急性低盐胁迫下红鳍东方鲀幼鱼IgM、NKCC1和Hsp70基因的表达
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摘要
为探讨红鳍东方鲀(Takifugu rubripes)耐低盐的分子机制,以自然海水组为对照,利用实时荧光定量PCR(q RT-PCR)技术,分析红鳍东方鲀幼鱼在盐度16、12、8和4胁迫下,鳃和肾中免疫球蛋白M(immunoglobulin M,IgM)、钠钾氯协同转运蛋白1(Na-K-Cl cotransporter 1,NKCC1)和热休克蛋白70(heat shock protein 70,Hsp70)3个基因的表达情况。结果表明,3个基因在鳃和肾中均有表达,IgM和Hsp70基因在鳃和肾中的表达量均无显著性差异(P>0.05),而NKCC1基因在鳃中的表达量显著高于肾(P<0.05)。同一盐度下,随着时间的增加,鳃中IgM基因的表达量大致呈现先降低后升高的趋势,肾中则呈现先降低后升高再趋于平稳的趋势;同一时间内,鳃中低盐度组与对照组的IgM基因表达量差异比肾中差异更为明显。在鳃中,相同时间内NKCC1基因在各低盐度组的表达量低于对照组,尤其在6h和72h两个时间点时显著低于对照组(P<0.05);肾中各时间点的表达量基本都高于0h表达量。在相同时间内,3h、6h、24h、72h的各低盐度组,在鳃中,Hsp70基因的表达量均与同一时间对照组之间差异明显(P<0.05);在肾中,从6h开始,各低盐组与对照组之间差异性显著(P<0.05)。以上结果表明,红鳍东方鲀幼鱼IgM、NKCC1、Hsp70 3个基因的表达量在不同盐度不同时间下存在差异,由此推测这3个基因对红鳍东方鲀幼鱼的渗透压调节起重要作用。
Takifugu rubripes is a popular food in China, Japan, and Korea because of its fresh meat and high nutritional value. In recent years, desalination marine fish farming has aroused interest, and T. rubripes is a marine model organism for studying low-salinity aquaculture because of its ability to tolerate a wide range of environmental salinities. This fish species has been used to explore the molecular mechanisms of osmoregulation in marine teleosts due to its small genome, and>95% of the genome has been sequenced. Studies have shown that T. rubripes can survive in hypo-osmotic conditions such as 10%–25% saltwater, but the fish does not survive in freshwater. Therefore, to investigate the molecular mechanism of low-salinity tolerance in T. rubripes, the expression of three genes, including immunoglobulin M(IgM), Na-K-Cl cotransporter 1(NKCC1), and heat shock protein 70(Hsp70) was analyzed using real-time quantitative polymerase chain reaction(q RT-PCR) analysis of the gill and kidney from juvenile T. rubripes under low-salinity stress. T. rubripes juveniles were divided randomly into five groups and maintained in water with salinities of 32(control) 16, 12, 8, and 4 for 72 h. The results show that all three genes were expressed in gill and kidney. IgM and Hsp70 expression levels were not different between the two tissues(P>0.05), but NKCC1 expression level in the gill was significantly higher than that in the kidney(P<0.05). IgM expression decreased initially and then increased in the gill, whereas it decreased initially, increased, and finally tended to stabilize in the kidney in fish under acute low-salinity stress. More notable differences were detected in the gill between the low-salinity and control groups compared with those in the kidney at the same time points. NKCC1 expression decreased in the gill but increased in the kidney under low-salinity stress, and NKCC1 expression levels in the gill were higher at all time points than that at 0 h. NKCC1 expression levels in the gill of fish in the low-salinity groups were significantly lower than those in the control group at 6 and 72 h(P<0.05). Significant differences in gill Hsp70 expression levels were observed in the low-salinity groups at 3 h, 6 h, 24 h, and 72 h compared with those in the control. Changes in kidney Hsp70 expression began at 6 h. Taken together, these results reveal that IgM, NKCC1, and Hsp70 expression levels in the gill and kidney changed when juvenile T. rubripes were maintained in different salinities and for various durations. These results suggest that these three genes play an important role in the low-salinity tolerance of T. rubripes and provide basic data for clarifying the molecular mechanisms of osmoregulation in T. rubripes.
Takifugu rubripes is a popular food in China, Japan, and Korea because of its fresh meat and high nutritional value. In recent years, desalination marine fish farming has aroused interest, and T. rubripes is a marine model organism for studying low-salinity aquaculture because of its ability to tolerate a wide range of environmental salinities. This fish species has been used to explore the molecular mechanisms of osmoregulation in marine teleosts due to its small genome, and>95% of the genome has been sequenced. Studies have shown that T. rubripes can survive in hypo-osmotic conditions such as 10%–25% saltwater, but the fish does not survive in freshwater. Therefore, to investigate the molecular mechanism of low-salinity tolerance in T. rubripes, the expression of three genes, including immunoglobulin M(IgM), Na-K-Cl cotransporter 1(NKCC1), and heat shock protein 70(Hsp70) was analyzed using real-time quantitative polymerase chain reaction(q RT-PCR) analysis of the gill and kidney from juvenile T. rubripes under low-salinity stress. T. rubripes juveniles were divided randomly into five groups and maintained in water with salinities of 32(control) 16, 12, 8, and 4 for 72 h. The results show that all three genes were expressed in gill and kidney. IgM and Hsp70 expression levels were not different between the two tissues(P>0.05), but NKCC1 expression level in the gill was significantly higher than that in the kidney(P<0.05). IgM expression decreased initially and then increased in the gill, whereas it decreased initially, increased, and finally tended to stabilize in the kidney in fish under acute low-salinity stress. More notable differences were detected in the gill between the low-salinity and control groups compared with those in the kidney at the same time points. NKCC1 expression decreased in the gill but increased in the kidney under low-salinity stress, and NKCC1 expression levels in the gill were higher at all time points than that at 0 h. NKCC1 expression levels in the gill of fish in the low-salinity groups were significantly lower than those in the control group at 6 and 72 h(P<0.05). Significant differences in gill Hsp70 expression levels were observed in the low-salinity groups at 3 h, 6 h, 24 h, and 72 h compared with those in the control. Changes in kidney Hsp70 expression began at 6 h. Taken together, these results reveal that IgM, NKCC1, and Hsp70 expression levels in the gill and kidney changed when juvenile T. rubripes were maintained in different salinities and for various durations. These results suggest that these three genes play an important role in the low-salinity tolerance of T. rubripes and provide basic data for clarifying the molecular mechanisms of osmoregulation in T. rubripes.
引文
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[11]Motohashi E,Hasegawa S,Mishiro K,et al.Osmoregulatory responses of expression of vasotocin,isotocin,prolactin and growth hormone genes following hypoosmotic challenge in a stenohaline marine teleost,tiger puffer(Takifugu rubripes)[J].Comp Biochem Physiol,2009,154(3):353-359.
[12]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[13]Su B,Wang D,Liu H B.Research advances in immunoglobulin classification and diversity mechanism in fish[J].Chinese Journal of Fisheries,2013,26(1):60-64.[宿斌,王荻,刘红柏.硬骨鱼类免疫球蛋白分类及多样性产生机制研究进展[J].水产学杂志,2013,26(1):60-64.]
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[15]Yu N,Li J E,Ou Y J,et al.Structural changes in gill and kidney of juvenile grey mullet under different salinity[J].Ecological Science,2012,31(4):424-428.[于娜,李加儿,区又军,等.不同盐度下鲻鱼幼鱼鳃和肾组织结构变化[J].生态科学,2012,31(4):424-428.]
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[17]Hebert S C,Mount D B,Gamba G.Molecular physiology of cationcoupled Cl-cotransport:the SLC12 family[J].Pflügers Arch,2004,447(5):580-593.
[18]Marshall W S,Grosell M.Ion transport,osmoregulation,and acidbase balance[M]//Evans D H,Claiborne J B.The Physiology of Fishes.Boca Raton:CRC Taylor and Franis,2006:177-230.
[19]Inokuchi M,Hiroi J,Watanabe S,et al.Gene expression and morphological localization of NHE3,NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia(Oreochromis mossambicus)acclimated to a wide range of salinities[J].Comp Biochem Physiol,2008,151(2):151-158.
[20]Wang B,Fan W J,Li S F.Tissue-specific changes of NKCC1a m RNA expression by salinity in“GILI”tilapia[J].Journal of Fishery Sciences of China,2011,18(3):515-522.[王兵,范武江,李思发.不同盐度下“吉丽”罗非鱼(尼罗罗非鱼♀×萨罗罗非鱼♂)NKCC1a m RNA的组织特异性表达[J].中国水产科学,2011,18(3):515-522.]
[21]Cutler C P,Cramb G.Two isoforms of the Na+/K+/2Cl-cotransporter are expressed in the European eel(Anguilla anguilla)[J].Biochim Biophys Acta,2002,1566(1-2):92-103.
[22]Tipsmark C K,Madsen S S,Borski R J.Effect of salinity on expression of branchial ion transporters in striped bass(Morone saxatilis)[J].J Exp Zool A:Comp Exp Biol,2004,301(12):979-991.
[23]Wang Y N,Wang H,Luo M M,et al.Effects of simultaneous variation in temperature and salinity on the expression of mantle Hsp70 gene in Pinctada fucata[J].Journal of Guangdong Ocean University,2012,32(3):35-41.[王亚男,王辉,罗明明,等.温度、盐度对马氏珠母贝外套膜Hsp70基因表达量的联合影响[J].广东海洋大学学报,2012,32(3):35-41.]
[24]Deane E E,Kelly S P,Luk J C Y,et al.Chronic salinity adaptation modulates hepatic heat shock protein and insulin-like growth factor I expression in black sea bream[J].Mar Biotechnol,2002,4(2):193-205.
[25]Fangue N A,Myriam H,Schulte P M.Intraspecific variation in thermal tolerance and heat shock protein gene expression in common killifish,Fundulus heteroclitus[J].J Exp Biol,2006,209(15):2859-2872.
[26]Pan F,Zarate J M,Tremblay G C,et al.Cloning and characterization of salmon hsp90 c DNA upregulation by thermal and hyperosmotic stress[J].J Exp Zool,2000,287(3):199-212.
[27]Yang M W,Huang W T,Tsai M J,et al.Transient response of brain heat shock proteins 70 and 90 to acute osmotic stress in Tilapia(Oreochromis mossambicus)[J].Zool Stud,2009,48(6):723-736.
[28]Feder M,Hofmann G.Heat shock proteins,molecular chaperones,and the stress response:Evolutionary and ecological physiology[J].Annu Rev Physiol,1999,61:243-282.
[29]Geething M J,Sambrook J.Protein folding in the cell[J].Nature,1992,355(6355):33-45.
[2]Ma A J,Li W Y,Wang X A.Reasearch progress and outlook of Takifugu rubripes culture techniques[J].Marine Sciences,2014,38(2):116-121.[马爱军,李伟业,王新安.红鳍东方鲀养殖技术研究现状及展望[J].海洋科学,2014,38(2):116-121.]
[3]Shi Z H,Huang X X,Fu R B,et al.Salinity stress on embryos and early larval stages of the pomfret Pampus punctatissimus[J].Aquaculture,2008,275(1-4):306-310.
[4]Tong Y,Chen L Q,Zhuang P,et al.Cortisol,metabolism response and osmoregulation of juvenile Acipenser schrenckii to ambient salinity stress[J].Journal of Fisheries of China,2007,31(z1):38-44.[童燕,陈立侨,庄平,等.急性盐度胁迫对施氏鲟的皮质醇、代谢反应及渗透调节的影响[J].水产学报,2007,31(z1):38-44.]
[5]Lee K M,Kaneko T,Kato F,et al.Prolactin gene expression and gill chloride cell activity in fugu Takifugu rubripes exposed to a hypoosmotic environment[J].Gen Comp Endocrinol,2006,149(3):285-293.
[6]Bodinier C,Nebel C L,Charmantier G,et al.Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of juvenile Dicentrarchus labrax exposed to seawater and freshwater[J].Comp Biochem Physiol,2009,153(3):345–351.
[7]Larsen P F,Nielsen E E,Meier K,et al.Differences in salinity tolerance and gene expression between two populations of Atlantic Cod(Gadus morhua)in response to salinity stress[J].Biochem Genet,2012,50(5–6):454–466.
[8]Huang Z H,Ma A J,Wang X A,et al.Interaction of temperature and salinity on the expression of immunity factors in different tissues of juvenile turbot Scophthalmus maximus based on response surface methodology[J].Chin J Oceanol Limnol,2015,33(1):28-36.
[9]Kang C K,Tsai H J,Liu C C,et al.Salinity-dependent ex-pression of a Na+、K+、2Cl-cotransporter in gills of the brackish medaka Oryzias dancena:A molecular correlate for hyposmoregulatory endurance[J].Comp Biochem Physiol,2010,157(1):7-18.
[10]Lee K M,Kaneko T,Aida K.Prolactin and prolactin receptor expressions in a marine teleost,pufferfish Takifugu rubripes[J].Gen Comp Endocrinol,2006,146(3):318–328.
[11]Motohashi E,Hasegawa S,Mishiro K,et al.Osmoregulatory responses of expression of vasotocin,isotocin,prolactin and growth hormone genes following hypoosmotic challenge in a stenohaline marine teleost,tiger puffer(Takifugu rubripes)[J].Comp Biochem Physiol,2009,154(3):353-359.
[12]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[13]Su B,Wang D,Liu H B.Research advances in immunoglobulin classification and diversity mechanism in fish[J].Chinese Journal of Fisheries,2013,26(1):60-64.[宿斌,王荻,刘红柏.硬骨鱼类免疫球蛋白分类及多样性产生机制研究进展[J].水产学杂志,2013,26(1):60-64.]
[14]LüA J,Hu X C,Zhang Y H,et al.Several new immunoglobulins in fish[J].Fisheries Science,2011,30(7):425-428.[吕爱军,胡秀彩,张艳华.鱼类中新发现的免疫球蛋白[J].水产科学,2011,30(7):425-428.]
[15]Yu N,Li J E,Ou Y J,et al.Structural changes in gill and kidney of juvenile grey mullet under different salinity[J].Ecological Science,2012,31(4):424-428.[于娜,李加儿,区又军,等.不同盐度下鲻鱼幼鱼鳃和肾组织结构变化[J].生态科学,2012,31(4):424-428.]
[16]Gamba G.Molecular physiology and pathophysiology of electroneutral cation chloride cotransporters[J].Physiol Rev,2005,85(2):423-493.
[17]Hebert S C,Mount D B,Gamba G.Molecular physiology of cationcoupled Cl-cotransport:the SLC12 family[J].Pflügers Arch,2004,447(5):580-593.
[18]Marshall W S,Grosell M.Ion transport,osmoregulation,and acidbase balance[M]//Evans D H,Claiborne J B.The Physiology of Fishes.Boca Raton:CRC Taylor and Franis,2006:177-230.
[19]Inokuchi M,Hiroi J,Watanabe S,et al.Gene expression and morphological localization of NHE3,NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia(Oreochromis mossambicus)acclimated to a wide range of salinities[J].Comp Biochem Physiol,2008,151(2):151-158.
[20]Wang B,Fan W J,Li S F.Tissue-specific changes of NKCC1a m RNA expression by salinity in“GILI”tilapia[J].Journal of Fishery Sciences of China,2011,18(3):515-522.[王兵,范武江,李思发.不同盐度下“吉丽”罗非鱼(尼罗罗非鱼♀×萨罗罗非鱼♂)NKCC1a m RNA的组织特异性表达[J].中国水产科学,2011,18(3):515-522.]
[21]Cutler C P,Cramb G.Two isoforms of the Na+/K+/2Cl-cotransporter are expressed in the European eel(Anguilla anguilla)[J].Biochim Biophys Acta,2002,1566(1-2):92-103.
[22]Tipsmark C K,Madsen S S,Borski R J.Effect of salinity on expression of branchial ion transporters in striped bass(Morone saxatilis)[J].J Exp Zool A:Comp Exp Biol,2004,301(12):979-991.
[23]Wang Y N,Wang H,Luo M M,et al.Effects of simultaneous variation in temperature and salinity on the expression of mantle Hsp70 gene in Pinctada fucata[J].Journal of Guangdong Ocean University,2012,32(3):35-41.[王亚男,王辉,罗明明,等.温度、盐度对马氏珠母贝外套膜Hsp70基因表达量的联合影响[J].广东海洋大学学报,2012,32(3):35-41.]
[24]Deane E E,Kelly S P,Luk J C Y,et al.Chronic salinity adaptation modulates hepatic heat shock protein and insulin-like growth factor I expression in black sea bream[J].Mar Biotechnol,2002,4(2):193-205.
[25]Fangue N A,Myriam H,Schulte P M.Intraspecific variation in thermal tolerance and heat shock protein gene expression in common killifish,Fundulus heteroclitus[J].J Exp Biol,2006,209(15):2859-2872.
[26]Pan F,Zarate J M,Tremblay G C,et al.Cloning and characterization of salmon hsp90 c DNA upregulation by thermal and hyperosmotic stress[J].J Exp Zool,2000,287(3):199-212.
[27]Yang M W,Huang W T,Tsai M J,et al.Transient response of brain heat shock proteins 70 and 90 to acute osmotic stress in Tilapia(Oreochromis mossambicus)[J].Zool Stud,2009,48(6):723-736.
[28]Feder M,Hofmann G.Heat shock proteins,molecular chaperones,and the stress response:Evolutionary and ecological physiology[J].Annu Rev Physiol,1999,61:243-282.
[29]Geething M J,Sambrook J.Protein folding in the cell[J].Nature,1992,355(6355):33-45.