沙葱萤叶甲热激蛋白基因GdHsp10a的克隆、分子特征与表达分析
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摘要
为明确热激蛋白HSP10在沙葱萤叶甲Galeruca daurica生长发育过程中的作用,采用RTPCR及RACE技术克隆沙葱萤叶甲Hsp10基因cDNA的全长序列,采用生物信息学方法分析其序列特征,并利用qPCR技术对该基因在沙葱萤叶甲不同发育阶段、成虫羽化后不同时期以及不同温度下的表达谱进行分析。结果表明,克隆获得1条新的沙葱萤叶甲Hsp10基因,命名为GdHsp10a,GenBank登录号为MG460308,cDNA序列全长为526 bp,开放阅读框为333 bp,编码蛋白含110个氨基酸,预测分子量为11.97 kD,等电点为9.74;无信号肽及跨膜结构。同源比对及系统进化分析表明,GdHSP10a与光肩星天牛Anoplophora glabripennis HSP10亲缘关系最近,氨基酸序列一致性为53.15%。qPCR结果表明,GdHsp10a在沙葱萤叶甲各发育阶段均有表达,其中在卵期和成虫期的表达量显著高于幼虫期、预蛹期及蛹期;成虫羽化后不同时期表达量差异显著,25 d时表达量最高,其次为100、10和7 d时的表达量;温度对GdHsp10a表达量有显著影响,30℃下表达量最高,35℃下表达量次之,15、20、25及40℃下表达量最低且无显著差异。表明GdHsp10a可能在沙葱萤叶甲生长发育及成虫越夏中起着多重作用。
In order to investigate the functions of HSP10 in the development of Galeruca daurica, a fulllength cDNA encoding HSP10 was cloned from G. daurica by RT-PCR and RACE technologies. Bioinformatics programs were applied to analyze its sequence characteristics. qPCR was used to detect its mRNA expression levels at various developmental stages of G. daurica, different periods after the adult eclosion, and different temperature stresses. The results showed that the complete cDNA of the gene encoding heat shock protein HSP10 was obtained from G. daurica, and named GdHsp 10a(GenBank accession: MG460308). Its full-length cDNA was 526 bp containing a 333 bp open reading frame that encodes 110 amino acids with the predicted molecular weight of 11.97 k D and pI of 9.74. The deduced protein contained no signal peptide and transmembrane domain. Homology and phylogenetic analyses showed that the amino acid sequence of GdHSP10 a had the highest similarity(53.15%) with that of Asian longhorn beetle Anoplophora glabripennis HSP10. GdHsp10 a was expressed at all developmental stages of G. daurica, and the expression levels in eggs and adults were significantly higher than those in the larva,pre-pupa and pupa. The expression levels were significantly different among the different periods after the adult eclosion, and reached the highest on the 25 th day. Temperature had a significant effect on the expression of GdHsp10 a, with the maximum at 30℃, followed by at 35℃, and with the minimum at 15, 20,25 and 40℃, among which no significant differences existed. The results suggested that GdHsp10 a might play multiple roles in the development of G. daurica and its adult oversummering.
In order to investigate the functions of HSP10 in the development of Galeruca daurica, a fulllength cDNA encoding HSP10 was cloned from G. daurica by RT-PCR and RACE technologies. Bioinformatics programs were applied to analyze its sequence characteristics. qPCR was used to detect its mRNA expression levels at various developmental stages of G. daurica, different periods after the adult eclosion, and different temperature stresses. The results showed that the complete cDNA of the gene encoding heat shock protein HSP10 was obtained from G. daurica, and named GdHsp 10a(GenBank accession: MG460308). Its full-length cDNA was 526 bp containing a 333 bp open reading frame that encodes 110 amino acids with the predicted molecular weight of 11.97 k D and pI of 9.74. The deduced protein contained no signal peptide and transmembrane domain. Homology and phylogenetic analyses showed that the amino acid sequence of GdHSP10 a had the highest similarity(53.15%) with that of Asian longhorn beetle Anoplophora glabripennis HSP10. GdHsp10 a was expressed at all developmental stages of G. daurica, and the expression levels in eggs and adults were significantly higher than those in the larva,pre-pupa and pupa. The expression levels were significantly different among the different periods after the adult eclosion, and reached the highest on the 25 th day. Temperature had a significant effect on the expression of GdHsp10 a, with the maximum at 30℃, followed by at 35℃, and with the minimum at 15, 20,25 and 40℃, among which no significant differences existed. The results suggested that GdHsp10 a might play multiple roles in the development of G. daurica and its adult oversummering.
引文
Bakthisaran R,Tangirala R,Rao CM.2015.Small heat shock proteins:role in cellular functions and pathology.Biochimica et Biophysica Acta,1854(4):291-319
Franck E,Madsen O,van Rheede T,Ricard G,Huynen MA,de Jong WW.2004.Evolutionary diversity of vertebrate small heat shock proteins.Journal of Molecular Evolution,59(6):792-805
Fremdt H,Amendt J,Zehner R.2014.Diapause-specific gene expression in Calliphora vicina(Diptera:Calliphoridae)-a useful diagnostic tool for forensic entomology.International Journal of Legal Medicine,128(6):1001-1011
Gao JC,Zhou XR,Pang BP,Bao X,Luo JP,Eerdengqimuge.2015.Effects of low temperature on the survivorship and development of overwintering eggs of Galeruca daurica(Coleoptera:Chrysomelidae).Acta Entomologia Sinica,52(8):881-886(in Chinese)[高靖淳,周晓榕,庞保平,包祥,罗建平,额尔登其木格.2015.低温对沙葱萤叶甲越冬卵存活和发育的影响.昆虫学报,52(8):881-886]
Garczynski SF,Unruh TR,Guedot C,Neven LG.2011.Characterization of three transcripts encoding small heat shock proteins expressed in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae).Insect Science,18(5):473-483
Gu J,Huang LX,Shen Y,Huang LH,Feng QL.2012.Hsp70 and small Hsps are the major heat shock protein members involved in midgut metamorphosis in the common cutworm,Spodoptera litura.Insect Molecular Biology,21(5):535-543
Hao X,Zhou XR,Pang BP,Zhang ZR,Bao X.2015.Morphological and biological characteristics of Galeruca daurica Joannis.Acta Agrestia Sinica,23(5):1106-1108(in Chinese)[昊翔,周晓榕,庞保平,张卓然,包祥.2015.沙葱萤叶甲的形态特征和生物学特性研究.草地学报,23(5):1106-1108]
Hayer-Hartl M,Bracher A,Hartl FU.2016.The GroEL-GroES chaperonin machine:a nano-cage for protein folding.Trends in Biochemical Sciences,41(1):62-76
Hayward SA,Pavlides SC,Tammariello SP,Rinehart JP,Denlinger DL.2005.Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through postdiapause quiescence.Journal of Insect Physiology,51(6):631-640
Hoffmann AA,Sorensen JG,Loeschcke V.2003.Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches.Journal of Thermal Biology,28(3):175-216
Huang LH,Chen B,Kang L.2007.Impact of mild temperature hardening on thermotolerance,fecundity,and Hsp gene expression in Liriomyza huidobrensis.Journal of Insect Physiology,53(12):1199-1205
Jiang J,Li XR,Zhang YH,Guo WC,Cheng DF,Guo QY.2013.c DNAcloning of heat shock protein 90(Ld-hsp90)in Leptinotarsa decemlineata(Coleoptera:Chrysomelidae)and its expression under temperature stress.Journal of Plant Protection,40(5):399-406(in Chinese)[蒋健,李祥瑞,张云慧,郭文超,程登发,郭青云.2013.马铃薯甲虫热激蛋白基因Ldhsp90的克隆及温度胁迫下的表达.植物保护学报,40(5):399-406]
King AM,MacRae TH.2015.Insect heat shock proteins during stress and diapause.Annual Review of Entomology,60:59-75
Li YT,Zhao Q,Fang B,Chen MH.2017.Molecular cloning and expression analysis of the Hsp90 gene in the bird cherry-oat aphid,Rhopalosiphum padi.Journal of Plant Protection,44(1):16-23(in Chinese)[李玉婷,赵奇,房冰,陈茂华.2017.禾谷缢管蚜热激蛋白Hsp90基因的克隆和表达分析.植物保护学报,44(1):16-23]
Li ZW,Li X,Yu QY,Xiang ZH,Kishino H,Zhang Z.2009.The small heat shock protein(sHSP)genes in the silkworm,Bombyx mori,and comparative analysis with other insect sHSP genes.BMCEvolutionary Biology,9:215
Livak KJ,Schmittgen TD.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod.Methods,25(4):402-408
Lu MX,Hua J,Cui YD,Du YZ.2014.Five small heat shock protein genes from Chilo suppressalis:characteristics of gene,genomic organization,structural analysis,and transcription profiles.Cell Stress and Chaperones,19(1):91-104
Lu MX,Xu J,Du YZ.2015.Progress in research on insect small heat shock proteins.Chinese Journal of Applied Entomology,52(6):1326-1332(in Chinese)[陆明星,徐静,杜予州.2015.昆虫小分子量热激蛋白的研究进展.应用昆虫学报,52(6):1326-1332]
Martín-Folgar R,de la Fuente M,Morcillo G,Martínez-Guitarte JL.2015.Characterization of six small HSP genes from Chironomus riparius(Diptera,Chironomidae):differential expression under conditions of normal growth and heat-induced stress.Comparative Biochemistry and Physiology,Part A:Molecular&Integrative Physiology,188:76-86
Martínez-Paz P,Morales M,Martín R,Martínez-Guitarte JL,Morcillo G.2014.Characterization of the small heat shock protein Hsp27gene in Chironomus riparius(Diptera)and its expression profile in response to temperature changes and xenobiotic exposures.Cell Stress and Chaperones,19(4):529-540
Michaud S,Morrow G,Marchand J,Tanguay RM.2002.Drosophila small heat shock proteins:cell and organelle-specific chaperones?Progress in Molecular and Subcellular Biology,28:79-101
Pan DD,Lu MX,Li QY,Du YZ.2018.Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from Chilo suppressalis.Cell Stress and Chaperones,23(1):55-64
Rinehart JP,Li A,Yocum GD,Robich RM,Hayward SAL,Denlinger DL.2007.Up-regulation of heat shock proteins is essential for cold survival during insect diapause.Proceedings of the National Academy of Sciences of the United States of America,104(27):11130-11137
Shen Y,Gu J,Huang LH,Zheng SC,Liu L,Xu WH,Feng QL,Kang L.2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura.Journal of Insect Physiology,57(7):908-914
Shi XK,Kou LH,Zhang YP,Ma EB,Zhang JZ,Wu HH.2016.Effects of temperature on the expression patterns of OcsHSP genes in Oxya chinensis.Chinese Journal of Applied Entomology,53(6):1267-1273(in Chinese)[史学凯,寇利花,张育平,马恩波,张建珍,吴海花.2016.温度对中华稻蝗小分子热休克蛋白基因表达的影响.应用昆虫学报,53(6):1267-1273]
S?rensen JG,Kristensen TN,Loeschcke V.2003.The evolutionary and ecological role of heat shock proteins.Ecology Letters,6(11):1025-1037
Sun Y,MacRae TH.2005.Small heat shock proteins:molecular structure and chaperone function.Cellular and Molecular Life Sciences,62(21):2460-2476
Takahashi KH,Rako L,Takano-Shimizu T,Hoffmann AA,Lee SF.2010.Effects of small Hsp genes on developmental stability and microenvironmental canalization.BMC Evolutionary Biology,10:284
Tan Y,Zhang Y,Huo ZJ,Zhou XR,Pang BP.2017a.Molecular cloning of heat shock protein 10(hsp10)and 60(hsp60)c DNAs from Galeruca daurica(Coleoptera:Chrysomelidae)and their expression analysis.Bulletin of Entomological Research,108(4):510-512
Tan Y,Zhang Y,Huo ZJ,Zhou XR,Shan YM,Pang BP.2017.Molecular cloning and expression profiling of the heat shock protein gene GdHsp70 in Galeruca daurica Joannis(Coleoptera:Chrysomelidae).Acta Entomologia Sinica,60(8):865-875(in Chinese)[谭瑶,张玉,霍志家,周晓榕,单艳敏,庞保平.2017.沙葱萤叶甲热激蛋白基因GdHsp70的克隆与表达模式分析.昆虫学报,60(8):865-875]
Tan Y,Zhou XR,Pang BP.2017b.Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica(Joannis).Bulletin of Entomological Research,107(3):359-368
Wang HH,Lei ZR.2005.Current developments of heat shock proteins in insects.Scientia Agricultura Sinica,38(10):2023-2034(in Chinese)[王海鸿,雷仲仁.2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034]
Wei CP,Jiang J,Li XR,Zhang YH,Zhang FM,Cheng DF,Guo WC,Liu H.2017.Cloning and sequence analysis of heat shock protein gene Ld-HSP60 in Leptinotarsa decemlineata(Coleoptera:Chrysomelidae)and its expression under temperature stress.Acta Entomologia Sinica,60(5):523-532(in Chinese)[魏长平,蒋健,李祥瑞,张云慧,张方梅,程登发,郭文超,刘怀.2017.马铃薯甲虫热激蛋白基因Ld-HSP60的克隆序列分析及温度胁迫下的表达.昆虫学报,60(5):523-532]
Yocum GD,Joplin KH,Denlinger DL.1998.Up-regulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly Sarcophaga crassipalpis.Insect Biochemistry and Molecular Biology,28(9):677-682
Zhang YY,Liu YL,Guo XL,Li YL,Gao HG,Guo XQ,Xu BH.2014.sHsp22.6,an intronless small heat shock protein gene,is involved in stress defense and development in Apis cerana cerana.Insect Biochemistry and Molecular Biology,53:1-12
Zhou XR,Gao JC,Pang BP.2016.Effects of temperature on the termination of egg diapause and post-diapause embryonic development of Galeruca daurica(Coleoptera:Chrysomelidae).Environmental Entomology,45(4):1076-1080
Franck E,Madsen O,van Rheede T,Ricard G,Huynen MA,de Jong WW.2004.Evolutionary diversity of vertebrate small heat shock proteins.Journal of Molecular Evolution,59(6):792-805
Fremdt H,Amendt J,Zehner R.2014.Diapause-specific gene expression in Calliphora vicina(Diptera:Calliphoridae)-a useful diagnostic tool for forensic entomology.International Journal of Legal Medicine,128(6):1001-1011
Gao JC,Zhou XR,Pang BP,Bao X,Luo JP,Eerdengqimuge.2015.Effects of low temperature on the survivorship and development of overwintering eggs of Galeruca daurica(Coleoptera:Chrysomelidae).Acta Entomologia Sinica,52(8):881-886(in Chinese)[高靖淳,周晓榕,庞保平,包祥,罗建平,额尔登其木格.2015.低温对沙葱萤叶甲越冬卵存活和发育的影响.昆虫学报,52(8):881-886]
Garczynski SF,Unruh TR,Guedot C,Neven LG.2011.Characterization of three transcripts encoding small heat shock proteins expressed in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae).Insect Science,18(5):473-483
Gu J,Huang LX,Shen Y,Huang LH,Feng QL.2012.Hsp70 and small Hsps are the major heat shock protein members involved in midgut metamorphosis in the common cutworm,Spodoptera litura.Insect Molecular Biology,21(5):535-543
Hao X,Zhou XR,Pang BP,Zhang ZR,Bao X.2015.Morphological and biological characteristics of Galeruca daurica Joannis.Acta Agrestia Sinica,23(5):1106-1108(in Chinese)[昊翔,周晓榕,庞保平,张卓然,包祥.2015.沙葱萤叶甲的形态特征和生物学特性研究.草地学报,23(5):1106-1108]
Hayer-Hartl M,Bracher A,Hartl FU.2016.The GroEL-GroES chaperonin machine:a nano-cage for protein folding.Trends in Biochemical Sciences,41(1):62-76
Hayward SA,Pavlides SC,Tammariello SP,Rinehart JP,Denlinger DL.2005.Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through postdiapause quiescence.Journal of Insect Physiology,51(6):631-640
Hoffmann AA,Sorensen JG,Loeschcke V.2003.Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches.Journal of Thermal Biology,28(3):175-216
Huang LH,Chen B,Kang L.2007.Impact of mild temperature hardening on thermotolerance,fecundity,and Hsp gene expression in Liriomyza huidobrensis.Journal of Insect Physiology,53(12):1199-1205
Jiang J,Li XR,Zhang YH,Guo WC,Cheng DF,Guo QY.2013.c DNAcloning of heat shock protein 90(Ld-hsp90)in Leptinotarsa decemlineata(Coleoptera:Chrysomelidae)and its expression under temperature stress.Journal of Plant Protection,40(5):399-406(in Chinese)[蒋健,李祥瑞,张云慧,郭文超,程登发,郭青云.2013.马铃薯甲虫热激蛋白基因Ldhsp90的克隆及温度胁迫下的表达.植物保护学报,40(5):399-406]
King AM,MacRae TH.2015.Insect heat shock proteins during stress and diapause.Annual Review of Entomology,60:59-75
Li YT,Zhao Q,Fang B,Chen MH.2017.Molecular cloning and expression analysis of the Hsp90 gene in the bird cherry-oat aphid,Rhopalosiphum padi.Journal of Plant Protection,44(1):16-23(in Chinese)[李玉婷,赵奇,房冰,陈茂华.2017.禾谷缢管蚜热激蛋白Hsp90基因的克隆和表达分析.植物保护学报,44(1):16-23]
Li ZW,Li X,Yu QY,Xiang ZH,Kishino H,Zhang Z.2009.The small heat shock protein(sHSP)genes in the silkworm,Bombyx mori,and comparative analysis with other insect sHSP genes.BMCEvolutionary Biology,9:215
Livak KJ,Schmittgen TD.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod.Methods,25(4):402-408
Lu MX,Hua J,Cui YD,Du YZ.2014.Five small heat shock protein genes from Chilo suppressalis:characteristics of gene,genomic organization,structural analysis,and transcription profiles.Cell Stress and Chaperones,19(1):91-104
Lu MX,Xu J,Du YZ.2015.Progress in research on insect small heat shock proteins.Chinese Journal of Applied Entomology,52(6):1326-1332(in Chinese)[陆明星,徐静,杜予州.2015.昆虫小分子量热激蛋白的研究进展.应用昆虫学报,52(6):1326-1332]
Martín-Folgar R,de la Fuente M,Morcillo G,Martínez-Guitarte JL.2015.Characterization of six small HSP genes from Chironomus riparius(Diptera,Chironomidae):differential expression under conditions of normal growth and heat-induced stress.Comparative Biochemistry and Physiology,Part A:Molecular&Integrative Physiology,188:76-86
Martínez-Paz P,Morales M,Martín R,Martínez-Guitarte JL,Morcillo G.2014.Characterization of the small heat shock protein Hsp27gene in Chironomus riparius(Diptera)and its expression profile in response to temperature changes and xenobiotic exposures.Cell Stress and Chaperones,19(4):529-540
Michaud S,Morrow G,Marchand J,Tanguay RM.2002.Drosophila small heat shock proteins:cell and organelle-specific chaperones?Progress in Molecular and Subcellular Biology,28:79-101
Pan DD,Lu MX,Li QY,Du YZ.2018.Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from Chilo suppressalis.Cell Stress and Chaperones,23(1):55-64
Rinehart JP,Li A,Yocum GD,Robich RM,Hayward SAL,Denlinger DL.2007.Up-regulation of heat shock proteins is essential for cold survival during insect diapause.Proceedings of the National Academy of Sciences of the United States of America,104(27):11130-11137
Shen Y,Gu J,Huang LH,Zheng SC,Liu L,Xu WH,Feng QL,Kang L.2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura.Journal of Insect Physiology,57(7):908-914
Shi XK,Kou LH,Zhang YP,Ma EB,Zhang JZ,Wu HH.2016.Effects of temperature on the expression patterns of OcsHSP genes in Oxya chinensis.Chinese Journal of Applied Entomology,53(6):1267-1273(in Chinese)[史学凯,寇利花,张育平,马恩波,张建珍,吴海花.2016.温度对中华稻蝗小分子热休克蛋白基因表达的影响.应用昆虫学报,53(6):1267-1273]
S?rensen JG,Kristensen TN,Loeschcke V.2003.The evolutionary and ecological role of heat shock proteins.Ecology Letters,6(11):1025-1037
Sun Y,MacRae TH.2005.Small heat shock proteins:molecular structure and chaperone function.Cellular and Molecular Life Sciences,62(21):2460-2476
Takahashi KH,Rako L,Takano-Shimizu T,Hoffmann AA,Lee SF.2010.Effects of small Hsp genes on developmental stability and microenvironmental canalization.BMC Evolutionary Biology,10:284
Tan Y,Zhang Y,Huo ZJ,Zhou XR,Pang BP.2017a.Molecular cloning of heat shock protein 10(hsp10)and 60(hsp60)c DNAs from Galeruca daurica(Coleoptera:Chrysomelidae)and their expression analysis.Bulletin of Entomological Research,108(4):510-512
Tan Y,Zhang Y,Huo ZJ,Zhou XR,Shan YM,Pang BP.2017.Molecular cloning and expression profiling of the heat shock protein gene GdHsp70 in Galeruca daurica Joannis(Coleoptera:Chrysomelidae).Acta Entomologia Sinica,60(8):865-875(in Chinese)[谭瑶,张玉,霍志家,周晓榕,单艳敏,庞保平.2017.沙葱萤叶甲热激蛋白基因GdHsp70的克隆与表达模式分析.昆虫学报,60(8):865-875]
Tan Y,Zhou XR,Pang BP.2017b.Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica(Joannis).Bulletin of Entomological Research,107(3):359-368
Wang HH,Lei ZR.2005.Current developments of heat shock proteins in insects.Scientia Agricultura Sinica,38(10):2023-2034(in Chinese)[王海鸿,雷仲仁.2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034]
Wei CP,Jiang J,Li XR,Zhang YH,Zhang FM,Cheng DF,Guo WC,Liu H.2017.Cloning and sequence analysis of heat shock protein gene Ld-HSP60 in Leptinotarsa decemlineata(Coleoptera:Chrysomelidae)and its expression under temperature stress.Acta Entomologia Sinica,60(5):523-532(in Chinese)[魏长平,蒋健,李祥瑞,张云慧,张方梅,程登发,郭文超,刘怀.2017.马铃薯甲虫热激蛋白基因Ld-HSP60的克隆序列分析及温度胁迫下的表达.昆虫学报,60(5):523-532]
Yocum GD,Joplin KH,Denlinger DL.1998.Up-regulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly Sarcophaga crassipalpis.Insect Biochemistry and Molecular Biology,28(9):677-682
Zhang YY,Liu YL,Guo XL,Li YL,Gao HG,Guo XQ,Xu BH.2014.sHsp22.6,an intronless small heat shock protein gene,is involved in stress defense and development in Apis cerana cerana.Insect Biochemistry and Molecular Biology,53:1-12
Zhou XR,Gao JC,Pang BP.2016.Effects of temperature on the termination of egg diapause and post-diapause embryonic development of Galeruca daurica(Coleoptera:Chrysomelidae).Environmental Entomology,45(4):1076-1080