Glutathione S-transferase gene expression in transformant Drosophila expressing ocular albinism type 1 from gypsy moth and its response to deltamethrin stress
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摘要: 舞毒蛾是重要林业食叶害虫,揭示其G蛋白偶联受体介导G蛋白对GSTs的调控,这对于挖掘分子靶标开发新型杀虫剂具有重要意义。本文构建转LdOA1基因果蝇载体,获得表达LdOA1基因果蝇品系,分析了转基因果蝇GSTs基因表达量及对溴氰菊酯胁迫的响应,为明确昆虫OA1基因功能提供理论依据。通过传统的酶切-连接方法构建重组载体pUAST-attB-LdOA1,采用转基因技术构建表达LdOA1基因的纯合果蝇品系,利用PCR技术验证转基因果蝇品系,并运用实时荧光定量RT-PCR技术测定转基因果蝇GSTs Delta家族基因表达量及低浓度溴氰菊酯胁迫对GSTs基因表达量的影响。PCR扩增条带显示转LdOA1基因果蝇品系均能检测到453bp目的基因条带。与非转基因果蝇相比,转LdOA1基因果蝇品系中GSTs Delta家族基因(除GSTd4和GSTd7)均上调表达,为非转基因组1.01~3.27倍。低浓度溴氰菊酯胁迫6h,非转基因果蝇GSTd6和GSTd10基因被显著诱导激活,而其他GSTs基因被显著抑制,抑制率为6.48%~95.84%,胁迫12~72h,GSTs基因(除GSTd1和GSTd10外)均被显著抑制。低浓度溴氰菊酯胁迫转基因果蝇GSTs表达量变化趋势与非转基因果蝇基本一致,但转基因果蝇品系GSTs表达量显著高于非转基因果蝇品系(除12~48h GSTd1和GSTd10),且胁迫6h表达量最大。这些结果表明LdOA1基因可能介导G蛋白信号通路调控下游GSTs Delta亚家族基因表达响应溴氰菊酯胁迫。Abstract: The Asian gypsy moth (Lymantria dispar) is a key forest defoliator throughout the world. Study on regulation of G protein for GST genes mediated by G protein coupled receptor contributes to exploring molecular targets for developing novel pesticides to control gypsy moth. In the present study, transformant Drosophila vector was constructed and transformant Drosophila lines expressing LdOA1 were successfully obtained, GSTs gene expression levels in transformant Drosophila and its response to deltamethrin stress were analyzed. These results provided basic information for elucidating insect OA1 gene functions. The recombinant vector pUAST-attB-LdOA1 was constructed by traditional enzyme digestion and ligation technology. The transformant Drosophila lines were obtained by transgenic technology and validated using PCR technology. The expression levels of GSTs Delta family genes and the effects of deltamethrin on GSTs gene expressions in transformant Drosophila were analyzed by real-time RT-PCR technology. The RT-PCR results showed that the 453bp band of LdOA1 gene was detected in transformant Drosophila lines expressing LdOA1 gene. Compared with untransformant Drosophila, the expression levels of GSTs Delta family genes, except for GSTd4 and GSTd7 genes, in transformant Drosophila lines were up-regulated with 1.01- to 3.27-fold of untransformant Drosophila using qRT-PCR analysis. Under low concentration of deltamethrin stress for 6h, the GSTd6 and GSTd10 were obviously induced while other GSTs genes were significantly inhibited in untransformant Drosophila, and the inhibition ratios were 6.48%-95.84%. Under deltamethrin stress for 12-72h, the expressions of GST genes, except for GSTd1 and GSTd10, were significantly inhibited in untransformant Drosophila. Compared with untransformant Drosophila, the expressions of GSTs genes in transformant Drosophila varied in similarity under low concentration of deltamethrin stress. However, the GST gene expressions in transformant Drosophila were significantly higher than those in untransformant Drosophila except for GSTd1 and GSTd10 for 12-48h, and the GST gene expressions in transformant Drosophila were highest under low concentration of deltamethrin stress for 6h. These results indicated LdOA1 may mediate G protein signal transduction to regulate GSTs Delta subfamily genes in response to deltamethrin stress.
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Keywords:
- transformant Drosophila /
- gypsy moth /
- LdOA1 /
- GSTs
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[1] ZHOU Q, YU G C, JIANG A A. Research progress in genetic basis of ocular albinism type Ⅰ [J]. Medical recapitulate, 2013,19(24): 4433-4435.
[1] SIMONET G, POELS J, CLAEYS I, et al. Neuroendocrinological and molecular aspects of insect reproduction [J]. Journal of Neuroendocrinology, 2004, 16(8): 649-659.
[2] CAO C W, SUN L L, WEN R R, et al. Cloning and analysis of LdOA 1 in Lymantria dispar and its response to the stress of three kinds of insecticides [J]. Scientia Silvae Sinicae, 2014, 50(8): 102-107.
[2] HAUSER F, CAZZAMALI G, WILLIAMSON M, et al. A review of neurohormone GPCRs present in the fruit fly Drosophila melanogaster and the honey bee Apis mellifera [J]. Progress in Neurobiology, 2006, 80(1): 1-19.
[3] WU W Q, LI H Y, ZHENG H. Molecular pathophysiological basis of the ocular albinism type 1 [J]. Chinese Journal of Pathophysiology, 2004, 20(2): 278-282.
[4] SPIT J, BADISCO L, VERLINDEN H, et al. Peptidergic control of food intake and digestion in insects [J]. Canadian Journal of Zoology, 2012, 90(4): 489-506.
[5] KOSTAROPOULOS I, PAPADOPOULOS A I, METAXAKIS A, et al. Glutathione S-transferase in the defence against pyrethroids in insects [J]. Insect Biochemistry Molecular Biology, 2001, 31(4-5):313-319.
[6] LOW W Y, FEIL S C, NG H L, et al. Recognition and detoxification of the insecticide DDT by Drosophila melanogaster glutathione S-transferase Dl [J]. Journal of Molecular Biology, 2010, 399(3): 358-366.
[7] VONTAS J G, SMALL G J, HEMINGWAY J. Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilapartbta lugens [J]. Biochemical Journal, 2001, 357: 65-72.
[8] BERRUECO R, RIVES S, CAM��S M, et al. Syndromic albinism and haemophagocytosis [J]. British Journal of Haematology, 2010, 148(6) [2015-10-09]. doi: 10.1111/j.1365-2141.2009.07885.x.
[9] MARTINEZ-GARCIA M, RIVEIRO-ALVAREZ R, VILLAVERDE-MONTERO C, et al. Identification of a novel deletion in the OA1 gene: report of the first Spanish family with X-linked ocular albinism [J]. Clinical and Experimental Ophthalmology, 2010, 38(5):489-495.
[10] FALLETTA P, BAGNATO P, BONO M, et al. Melanosome-autonomous regulation of size and number: the OA1 receptor sustains PMEL expression [J]. Pigment Cell & Melanoma Research, 2014, 27(4): 565-579.
[11] SCHIAFFINO M V. Signaling pathways in melanosome biogenesis and pathology [J]. International Journal of Biochemistry & Cell Biology, 2010, 42(7): 1094-1104.
[12] WITTKOPP P J, WILLIAMS B L, SELEGUE J E, et al. Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes [J]. Proceedings of the National Academy of Sciences of the USA,2003, 100(4): 1808-1813.
[13] 周琦, 余国春, 蒋岸岸. 眼白化病1型的遗传基础研究进展 [J]. 医学综述, 2013, 19(24): 4433-4435. [14] 曹传旺, 孙丽丽, 问荣荣, 等. 舞毒蛾 LdOA 1基因克隆分析及对3种杀虫剂胁迫的响应 [J].林业科学, 2014, 50(8): 102-107. [15] SUN L L, WANG Z Y , WU H Q, et al. Role of ocular albinism type 1 (OA1) GPCR in Asian gypsy moth development and transcriptional expression of heat-shock protein genes [J]. Pesticide Biochemistry and Physiology, 2016, 126:35-41.
[16] ZHU F, PARTHASARATHY R, BAI H, et al. A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(19): 8557-8562.
[17] WILLIS D K, WANG J, LINDHOLM J R, et al. Microarray analysis of juvenile hormone response in Drosophila melanogaster S2 cells [J]. Journal of Insect Science, 2010,10 [2015-10-02]. doi: 10.1673/031.010.6601.
[18] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 - ΔΔCT method [J]. Methods, 2001, 25(4): 402-408.
[19] HILL C A, FOX A N, PITTS R J, et al. G protein-coupled receptors in Anopheles gambiae [J]. Science, 2002, 298:176-178.
[20] LI T, CAO, C W, YANG T, et al. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus [J]. Scientific Reports, 2015, 5 [2015-10-01]. doi: 10.1038/srep17772.
[21] MEYER J M, EJENDAL K F K, AVRAMOVA L V, et al. A “genome-to-lead” approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1)-like dopamine receptors [J]. PLoS Neglected Tropical Diseases, 2012, 6(1) [2015-10-05]. doi: 10.1371/journal.pntd.0001478.
[22] HILL C A, MEYER J M, EJENDAL K F K, et al. Re-invigorating the insecticide discovery pipeline for vector control: GPCRs as targets for the identification of next gen insecticides [J]. Pesticide Biochemistry and Physiology, 2013, 106(3): 141-148.
[23] SHEN B, SAMARAWEERA P, ROSENBERG B, et al. Ocular albinism type 1: more than meets the eye [J]. Pigment Cell Research, 2001, 14(4): 243-248.
[24] LOPEZ V M, DECATUR C L, STAMER W D, et al. I-DOPA is an endogenous ligand for OA1 [J]. PLoS Biology, 2008, 6(9) [2015-10-10]. doi: 10.1371/journal.pbio.0060236.
[25] GHOSH A, SONAVANE U, ANDHIRKA S K, et al. Structural insights into human GPCR protein OA1: a computational perspective [J]. Journal of Molecular Modeling, 2012, 18(5): 2117-2133.
[26] 吴维青, 李洪义, 郑辉. 眼白化病1型的分子病理生理基础 [J].中国病理生理杂志, 2004, 20(2):278-282. [27] ENAYATI A A, RANSON H, HEMINGWAY J. Insect glutathione transferases and insecticide resistance [J]. Insect Molecular Biology, 2005, 14(1): 3-8.
[28] OAKLEY A J, JIRAJAROERU'AT K , HAMNOI T, et al. Crystallization of two glutathione S-transferases from an unusual gene family [J]. Acta Crystallographica Section D: Biological Crystallography, 2001, 57(6): 870-872.
[29] CHEN L, HALL P R, ZHOU X E, et al. Structure of an insect δ-class glutathione S-transferase from a DDT-resistant strain of the malaria vector Anopheles gambiae [J]. Acta Crystallographica Section D Biological Crystallography, 2003, 59(12): 2211-2217.
[30] UDOMSINPRASERT R, PONGJAROENKIT S, WONGSANTICHON J, et al. Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme [J]. Biochemistry Journal, 2005, 388(3): 763-771.
[31] WANG Y J, QIU L, RANSON H, et al. Structure of an insect epsilon class glutathione S-transferase from the malaria vector Anopheles gambiae provides an explanation for the high DDT-detoxifying activity [J]. Journal of Structural Biology, 2008, 164(2): 228-235.
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