中國農業科學 ?? 2014, Vol. 47 ?? Issue (16): 3184-3194.doi: 10.3864/j.issn.0578-1752.2014.16.007

? 植物保護 ? 上一篇    下一篇

橘小實蠅谷氨酸脫羧酶的生化及分子特性

 魏冬, 王濤, 豆威, 王進軍   

  1. 西南大學植物保護學院/昆蟲學及害蟲控制工程重慶市市級重點實驗室,重慶 400716
  • 收稿日期:2014-02-19 出版日期:2014-08-18 發布日期:2014-03-28
  • 通訊作者: 王進軍,Tel:023-68250255;E-mail:[email protected] E-mail:[email protected]
  • 作者簡介:魏冬,Tel:023-68250653;E-mail:[email protected]。王濤,Tel:023-68250653;E-mail:[email protected]。魏冬與王濤為同等貢獻作者
  • 基金資助:

    重慶市自然科學基金重點項目(CSTC, 2013jjB0176)、中央高校基本科研業務費(XDJK2013A017)、公益性行業(農業)科研專項(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】在測定橘小實蠅(Bactrocera dorsalis)γ-氨基丁酸(GABA)含量和谷氨酸脫羧酶(GAD)活性的基礎上,克隆獲得橘小實蠅GAD基因(BdGAD1)全長序列,進一步解析GABA、GAD以及BdGAD1在橘小實蠅各發育階段、成蟲不同體段以及經阿維菌素刺激后的表達模式,分析橘小實蠅GAD對阿維菌素作用的應激反應,為系統解析GABA介導的阿維菌素抗藥性機制提供基礎數據。【方法】采用高效液相色譜法測定橘小實蠅體內GABA含量,分析阿維菌素刺激對GABA含量的劑量和時間效應;以谷氨酸為底物,采用微量滴度酶標板法測定橘小實蠅經阿維菌素刺激后GAD活力的變化;通過同源序列比對的方法,從橘小實蠅轉錄組數據中篩選出1條GAD基因片段,利用cDNA末端快速擴增技術(RACE)獲得該基因的全長cDNA序列;應用生物信息學分析軟件對該基因的開放閱讀框、編碼的氨基酸序列、分子量等信息進行預測,并基于最大似然法構建該基因與其他昆蟲相關基因序列的系統發育樹,明確其系統進化關系。此外,分別提取橘小實蠅各發育階段和成蟲不同體段的RNA,以表達穩定的α-Tubulin為內參基因,應用qPCR技術,解析BdGAD1在橘小實蠅各發育階段(卵、幼蟲、蛹和成蟲)和成蟲不同體段(頭、胸、腹)以及經阿維菌素刺激后的表達模式。【結果】經阿維菌素刺激后,橘小實蠅體內GABA含量升高,且與阿維菌素劑量及處理時間呈正相關,暗示橘小實蠅可能通過調節GABA含量以抵御阿維菌素的毒害。同時,橘小實蠅體內GAD的比活力也隨藥劑劑量增加而升高。通過RACE擴增,獲得了橘小實蠅BdGAD1的cDNA全序列,長度1 755 bp,開放閱讀框1 197 bp,編碼398個氨基酸,GenBank登錄號為KC763804。基于最大似然法構建的系統發育樹顯示,該基因編碼的蛋白質與岡比亞按蚊的GAD親緣關系最近,序列一致性高達97%。qPCR分析結果表明,BdGAD1在幼蟲期表達量最高,不同體段間相比較發現該基因在成蟲腹部的表達量最高。經阿維菌素刺激后,BdGAD1表達水平上調。【結論】BdGAD1的表達具有發育階段和體段特異性。阿維菌素能夠刺激橘小實蠅體內BdGAD1表達水平上升,進而引起GAD活力增加促使蟲體合成產生大量的GABA,這可能是橘小實蠅抵御阿維菌素毒害甚至產生抗藥性的原因。

關鍵詞: 橘小實蠅 , 谷氨酸脫羧酶 , &gamma, -氨基丁酸 , 阿維菌素 , 抗藥性

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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