马氏珠母贝(Pinctada fucata martensii)Pm-HSP70基因的克隆及其对温度胁迫的响应
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摘要
本实验利用RACE技术克隆获得了马氏珠母贝HSP70 (Pm-HSP70)基因,并对其基因和氨基酸序列结构特征进行了生物信息学分析,同时采用荧光定量PCR技术分析了该基因在不同组织的表达模式及不同温度下的时序表达模式。序列分析表明,Pm-HSP70 cDNA序列全长为2 215 bp,其中开放式阅读框1 899 bp,5'UTR 107 bp,3'UTR 209 bp,编码632个氨基酸,理论蛋白分子量为69.44 kD,理论等电点为5.61。该蛋白具有HSP70家族典型的结构域HSP70,以及ATP结合位点、细胞质特征性保守序列和3个HSP70家族标签。多序列比对结果表明Pm-HSP70与菲律宾蛤仔HSP70同源性最高,为80%;系统进化分析发现,Pm-HSP70与菲律宾蛤仔等贝类HSP70聚为一支。组织表达定量分析结果显示,Pm-HSP70在马氏珠母贝多个组织中均有表达,在肝胰腺中表达量最高,其次是性腺;对不同温度下鳃组织中Pm-HSP70的时序表达分析发现,在处理后各时间点高温组(32℃)基因表达水平最高,且均显著高于对照组(22℃)和低温组(17℃)。以上结果表明Pm-HSP70可能参与马氏珠母贝的高温胁迫响应。该研究为进一步探索Pm-HSP70在马氏珠母贝温度适应性中的作用提供了基础资料。
In this study, HSP70(Pm-HSP70) gene of Pinctada fucata martensii was cloned by using RACE technology and the structural characteristics of the gene and amino acid sequence were analyzed by bioinformatics, and the expression patterns of the gene(Pm-HSP70) in different tissues as well as its temporal expression patterns under different temperatures were analyzed by using q RT-PCR technology. Sequence analysis showed that the full length of Pm-HSP70 cDNA sequence was 2 215 bp, containing 1 899 bp of the open reading frame(ORF), 107 bp of 5'UTR and 209 bp of 3'UTR, which encoded 632 amino acids. The theoretical protein molecular weight was69.44 kD and the theoretical isoelectric point was 5.61. The protein had typical structure domain HSP70 of HSP70 family, ATP binding site, cytoplasmic characteristic conservative sequences and three HSP70 family tags. Multiple sequence alignment results showed that Pm-HSP70 had the highest homology(80%) with HSP70 in Ruditapes philippinarum. Phylogenetic analysis showed that Pm-HSP70 was clustered with molluscs HSP70 as a branch, such as Ruditapes philippinarum. The results of the tissue expression quantitative analysis showed that Pm-HSP70 was expressed in many tissues of Pinctada fucata martensii, with the most expressive in hepatopancreas, followed by the gonads. And the analysis results of Pm-HSP70 temporal expression at different temperatures in gill indicated that the gene expression level in the high temperature group(32℃) was the highest at each time point after treatment,which was significantly higher than it in the control group(22℃) and hypothermia group(17℃). The above results suggested that Pm-HSP70 might be involved in the resistance to high-temperature stress of Pinctada fucata martensii. This study would provide basic data for the further exploring the role of Pm-HSP70 in temperature adaptability of Pinctada fucata martensii.
In this study, HSP70(Pm-HSP70) gene of Pinctada fucata martensii was cloned by using RACE technology and the structural characteristics of the gene and amino acid sequence were analyzed by bioinformatics, and the expression patterns of the gene(Pm-HSP70) in different tissues as well as its temporal expression patterns under different temperatures were analyzed by using q RT-PCR technology. Sequence analysis showed that the full length of Pm-HSP70 cDNA sequence was 2 215 bp, containing 1 899 bp of the open reading frame(ORF), 107 bp of 5'UTR and 209 bp of 3'UTR, which encoded 632 amino acids. The theoretical protein molecular weight was69.44 kD and the theoretical isoelectric point was 5.61. The protein had typical structure domain HSP70 of HSP70 family, ATP binding site, cytoplasmic characteristic conservative sequences and three HSP70 family tags. Multiple sequence alignment results showed that Pm-HSP70 had the highest homology(80%) with HSP70 in Ruditapes philippinarum. Phylogenetic analysis showed that Pm-HSP70 was clustered with molluscs HSP70 as a branch, such as Ruditapes philippinarum. The results of the tissue expression quantitative analysis showed that Pm-HSP70 was expressed in many tissues of Pinctada fucata martensii, with the most expressive in hepatopancreas, followed by the gonads. And the analysis results of Pm-HSP70 temporal expression at different temperatures in gill indicated that the gene expression level in the high temperature group(32℃) was the highest at each time point after treatment,which was significantly higher than it in the control group(22℃) and hypothermia group(17℃). The above results suggested that Pm-HSP70 might be involved in the resistance to high-temperature stress of Pinctada fucata martensii. This study would provide basic data for the further exploring the role of Pm-HSP70 in temperature adaptability of Pinctada fucata martensii.
引文
Ahn H.J.,K im S.,and Nam H.W.,2003,Molecular cloning of the 82-kD heat shock protein(HSP90)of Toxoplasma gondii associated with the entry into and growth in host cells,Biochemical&Biophysical Research Communications,311(3):654-659
Besson M.,Vandeputte M.,van Arendonk J.A.M.,Aubin J.,Boer I.J.M.D.,Quillet E.,and Komen H.,2016,Influence of water temperature on the economic value of growth rate in fish farming:The case of sea bass(Dicentrarchus labrax)cage farming in the Mediterranean,Aquaculture,462:47-55
Cheng P.,Liu X.,Zhang G.,and He J.,2007,Cloning and expression analysis of a HSP70 gene from Pacific abalone(Haliotis discus hannai),Fish&Shellfish Immunology,22(1-2):77-87
Daugaard M.,Rohde M.,and JaattelaM.,2007,The heat shock protein 70 family:Highly homologous proteins with overlapping and distinct functions,FEBS Letters,581(19):3702-3710
Deane E.E.,and Woo N.Y.,2005,Cloning and characterization of the hsp70 multigene family from silver sea bream:Modulated gene expression between warm and cold temperature acclimation,Biochem.Biophys.Res.Commun.,330(3):776-783
Farcy E.,Serpentini A.,Fievet B.,and Lebel J.,2007,Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata:Transcriptional induction in response to thermal stress in hemocyte primary culture,Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,146(4):540-550
Franzellitti S.,and Fabbri E.,2005,Differential HSP70 gene expression in the mediterranean mussel exposed to various stressors,Biochemical&Biophysical Research Communications,336(4):1157-1163
Green T.J.,Montagnani C.,Benkendorff K.,Robinson N.,and Speck P.,2014,Ontogeny and water temperature influences the antiviral response of the Pacific oyster,Crassostrea gigas,Fish&Shellfish Immunology,36(1):151-157
Gupta R.S.,and Singh B.,1994,Phylogenetic analysis of 70 kDheat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus,Current Biology Cb,4(12):1104-1114
Hamdoun A.M.,and Cherr G.N.,2003,Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific Oyster(Crassostrea gigas):implications for thermal limits and induction of thermal tolerance,Biological Bulletin,205(2):160-169
Han Y.L.,Yang W.X.,Long L.L.,Sheng Z.,Zhou Y.,Zhao Y.Q.,Wang Y.F.,and Zhu J.Q.,2016,Molecular cloning,expression pattern,and chemical analysis of heat shock protein 70(HSP70)in the mudskipper Boleophthalmus pectinirostris:Evidence for its role in regulating spermatogenesis,Gene,575(2):331-338
Hao R.J.,Zheng Z.,Wang Q.H.,Deng Y.W.,Jiao Y.,and Du X.D.,2015,Molecular characterization and expression analysis of PmCHST9 gene from sulfotransferase of Pinctada martensii,Jiyinzuexue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2387-2394(郝瑞娟,郑哲,王庆恒,邓岳文,焦钰,杜晓东,2015,马氏珠母贝磺基转移酶PmCHST9基因的分子特征与表达分析,基因组学与应用生物学,34(11):2387-2394)
Hightower L.E.,1991,Heat shock,stress proteins,chaperones,and proteotoxicity,Cell,66(2):191-197
Kiang J.G.,and Tsokos G.C.,1998,Heat shock protein 70 k D:molecular biology,biochemistry,and physiology,Pharmacology&Therapeutics,80(2):183-201
Li J.,Zhang H.,Zhang X.,Yang S.,Yan T.,and Song Z.,2015,Molecular cloning and expression of two heat-shock protein genes(HSC70/HSP70)from Prenant's schizothoracin(Schizothorax prenanti),Fish Physiology&Biochemistry,41(2):573-585
Liu T.,Pan L.,Cai Y.,and Miao J.,2015,Molecular cloning and sequence analysis of heat shock proteins 70(HSP70)and 90(HSP90)and their expression analysis when exposed to benzo(a)pyrene in the clam Ruditapes philippinarum,Gene,555(2):108-118
Madeira D.,Narciso L.,Cabral H.N.,Diniz M.S.,and Vinagre C.,2014,Role of thermal niche in the cellular response to thermal stress:Lipid peroxidation and HSP70 expression in coastal crabs,Ecological Indicators,36(1):601-606
Mallouk Y.,Vayssier-taussat M.,Bonventre J.V.,and Polla B.S.,1999,Heat shock protein 70 and ATP as partners in cell homeostasis(Review),International Journal of Molecular Medicine,4(5):463-474
Mohamed B.A.,Barakat A.Z.,Zimmermann W.H.,Bittner R.E.,Mühlfeld C.,Hünlich M.,Engel W.,Maier L.S.,and Adham I.M.,2012,Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis,Journal of Molecular&Cellular Cardiology,53(4):459-468
Molina A.,Biemar F.,Müller F.,Iyengar A.,Prunet P.,Maclean N.,Martial J.A.,and Muller M.,2000,Cloning and expression analysis of an inducible HSP70 gene from tilapia fish,FEBS Letters,474(1):5-10
Morales M.,PlanellóR.,Martínezpaz P.,Herrero O.,Cortés E.,Martínezguitarte J.L.,and Morcillo G.,2011,Characterization of Hsp70 gene in Chironomus riparius:expression in response to endocrine disrupting pollutants as a marker of ecotoxicological stress,Comparative Biochemistry&Physiology Part C,153(1):150-158
Rui S.,2012,A Dancer Caught midstep:the structure of ATP-Bound Hsp70,Molecular Cell,48(6):821-823
Uhlinger K.R.,Jackson S.A.,and Cherr G.N.,1998,Induced thermotolerance and the heat shock protein-70 family in the Pacific oyster Crassostrea gigas,Molecular Marine Biology&Biotechnology,7(1):21-30
Valenzuela-Castillo A.,Sánchezpaz A.,Castrolongoria R.,López-Torres M.A.,and Grijalva-Chon J.M.,2015,Seasonal changes in gene expression and polymorphism of hsp70 in cultivated oysters(Crassostrea gigas)at extreme temperatures,Marine Environmental Research,110:25-32
Vos M.J.,Hageman J.,Carra S.,and Kampinga H.H.,2008,Structural and functional diversities between members of the human HSPB,HSPH,HSPA,and DNAJ chaperone families,Biochemistry,47(27):7001-7011
Xu Y.,Zheng G.,Dong S.,Liu G.,and Yu X.,2014,Molecular cloning,characterization and expression analysis of HSP60,HSP70 and HSP90 in the golden apple snail,Pomacea canaliculata,Fish&Shellfish Immunology,41(2):643-653
Besson M.,Vandeputte M.,van Arendonk J.A.M.,Aubin J.,Boer I.J.M.D.,Quillet E.,and Komen H.,2016,Influence of water temperature on the economic value of growth rate in fish farming:The case of sea bass(Dicentrarchus labrax)cage farming in the Mediterranean,Aquaculture,462:47-55
Cheng P.,Liu X.,Zhang G.,and He J.,2007,Cloning and expression analysis of a HSP70 gene from Pacific abalone(Haliotis discus hannai),Fish&Shellfish Immunology,22(1-2):77-87
Daugaard M.,Rohde M.,and JaattelaM.,2007,The heat shock protein 70 family:Highly homologous proteins with overlapping and distinct functions,FEBS Letters,581(19):3702-3710
Deane E.E.,and Woo N.Y.,2005,Cloning and characterization of the hsp70 multigene family from silver sea bream:Modulated gene expression between warm and cold temperature acclimation,Biochem.Biophys.Res.Commun.,330(3):776-783
Farcy E.,Serpentini A.,Fievet B.,and Lebel J.,2007,Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata:Transcriptional induction in response to thermal stress in hemocyte primary culture,Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,146(4):540-550
Franzellitti S.,and Fabbri E.,2005,Differential HSP70 gene expression in the mediterranean mussel exposed to various stressors,Biochemical&Biophysical Research Communications,336(4):1157-1163
Green T.J.,Montagnani C.,Benkendorff K.,Robinson N.,and Speck P.,2014,Ontogeny and water temperature influences the antiviral response of the Pacific oyster,Crassostrea gigas,Fish&Shellfish Immunology,36(1):151-157
Gupta R.S.,and Singh B.,1994,Phylogenetic analysis of 70 kDheat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus,Current Biology Cb,4(12):1104-1114
Hamdoun A.M.,and Cherr G.N.,2003,Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific Oyster(Crassostrea gigas):implications for thermal limits and induction of thermal tolerance,Biological Bulletin,205(2):160-169
Han Y.L.,Yang W.X.,Long L.L.,Sheng Z.,Zhou Y.,Zhao Y.Q.,Wang Y.F.,and Zhu J.Q.,2016,Molecular cloning,expression pattern,and chemical analysis of heat shock protein 70(HSP70)in the mudskipper Boleophthalmus pectinirostris:Evidence for its role in regulating spermatogenesis,Gene,575(2):331-338
Hao R.J.,Zheng Z.,Wang Q.H.,Deng Y.W.,Jiao Y.,and Du X.D.,2015,Molecular characterization and expression analysis of PmCHST9 gene from sulfotransferase of Pinctada martensii,Jiyinzuexue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2387-2394(郝瑞娟,郑哲,王庆恒,邓岳文,焦钰,杜晓东,2015,马氏珠母贝磺基转移酶PmCHST9基因的分子特征与表达分析,基因组学与应用生物学,34(11):2387-2394)
Hightower L.E.,1991,Heat shock,stress proteins,chaperones,and proteotoxicity,Cell,66(2):191-197
Kiang J.G.,and Tsokos G.C.,1998,Heat shock protein 70 k D:molecular biology,biochemistry,and physiology,Pharmacology&Therapeutics,80(2):183-201
Li J.,Zhang H.,Zhang X.,Yang S.,Yan T.,and Song Z.,2015,Molecular cloning and expression of two heat-shock protein genes(HSC70/HSP70)from Prenant's schizothoracin(Schizothorax prenanti),Fish Physiology&Biochemistry,41(2):573-585
Liu T.,Pan L.,Cai Y.,and Miao J.,2015,Molecular cloning and sequence analysis of heat shock proteins 70(HSP70)and 90(HSP90)and their expression analysis when exposed to benzo(a)pyrene in the clam Ruditapes philippinarum,Gene,555(2):108-118
Madeira D.,Narciso L.,Cabral H.N.,Diniz M.S.,and Vinagre C.,2014,Role of thermal niche in the cellular response to thermal stress:Lipid peroxidation and HSP70 expression in coastal crabs,Ecological Indicators,36(1):601-606
Mallouk Y.,Vayssier-taussat M.,Bonventre J.V.,and Polla B.S.,1999,Heat shock protein 70 and ATP as partners in cell homeostasis(Review),International Journal of Molecular Medicine,4(5):463-474
Mohamed B.A.,Barakat A.Z.,Zimmermann W.H.,Bittner R.E.,Mühlfeld C.,Hünlich M.,Engel W.,Maier L.S.,and Adham I.M.,2012,Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis,Journal of Molecular&Cellular Cardiology,53(4):459-468
Molina A.,Biemar F.,Müller F.,Iyengar A.,Prunet P.,Maclean N.,Martial J.A.,and Muller M.,2000,Cloning and expression analysis of an inducible HSP70 gene from tilapia fish,FEBS Letters,474(1):5-10
Morales M.,PlanellóR.,Martínezpaz P.,Herrero O.,Cortés E.,Martínezguitarte J.L.,and Morcillo G.,2011,Characterization of Hsp70 gene in Chironomus riparius:expression in response to endocrine disrupting pollutants as a marker of ecotoxicological stress,Comparative Biochemistry&Physiology Part C,153(1):150-158
Rui S.,2012,A Dancer Caught midstep:the structure of ATP-Bound Hsp70,Molecular Cell,48(6):821-823
Uhlinger K.R.,Jackson S.A.,and Cherr G.N.,1998,Induced thermotolerance and the heat shock protein-70 family in the Pacific oyster Crassostrea gigas,Molecular Marine Biology&Biotechnology,7(1):21-30
Valenzuela-Castillo A.,Sánchezpaz A.,Castrolongoria R.,López-Torres M.A.,and Grijalva-Chon J.M.,2015,Seasonal changes in gene expression and polymorphism of hsp70 in cultivated oysters(Crassostrea gigas)at extreme temperatures,Marine Environmental Research,110:25-32
Vos M.J.,Hageman J.,Carra S.,and Kampinga H.H.,2008,Structural and functional diversities between members of the human HSPB,HSPH,HSPA,and DNAJ chaperone families,Biochemistry,47(27):7001-7011
Xu Y.,Zheng G.,Dong S.,Liu G.,and Yu X.,2014,Molecular cloning,characterization and expression analysis of HSP60,HSP70 and HSP90 in the golden apple snail,Pomacea canaliculata,Fish&Shellfish Immunology,41(2):643-653