using rapid amplification of cDNA ends (RACE). The open reading frame (ORF), deduced amino acid sequence, and molecular weight were predicted, and a phylogenetic tree with GAD genes from other insects was constructed using maximum likelihood method to clarify its phylogenetic relationship. Besides, the RNA was extracted from different developmental stages (egg, larva, pupa, and adult) and different tagmata (head, thorax, and abdomen) of adult. Based on the reference evaluation, α-Tubulin was used as housekeeping gene for qPCR to analyze the expression profiles of different developmental stages, tagmata, and stimulated by avermectin. 【Result】 The GABA contents of B. dorsalis increased under the stimuli of avermectin, and there was a positive correlation between GABA content and the avermectin dose and treatment duration, suggesting that B. dorsalis may mediate the content of GABA to avoid the damage of avermectin. Moreover, the specific activity of GAD in B. dorsalis also increased with the increase of treatment doses. A complete sequence of BdGAD1 was cloned by RACE amplification with a full length of 1 755 bp, and ORF of 1 197 bp encoding 398 amino acids. The GenBank accession number was KC763804. This gene exhibited a close relationship with the gene from Anopheles gambiae based on maximum likelihood phylogenetic tree. The amino acid identity was up to 97%. The expression level of BdGAD1 was the highest in larva among different developmental stages, and was the highest in abdomen among different tagmata. Under the stimuli of avermectin, the expression of BdGAD1 was also unregulated.【Conclusion】 Avermectin could increase GABA content by increasing the expression level of BdGAD1 and specific activity of GAD resulting in more GABA synthesis. This might be one reason for the resistance of B. dorsalis against avermectin.

Key words: Bactrocera dorsalis , glutamate acid decarboxylase , γ-aminobutyric acid , avermectin , resistance

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