高温应激对大口黑鲈crh及其受体基因表达和皮质醇浓度的影响  

Effects of heat stress on the expression of crh and its receptor genes and the concentration of cortisol in largemouth bass(Micropterus salmoides)

作　　者：李铭 杜金星 宋红梅[2] 朱涛 雷彩霞 吴广州 李胜杰[2] LI Ming;DU Jin-xing;SONG Hong-mei;ZHU Tao;LEI Cai-xia;WU Guang-zhou;LI Sheng-jie(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Tropical and Subtropical Fishery Resource Application and Cultivation,Ministry of Agriculture and Rural Affairs/Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou,Guangdong 510380,China;Jining Fishery Development and Resource Conservation Center,Jining,Shandong 272000,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]农业农村部热带亚热带水产资源利用与养殖重点实验室/中国水产科学研究院珠江水产研究所,广东广州510380 [3]济宁市渔业发展和资源养护中心,山东济宁272000

出　　处：《南方农业学报》2024年第12期3748-3757,共10页Journal of Southern Agriculture

基　　金：广东省乡村振兴战略专项(2022SPY00003);中国水产科学研究院基本科研业务费专项(2023TD95);山东省现代农业鱼类产业技术体系建设专项(SDAIT-12)。

摘　　要：【目的】明确急性高温应激对大口黑鲈促肾上腺皮质激素释放激素基因(crh)及其受体基因(crhr1和crhr2)表达和皮质醇浓度的影响,深入分析crh、crhr1和crhr2基因与皮质醇间的作用关系,为揭示大口黑鲈应激反应的分子调控机制打下基础。【方法】通过PCR扩增大口黑鲈crh、crhr1和crhr2基因开放阅读框(ORF),采用实时荧光定量PCR检测这3种基因在大口黑鲈不同组织中的表达分布;对大口黑鲈进行急性高温应激胁迫后分别采集血样及脑组织,以25.0℃为初始水温[设为对照(CK)],测定不同时间点大口黑鲈脑组织中的crh、crhr1和crhr2基因相对表达量及血清皮质醇浓度。【结果】大口黑鲈crh、crhr1、crhr2基因ORF序列分别长510、1392和1245 bp,对应编码169、463和414个氨基酸残基;基于crh、crhr1和crhr2基因核苷酸序列相似性构建的系统发育进化树显示,大口黑鲈与鲈形目鱼类聚为一支,尤其与小口黑鲈的亲缘关系最近。crh、crhr1和crhr2基因在大口黑鲈心脏、肝脏、肠道、肾脏、脑、鳃和肌肉等组织均有表达,且以脑组织中的相对表达量较高。急性高温应激后,大口黑鲈脑组织中crh、crhr1和crhr2基因相对表达量及血清皮质醇浓度均随时间推移呈先上升后下降的变化趋势,其中,crh和crhr1基因相对表达量及血清皮质醇浓度在高温应激后3 h(33.0℃)时达峰值,crhr2基因表达变化存在一定滞后性,其相对表达量在高温应激后6 h(30.4℃)时才达峰值。【结论】急性高温应激后大口黑鲈crh和crhr1基因的表达与血清皮质醇浓度变化趋势一致,而crhr2基因的表达滞后于皮质醇浓度变化,由此推测crh和crhr1基因参与皮质醇合成与分泌过程,与应激启动有关,而crhr2基因参与皮质醇的分解,与应激恢复相关。【Objective】To investigate the effects of heat stress on the expressions of the corticotropin-releasing hormone gene(crh)and its receptor genes(crhr1 and crhr2)and the concentration of cortisol in largemouth bass(Micropterus salmoides),and to further analyze the interaction between crh,crhr1 and crhr2 genes and cortisol,which would lay a foundation for revealing the molecular regulatory mechanism of stress response in largemouth bass.【Method】The open reading frames(ORF)of crh,crhr1 and crhr2 genes were amplified by PCR amplification,and the expression of the 3 genes in different tissues of largemouth bass was detected by real-time fluorescence quantitative PCR(qRT-PCR).The blood samples and brain tissues of largemouth bass were collected after acute heat stress,the relative expressions levels of crh,crhr1 and crhr2 genes in brain tissues and the concentration of serum cortisol of largemouth bass were measured at different treatment time points,with 25.0℃as the initial water temperature[set as control(CK)].【Result】The ORF sequences of the crh,crhr1 and crhr2 genes of largemouth bass were obtained as 510,1392 and 1245 bp,encoding 169,463 and 414 amino acids respectively.The phylogenetic tree based on the nucleotide sequence similarity of crh,crhr1,and crhr2 genes showed that largemouth bass was clustered with percoid fish,especially close to smallmouth bass.The genes of crh,crhr1 and crhr2 were expressed in heart,liver,intestine,kidney,brain,gill and muscle,and the relative expressions were higher in brain.After acute heat stress,the relative expressions of crh,crhr1 and crhr2 genes in brain and the concentration of serum cortisol increased first and then decreased with time.Among these,the relative expressions of crh and crhr1 genes and the concentration of serum cortisol reached the peak at 3 h post heat(33.0℃).There was a lag in the expression of crhr2 gene,and its relative expression reached its peak at 6 h(30.4℃)stress.【Conclusion】After acute heat stress in largemouth bass,the expressions

关 键 词：大口黑鲈 高温应激 crh基因 crhr1基因 crhr2基因 皮质醇 

分 类 号：S965.211[农业科学—水产养殖]