基于转录组和基因表达谱的葱蝇分泌组挖掘及其在滞育发育中的可能作用分析（英文）
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摘要
【目的】昆虫滞育是一种重要的季节性适应生存的环境生理现象,但是调控葱蝇Delia antiqua夏滞育和冬滞育的分子机理却知之甚少。本研究旨在鉴定与葱蝇蛹滞育相关的候选基因。【方法】利用RNA-seq和数字基因表达谱数据,通过生物信息学的方法预测分析葱蝇滞育相关的分泌蛋白,并进行了氨基酸序列相似性、基本理化性质分析;从表达谱中选取12个差异表达基因,利用qRT-PCR验证了它们在非滞育蛹以及夏滞育和冬滞育蛹的不同发育时期的表达特点。【结果】共鉴定出38个在葱蝇夏滞育蛹和冬滞育蛹之间差异表达的可能的分泌蛋白。同源基因注释表明,这些基因可能在气味分子结合、几丁质和脂类代谢、免疫及发育等方面发挥作用,其中脂类和几丁质代谢过程的调节似乎在滞育发育过程起到重要作用。qRT-PCR分析结果表明,12个被选择基因的表达谱与RNA-seq结果相吻合(R=0.862,P=0.001)。【结论】本研究为基于高通量测序的分泌组挖掘及滞育相关基因的鉴定提供了强有力地分析策略。
【Aim】Insect diapause is a critical eco-physiological adaptation for seasonal survival,but little is known about the molecular mechanisms of summer and winter diapause of Delia antiqua.Here,our main goal is to identify candidate genes linked to pupal diapause of D.antiqua.【Methods 】A straightforward in silico approach was performed to predict the secreted diapause-related proteins based on the RNA-seq and digital gene expression datasets.Amino acid sequence similarity and basic physiochemical characteristics analysis were conducted.Gene expression levels of 12 selected genes from RNA-seq data were further validated in nondiapause pupae,and different developmental stages of summer and winter diapause pupae by qRT-PCR.【Results】A total of 38 putative secreted proteins were differentially regulated between the summer and winter diapause pupae of D.antiqua.Gene Ontology analysis revealed differentially expressed genes to be associated with odorant binding,chitin and lipid metabolic processes,innate immunity and development,etc.Among them,regulation of lipid mobilization and chitin-related metabolism was found to be important during diapause development.qRT-PCR analyses showed that the expression profiles of the 12 selected genes were in good agreement with results from RNA-seq(R = 0.862,P = 0.001).【Conclusion】This study establishes a robust pipeline for the discovery of diapause-related genes by secretome mining based on the high-throughput sequencing.
【Aim】Insect diapause is a critical eco-physiological adaptation for seasonal survival,but little is known about the molecular mechanisms of summer and winter diapause of Delia antiqua.Here,our main goal is to identify candidate genes linked to pupal diapause of D.antiqua.【Methods 】A straightforward in silico approach was performed to predict the secreted diapause-related proteins based on the RNA-seq and digital gene expression datasets.Amino acid sequence similarity and basic physiochemical characteristics analysis were conducted.Gene expression levels of 12 selected genes from RNA-seq data were further validated in nondiapause pupae,and different developmental stages of summer and winter diapause pupae by qRT-PCR.【Results】A total of 38 putative secreted proteins were differentially regulated between the summer and winter diapause pupae of D.antiqua.Gene Ontology analysis revealed differentially expressed genes to be associated with odorant binding,chitin and lipid metabolic processes,innate immunity and development,etc.Among them,regulation of lipid mobilization and chitin-related metabolism was found to be important during diapause development.qRT-PCR analyses showed that the expression profiles of the 12 selected genes were in good agreement with results from RNA-seq(R = 0.862,P = 0.001).【Conclusion】This study establishes a robust pipeline for the discovery of diapause-related genes by secretome mining based on the high-throughput sequencing.
引文
Andronopoulou E,Labropoulou V,Douris V,Woods DF,Biessmann H,Iatrou K,2006.Specific interactions among odorant-binding proteins of the African malaria vector anopheles gambiae.Insect Mol.Biol.,15(6):797-811.
Arai T,Kawasaki K,Kubo T,Natori S,1998.Cloning of c DNA for regenectin,a humoral C-type lectin of Periplaneta americana,and expression of the regenectin gene during leg regeneration.Insect Biochem.Molec.Biol.,28(12):987-994.
Chen L,Ma W,Wang X,Niu C,Lei C,2010.Analysis of pupal head proteome and its alteration in diapause pupae of Helicoverpa armigera.J.Insect Physiol.,56(3):247-252.
Daibo S,Kimura MT,Goto SG,2001.Upregulation of genes belonging to the drosomycin family in diapausing adults of Drosophila triauraria.Gene,278(1-2):177-184.
Denlinger DL,2002.Regulation of diapause.Annu.Rev.Entomol.,47(1):93-122.
Durand N,Carot-Sans G,Chertemps T,Bozzolan F,Party V,Renou M,Debernard S,Rosell G.Maibeche-Coisne,M,2010.Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.PLo S ONE,5(11):e15026.
Foret S,Maleszka R,2006.Function and evolution of a gene family encoding odorant binding-like proteins in a social insect,the honey bee(Apis mellifera).Genome Res.,16(11):1404-1413.
Fu DY,Si FL,Chen B,Xu YL,Hao YJ,2016.Transcriptome-guided identification of the cuticular protein gene family of Delia antiqua(Diptera:Anthomyiidae)and expression analysis under non-and winter-diapause conditions.Acta Entomol.Sin.,59(2):172-184.[付丹影,司风玲,陈斌,徐燕玲,郝友进,2016.基于转录组的葱蝇表皮蛋白基因家族的鉴定及在非滞育和冬滞育条件下的表达分析.昆虫学报,59(2):172-184]
Goto SG,Denlinger DL,2002.Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause.Gene,292(1-2):121-127.
Hao YJ,Zhang YJ,Si FL,Fu DY,He ZB,Chen B,2016.Insight into the possible mechanism of the summer diapause of Delia antiqua(Diptera:Anthomyiidae)through digital gene expression analysis.Insect Sci.,23(3):438-451.
Horabin JI,Walthall S,Vied C,Moses M,2003.A positive role for patched in hedgehog signaling revealed by the intracellular trafficking of sex-lethal,the Drosophila sex determination master switch.Development,130:6101-6109.
Jia QQ,Liu Y,Liu HH,Li S,2014.Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles.Sci.Rep.,4:7535.
Kalischuk-Tymensen L,2001.Isolation and Identification of Genes Expressed during Diapause in Horn Fly,Haematobia irritans(L).Ph D Dissertation,Faculty of Arts&Science,University of Lethbridge,Lethbridge,Alberta.
Kawaguchi N,Komano H,Natori S,1991.Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner.Dev.Biol.,144(1):86-93.
Kawamura K,Shibata T,Saget O,Peel D,Bryant PJ,1999.A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells.Development,126(2):211-219.
Leshko-Lindsay LA,Corces VG,1997.The role of selectins in Drosophila eye and bristle development.Development,124(1):169-180.
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods,25(4):402-408.
Macrae TH,2010.Gene expression,metabolic regulation and stress tolerance during diapause.Cell.Mol.Life Sci.,67(14):2405-2424.
Meyers PJ,Powell TH,Walden KK,Schieferecke AJ,Feder JL,Hahn DA,Robertson HM,Berlocher SH,Ragland GJ,2016.Divergence of the diapause transcriptome in apple maggot flies:winter regulation and post-winter transcriptional repression.J.Exp.Biol.,219(17):2613-2622.
Mitaka H,Matsuo T,Miura N,Ishikawa Y,2011.Identification of odorant-binding protein genes from antennal expressed sequence tags of the onion fly,Delia antiqua.Mol.Biol.Rep.,38(3):1787-1792.
Palli SR,Kothapalli R,Feng Q,Ladd T,Perera SC,Zheng SC,Gojtan K,Pang ASD,Primavera MJ,Tomkins WL,Retnakaran A,2001.Molecular analysis of overwintering diapause.In:Denlinger DL,Giebultowicz JM,Saunders DS eds.Insect Timing:Circadian Rhythmicity to Seasonality.Elsevier Science Inc.,Amsterdam,The Netherlands.133-144.
Qi XY,Ren XY,An T,Chen HY,Huang J,Zhang L,2016.Transcriptome analysis of diapause-associated genes of Coccinella septempunctata L.J.Environ.Entomol.,38(2):238-248.
Qi X,Zhang L,Han Y,Ren X,Huang J,Chen H,2015.De novo transcriptome sequencing and analysis of Coccinella septempunctata L.in non-diapause,diapause and diapause-terminated states to identify diapause-associated genes.BMC Genom.,16(1):1086.
Robich RM,Denlinger DL,2005.Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony.Proc.Natl.Acad.Sci.USA,102(44):15912-15917.
Yocum GD,Rinehart JP,Larson ML,2009.Down regulation of gene expression between the diapause initiation and maintenance phases of the Colorado potato beetle,Leptinotarsa decemlineata.Eur.J.Entomol.,106(4):471-476.
Zhang YJ,Hao YJ,Si F,Ren S,Hu G,Shen L,Chen B,2014.The de novo transcriptome and its analysis in the worldwide vegetable pest,Delia antiqua(Diptera:Anthomyiidae).G3,4(5):851-859.
Zhao JY,Zhao XT,Sun JT,Zou LF,Yang SX,Han X,Zhu WC,Yin Q,Hong XY,2017.Transcriptome and proteome analyses reveal complex mechanisms of reproductive diapause in the two-spotted spider mite,Tetranychus urticae.Insect Mol.Biol.,26(2):215-232.
Arai T,Kawasaki K,Kubo T,Natori S,1998.Cloning of c DNA for regenectin,a humoral C-type lectin of Periplaneta americana,and expression of the regenectin gene during leg regeneration.Insect Biochem.Molec.Biol.,28(12):987-994.
Chen L,Ma W,Wang X,Niu C,Lei C,2010.Analysis of pupal head proteome and its alteration in diapause pupae of Helicoverpa armigera.J.Insect Physiol.,56(3):247-252.
Daibo S,Kimura MT,Goto SG,2001.Upregulation of genes belonging to the drosomycin family in diapausing adults of Drosophila triauraria.Gene,278(1-2):177-184.
Denlinger DL,2002.Regulation of diapause.Annu.Rev.Entomol.,47(1):93-122.
Durand N,Carot-Sans G,Chertemps T,Bozzolan F,Party V,Renou M,Debernard S,Rosell G.Maibeche-Coisne,M,2010.Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.PLo S ONE,5(11):e15026.
Foret S,Maleszka R,2006.Function and evolution of a gene family encoding odorant binding-like proteins in a social insect,the honey bee(Apis mellifera).Genome Res.,16(11):1404-1413.
Fu DY,Si FL,Chen B,Xu YL,Hao YJ,2016.Transcriptome-guided identification of the cuticular protein gene family of Delia antiqua(Diptera:Anthomyiidae)and expression analysis under non-and winter-diapause conditions.Acta Entomol.Sin.,59(2):172-184.[付丹影,司风玲,陈斌,徐燕玲,郝友进,2016.基于转录组的葱蝇表皮蛋白基因家族的鉴定及在非滞育和冬滞育条件下的表达分析.昆虫学报,59(2):172-184]
Goto SG,Denlinger DL,2002.Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause.Gene,292(1-2):121-127.
Hao YJ,Zhang YJ,Si FL,Fu DY,He ZB,Chen B,2016.Insight into the possible mechanism of the summer diapause of Delia antiqua(Diptera:Anthomyiidae)through digital gene expression analysis.Insect Sci.,23(3):438-451.
Horabin JI,Walthall S,Vied C,Moses M,2003.A positive role for patched in hedgehog signaling revealed by the intracellular trafficking of sex-lethal,the Drosophila sex determination master switch.Development,130:6101-6109.
Jia QQ,Liu Y,Liu HH,Li S,2014.Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles.Sci.Rep.,4:7535.
Kalischuk-Tymensen L,2001.Isolation and Identification of Genes Expressed during Diapause in Horn Fly,Haematobia irritans(L).Ph D Dissertation,Faculty of Arts&Science,University of Lethbridge,Lethbridge,Alberta.
Kawaguchi N,Komano H,Natori S,1991.Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner.Dev.Biol.,144(1):86-93.
Kawamura K,Shibata T,Saget O,Peel D,Bryant PJ,1999.A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells.Development,126(2):211-219.
Leshko-Lindsay LA,Corces VG,1997.The role of selectins in Drosophila eye and bristle development.Development,124(1):169-180.
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods,25(4):402-408.
Macrae TH,2010.Gene expression,metabolic regulation and stress tolerance during diapause.Cell.Mol.Life Sci.,67(14):2405-2424.
Meyers PJ,Powell TH,Walden KK,Schieferecke AJ,Feder JL,Hahn DA,Robertson HM,Berlocher SH,Ragland GJ,2016.Divergence of the diapause transcriptome in apple maggot flies:winter regulation and post-winter transcriptional repression.J.Exp.Biol.,219(17):2613-2622.
Mitaka H,Matsuo T,Miura N,Ishikawa Y,2011.Identification of odorant-binding protein genes from antennal expressed sequence tags of the onion fly,Delia antiqua.Mol.Biol.Rep.,38(3):1787-1792.
Palli SR,Kothapalli R,Feng Q,Ladd T,Perera SC,Zheng SC,Gojtan K,Pang ASD,Primavera MJ,Tomkins WL,Retnakaran A,2001.Molecular analysis of overwintering diapause.In:Denlinger DL,Giebultowicz JM,Saunders DS eds.Insect Timing:Circadian Rhythmicity to Seasonality.Elsevier Science Inc.,Amsterdam,The Netherlands.133-144.
Qi XY,Ren XY,An T,Chen HY,Huang J,Zhang L,2016.Transcriptome analysis of diapause-associated genes of Coccinella septempunctata L.J.Environ.Entomol.,38(2):238-248.
Qi X,Zhang L,Han Y,Ren X,Huang J,Chen H,2015.De novo transcriptome sequencing and analysis of Coccinella septempunctata L.in non-diapause,diapause and diapause-terminated states to identify diapause-associated genes.BMC Genom.,16(1):1086.
Robich RM,Denlinger DL,2005.Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony.Proc.Natl.Acad.Sci.USA,102(44):15912-15917.
Yocum GD,Rinehart JP,Larson ML,2009.Down regulation of gene expression between the diapause initiation and maintenance phases of the Colorado potato beetle,Leptinotarsa decemlineata.Eur.J.Entomol.,106(4):471-476.
Zhang YJ,Hao YJ,Si F,Ren S,Hu G,Shen L,Chen B,2014.The de novo transcriptome and its analysis in the worldwide vegetable pest,Delia antiqua(Diptera:Anthomyiidae).G3,4(5):851-859.
Zhao JY,Zhao XT,Sun JT,Zou LF,Yang SX,Han X,Zhu WC,Yin Q,Hong XY,2017.Transcriptome and proteome analyses reveal complex mechanisms of reproductive diapause in the two-spotted spider mite,Tetranychus urticae.Insect Mol.Biol.,26(2):215-232.