Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (16): 3184-3194.doi: 10.3864/j.issn.0578-1752.2014.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

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

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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

[1]潘志萍, 曾玲, 陆永跃. 华南地区桔小实蝇对几种农药的抗药性研究. 华南农业大学学报, 2005, 26(4): 23-26.

Pan Z P, Zeng L, Lu Y Y. Monitoring of resistance of oriental fruit fly adults to insecticides in South China. Journal of South China Agricultural University, 2005, 26(4): 23-26. (in Chinese)

[2]Follett P A, Neven L G. Current trends in quarantine Entomology. Annual Review of Entomology, 2006, 51: 359-385.

[3]Clarke A R, Armstrong K F, Carmichael A E, Milne J R, Raghu S, Roderick G K, Yeates D K. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: the Bactrocera dorsalis complex of fruit flies. Annual Review of Entomology, 2005, 50: 293-319.

[4]Hsu J C, Feng H T, Wu W J. Resistance and synergistic effects of insecticides in Bactrocera dorsalis (Diptera: Tephritidae) in Taiwan. Journal of Economic Entomology, 2004, 97(5): 1682-1688.

[5]章玉苹, 曾玲, 陆永跃, 梁广文. 桔小实蝇对敌百虫抗性稳定性及再增长趋势. 昆虫学报, 2008, 51(10): 1044-1049.

Zhang Y P, Zeng L, Lu Y Y, Liang G W. Resistance stability and re-growth in adults of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) to trichlorphon. Acta Entomologica Sinica, 2008, 51(10): 1044-1049. (in Chinese)

[6]章玉苹, 曾玲, 陆永跃, 梁广文. 华南地区桔小实蝇抗药性动态监测. 华南农业大学学报, 2007, 28(3): 20-23.

Zhang Y P, Zeng L, Lu Y Y, Liang G W. Monitoring of insecticide resistance of Bactrocera dorsalis adults in South China. Journal of South China Agricultural University, 2007, 28(3): 20-23. (in Chinese)

[7]潘志萍, 陆永跃, 曾玲, 曾鑫年. 桔小实蝇实验种群对敌百虫、高效氯氰菊酯和阿维菌素的抗性增长规律. 昆虫学报, 2008, 51(6): 609-617.

Pan Z P, Lu Y Y, Zeng L, Zeng X N. Development of resistance to trichlorophon, alphamethrin, and abamectin in laboratory populations of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). Acta Entomologica Sinica, 2008, 51(6): 609-617. (in Chinese)

[8]Jin T, Zeng L, Lin Y Y, Lu Y Y, Liang G W. Insecticide resistance of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in mainland China. Pest Management Science, 2011, 67(3): 370-376.

[9]Narayan V S, Nair P M. Metabolism, enzymology and possible roles of 4-aminobutyrate in higher plants. Phytochemistry, 1990, 29(2): 367-375.

[10]Ffrench-Constant R H, Steichen J C, Rocheleau T A, Aronstein K, Roush R T. A single-amino acid substitution in a γ-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations. Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(5): 1957-1961.

[11]李婷, 龙虎, 温海林, 隗汶校, 李坤明, 刘忠俊, 王晔. 农药阿维菌素毒理学机制及中毒治疗. 医学综述, 2010, 16(10): 1554-1556.

Li T, Long H, Wen H L, Wei W X, Li K M, Liu Z J, Wang Y. Toxicological mechanisms and treatment of avermectins intoxication. Medical Recapitulate, 2010, 16(10): 1554-1556. (in Chinese)

[12]Cully D F, Wilkinson H, Vassilatis D K, Etter A, Arena J P. Molecular biology and electrophysiology of glutamategated chloride channels of invertebrates. Parasitology, 1996. 113(S1): S191-S200.

[13]Rufingier C, Pasteur N, Lagnel J, Martin C, Navajas M. Mechanisms of insecticide resistance in the aphid Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae) from France. Insect Biochemistry and Molecular Biology, 1999, 29(4): 385-391.

[14]Gao J R, Kozaki T, Leichter C A, Rinkevich F D, Shono T, Scott J G. The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA. Pesticide Biochemistry and Physiology, 2007, 88(1): 66-70.

[15]Nakao T, Naoi A, Kawahara N, Hirase K. Mutation of the GABA receptor associated with fipronil resistance in the whitebacked planthopper, Sogatella furcifera. Pesticide Biochemistry and Physiology, 2010, 97(3): 262-266.

[16]Wolff M A, Wingate V P. Characterization and comparative pharmacological studies of a functional γ-aminobutyric acid (GABA) receptor cloned from the tobacco budworm, Heliothis virescens (Noctuidae: Lepidoptera). Invertebrate Neuroscience, 1998, 3(4): 305-315.

[17]Wondji C S, Dabire R K, Tukur Z, Irving H, Djouaka R, Morgan J C. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa. Insect Biochemistry and Molecular Biology, 2011, 41(7): 484-491.

[18]Zhu X J, Lu W C, Feng Y N, He L. High γ-aminobutyric acid content, a novel component associated with resistance to abamectin in Tetranychus cinnabarinus (Boisduval). Journal of Insect Physiology, 2010, 56(12): 1895-1900.

[19]Erlander M G, Tillakaratne N J, Feldblum S, Patel N, Tobin A J. Two genes encode distinct glutamate decarboxylases. Neuron, 1991, 7(1): 91-100.

[20]Donner J, Sipilä T, Ripatti S, Kananen L, Chen X, Kendler K S, Lönnqvist J, Pirkola S, Hettema J M, Hovatta I. Support for involvement of glutamate decarboxylase 1 and neuropeptide Y in anxiety susceptibility. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 2012, 159(3): 316-327.

[21]Occhialini A, de Bagüés M P J, Saadeh B, Bastianelli D, Hanna N, De Biase D, Köhler S. The glutamic acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and to oral infection of mice. The Journal of Infectious Diseases, 2012, 206(9): 1424-1432.

[22]Kim Y B, Lee M K, Kim S J, Kim H H, Chung E, Lee J H, Park S U. Accumulation of γ-aminobutyric acid and transcription of glutamate decarboxylase in Brassica juncea (L.) Czern. Plant Omics Journal, 2013, 6(4): 263-267.

[23]Ilg T, Berger M, Noack S, Rohwer A, Gaßel M. Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse. Insect Biochemistry and Molecular Biology, 2013, 43: 162-177.

[24]Jackson F R, Newby L M, Kulkarni S J. Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxylase. Journal of Neurochemistry, 1990, 54(3): 1068-1078.

[25]Phillips A M, Salkoff L B, Kelly L E. A neural gene from Drosophila melanogaster with homology to vertebrate and invertebrate glutamate decarboxylases. Journal of Neurochemistry, 1993, 61(4): 1291-1301.

[26]Wang J J, Wei D, Dou W, Hu F, Liu W F, Wang J J. Toxicities and synergistic effects of several insecticides against the oriental fruit fly (Diptera: Tephritidae). Journal of Economic Entomology, 2013, 106(2): 970-978.

[27]Shen G M, Huang Y, Jiang X Z, Dou W, Wang J J. Effect of β-cypermethrin exposure on the stability of nine housekeeping genes in Bactrocera dorsalis (Diptera: Tephritidae). Florida Entomologist, 2013, 96(2): 442-450.

[28]房克敏, 李再贵, 袁汉成, 孟颖. HPLC法测定发芽糙米中γ-氨基丁酸含量. 食品科学, 2006, 27(4): 208-211.

Fang K M, Li Z G, Yuan H C, Meng Y. Determination of γ-aminobutyric acid in germinated brown rice by HPLC. Food Science, 2006, 27(4): 208-211. (in Chinese)

[29]穆小民, 沈黎明, 吴显荣. 林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究. 生物化学杂志, 1997, 13(2): 181-185.

Mu X M, Shen L M, Wu X R. Studies on the purification and properties of glutamic acid decarboxylase from flat pea. Chinese Biochemical Journal, 1997, 13(2): 181-185. (in Chinese)

[30]Satyanarayan V, Nair P M. Purification and characterization of glutamate decarboxylase from Solanum tuberosum. European Journal of Biochemistry, 1985, 150(1): 53-60.

[31]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72(1/2): 248-254.

[32]Shen G M, Dou W, Niu J Z, Jiang H B, Yang W J, Jia F X, Hu F, Cong L, Wang J J. Transcriptome analysis of the oriental fruit fly (Bactrocera dorsalis). PLoS ONE, 2011, 6(12): e29127.

[33]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[34]Shen G M, Jiang H B, Wang X N, Wang J J. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Molecular Biology, 2010, 11(1): 76.

[35]Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 2001, 29(9): 2002-2007.

[36]Scott J, Georghiou G. The biochemical genetics of permethrin resistance in the Learn-PyR strain of house fly. Biochemical Genetics, 1986, 24(1/2): 25-37.

[37]梁沛, 高希武, 郑炳宗, 戴洪波. 小菜蛾对阿维菌素的抗性机制及交互抗性研究. 农药学学报, 2001, 3(1): 41-45.

Liang P, Gao X W, Zheng B Z, Dai H B. Study on resistance machanisms and cross-resistance of abamectin in diamondback moth Plutella oxylostella (L.). Chinese Journal of Pesticide Science, 2001, 3(1): 41-45. (in Chinese)

[38]Goodwin S, Herron G, Gough N, Wellham T, Rophail J, Parker R. Relationship between insecticide-acaricide resistance and field control in Tetranychus urticae (Acari: Tetranychidae) infesting roses. Journal of Economic Entomology, 1995, 88(5): 1106-1112.

[39]Wolf R, Klemisch H. Adaptation of an enzymatic fluorescence assay for L-glutamic acid decarboxylase. Analytical Biochemistry, 1991, 192(1): 78-81.

[40]Kang T J, Ho N A T, Pack S P. Buffer-free production of gamma- aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli. Enzyme and Microbial Technology, 2013, 53(3): 200-205.

[41]Malter M P, Helmstaedter C, Urbach H, Vincent A, Bien C G. Antibodies to glutamic acid decarboxylase define a form of limbic encephalitis. Annals of Neurology, 2010, 67(4): 470-478.

[42]Pehrson A L, Bondi C O, Totah N K, Moghaddam B. The influence of NMDA and GABAA receptors and glutamic acid decarboxylase (GAD) activity on attention. Psychopharmacology, 2013, 225(1): 31-39.

[43]Luo L, Sun Y J, Wu Y J. Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways. Insect Biochemistry and Molecular Biology, 2013, 43: 627-634.
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