热应激对银鲫生化指标和HSP70 mRNA表达的影响
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摘要
本研究旨在探讨热应激对银鲫(Carassius auratus gibelio)生化指标及肝脏HSP70 mRNA表达的影响,选取平均体重为(72.8±10.1) g的银鲫,分别在热应激前(25℃,对照组)、热应激后(32℃,0 h、2 h、6 h、10 h)和恢复适温后6 h (25℃,恢复组)的条件下,测定机体血浆乳酸(LD)、血浆葡萄糖(Glu)、肝糖原(Gly)、己糖激酶(HK)、丙酮酸激酶(PK)和肝脏HSP70 m RNA表达量变化。研究表明,实验组乳酸含量从热应激开始至恢复期,差异始终不显著(P>0.05);实验组血浆葡萄糖水平在热应激后0 h、2 h、6 h,极显著高于对照组(P<0.01);实验组肝糖原水平在热应激0 h时,极显著高于对照组(P<0.01),其余时间点差异不显著(P>0.05);己糖激酶活力在热应激后2 h,显著高于对照组(P>0.05);丙酮酸激酶活力在热应激后6h,极显著高于对照组(P<0.01);热应激后2h,肝脏中HSP70 m RNA表达量达到最高(P<0.01),恢复至适温后,表达量与应激前无差异(P>0.05)。本研究为进一步探究银鲫的高温应激作用机制及调控机理提供参考数据,同时,也为银鲫养殖和育种等生产实践提供相应参考依据。
The aim of the present study was to assess the effects of heat stress and recovery on biochemical indices and HSP70 mRNA expression in gibel carp(Carassius auratus gibelio). The fishes were selected as experimental animals with an average weight of(72.8±10.1) g under laboratory conditions. Samples used to determine lactic acid(LD) content, glucose(Glu) content, glycogen(Gly) content, hexokinase(HK) activity, pyruvate kinase(PK) activity, and HSP70 m RNA expression were obtained before heat stress(25℃, the control group), 0, 2, 6, and 10 h after stress(32℃), and heat stress recovery(25℃, HSR), respectively. The results revealed no significant difference in LD content compared with that in the control group(P>0.05). Glu content was significantly increased at 0, 2, and 6 h(P<0.01). Gly was significantly increased at 0 h(P<0.01). HK activity was significantly increased at 2 h(P<0.01). PK activity significantly increased at 6 h(P<0.01). Meanwhile, the highest HSP70 m RNA expression was observed at 2 h under heat stress(P<0.01). Following HSR(25℃, 6 h), the HSP70 mRNA expression level recovered to the pre-stress levels(P>0.05). The results provide basic data for research on the mechanism of high temperature adaption in gibel carp, as well as a scientific basis for acute temperature change in aquaculture and breeding.
The aim of the present study was to assess the effects of heat stress and recovery on biochemical indices and HSP70 mRNA expression in gibel carp(Carassius auratus gibelio). The fishes were selected as experimental animals with an average weight of(72.8±10.1) g under laboratory conditions. Samples used to determine lactic acid(LD) content, glucose(Glu) content, glycogen(Gly) content, hexokinase(HK) activity, pyruvate kinase(PK) activity, and HSP70 m RNA expression were obtained before heat stress(25℃, the control group), 0, 2, 6, and 10 h after stress(32℃), and heat stress recovery(25℃, HSR), respectively. The results revealed no significant difference in LD content compared with that in the control group(P>0.05). Glu content was significantly increased at 0, 2, and 6 h(P<0.01). Gly was significantly increased at 0 h(P<0.01). HK activity was significantly increased at 2 h(P<0.01). PK activity significantly increased at 6 h(P<0.01). Meanwhile, the highest HSP70 m RNA expression was observed at 2 h under heat stress(P<0.01). Following HSR(25℃, 6 h), the HSP70 mRNA expression level recovered to the pre-stress levels(P>0.05). The results provide basic data for research on the mechanism of high temperature adaption in gibel carp, as well as a scientific basis for acute temperature change in aquaculture and breeding.
引文
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Guo B,Wang F,Hou CQ,et al.Effects of acute temperature fluctuation on HK and PK activity,HSP70 relative content in Litopenaeus vannamei.Journal of Fishery Sciences of China,2008,15(5):885-889[郭彪,王芳,侯纯强,等.温度突变对凡纳滨对虾己糖激酶和丙酮酸激酶活力以及热休克蛋白表达的影响.中国水产科学,2008,15(5):885-889]
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Hyndman CA,Kieffer JD and Benfey TJ.The physiological response of diploid and triploid brook trout to exhaustive exercise.Comparative Biochemistry and Physiology Part A:Molecular and Integrative Physiology,2003,134(1):167-179
Li HJ,Tian XQ,Xu MJ.Recent advance in animal stress research.Feed Research,2006(10):6-11[李红军,田夏清,徐美娟.动物应激的研究进展.饲料研究,2006(10):6-11]
Liu FH,Wu GJ,Wang ZH,et al.Effect on chicken serum biochemical and immune parameters in acute heat-stress.Journal of Beijing Agricultural College,2002,17(4):51-54[刘凤华,吴国娟,王占贺,等.热应激对仔鸡血清生化及免疫指标的影响.北京农学院学报,2002,17(4):51-54]
Liu XR.Gene cloning,sequence analysis and cold adaptation studies on Hsp70 and Hsp90βgenes from Chionodraco hamatus and Trematomus bernacchii.Master′s Thesis of Shanghai Ocean University,2015[刘秀荣.独角雪冰鱼和伯氏肩孔南极鱼HSP70和HSP90β基因的克隆、序列分析以及低温适应性研究.上海海洋大学硕士研究生学位论文,2015]
Ma B,Huo TB,Li J,et al.Comparative analysis between two types of female silver crucian carp Carassius auratus gibelio in the Irtysh River using morphology and mitochondrial control regions.Journal of Fishery Sciences of China,2013,20(1):157-165[马波,霍堂斌,李喆,等.额尔齐斯河2种类型雌性银鲫的形态特征及D-loop基因序列比较.中国水产科学,2013,20(1):157-165]
Metón I,Fernández F and Baanante IV.Short-and long-term effects of refeeding on key enzyme activities in glycolysisgluconeogenesis in the liver of gilthead seabream(Sparus aurata).Aquaculture,2003,225(1-4):99-107
Ming JH,Xie J,Xu P,et al.Molecular cloning and expression of two HSP70 genes in the Wuchang bream(Megalobrama amblycephala Yih).Fish and Shellfish Immunology,2010,28(3):407-418
Morimoto RI.Cells in stress:Transcriptional activation of heat shock genes.Science,1993,259(5100):1409-1410
Ning JH,Qin YB,Hu LC,et al.Effects of abrupt and gradual decreases in water temperature on blood physiological and biochemical parameters in dusty rabbit fish Siganus fuscescens.Journal of Dalian Ocean University,2017,32(3):294-301[宁军号,秦宇博,胡伦超,等.水温骤降和缓降胁迫对褐篮子鱼血液生理生化指标的影响.大连海洋大学学报,2017,32(3):294-301]
Qu LY,Xiang JH,Sun XQ,et al.Expression analysis of HSP70in various tissues of Chlamys farreri under thermal stress.Chinese High Technology Letters,2005,15(5):96-100[曲凌云,相建海,孙修勤,等.温度刺激下栉孔扇贝不同组织热休克蛋白HSP70的表达研究.高技术通讯,2005,15(5):96-100]
Shao YX,Chen C,Li YL,et al.Effects of low temperature stress on serum biochemical indices of the juvenile hybrid of Epinephelus moara♀×Epinephelus lanceolatus♂.Progress in Fishery Sciences,2017,38(2):70-76[邵彦翔,陈超,李炎璐,等.低温胁迫对云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交后代血清生化指标的影响.渔业科学进展,2017,38(2):70-76]
Sheng AZ,Ju HM,Li GY,et al.Expression patterns of Hsp90 of Paralichthys olivaceus in response to temperature treatment and Vibrio anguillarum infection.Progress in Fishery Sciences,2016,37(5):1-8[生安志,鞠辉明,李贵阳,等.牙鲆(Paralichthys olivaceus)Hsp90 m RNA在温度刺激和鳗弧菌感染下的表达特征.渔业科学进展,2016,37(5):1-8]
Silbergeld EK.Blood glucose:A sensitive indicator of environmental stress in fish.Bulletin of Environmental Contamination and Toxicology,1974,11(1):20-25
Su L,Li SW,Wang D,et al.Semi-quantitative RT-PCR method of detection of heat stress on fish liver content of HSP70mRNA.Acta Agriculturae Boreali-Sinica,2010,25(Sup):100-104[苏岭,李绍戊,王荻,等.半定量RT-PCR方法检测热应激对鲫鱼肝脏中HSP70 mRNA含量的影响.华北农学报,2010,25(Sup):100-104]
Sun LT,Chen GR,Chang CF.The physiology responses of tilapia exposed to low temperature.Journal of Thermal Biology,1992,17(3):149-153
Vijayan M.Acute handling stress alter hepatic glycogen metabolism in food-deprived rainbow trout(Oncorhynchus mykiss).Canadian Journal of Fisheries and Aquatic Sciences,1992,49:2260-2266
Vijayan MM,Pereira C,Grau EG,et al.Metabolic responses associated with confinement stress in tilapia:The role of cortisol.Comparative Biochemistry and Physiology Part C:Pharmacology Toxicology and Endocrinology,1997,116(1):89-95
Wang QJ,Zhang HF,Lu QP,et al.Effect of heat exposure on body fat deposition and some biochemical indices of broiler blood.Journal of Anhui Agricultural Science,2006,34(9):1886-1887,1890[王启军,张宏福,卢庆萍,等.热应激对肉仔鸡脂肪沉积及血液生化指标的影响.安徽农业科学,2006,34(9):1886-1887,1890]
Wang YN,Liu Z,Kang YJ,et al.Effects of heat stress on some non-specific immunity parameters in rainbow trout(Oncorhynchus mykiss).Journal of Agricultural Biotechnology,2015,23(5):634-642[王艳妮,刘哲,康玉军,等.热应激对虹鳟部分非特异性免疫指标的影响.农业生物技术学报,2015,23(5):634-642]
Wen HS,Lv LK,Li LM,et al.Effect of temperature on physiological and biochemical parameters and gene expression of male Sebastes schlegelii.Periodical of Ocean University of China,2016,46(11):44-51[温海深,吕里康,李兰敏,等.急性高温胁迫对雄性许氏平鲉血液生理生化及相关基因表达的影响.中国海洋大学学报,2016,46(11):44-51]
Wendelaar Bonga SE.The stress response in fish.Physiological Reviews,1997,77(3):591-625
Zhang CY,Xing W,Li TL,et al.Effects of heat stress on nonspecific immune parameters and HSP70 gene expression in Taisho Sanke koi(Cyprinus carpio).Journal of Fisheries of China,2012,36(3):336-342[张崇英,邢薇,李铁梁,等.热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响.水产学报,2012,36(3):336-342]
Zhu JL,Wang GL.The research progress on HSP70 in fish species.Journal of Ningbo University(NSEE),2007,20(4):446-450[祝璟琳,王国良.鱼类HSP70的研究进展.宁波大学学报(理工版),2007,20(4):446-450]
Zou SX.Animal biochemistry.Beijing:China Agriculture Press,2012[邹思湘.动物生物化学.北京:中国农业出版社,2012]
Basu,N,Todgham AE,Ackerman PA,et al.Heat shock protein genes and their functional significance in fish.Gene,2002,295(2):173-183
Fanouraki E,Mylonas CC,Papandroulakis N,et al.Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture.General and Comparative Endocrinology,2011,173(2):313-322
Franzellitti S,Fabbri E.Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors.Biochemical and Biophysical Research Communications,2005,336(4):1157-1163
Guo B,Wang F,Hou CQ,et al.Effects of acute temperature fluctuation on HK and PK activity,HSP70 relative content in Litopenaeus vannamei.Journal of Fishery Sciences of China,2008,15(5):885-889[郭彪,王芳,侯纯强,等.温度突变对凡纳滨对虾己糖激酶和丙酮酸激酶活力以及热休克蛋白表达的影响.中国水产科学,2008,15(5):885-889]
He FL,Xiang JG,Li CJ,et al.Preliminary study on the effect of water temperature on hematology indices of rainbow trout.Acta Hydrobiologica Sinica,2007,31(3):363-369[何福林,向建国,李常健,等.水温对虹鳟血液学指标影响的初步研究.水生生物学报,2007,31(3):363-369]
Huang ZLY,Chen SJ,Zhang ZS,et al.Effects of heat stress on some non-specific immune factors and blood cell apoptosis in Schizothorax prenati.Acta Hydrobiologica Sinica,2016,40(6):1152-1157[黄正澜懿,陈世静,张争世,等.热应激对齐口裂腹鱼非特异性免疫功能及细胞凋亡的影响.水生生物学报,2016,40(6):1152-1157]
Hyndman CA,Kieffer JD and Benfey TJ.The physiological response of diploid and triploid brook trout to exhaustive exercise.Comparative Biochemistry and Physiology Part A:Molecular and Integrative Physiology,2003,134(1):167-179
Li HJ,Tian XQ,Xu MJ.Recent advance in animal stress research.Feed Research,2006(10):6-11[李红军,田夏清,徐美娟.动物应激的研究进展.饲料研究,2006(10):6-11]
Liu FH,Wu GJ,Wang ZH,et al.Effect on chicken serum biochemical and immune parameters in acute heat-stress.Journal of Beijing Agricultural College,2002,17(4):51-54[刘凤华,吴国娟,王占贺,等.热应激对仔鸡血清生化及免疫指标的影响.北京农学院学报,2002,17(4):51-54]
Liu XR.Gene cloning,sequence analysis and cold adaptation studies on Hsp70 and Hsp90βgenes from Chionodraco hamatus and Trematomus bernacchii.Master′s Thesis of Shanghai Ocean University,2015[刘秀荣.独角雪冰鱼和伯氏肩孔南极鱼HSP70和HSP90β基因的克隆、序列分析以及低温适应性研究.上海海洋大学硕士研究生学位论文,2015]
Ma B,Huo TB,Li J,et al.Comparative analysis between two types of female silver crucian carp Carassius auratus gibelio in the Irtysh River using morphology and mitochondrial control regions.Journal of Fishery Sciences of China,2013,20(1):157-165[马波,霍堂斌,李喆,等.额尔齐斯河2种类型雌性银鲫的形态特征及D-loop基因序列比较.中国水产科学,2013,20(1):157-165]
Metón I,Fernández F and Baanante IV.Short-and long-term effects of refeeding on key enzyme activities in glycolysisgluconeogenesis in the liver of gilthead seabream(Sparus aurata).Aquaculture,2003,225(1-4):99-107
Ming JH,Xie J,Xu P,et al.Molecular cloning and expression of two HSP70 genes in the Wuchang bream(Megalobrama amblycephala Yih).Fish and Shellfish Immunology,2010,28(3):407-418
Morimoto RI.Cells in stress:Transcriptional activation of heat shock genes.Science,1993,259(5100):1409-1410
Ning JH,Qin YB,Hu LC,et al.Effects of abrupt and gradual decreases in water temperature on blood physiological and biochemical parameters in dusty rabbit fish Siganus fuscescens.Journal of Dalian Ocean University,2017,32(3):294-301[宁军号,秦宇博,胡伦超,等.水温骤降和缓降胁迫对褐篮子鱼血液生理生化指标的影响.大连海洋大学学报,2017,32(3):294-301]
Qu LY,Xiang JH,Sun XQ,et al.Expression analysis of HSP70in various tissues of Chlamys farreri under thermal stress.Chinese High Technology Letters,2005,15(5):96-100[曲凌云,相建海,孙修勤,等.温度刺激下栉孔扇贝不同组织热休克蛋白HSP70的表达研究.高技术通讯,2005,15(5):96-100]
Shao YX,Chen C,Li YL,et al.Effects of low temperature stress on serum biochemical indices of the juvenile hybrid of Epinephelus moara♀×Epinephelus lanceolatus♂.Progress in Fishery Sciences,2017,38(2):70-76[邵彦翔,陈超,李炎璐,等.低温胁迫对云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交后代血清生化指标的影响.渔业科学进展,2017,38(2):70-76]
Sheng AZ,Ju HM,Li GY,et al.Expression patterns of Hsp90 of Paralichthys olivaceus in response to temperature treatment and Vibrio anguillarum infection.Progress in Fishery Sciences,2016,37(5):1-8[生安志,鞠辉明,李贵阳,等.牙鲆(Paralichthys olivaceus)Hsp90 m RNA在温度刺激和鳗弧菌感染下的表达特征.渔业科学进展,2016,37(5):1-8]
Silbergeld EK.Blood glucose:A sensitive indicator of environmental stress in fish.Bulletin of Environmental Contamination and Toxicology,1974,11(1):20-25
Su L,Li SW,Wang D,et al.Semi-quantitative RT-PCR method of detection of heat stress on fish liver content of HSP70mRNA.Acta Agriculturae Boreali-Sinica,2010,25(Sup):100-104[苏岭,李绍戊,王荻,等.半定量RT-PCR方法检测热应激对鲫鱼肝脏中HSP70 mRNA含量的影响.华北农学报,2010,25(Sup):100-104]
Sun LT,Chen GR,Chang CF.The physiology responses of tilapia exposed to low temperature.Journal of Thermal Biology,1992,17(3):149-153
Vijayan M.Acute handling stress alter hepatic glycogen metabolism in food-deprived rainbow trout(Oncorhynchus mykiss).Canadian Journal of Fisheries and Aquatic Sciences,1992,49:2260-2266
Vijayan MM,Pereira C,Grau EG,et al.Metabolic responses associated with confinement stress in tilapia:The role of cortisol.Comparative Biochemistry and Physiology Part C:Pharmacology Toxicology and Endocrinology,1997,116(1):89-95
Wang QJ,Zhang HF,Lu QP,et al.Effect of heat exposure on body fat deposition and some biochemical indices of broiler blood.Journal of Anhui Agricultural Science,2006,34(9):1886-1887,1890[王启军,张宏福,卢庆萍,等.热应激对肉仔鸡脂肪沉积及血液生化指标的影响.安徽农业科学,2006,34(9):1886-1887,1890]
Wang YN,Liu Z,Kang YJ,et al.Effects of heat stress on some non-specific immunity parameters in rainbow trout(Oncorhynchus mykiss).Journal of Agricultural Biotechnology,2015,23(5):634-642[王艳妮,刘哲,康玉军,等.热应激对虹鳟部分非特异性免疫指标的影响.农业生物技术学报,2015,23(5):634-642]
Wen HS,Lv LK,Li LM,et al.Effect of temperature on physiological and biochemical parameters and gene expression of male Sebastes schlegelii.Periodical of Ocean University of China,2016,46(11):44-51[温海深,吕里康,李兰敏,等.急性高温胁迫对雄性许氏平鲉血液生理生化及相关基因表达的影响.中国海洋大学学报,2016,46(11):44-51]
Wendelaar Bonga SE.The stress response in fish.Physiological Reviews,1997,77(3):591-625
Zhang CY,Xing W,Li TL,et al.Effects of heat stress on nonspecific immune parameters and HSP70 gene expression in Taisho Sanke koi(Cyprinus carpio).Journal of Fisheries of China,2012,36(3):336-342[张崇英,邢薇,李铁梁,等.热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响.水产学报,2012,36(3):336-342]
Zhu JL,Wang GL.The research progress on HSP70 in fish species.Journal of Ningbo University(NSEE),2007,20(4):446-450[祝璟琳,王国良.鱼类HSP70的研究进展.宁波大学学报(理工版),2007,20(4):446-450]
Zou SX.Animal biochemistry.Beijing:China Agriculture Press,2012[邹思湘.动物生物化学.北京:中国农业出版社,2012]