Screening the Differential Expressed Proteins by Protein Chip in Gill of Red Tilapia (Oreochromis mossambicus ♀ × O. niloticus ♂) Under Alkalinity Stress
Abstract：Euryhalinous teleosts can survive in certain range saline-alkali water environment. The Gill is considered to be the main organ which is responsible for ion transport , maintain osmotic pressure balance and acid-base equilibrium.This study was to screen and identify the differential expressed proteins in gill of the Red tilapia (Oreochromis mossambicus ♀× O. niloticus ♂) by protein chips and mass spectrometry techniques, then verified by using immunohistochemistry and Western blot techniques, and to provide protein theoretical basis for alkali-resistant mechanism. There were 229 differential expressed protein spots detected (change multiple≥1.5), which contains 41 up-regulated proteins and 49 down-regulated proteins. Among them, 2 proteins were identified by mass spectrometry from 5 differential expressed proteins: 14-3-3 protein beta/alpha-A-like isoform X1 (14-3-3), Dna J homolog subfamily β member 11-like (Dna J).The immunohistochemistry results showed that 14-3-3 and Dna J were expressed in the gill base of Red tilapia at fresh water and alkalinity water, and showed a trend of first decrease and then the positive reaction increased significantly with increasing alkalinity. The results of Western blot showed that in the fresh water and alkali water, 14-3-3 and Dna J proteins were both expressed in the gill. With increasing alkalinity, the protein expression was the same as the immunohistochemistry. The expression of 14-3-3 protein showed a trend of increase with the increase of alkalinity concentration, and the expression of Dna J protein was significantly increased at alkalinity water which compare with fresh water. In the 4 alkalinity group , 14-3-3 protein reduced in the early stage of stress,then increased, and reached the lowest value at 12 h, and the peak value rised at 72 h. While the expression of Dna J protein was significantly increased at 96 h (P＜0.05). The results suggest that red tilapia is able to cope with protein activitity and stability in the body by activating the protein 14-3-3 and Dna J protein in the gill tissue, thereby maintaining the body protein normal function stability.The expression of 14-3-3 and Dna J proteins in gill of red tilapia was increased under alkalinity stress. Thus it would have great significance to support that 14-3-3 and Dna J proteins are both participated in the response of alkalinity stress.The present study gives more information to learn the mechanism of alkalinity stress response in red tilapia.
郭义敏, 刘颖, 陈历明. 近端肾小管碳酸氢根重吸收的分子机制及代谢性酸中毒[J]. 生理学报, 2014, 66: 398-414.(Guo Y M, Liu Y, Chen L M, et al. Bicarbonate reabsorption in proximal renal tubule: molecular mechanisms and metabolic acidosis[J]. Acta Physiologica Sinica, 2014, 66: 398-414. )何强,常玉梅,苏宝峰,等.碳酸盐碱度对达里湖瓦氏雅罗鱼耗氧率、氨氮排泄和排氨基因表达的影响[J]. 上海海洋大学学报, 2016, 25(4):551-558.(He Q, Chang Y M, Su B F, et al. Effects of carbonate alkalinities on oxygen consumption, ammonia excretionand ammonia excretion gene expression in Leuciscus waleckii Dybowski[J]. Journal of Shanghai Ocean University, 2016, 25(4):551-558. )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.(Hu 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. )黄思颖,赵金良,王燕,等.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[J].中国水产科学,2018,25(1):1-8.(Huang S Y, Zhao J N, Wang Y,et al.Screening the differential expressed proteins by protein chip in gill of Re d tilapia（Oreochromis mossambicus× O. niloticus） under salinity stress [J]. Journal of Fishery Sciences of China, 2018, 25(1): 1-8. )蒋玫,李磊,沈新强, 等. 鲻鱼鳃组织14-3-3a、NKCCla、Apo-14和Na+-K+-ATPaseβ基因表达对盐度变化的响应[J]. 生态学杂志, 2014,33(9):2429-2435.(Jiang M, Li L, Shen X Q, et al. Effects of abrupt salinity stress on gill 14-3-3a, NKCCla, Apo-14 and Na+-K+-ATPaseβ expression of Mugil cephalus[J]. Chinese Journal of Ecology, 2014,33(9):2429-2435. )梁从飞, 赵金良, 甘远迪,等. 盐碱胁迫对尼罗罗非鱼鳃Na+/HCO3-共转运子、碳酸酐酶基因表达的影响[J]. 中国水产科学, 2016, 23(2):274-283.(Liang C F, Zhao J N, Gan Y D, et al. Effects of salinity and alkalinity on mRNA expression of Na+/HCO3- cotransporter and carbonic anhydrase genes from Oreochromis niloticus.[J]. Journal of Fishery Sciences of China, 2016, 23(2):274-283. )梁利群,任波,常玉梅,等.中国内陆咸(盐碱)水资源及渔业综合开发利用[J].中国渔业经济, 2013,31(4):138-145.(Liang L Q, Ren B, Chang Y M, et al. Inland brackish (alkaline-saline) water resources and fisheries utilization in China[J].Chinese Fisheries Economics, 2013,31(4):138-145. )刘济源, 么宗利, 来琦芳,等. 盐碱胁迫对青海湖裸鲤耗氧率、血浆渗透浓度和离子浓度的影响[J]. 生态学杂志, 2012, 31(3): 664-669.(Liu J Y, Me Z L, Lai Q F, et al. Effects of saline-alkali stress on the oxygen consumption and plasma osmolality and ion concentrations of Gymnocypris przewalskii[J].Chinese Journal of Ecology, 2012, 31(3): 664-669. )刘永新,方辉,来琦芳,等. 我国盐碱水渔业现状与发展对策[J]. 中国工程科学, 2016,18(3):74-78.(Liu Y Y, Fang H, Lai Q F, et al. The current state and development strategy for China’s saline-alkaline fisheries[J]. Engineering Sciences, 2016,18(3): 74-78.)柳斌,杨金亮,宋鑫,等.应用蛋白印迹-免疫沉淀-质谱法筛选卵巢癌肿瘤抗原[J]. 癌症, 2005, 24(7):890-892.(Liu B, Yang J L, Song X, et al. Screening tumor antigens of Ovarian Carcinoma by Western blot, Immunoprecipitation, and Mass Spectrometry [J].Chinese Journal of Cancer, 2005,24(7): 890-892. )唐韬,孟峻. 14-3-3蛋白功能的研究进展[J]. 检验医学与临床, 2015,12(11):1628-1631.(Tang T, Meng J. Research progress of 14-3-3 protein function[J]. Laboratory Medicine and Clinic, 2015, 12(11): 1628-1631. )王书平,孔祥会. 鱼类14-3-3基因家族研究进展[J]. 河南农业科学, 2010, 39(10): 138-142.(Wang S P, Kong X H. Research progress of 14-3-3 gene family in fish[J]. Journal of Henan Agricultural Sciences, 2010, 39(10): 138-142. )王燕, 赵金良,赵岩,等. 碱度对尼罗罗非鱼血清渗透压、离子浓度及离子转运酶活力的影响[J]. 生态科学, 2017, 36(4): 12-20.(Wang Y, Zhao J N, Zhao Y, et al. Influence of alkalinity on serum osmolality, ion concentration and ion transport enzymes activity of Oreochromis niloticus[J]. Ecological Science, 2017, 36(4): 12-20. )王芸, 李健, 张喆,等. pH、氨氮胁迫对中国对虾HSP90基因表达的影响[J]. 渔业科学进展, 2013, 34(5):43-50.(Wang Y, Li J, Zhang Z, et al. Effects of pH and ammonia-N stresses on HSP90 gene expression of Fenneropenaeus chinensis[J]. Progress in Fishery Sciences, 2013, 34(5):43-5. )吴俊伟. 尼罗罗非鱼耐盐碱选育二代耐受性、生长性能及碱胁迫下氨代谢基因表达变化[D]. 硕士学位论文，上海海洋大学, 上海. 2016.(Wu J W. Tolerance, growth of the second selected generation of Oreochromis niloticus in saline and alkaline water and ammonia metabolism genes expression change under alkalinity stress [D]. Shanghai Ocean Univercity, Shanghai. 2016.)赵丽慧,赵金良,Thammaratsuntorn Jeerawat,等. 盐碱胁迫对尼罗罗非鱼血清渗透压、离子浓度及离子转运酶基因表达的影响[J]. 水产学报, 2014, 38(10):1696-1704.(Zhao L H, Zhao J N, Thammaratsuntorn J, et al. Effects of salinity-alkalinity on serum osmolality，ion concentration and mRNA expression of ion transport enzymes of Oreochromis niloticus. [J]. Journal of Fisheries of China, 2014, 38(10):1696-1704. )赵岩,吴俊伟,孟森,等. 碳酸盐碱度胁迫对尼罗罗非鱼血清pH、游离氨浓度及相关基因表达的影响[J]. 南方农业学报, 2016,47(6):1032-1038.(Zhao Y, Wu J W, Meng S, et al. Effects of carbonate alkalinity on serum pH, ammonia concentration and related gene expression in Oreochromis niloticus[J]. Journal of Southern Agriculture, 2016,47(6):1032-1038. )赵志常,张建军,张皖蓉,等. 拟南芥Dna J蛋白的过量表达对细菌耐盐性的影响[J]. 广西师范大学学报(自然科学版), 2010, 28(1):54-57.(Zhao Z C, Zhang J J, Zhang W R, et al. Over-expression of Arabidopsis DnaJ (Hsp40) contributes to NaCl-stress tolerance of Escherichia coli[J]. Journal of Guangxi Normal University(Natural Science Edition), 2010, 28(1):54-57. )郑伟刚,张兆琪,张美昭,等. 盐度与碱度对花鲈幼鱼的毒性研究[J]. 中国农业生态学报, 2005, 13(3): 116-118 .(Zheng W G, Zhang Z Q, Zhang M Z, et al. Toxicity of salinity and alkalinity to Lateolabrax japonicus fingerlings. [J]. Chinese Journal of Eco-Agriculture, 2005, 13(3): 116-118. )Besser J, Bagowski C P, SalasVidal E, et al. Expression analysis of the family of 14-3-3 proteins in zebrafish development[J]. Gene Expression Patterns, 2007, 7(4):511-520. Caputo E, Lombardi M L, Luongo V. et al. Peptide profiling in epithelial tumor plasma by the emerging proteomic techniques[J]. Journal of Chromatography B Analytical Technologies in the Biomedical & Life Sciences 2005, 819: 59.Craig A, Huang P, Aron R, et al. The diverse roles of J proteins, the obligate Hsp70 co-chaperone[J]. Reviews of Physiology Biochemistry and Pharmacology, 2006, 156: 1-21.Chen Kunming, Holmstrom R W, Suorsa M, et al. Small chloroplast-targeted Dna J proteins are involved in optimization of photosynthetic reactions in Arabidopsis thaliana[J]. BMC Plant Biology, 2010, 10: 43.Fu H，Subramanian R R，Masters S C. 14-3-3 proteins: Structure, function, and regulation[J]. Annual Review of Pharmacology and Toxicology, 2000,40(1): 617-647.Hwang P P, Lee T H, Lin L Y. Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[J].Am J Physiol-Regul Integr Comp Physiol, 2011, 301(1):R28–R47.Hiroi J, McCormick 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.Kaur J, Bachhawat A K. A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein[J]. Analytical Biochemistry, 2009, 384(2):348.Kohn A, Chakravarty D, Kültz D. Teleost Fh14‐3‐3a protein protects xenopus oocytes from hyperosmolality[J]. Journal of Experimental Zoology Part A Ecological Genetics & Physiology, 2010, 299A(2):103-109.Koskinen H, Krasnov A, Rexroad C, et al. The 14-3-3 proteins in the teleost fish rainbow trout (Oncorhynchus mykiss)[J]. The Journal of Experimental Biology, 2004, 207(19):3361-3368.Kǜltz D, Chakravary D, Adilakshmi T. A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus[J].The Journal of Experimental Biology, 2001, 204: 2975-2985.Lam S H, Lui E Y, Li Z, et al. Differential transcriptomic analyses revealed genes and signaling pathways involved in iono-osmoregulation and cellular remodeling in the gills of euryhaline Mozambique tilapia, Oreochromis mossambicus[J]. BMC Genomics, 2014, 15(1):1-23.Marshall W S. Na+, Cl–, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis[J]. Journal of Experimental Zoology Part A Ecological Genetics & Physiology,2002, 293(3): 264–283．Miernyk J A. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones[J]. Cell Stress & Chaperones, 2001, 6(3): 209-218. Morimoto R I. Cells in stress: transcriptional activation of heat shock genes.[J]. Science, 1993, 259(5100):1409-10.Ohana E, Yang D, Shcheynikov N, et al. 2009. Diverse transport modes by the solute carrier 26 family of anion transporters[J]. Journal of Physiology, 2009, 587: 2179-2185.Parker M D, Boron W F. 2013. The divergence, actions, roles, and relatives of sodium coupled bicarbonate transporters[J]. Physiol Rev, 2013, 93: 803-959.Romero M F, Fulton C M, Boron W F. 2004. The SLC4 family of HCO3- transporters[J]. Pflügers Archiv: European Journal of Physiology, 2004,447(5): 495-509.Shikano S, Coblitz B, Wu M, et al. 14-3-3 proteins: regulation of endoplasmic reticulum localization and surface expression of membrane proteins[J]. Trends in Cell Biology, 2006,16(7): 370-375 T?R?K Z, Horv? I, Goloubinoff P. et al. Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions[J]. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94: 2192-2197.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.Wood C M. Toxic response of the gill. In: SCHLENK, D.; BENSON, W.H. Target organ toxicity in marine and freshwater teleosts[M]. London: Taylor & Francis, 2001: 1-89.Warapond W, Caird E R, Yao J B. Identification of a functional splice variant of 14-3-3 E1 in rainbow trout[ J]. Mar Biotechnol, 2010, 12(1):70-80.Xu Z, Gan L, Li T, et al. Transcriptome profiling and molecular pathway analysis of genes in association with salinity adaptation in Nile tilapia Oreochromis niloticus[J]. Plos One, 2015, 10(8):e0136506.
