利用蛋白抗体芯片筛选盐胁迫下红罗非鱼鳃差异表达蛋白
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摘要
为从蛋白角度探索红罗非鱼(Oreochromis mossambicus♀×O.niloticus♂)在盐胁迫环境下的调节适应机制,本研究采用蛋白抗体芯片结合串联质谱技术初步筛选了盐胁迫下红罗非鱼鳃组织的差异表达蛋白,并通过免疫组化和免疫印迹技术对候选差异蛋白进行了验证。结果显示,共筛选获得181个表达量有显著差异的蛋白质(变化倍数≥1.5),包含上调蛋白142个,下调蛋白39个。挑选5个蛋白进行质谱鉴定得到3个蛋白质:中间丝蛋白(intermediate filament protein,IF)、转运蛋白63(translocation protein 63,SEC63)、蛋白质二硫键异构酶A3(protein disulfide-isomerase A3,PDIA3)。免疫组化结果显示,在淡水、盐度组中,IF和PDIA3在鳃小片基部均有阳性反应,且随盐度升高,阳性反应呈先降低后增强的趋势,SEC63无阳性反应。Western blot结果显示,在淡水和盐度组中,IF和PDIA3两种蛋白均有表达,并且随盐度升高呈现先降低、再升高的变化趋势,SEC63蛋白无明显目的条带;在30盐度组中,胁迫早期,IF蛋白表达量降低,48 h达到最低值,72 h有明显回升,PDIA3蛋白表达量在胁迫后96 h显著升高(P<0.05)。根据研究结果推测,IF和PDIA3是在盐胁迫环境下红罗非鱼鳃组织的响应蛋白,它们分别在细胞骨架、内质网功能维持中发挥重要调节作用。
Due to its rapid growth rate and excellent adaptability to saline environments, the red tilapia(Oreochromis mossambicus♀ ×O. niloticus♂) could be a suitable subject for studies on saline tolerance. In this study, to provide a theoretical basis for saline tolerance mechanisms, we aimed to screen and identify differentially expressed proteins in the gill of Red tilapia using protein chips and mass spectrometry, which were subsequently verified by using immunohistochemistry and western blot techniques. There were 181 differentially expressed protein spots detected(change multiple ≥ 1.5), which included 142 up-regulated proteins and 39 down-regulated proteins. Among these, three proteins were identified by mass spectrometry from five differentially expressed proteins: intermediate filament protein(IF), translocation protein 63(SEC63), and disulfide-isomerase A3(PDIA3). The immunohistochemistry results showed that IF and PDIA3 are expressed in the gill base of red tilapia under both freshwater and saline water conditions, and showed a trend of initial decrease and then increase with an increase of saline concentration. However, no positive reaction was detected for SEC63. The western blot results showed IF and PDIA3 expression level changes in different saline waters similar to those observed using immunohistochemistry. In the 30 g/L salinity group, the expression of IF protein decreased during the early stage of stress, reached the lowest value at 48 h, and thereafter began to rise significantly at 72 h, The expression of PDIA3 protein was significantly increased after 96 h(P < 0.05). These results suggest that IF and PDIA3 are positively responsive proteins in the gill tissues of red tilapia under salinity stress, and play important roles in the regulation of cytoskeleton and endoplasmic reticulum function.
Due to its rapid growth rate and excellent adaptability to saline environments, the red tilapia(Oreochromis mossambicus♀ ×O. niloticus♂) could be a suitable subject for studies on saline tolerance. In this study, to provide a theoretical basis for saline tolerance mechanisms, we aimed to screen and identify differentially expressed proteins in the gill of Red tilapia using protein chips and mass spectrometry, which were subsequently verified by using immunohistochemistry and western blot techniques. There were 181 differentially expressed protein spots detected(change multiple ≥ 1.5), which included 142 up-regulated proteins and 39 down-regulated proteins. Among these, three proteins were identified by mass spectrometry from five differentially expressed proteins: intermediate filament protein(IF), translocation protein 63(SEC63), and disulfide-isomerase A3(PDIA3). The immunohistochemistry results showed that IF and PDIA3 are expressed in the gill base of red tilapia under both freshwater and saline water conditions, and showed a trend of initial decrease and then increase with an increase of saline concentration. However, no positive reaction was detected for SEC63. The western blot results showed IF and PDIA3 expression level changes in different saline waters similar to those observed using immunohistochemistry. In the 30 g/L salinity group, the expression of IF protein decreased during the early stage of stress, reached the lowest value at 48 h, and thereafter began to rise significantly at 72 h, The expression of PDIA3 protein was significantly increased after 96 h(P < 0.05). These results suggest that IF and PDIA3 are positively responsive proteins in the gill tissues of red tilapia under salinity stress, and play important roles in the regulation of cytoskeleton and endoplasmic reticulum function.
引文
[1]Hwang P P,Lee T H.New insights into fish ion regulation and mitochondrion-rich cells[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2007,148(3):479-497.
[2]Tang C H,Hwang L Y,Lee T H.Chloride channel Cl C-3 in gills of the euryhaline teleost,Tetraodon nigroviridis:expression,localization and the possible role of chloride absorption[J].Journal of Experimental Biology,2010,213(5):683-693.
[3]Kültz D,Bastrop R,Jürss K,et al.Mitochondria-rich(MR)cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,102(2):293-301.
[4]Hiroi J,Mc Cormick S D.New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish[J].Respiratory Physiology&Neurobiology,2012,184(3):257-268.
[5]Chang I C,Lee T H,Yang C H,et al.Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments[J].Physiological&Biochemical Zoology,2001,74(1):111-119.
[6]Evans D H,Piermarini P M,Choe K P.The multifunctional fish gill:dominant site of gas exchange,osmoregulation,acid-base regulation,and excretion of nitrogenous waste[J].Physiological Reviews,2005,85(1):97-177.
[7]Hiroi J,Mc Cormick S D,Ohtani-Kaneko R,et al.Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia(Oreochromis mossambicus)embryos,by means of triple immunofluorescence staining for Na+/K+-ATPase,Na+/K+/2Cl–cotransporter and CFTR anion channel[J].Journal of Experimental Biology,2005,208(Pt11):2023-2036.
[8]Marshall W S.Mechanosensitive signalling in fish gill and other ion transporting epithelia[J].Acta Physiologica,2011,202(3):487-499.
[9]Singer T D,Tucker S J,Marshall W S,et al.A divergent CFTR homologue:highly regulated salt transport in the euryhaline teleost F.heteroclitus[J].American Journal of Physiology-Cell Physiology,1998,274(3 Pt 1):C715-723.
[10]Wu Y C,Lin L Y,Lee T H.Na+,K+,2Cl–cotransporter:a novel marker for identifying freshwater-and seawater-type mitochondria-rich cells in gills of the euryhaline tilapia,Oreochromis mossambicus[J].Zoological Studies,2003,42(1):186-192.
[11]Evans T G,Somero G N.A microarray-based transcriptomic time-course of hyper-and hypo-osmotic stress signaling events in the euryhaline fish Gillichthys mirabilis:osmosensors to effectors[J].Journal of Experimental Biology,2008,211(Pt22):3636-3649.
[12]Lin C H,Huang C L,Yang C H,et al.Time-course changes in the expression of Na,K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia(Oreochromis mossambicus)during freshwater acclimation[J].Journal of Experimental Zoology Part A:Comparative Experimental Biology,2004,301(1):85-96.
[13]Scott G R,Richards J G,Forbush B,et al.Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer[J].American Journal of Physiology-Cell Physiology,2004,287(2):C300-C309.
[14]Xu Z L,Gan L,Li T Y,et al.Transcriptome profiling and molecular pathway analysis of genes in association with salinity adaptation in Nile tilapia Oreochromis niloticus[J].PLo S ONE,2015,10(8):e0136506.
[15]Zhang C X,Liu H P,Tang Z M,et al.Cell detection based on protein array using modified glass slides[J].Electrophoresis,2003,24(18):3279-3283.
[16]Zhu J,Zhu Y M,Han J X.Application of protein chip technology in the proteomics research[J].International Journal of Laboratory Medicine,2004,25(3):223-225.[胡洁,朱有名,韩金祥.蛋白质芯片技术在蛋白质组研究领域的应用[J].国际检验医学杂志,2004,25(3):223-225.]
[17]Zhu D,Zhang C H,Huang H X,et al.Effect of Jinhuang Fuzheng powder on cytokines of immunosuppressive mice with protein antibody micro-array[J].China Journal of Chinese Materia,2012,37(23):3624-3627.[朱丹,张春花,黄慧学,等.蛋白质抗体芯片技术检测金黄扶正散对免疫抑制小鼠细胞因子的影响[J].中国中药杂志,2012,37(23):3624-3627.]
[18]Thammaratsuntorn J.Biochemical,cellular and molecular responses of red tilapia(Oreochromis niloticus♀×O.mossambicus♂)to salinity and alkalinity stresses[D].Shanghai:Shanghai Ocean University,2015:20-54.
[19]He L,Wu K C,Hui X L,et al.Screening of receptors of the peptide CGNSNPKSC that bind specifilcally to tumor vascular endothelium by immunoprecipitation and mass spectromethy[J].Journal of Modern Oncology,2008,16(8):1259-1263.[贺莉,吴开春,惠晓莉,等.应用免疫沉淀-质谱法筛选肿瘤血管靶向肽CGNSNPKSC的结合受体[J].现代肿瘤医学,2008,16(8):1259-1263.]
[20]Zhai Z H.Cell Biology[M].4th Edition.Beijing:Higher Education Press,2011.[翟中和.细胞生物学[M].第四版.北京:高等教育出版社,2011.]
[21]Xue B G.Research of hereditary feature and serum biochemical indexes of character related to cold-temperature tolerance of the turbot[D].Shanghai:Shanghai Ocean University,2011:46-63.[薛宝贵.大菱鲆耐低温性状相关表型遗传特征及生理生化指标研究[D].上海:上海海洋大学,2011:46-63.]
[22]Li M Y,Ji D W,Wu H Q,et al.2-DE Analysis in liver of Pseudosciaena crocea during low temperature stress[J].Journal of Agricultural Biotechnology,2010,18(2):323-328.[李明云,冀德伟,吴海庆,等.低温胁迫下大黄鱼肝脏蛋白质组双向电泳分析[J].农业生物技术学报,2010,18(2):323-328.]
[23]Liao I C,Chang S L.Studies on the feasibility of red tilapia culture in saline water[C]//Proceedings,International Symposium on Tilapia in Aquaculture Nazareth,Israel:Tel Aviv University,1983:524-533.
[24]Farmer G J,Beamish F W H.Oxygen consumption of Tilapia nilotica in relation to swimming speed and salinity[J].Journal of the Fisheries Research Board of Canada,1969,26(11):2807-2821.
[25]Martinez-Palacios C A,Ross L G,Rosado-Vallado M.The effects of salinity on the survival and growth of juvenile Cichlasoma urophthalmus[J].Aquaculture,1990,91(1-2):65-75.
[26]Kaufmann R,Kirsch D,Spengler B.Sequenching of peptides in a time-of-flight mass spectrometer:evaluation of postsource decay following matrix-assisted laser desorption ionisation(MALDI)[J].International Journal of Mass Spectrometry and Ion Processes,1994,131:355-385.
[27]Noiva R,Lennarz W J.Protein disulfide isomerase.A multifunctional protein resident in the lumen of the endoplasmic reticulum[J].Journal of Biological Chemistry,1992,267(6):3553-3556.
[28]Jiang H.Copmarative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to stresses[D].Qingdao:The Institute of Oceanology,Chinese Academy of Sciences,2009:58-106.[蒋昊.中国明对虾在胁迫条件下肝胰脏的差异蛋白质组学研究[D].青岛:中国科学院海洋研究所,2009:58-106.]
[2]Tang C H,Hwang L Y,Lee T H.Chloride channel Cl C-3 in gills of the euryhaline teleost,Tetraodon nigroviridis:expression,localization and the possible role of chloride absorption[J].Journal of Experimental Biology,2010,213(5):683-693.
[3]Kültz D,Bastrop R,Jürss K,et al.Mitochondria-rich(MR)cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,102(2):293-301.
[4]Hiroi J,Mc Cormick S D.New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish[J].Respiratory Physiology&Neurobiology,2012,184(3):257-268.
[5]Chang I C,Lee T H,Yang C H,et al.Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments[J].Physiological&Biochemical Zoology,2001,74(1):111-119.
[6]Evans D H,Piermarini P M,Choe K P.The multifunctional fish gill:dominant site of gas exchange,osmoregulation,acid-base regulation,and excretion of nitrogenous waste[J].Physiological Reviews,2005,85(1):97-177.
[7]Hiroi J,Mc Cormick S D,Ohtani-Kaneko R,et al.Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia(Oreochromis mossambicus)embryos,by means of triple immunofluorescence staining for Na+/K+-ATPase,Na+/K+/2Cl–cotransporter and CFTR anion channel[J].Journal of Experimental Biology,2005,208(Pt11):2023-2036.
[8]Marshall W S.Mechanosensitive signalling in fish gill and other ion transporting epithelia[J].Acta Physiologica,2011,202(3):487-499.
[9]Singer T D,Tucker S J,Marshall W S,et al.A divergent CFTR homologue:highly regulated salt transport in the euryhaline teleost F.heteroclitus[J].American Journal of Physiology-Cell Physiology,1998,274(3 Pt 1):C715-723.
[10]Wu Y C,Lin L Y,Lee T H.Na+,K+,2Cl–cotransporter:a novel marker for identifying freshwater-and seawater-type mitochondria-rich cells in gills of the euryhaline tilapia,Oreochromis mossambicus[J].Zoological Studies,2003,42(1):186-192.
[11]Evans T G,Somero G N.A microarray-based transcriptomic time-course of hyper-and hypo-osmotic stress signaling events in the euryhaline fish Gillichthys mirabilis:osmosensors to effectors[J].Journal of Experimental Biology,2008,211(Pt22):3636-3649.
[12]Lin C H,Huang C L,Yang C H,et al.Time-course changes in the expression of Na,K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia(Oreochromis mossambicus)during freshwater acclimation[J].Journal of Experimental Zoology Part A:Comparative Experimental Biology,2004,301(1):85-96.
[13]Scott G R,Richards J G,Forbush B,et al.Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer[J].American Journal of Physiology-Cell Physiology,2004,287(2):C300-C309.
[14]Xu Z L,Gan L,Li T Y,et al.Transcriptome profiling and molecular pathway analysis of genes in association with salinity adaptation in Nile tilapia Oreochromis niloticus[J].PLo S ONE,2015,10(8):e0136506.
[15]Zhang C X,Liu H P,Tang Z M,et al.Cell detection based on protein array using modified glass slides[J].Electrophoresis,2003,24(18):3279-3283.
[16]Zhu J,Zhu Y M,Han J X.Application of protein chip technology in the proteomics research[J].International Journal of Laboratory Medicine,2004,25(3):223-225.[胡洁,朱有名,韩金祥.蛋白质芯片技术在蛋白质组研究领域的应用[J].国际检验医学杂志,2004,25(3):223-225.]
[17]Zhu D,Zhang C H,Huang H X,et al.Effect of Jinhuang Fuzheng powder on cytokines of immunosuppressive mice with protein antibody micro-array[J].China Journal of Chinese Materia,2012,37(23):3624-3627.[朱丹,张春花,黄慧学,等.蛋白质抗体芯片技术检测金黄扶正散对免疫抑制小鼠细胞因子的影响[J].中国中药杂志,2012,37(23):3624-3627.]
[18]Thammaratsuntorn J.Biochemical,cellular and molecular responses of red tilapia(Oreochromis niloticus♀×O.mossambicus♂)to salinity and alkalinity stresses[D].Shanghai:Shanghai Ocean University,2015:20-54.
[19]He L,Wu K C,Hui X L,et al.Screening of receptors of the peptide CGNSNPKSC that bind specifilcally to tumor vascular endothelium by immunoprecipitation and mass spectromethy[J].Journal of Modern Oncology,2008,16(8):1259-1263.[贺莉,吴开春,惠晓莉,等.应用免疫沉淀-质谱法筛选肿瘤血管靶向肽CGNSNPKSC的结合受体[J].现代肿瘤医学,2008,16(8):1259-1263.]
[20]Zhai Z H.Cell Biology[M].4th Edition.Beijing:Higher Education Press,2011.[翟中和.细胞生物学[M].第四版.北京:高等教育出版社,2011.]
[21]Xue B G.Research of hereditary feature and serum biochemical indexes of character related to cold-temperature tolerance of the turbot[D].Shanghai:Shanghai Ocean University,2011:46-63.[薛宝贵.大菱鲆耐低温性状相关表型遗传特征及生理生化指标研究[D].上海:上海海洋大学,2011:46-63.]
[22]Li M Y,Ji D W,Wu H Q,et al.2-DE Analysis in liver of Pseudosciaena crocea during low temperature stress[J].Journal of Agricultural Biotechnology,2010,18(2):323-328.[李明云,冀德伟,吴海庆,等.低温胁迫下大黄鱼肝脏蛋白质组双向电泳分析[J].农业生物技术学报,2010,18(2):323-328.]
[23]Liao I C,Chang S L.Studies on the feasibility of red tilapia culture in saline water[C]//Proceedings,International Symposium on Tilapia in Aquaculture Nazareth,Israel:Tel Aviv University,1983:524-533.
[24]Farmer G J,Beamish F W H.Oxygen consumption of Tilapia nilotica in relation to swimming speed and salinity[J].Journal of the Fisheries Research Board of Canada,1969,26(11):2807-2821.
[25]Martinez-Palacios C A,Ross L G,Rosado-Vallado M.The effects of salinity on the survival and growth of juvenile Cichlasoma urophthalmus[J].Aquaculture,1990,91(1-2):65-75.
[26]Kaufmann R,Kirsch D,Spengler B.Sequenching of peptides in a time-of-flight mass spectrometer:evaluation of postsource decay following matrix-assisted laser desorption ionisation(MALDI)[J].International Journal of Mass Spectrometry and Ion Processes,1994,131:355-385.
[27]Noiva R,Lennarz W J.Protein disulfide isomerase.A multifunctional protein resident in the lumen of the endoplasmic reticulum[J].Journal of Biological Chemistry,1992,267(6):3553-3556.
[28]Jiang H.Copmarative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to stresses[D].Qingdao:The Institute of Oceanology,Chinese Academy of Sciences,2009:58-106.[蒋昊.中国明对虾在胁迫条件下肝胰脏的差异蛋白质组学研究[D].青岛:中国科学院海洋研究所,2009:58-106.]