基于iTRAQ的定量蛋白质组学技术探索淡色库蚊越冬机制
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摘要
目的比较淡色库蚊越冬后与滞育准备阶段蛋白表达差异,探索淡色库蚊越冬休眠(滞育)的机制。方法采用同位素标记相对和绝对定量(iTRAQ)技术,对越冬滞育及准备阶段淡色库蚊进行定量蛋白质组学分析。结果淡色库蚊越冬滞育前后共鉴定出244种差异表达蛋白,其中上调蛋白126种、下调蛋白118种。生物信息学分析表明,这些差异表达蛋白与能量产生及转化、脂代谢、细胞骨架重塑、糖代谢、蛋白质转运、分子伴侣、应激耐受以及各种代谢酶等有关。结论本研究首次采用现代蛋白质组学工具鉴定淡色库蚊越冬滞育相关蛋白,初步揭示了与淡色库蚊越冬滞育相关的KEGG通路及蛋白,进一步扩展了对蚊媒越冬滞育机制的理解。
Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipiens pallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification(iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipiens pallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification(iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
引文
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[22]Kim M,Robich RM,Rinehart JP,et al.Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito,Culex pipiens[J].J Insect Physiol,2006,52(11/12):1226-1233.
[23]Ouellet F,Carpentier E,Cope MJ,et al.Regulation of a wheat actin168-depolymerizing factor during cold acclimation[J].Plant Physiol,2001,125(1):360-368.
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[25]Bursac S,Brdovcak MC,Pfannkuchen M,et al.Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress[J].Proc Natl Acad Sci U S A,2012,109(50):20467-20472.
[26]Naora H.Involvement of ribosomal proteins in regulating cell growth and aptosis:translational modulation or recruitment for extraribosomal activity?[J].Immunol Cell Biol,1999,77(3):197-205.
[27]Saito A,Koga K,Sakaguchi B.Changes in polysome content during development after diapause of Bombyx mori embryos[J].FEBS Lett,1982,150(2):449-453.
[28]Siva AB,Kameshwari DB,Singh V,et al.Proteomics-based study on asthenozoospermia:differential expression of proteasome alpha complex[J].Mol Hum Reprod,2010,16(7):452-462.
[2]Blitvich BJ,Rayms-Keller A,Blair CD,et al.Identification and sequence determination of mRNAs detected in dormant(diapausing)Aedes triseriatus mosquito embryos[J].DNA Seq,2001,12(3):197-202.
[3]Nasci RS,Savage HM,White DJ,et al.West Nile virus in overwintering,Culex,mosquitoes,New York City,2000[J].Emerg Infect Dis,2001,7(4):742-744.
[4]Benoit JB,Denlinger DL.Suppression of water loss during adult diapause in the northern house mosquito,Culex pipiens[J].J Exp Biol,2007,210(2):217-226.
[5]Denlinger DL,Armbruster PA.Mosquito diapause[J].Annu Rev Entomol,2014,59:73-93.
[6]Hand SC,Denlinger DL,Podrabsky JE,et al.Mechanisms of animal diapause:recent developments from nematodes,crustaceans,insects,and fish[J].Am J Physiol Regul Integr Comp Physiol,2016,310(11):R1193-R1211.Chin J Schisto Control 2019Vol.31No.2
[7]Liu LJ,Zhang BG,Cheng P,et al.Overwintering of Culex pipiens pallens(Diptera:Culicidae)in Shandong,China[J].J Entomol Sci,2016,51(4):314-320.
[8]Spielman A,Wong J.Environmental control of ovarian diapause in Culex pipiens[J].Ann Entomol Soc Ama,1973,66(4):905-907.
[9]Sim C,Denlinger DL.Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens[J].Proc Natl Acad Sci U S A,2008,105(18):6777-6781.
[10]Tatar M,Kopelman A,Epstein D,et al.A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function[J].Science,2001,292(5514):107-110.
[11]Williams KD,Busto M,Suster ML,et al.Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase[J].Proc Natl Acad Sci U S A,2006,103(43):15911-15915.
[12]Sim C,Denlinger DL.Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens[J].Proc Natl Acad Sci U S A,2008,105(18):6777-6781.
[13]Sim C,Denlinger DL.A shut-down in expression of an insulin-like peptide,ILP-1,halts ovarian maturation during the overwintering diapause of the mosquito Culex pipiens[J].Insect Mol Biol,2009,18(3):325-332.
[14]Westerman J,Wirtz KWA.The primary structure of the nonspecific lipid transfer protein(sterol carrier protein 2)from bovine liver[J].Biochem Biophys Res Commun,1985,127(1):333-338.
[15]Xu WH,Lu YX,Denlinger DL.Cross-talk between the fat body and brain regulates insect developmental arrest[J].Proc Natl Acad Sci U S A,2012,109(36):14687-14692.
[16]Rinehart JP,Robich RM,Denlinger DL.Isolation of diapause-regulated genes from the flesh fly,Sarcophaga crassipalpis by suppressive subtractive hybridization[J].J Insect Physiol,2010,56(6):603-609.
[17]Florian W,Gadau Jürgen,Karl-Wilhelm K.Deciphering proteomic signatures of early diapause in Nasonia[J].PLoS One,2009,4(7):e6394.
[18]Robich RM,Rinehart JP,Kitchen LJ,et al.Diapause-specific gene expression in the northern house mosquito,Culex pipiens L.identified by suppressive subtractive hybridization[J].J Insect Physiol,2007,53(3):235-245.
[19]Li AQ,Denlinger DL.Pupal cuticle protein is abundant during early adult diapause in the mosquito Culex pipiens[J].J Med Entomol,2009,46(6):1382-1386.
[20]Lee KY,Hiremath S,Denlinger DL.Expression of actin in the central nervous system is switched off during diapause in the gypsy moth,Lymantria dispar[J].J Insect Physiol,1998,44(3/4):221-226.
[21]Yocum GD,Kemp WP,Bosch J,et al.Temporal variation in overwintering gene expression and respiration in the solitary bee Megachile rotundata[J].J Insect Physiol,2005,51(6):621-629.
[22]Kim M,Robich RM,Rinehart JP,et al.Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito,Culex pipiens[J].J Insect Physiol,2006,52(11/12):1226-1233.
[23]Ouellet F,Carpentier E,Cope MJ,et al.Regulation of a wheat actin168-depolymerizing factor during cold acclimation[J].Plant Physiol,2001,125(1):360-368.
[24]Baker NE,Kale A.Mutations in ribosomal proteins:apoptosis,cell competition,and cancer[J].Mol Cel Oncol,2016,3(1):e1029065.
[25]Bursac S,Brdovcak MC,Pfannkuchen M,et al.Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress[J].Proc Natl Acad Sci U S A,2012,109(50):20467-20472.
[26]Naora H.Involvement of ribosomal proteins in regulating cell growth and aptosis:translational modulation or recruitment for extraribosomal activity?[J].Immunol Cell Biol,1999,77(3):197-205.
[27]Saito A,Koga K,Sakaguchi B.Changes in polysome content during development after diapause of Bombyx mori embryos[J].FEBS Lett,1982,150(2):449-453.
[28]Siva AB,Kameshwari DB,Singh V,et al.Proteomics-based study on asthenozoospermia:differential expression of proteasome alpha complex[J].Mol Hum Reprod,2010,16(7):452-462.