烟蚜茧蜂脂代谢相关的滞育相关蛋白差异表达分析

doi:10.16409/j.cnki.2095-039x.2015.06.001

摘要/Abstract

摘要： 利用滞育特性可显著延长烟蚜茧蜂扩繁产品的货架期,探究烟蚜茧蜂滞育相关蛋白的表达特点,可促进对天敌昆虫滞育机理的了解。本文利用以iTRAQ为核心的蛋白定量技术,对提取的烟蚜茧蜂全蛋白进行酶解、标记、质谱检测、查库鉴定及定量分析,并结合生物信息学对烟蚜茧蜂滞育前期和滞育期的差异蛋白进行分析。定量鉴定蛋白1268个,其中滞育组差异表达蛋白601个,上调表达蛋白278个,鉴定肽段物种主要集中在膜翅目,占56.5%;下调表达蛋白323个,鉴定肽段物种主要集中在双翅目,占52.9%。对差异表达倍数分析发现,滞育烟蚜茧蜂有4个上调表达8倍以上的蛋白与脂代谢相关：脂质运载/胞质脂肪酸结合蛋白(gi_322794737)、C型凝集素(gi_607305114)、肽基脯氨酰顺反异构酶(W4X351_ATTCE)、芳基贮存蛋白α亚基(gi_332018934)。iTRAQ技术结合LC-MS/MS作为一种快速准确的定量蛋白质组学研究方法,能有效地分析烟蚜茧蜂滞育相关蛋白;烟蚜茧蜂滞育相关蛋白差异表达较为复杂,可针对上调表达倍数高的蛋白进行后续功能分析,深入开展昆虫滞育机理研究。

关键词: 滞育, 烟蚜茧蜂, iTRAQ定量技术, 滞育相关蛋白, 脂代谢

Abstract: Diapause may contribute to the shelf life of mass-produced Aphidius gifuensis. To better understand the diapause mechanism of A. gifuensis, iTRAQ quantitative techniques combined with bioinformatics were used to analyze differentially expressed proteins between non-diapausing and diapausing A. gifuensis. A total of 1268 proteins were quantitatively identified and 601 of them were differentially expressed, with 278 up-regulated proteins and 323 down-regulated proteins. There were 4 prominently up-regulated proteins in diapausing A. gifuensis, which were associated with lipid metabolism: hypothetical protein SINV_00606, partial (gi_322794737), conserved uncharacterized protein (gi_607305114), peptidyl-prolyl cis-trans isomerase (W4X351_ATTCE), arylphorin subunit alpha (gi_332018934). These results showed that iTRAQ technique combined with LC-MS/MS is applicable to protein quantification in diapausing A. gifuensis. The results laid a foundation for function analysis of the significantly up-regulated proteins and for further study of the diapausing mechanism in insects.

Key words: diapause, Aphidius gifuensis, iTRAQ quantitative techniques, diapause associated protein, lipid metabolism

中图分类号:

S476.3

黄凤霞, 蒋莎, 任小云, 安涛, 陈红印, 张礼生. 烟蚜茧蜂脂代谢相关的滞育相关蛋白差异表达分析[J]. 中国生物防治学报, 2015, 31(6): 811-820.

HUANG Fengxia, JIANG Sha, REN Xiaoyun, AN Tao, CHEN Hongyin, ZHANG Lisheng. Differential Expression of Proteins Associated with Lipid Metabolism in Diapausing Aphidius gifuensis Ashmaed[J]. journal1, 2015, 31(6): 811-820.

参考文献

[1] 张礼生. 滞育和休眠在昆虫饲养中的应用//曾凡荣, 陈红印主编. 天敌昆虫饲养系统工程[M]. 北京: 中国农业科学技术出版社, 2009, 54-89.
[2] 张礼生. 蚜茧蜂的扩繁与应用//张礼生, 陈红印, 李保平主编. 天敌昆虫扩繁与应用[M]. 北京: 中国农业科学技术出版社, 2014, 148-169.
[3] Denlinger D L, Armbruster P A. Mosquito diapause[J]. Annual Review of Entomology, 2014, 59: 73-93.
[4] Hahn D A, Denlinger D L. Energetics of insect diapause[J]. Annual Review of Entomology, 2011, 56: 103-121.
[5] Christiea A E, Roncallia V, Lonab P B, et al. In silico characterization of the insect diapause-associated protein couch potato (CPO) in Calanus finmarchicus (Crustacea: Copepoda)[J]. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2013, 8(1): 45-57.
[6] 刘遥, 张礼生, 陈红印, 等. 苹果酸脱氢酶与异柠檬酸脱氢酶在滞育七星瓢虫中的差异表达[J]. 中国生物防治学报, 2014, 30(5): 593-599.
[7] Richard D U, John R G, Anthony D W, et al. Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS[J]. Nature Protocols Erecipes for Researchers, 2010, 5(9): 1574-1582.
[8] White F M. On the iTRAQ of kinase inhibitors[J]. Nature Biotechnology, 2007, 25(9): 994-996.
[9] Karp N A, Huber W, Pawel G S, et al. Addressing accuracy and precision issues in iTRAQ quantitation[J]. Molecular and Cellular Proteomics, 2010, 9(9): 1885-1897.
[10] 黄凤霞, 周阳, 张礼生, 等. 一种扩繁烟蚜茧蜂的载体植物适合性评价[J]. 环境昆虫学报, 2015, 37(4) : 827-833.
[11] 李玉艳, 张礼生, 陈红印, 等. 烟蚜茧蜂滞育诱导的温光周期反应[J]. 应用昆虫学报, 2013, 50(3): 718-726.
[12] 张礼生, 陈红印, 王孟卿, 等. 寄生蜂的滞育研究进展[J]. 中国生物防治学报, 2014, 30(2): 149-164.
[13] 徐珊珊, 阎春兰, 刘黎明, 等. 细胞裂解液对蛋白质定量方法的影响[J]. 浙江大学学报(医学版), 2008, 37(1): 45-50.
[14] 胡文霞. 葱蝇非滞育和夏滞育蛹的比较蛋白质组学研究[D]. 重庆: 重庆师范大学, 2011.
[15] Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing[J]. Journal of the Royal Statistical Society Series B: Statistical Methodology, 1995, 57: 289-300.
[16] Wu W W, Wang G, Baek S J, et al. Comparative study of three proteomic quantitative methods, DIGE, ICAT, and iTRAQ, using 2D Gel or LC- MALDI TOF/TOF[J]. Journal of Proteome Research, 2006, 5(3): 651-658.
[17] Philipp M, Namrata D U, Karl R C, et al. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics[J]. Molecular and Cellular Proteomics, 2012, 11(6): M111.008466.
[18] Ranju R, DeSouza L V, Matta A, et al. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry[J]. Molecular and Cellular Proteomics, 2008, 7(6): 1162-1173.
[19] Wolff S, Otto A, Albrecht D, et al. Gel- free and gel- based proteomics in Bacillus subtilis: A comparative study [J]. Molecular and Cellular Proteomics. 2006, 5(7): 1183-1192.
[20] Golovan S P, Hakimov H A, Verschoor C P, et al. Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines, and conventional and genetically enhanced lines[J]. Comparative Biochemistry and Physiology Part D, Genomics and Proteomics, 2008, 3(3): 234-242.
[21] Zhu M, Simons B, Zhu N, et al. Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging[J]. Journal of Proteomics, 2010, 73(4): 790-805.
[22] Zhu J Y, Wu G X, Ze S Z, et al. Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae[J]. Archives of Insect Biochemistry and Physiology, 2014, 66: 37-44.
[23] 李玉艳. 烟蚜茧蜂滞育诱导的温光周期反应及滞育生理研究[D]. 北京: 中国农业科学院, 2011.
[24] 张洁, 张礼生, 陈红印, 等. 大规模扩繁烟蚜茧蜂的蚜类寄主筛选研究[J]. 中国生物防治报, 2014, 30(1): 32-37.
[25] 黄继梅. 烟蚜茧蜂防治烟蚜研究及推广应用[D]. 长沙: 湖南农业大学, 2008.
[26] Denlinger D L. Why study diapause[J]. Entomological Research, 2008, 38(1): 1- 9.
[27] 张礼生, 陈红印. 生物防治作用物研发与应用的进展[J]. 中国生物防治学报, 2014, 30(5): 581-586.
[28] Denlinger D L. Regulation of diapause[J]. Annual Review of Entomology, 2002, 47(1): 93-122.
[29] Hahn D A, Denlinger D L. Meeting the energetic demands of insect diapause: nutrient storage and utilization[J]. Journal of Insect Physiology, 2007, 53(8): 760-773.
[30] Li A Q, Popova B A, Dean D H, et al. Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause[J]. Journal of Insect Physiology, 2007, 53(4): 385-391.
[31] Veerkamp J H, Zimmerman A W. Fatty acid-binding proteins of nervous tissue[J]. Journal of Molecular Neuroscience, 2001, 16(2-3): 133-142.
[32] 王丹, 唐田娟, 刘悦, 等. 家蚕亲环蛋白A基因(BmCyPA)的克隆与表达分析[J]. 蚕业科学, 2011, 37(5): 792-800.
[33] Brown J J, Chippendale G M. Juvenile hormone and a protein associated with the larval diapause of the southwestern corn borer, Diatraea grandiosella[J]. Insect Biochemistry, 1978, 8(5): 359-367.
[34] Sim C, Denlinger D L. Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens[J]. PNAS, 2008, 105(18): 6777-6781.
[35] Sim C, Denlinger D L. Juvenile hormone III suppresses forkhead of transcription factor in the fat body and reduces fat accumulation in the diapausing mosquito, Culex pipiens[J]. Insect Molecular Biology, 2013, 22(1): 1-11.