碳酸盐碱胁迫下尼罗罗非鱼氨代谢两种途径时序研究
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摘要
为了解罗非鱼在碱水环境适应过程中的氨代谢机制,将尼罗罗非鱼(Oreochromis niloticus)放在(2、4、6 g/L)碳酸盐(Na HCO3)碱水环境中进行急性胁迫。检测碱胁迫72 h内的血氨浓度,肝、肾、鳃组织及水体、尿液、血液尿素浓度变化,肝、脑、鳃谷氨酰胺(Gln)浓度,肝、脑谷氨酰胺合成酶(GS)活性,肝氨甲酰磷酸合成酶(CPS)活性,不同组织中GS、CPS、谷氨酰胺酶(GLS)的基因表达变化。结果显示:急性胁迫下尼罗罗非鱼血氨浓度上升,于12 h到达峰值。随着血氨升高,各组织中的尿素浓度0~6 h快速升高,CPS活性0~2 h快速升高,基因相对表达量0~24 h升高,表明尿素代谢途径0-6 h内启动。肝谷氨酰胺浓度0~6 h快速升高到达峰值,肝GS活性0~6 h和12~24 h快速升高,组织中GS、GLS基因相对表达量在0~24 h升高,表明谷氨酰胺代谢途径0~6 h内启动。结果表明,在碱胁迫条件下,尼罗罗非鱼在胁迫早期同时启动尿素代谢途径与谷氨酰胺代谢途径共同参与调节血氨浓度。
This research was conducted to comprehend the mechanism of ammonia metabolism during the adaptation of tilapia( Oreochromis niloticus) to alkaline environment( 2,4,6 g/L Na HCO3). The concentration of serum ammonia,the concentrations of urea in liver,kidney,gill,water,urine and blood,the concentration of glutamine( Gln) in liver,brain and gill,the activities of glutamine synthetase( GS) in liver,brain and the activities of carbamyl phosphate synthetase( CPS) in liver,the changes in gene expression of synthetase,carbamyl phosphate synthetase,glutaminase( GLS) in different tissues of O. niloticus were analyzed under acute stress in 72 h. The results showed that the blood ammonia concentration of O. niloticus increased under acute stress,reaching its peak at 12 h. The concentration of urea in each tissue substantially increased from 0 to 6 h with the increase of blood ammonia. There was a dramatic growth of the activity of carbamyl phosphate synthase from 0 to 2 h. The expression quantity of carbamyl phosphate synthase went up from 0 to 24 h,which indicating that the urea metabolization pathway was activated within 0-6 h. The concentration of glutamine in the liver grew rapidly from 0 to 6 h and reached its highest point. The activity of glutamine synthase in the liver soared from 0 to 6 h and from 12 to 24 h. The expression of glutamine synthase and glutaminase in each tissue was on a rise at 0 to 24 h. It illustrated that glutamine metabolism was also activated within 0-6 h. The results indicated that O. niloticus activated urea metabolism pathway and glutamine metabolism pathway simultaneously to adjust blood ammonia concentration in the early stage of stress under alkali stress conditions.
This research was conducted to comprehend the mechanism of ammonia metabolism during the adaptation of tilapia( Oreochromis niloticus) to alkaline environment( 2,4,6 g/L Na HCO3). The concentration of serum ammonia,the concentrations of urea in liver,kidney,gill,water,urine and blood,the concentration of glutamine( Gln) in liver,brain and gill,the activities of glutamine synthetase( GS) in liver,brain and the activities of carbamyl phosphate synthetase( CPS) in liver,the changes in gene expression of synthetase,carbamyl phosphate synthetase,glutaminase( GLS) in different tissues of O. niloticus were analyzed under acute stress in 72 h. The results showed that the blood ammonia concentration of O. niloticus increased under acute stress,reaching its peak at 12 h. The concentration of urea in each tissue substantially increased from 0 to 6 h with the increase of blood ammonia. There was a dramatic growth of the activity of carbamyl phosphate synthase from 0 to 2 h. The expression quantity of carbamyl phosphate synthase went up from 0 to 24 h,which indicating that the urea metabolization pathway was activated within 0-6 h. The concentration of glutamine in the liver grew rapidly from 0 to 6 h and reached its highest point. The activity of glutamine synthase in the liver soared from 0 to 6 h and from 12 to 24 h. The expression of glutamine synthase and glutaminase in each tissue was on a rise at 0 to 24 h. It illustrated that glutamine metabolism was also activated within 0-6 h. The results indicated that O. niloticus activated urea metabolism pathway and glutamine metabolism pathway simultaneously to adjust blood ammonia concentration in the early stage of stress under alkali stress conditions.
引文
[1]中国农业部渔业管理局.2016中国渔业统计年鉴[M].北京:中国农业出版社,2016:1-2.
[2]王苏民,窦鸿身.中国湖泊志[M].北京:科学出版社,1998:3-8,84-85.
[3]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.
[4]郭雯翡,么宗利,来琦芳,等.盐碱胁迫下青海湖裸鲤鳃基因表达差异[J].海洋渔业,2012,34(2):137-144.
[5]Ip Y K,Chew S F,Randall D J.Ammonia toxicity,tolerance and excretion[J].Fish Physiol,2001,20(1):109-148.
[6]Tsui T K,Randall D J,Chew S,et al.Accumulation of ammonia in the body and NH3volatilization from alkaline regions of the body surface during ammonia loading and exposure to air in the weather loach Misgurnus anguillicaudatus[J].J Exp Biol,2002,205(5):651-659.
[7]Benli A C K,K9ksal G,zkul A.Sublethal ammonia Exposure of Nile tilapia(Oreochromis niloticus L.):Effects on gill,liver and kidney histology[J].Chemosphere,2008,72(9):1355-1358.
[8]Bolner K,Baldisserotto B.Water p H and urinary excretion in silver catfish Rhamdia quelen[J].J Fish Biol,2007,70(1):50-64.
[9]许友卿,李伟峰,丁兆坤.谷氨酰胺对水生动物免疫的影响及机理[J].动物营养学报,2012,24(3):406-410.
[10]Wood C M.Toxic response of the gill//Schlenk D,Benson W H.Target organ toxicity in marine and freshwater teleosts[M].London:Taylor&Francis,2001:1-89.
[11]Al-Amoudi M M.Acclimation of commercially cultured Oreochromis species to sea water-an experimental study[J].Aquaculture,1987,65(3-4):333-342.
[12]Lemarie G,Baroiller J F,Clota F,et al.A simple test to estimate the salinity resistance of fish with specific application to O.niloticus and S.melanotheron[J].Aquaculture,2004,240(1-4):575-587.
[13]么宗利,李思发,李学军,等.尼罗罗非鱼和以色列红罗非鱼耐盐驯化初步报告[J].上海水产大学学报,2003,(2):97-101.
[14]邢宪利.盐碱地池塘养殖罗非鱼试验[J].河北渔业,2010,(6):19-20.
[15]赵岩,吴俊伟,孟森,等.碳酸盐碱度胁迫对尼罗罗非鱼血清p H、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.
[16]吴俊伟,赵金良,赵岩,等.高碳酸盐碱胁迫对尼罗罗非鱼氨代谢基因表达变化的影响[J].中国水产科学,2016,23(6):1290-1299.
[17]Wilkie M P,Wood C M.The adaptations of fish to extremely alkaline environments[J].Comp Biochem Physiol,1996,113(4):665-673.
[18]Parra J E G,Baldisserotto B.Effect of water p H and hardness on survival and growth of freshwater teleosts[M].USA:Science Publishers,2007:135-150
[19]Binstock L,Lecar H.Ammonium ion currents in the squid giant axon[J].J Gen Physiol,1969,53(3):342-361.
[20]Cooper J L,Plum F.Biochemistry and physiology of brain ammonia[J].Physiol Rev,1987,67:440-519
[21]Wright P A,Wood C M.Muscle ammonia stores are not determined by p H gradients[J].Fish Physiol Biochem,1988,5(3):159-162.
[22]Wilkie M P.Ammonia excretion and urea handling by fish gills:present understanding and future research challenges[J].J Exp Zool,2002,293(3):284-301.
[23]Braun M H,Perry S F.Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii:evidence for the involvement of Rh and UT proteins[J].Com Biochem Phys A,2010,157(4):405-415.
[24]Wood C M.Ammonia and Urea Metabolism and Excretion[M].Boca Raton:CRC Press.1993:1-20.
[25]Danulat E,Kempe S.Nitrogenous waste excretion and accumulation of urea and ammonia in Chalcalburnus tarichi(Cyprinidae),endemic to the extremely alkaline Lake Van(Eastern Turkey)[J].Fish Physiol Biochem,1992,9(5-6):377-386.
[26]Wilkie M P,Wright P A,Iwama G K,et al.The physiological responses of the Lahontan cutthroattrout(Oncorhynchus clarki henshawi),a resident of highly alkaline Pyramid Lake(p H 9.4),to challenge at p H 10[J].Exp Biol,1993,175(1):173-194
[27]衣晓飞,来琦芳,史建全,等.高碱环境下青海湖裸鲤氮废物排泄及相关基因的表达规律[J].中国水产科学,2017,24(4):681-689.
[28]李少飞.中国对虾氨氮代谢酶基因的c DNA克隆及其在氨氮解毒代谢过程中的作用[D].辽宁大连:大连海洋大学,2014.
[29]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].Physiol Rev,2005,85(1):97-177.
[30]Wright P A,Steele S L,Hvitema A,et al.Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia[J].J Exp Biol,2007,210(16):2905-2911.
[31]Jow L Y,Chew S F,Lim C B,et al.The marble goby Oxyeleotris marmoratus activates hepatic glutamine synthetase and detoxifies ammonia to glutamine during air exposure[J].J Exp Biol,1999,202(3):237-245.
[32]Kim M,Wischmeyer P E.Glutamine[J].World Rev Nutr Diet,2013,105:90-96.
[33]Yin F G,Jiang W M,Guan S,et al.Glutamine and animal immune function[J].Food Agric Environ,2010,8(1):135-141.
[34]Ip Y K,Tay A S,Lee K H,et al.Strategies for surviving high concentrations of environmental ammonia in the swamp eel Monopterus albus[J].Physiol Biochem Zool,2004,77(3):390-405.
[2]王苏民,窦鸿身.中国湖泊志[M].北京:科学出版社,1998:3-8,84-85.
[3]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.
[4]郭雯翡,么宗利,来琦芳,等.盐碱胁迫下青海湖裸鲤鳃基因表达差异[J].海洋渔业,2012,34(2):137-144.
[5]Ip Y K,Chew S F,Randall D J.Ammonia toxicity,tolerance and excretion[J].Fish Physiol,2001,20(1):109-148.
[6]Tsui T K,Randall D J,Chew S,et al.Accumulation of ammonia in the body and NH3volatilization from alkaline regions of the body surface during ammonia loading and exposure to air in the weather loach Misgurnus anguillicaudatus[J].J Exp Biol,2002,205(5):651-659.
[7]Benli A C K,K9ksal G,zkul A.Sublethal ammonia Exposure of Nile tilapia(Oreochromis niloticus L.):Effects on gill,liver and kidney histology[J].Chemosphere,2008,72(9):1355-1358.
[8]Bolner K,Baldisserotto B.Water p H and urinary excretion in silver catfish Rhamdia quelen[J].J Fish Biol,2007,70(1):50-64.
[9]许友卿,李伟峰,丁兆坤.谷氨酰胺对水生动物免疫的影响及机理[J].动物营养学报,2012,24(3):406-410.
[10]Wood C M.Toxic response of the gill//Schlenk D,Benson W H.Target organ toxicity in marine and freshwater teleosts[M].London:Taylor&Francis,2001:1-89.
[11]Al-Amoudi M M.Acclimation of commercially cultured Oreochromis species to sea water-an experimental study[J].Aquaculture,1987,65(3-4):333-342.
[12]Lemarie G,Baroiller J F,Clota F,et al.A simple test to estimate the salinity resistance of fish with specific application to O.niloticus and S.melanotheron[J].Aquaculture,2004,240(1-4):575-587.
[13]么宗利,李思发,李学军,等.尼罗罗非鱼和以色列红罗非鱼耐盐驯化初步报告[J].上海水产大学学报,2003,(2):97-101.
[14]邢宪利.盐碱地池塘养殖罗非鱼试验[J].河北渔业,2010,(6):19-20.
[15]赵岩,吴俊伟,孟森,等.碳酸盐碱度胁迫对尼罗罗非鱼血清p H、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.
[16]吴俊伟,赵金良,赵岩,等.高碳酸盐碱胁迫对尼罗罗非鱼氨代谢基因表达变化的影响[J].中国水产科学,2016,23(6):1290-1299.
[17]Wilkie M P,Wood C M.The adaptations of fish to extremely alkaline environments[J].Comp Biochem Physiol,1996,113(4):665-673.
[18]Parra J E G,Baldisserotto B.Effect of water p H and hardness on survival and growth of freshwater teleosts[M].USA:Science Publishers,2007:135-150
[19]Binstock L,Lecar H.Ammonium ion currents in the squid giant axon[J].J Gen Physiol,1969,53(3):342-361.
[20]Cooper J L,Plum F.Biochemistry and physiology of brain ammonia[J].Physiol Rev,1987,67:440-519
[21]Wright P A,Wood C M.Muscle ammonia stores are not determined by p H gradients[J].Fish Physiol Biochem,1988,5(3):159-162.
[22]Wilkie M P.Ammonia excretion and urea handling by fish gills:present understanding and future research challenges[J].J Exp Zool,2002,293(3):284-301.
[23]Braun M H,Perry S F.Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii:evidence for the involvement of Rh and UT proteins[J].Com Biochem Phys A,2010,157(4):405-415.
[24]Wood C M.Ammonia and Urea Metabolism and Excretion[M].Boca Raton:CRC Press.1993:1-20.
[25]Danulat E,Kempe S.Nitrogenous waste excretion and accumulation of urea and ammonia in Chalcalburnus tarichi(Cyprinidae),endemic to the extremely alkaline Lake Van(Eastern Turkey)[J].Fish Physiol Biochem,1992,9(5-6):377-386.
[26]Wilkie M P,Wright P A,Iwama G K,et al.The physiological responses of the Lahontan cutthroattrout(Oncorhynchus clarki henshawi),a resident of highly alkaline Pyramid Lake(p H 9.4),to challenge at p H 10[J].Exp Biol,1993,175(1):173-194
[27]衣晓飞,来琦芳,史建全,等.高碱环境下青海湖裸鲤氮废物排泄及相关基因的表达规律[J].中国水产科学,2017,24(4):681-689.
[28]李少飞.中国对虾氨氮代谢酶基因的c DNA克隆及其在氨氮解毒代谢过程中的作用[D].辽宁大连:大连海洋大学,2014.
[29]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].Physiol Rev,2005,85(1):97-177.
[30]Wright P A,Steele S L,Hvitema A,et al.Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia[J].J Exp Biol,2007,210(16):2905-2911.
[31]Jow L Y,Chew S F,Lim C B,et al.The marble goby Oxyeleotris marmoratus activates hepatic glutamine synthetase and detoxifies ammonia to glutamine during air exposure[J].J Exp Biol,1999,202(3):237-245.
[32]Kim M,Wischmeyer P E.Glutamine[J].World Rev Nutr Diet,2013,105:90-96.
[33]Yin F G,Jiang W M,Guan S,et al.Glutamine and animal immune function[J].Food Agric Environ,2010,8(1):135-141.
[34]Ip Y K,Tay A S,Lee K H,et al.Strategies for surviving high concentrations of environmental ammonia in the swamp eel Monopterus albus[J].Physiol Biochem Zool,2004,77(3):390-405.