【作者】 张宇；

【导师】 孙俊龙； 张国庆；

【作者基本信息】 海南大学 ， 渔业发展， 2024， 硕士

【摘要】 高体鰤(Seriola dumerili)是我国名贵海产经济鱼类,产于南海诸岛、海南岛和广东、福建沿海等地。然而,夏季连续高温天气使得南海水域的水温进一步升高,极易造成养殖高体鰤的死亡,这严重制约了高体鰤养殖产业的发展。因此,本研究设置了1个高温组(32℃),以28℃为对照组,开展高体鰤在高温胁迫下的生理响应及分子调控机制研究,为高体鰤的高温响应机制提供基础数据。主要结果如下:1.高温胁迫下高体鰤肠道组织结构变化的结果显示,随着胁迫时间增加,肠道组织结构遭受显著的病理性损害,特别是在胁迫48 h后,肠内绒毛间隙扩大,绒毛缩短,出现肠道空腔的现象,且在胁迫96 h肠道组织损伤进一步加重。此外,高温组肠道中淀粉酶活性在12 h和24 h显著上升(p<0.05);胰蛋白酶活性在高温胁迫8-48 h显著上升(p<0.05);脂肪酶活性在高温胁迫18-72 h显著上升(p<0.05)。肠道16s测序结果显示,高温对高体鰤肠道菌群多样性没有显著影响;门水平上,变形菌门、拟杆菌门、厚壁菌门、放线菌门、疣微菌门为主要优势菌群。属水平上,无色杆菌属、土地杆菌属、假单胞菌属、不动杆菌属、小海员菌属为主要优势菌群,高温48 h无色杆菌属丰度显著降低(p<0.05)、假单胞菌属丰度显著升高(p<0.05);高温96 h假单胞菌属显著升高(p<0.05)、小海员菌属丰度显著降低(p<0.05)。高温使得高体鰤肠道组织结构受到损伤,导致肠道消化酶活性出现显著差异,肠道微生物的组成也发生了重大变化。随着胁迫时间的延长,这些变化的程度进一步加剧。这表明高温胁迫干扰了高体鰤的消化系统功能,持续影响其肠道健康。2.高温胁迫下高体鰤代谢相关指标结果显示,高温组血清葡萄糖含量在24 h、48 h显著上升(p<0.05),在72-96 h显著下降(p<0.05);肝脏乳酸含量无明显变化;肝脏甘油三酯含量在18-72 h显著上升(p<0.05);己糖激酶在12-24 h显著上升(p<0.05);丙酮酸激酶在48-96 h显著上升(p<0.05);肉碱棕榈酰转移酶在4-72 h显著上升(p<0.05);肝脏磷酸烯醇式丙酮酸羧基酶、谷丙转氨酶、谷草转氨酶几乎整个胁迫期间都显著高于对照组(p<0.05);乳酸脱氢酶活性在前18 h显著上升(p<0.05)。非靶向代谢组学测序差异代谢物的鉴定结果显示,C48h组与H48h组相比,19个代谢物上调,42个代谢物;C96h组与H96h组,35个代谢物上调,35个代谢物下调;代谢通路富集显示,H48h组主要富集到三磷酸腺苷结合转运蛋白途径、间隙连接、精氨酸生物合成、mTOR信号通路、氨酰-tRNA生物合成等信号通路,H96h组主要富集到亚油酸代谢、D-精氨酸和D-鸟氨酸代谢、三磷酸腺苷结合转运蛋白途径、三羧酸循环、牛磺酸和低牛磺酸代谢、核糖和葡萄糖醛酸互相转化、嘧啶代谢、类固醇激素生物合成等信号通路。高温使得高体鰤代谢指标发生了不同程度的显著变化,同时对脂质代谢与氨基酸代谢等通路造成了显著影响,说明高温增加了高体鰤肝脏的代谢负担,从而干扰正常的肝脏代谢功能。3.高温胁迫下高体鰤肝脏组织结构随着胁迫时间的增加表现出不同程度和形式的损伤,于胁迫24 h后肝脏组织出现轻度脂肪蓄积,空泡变性,胁迫48 h后肝细胞出现广泛脂肪蓄积,空泡变性,细胞核溶解,肝细胞坏死,肝脏结构不规则,肝窦扩张,在胁迫96 h肝脏组织损伤进一步加深,甚至出现细胞坏死、凋亡。血清、肝脏组织免疫指标的结果显示,血清补体C3含量在18-96 h显著上升(p<0.05);补体C4含量在8-96 h显著下降(p<0.05);免疫球蛋白M含量在0-72 h显著上升(p<0.05),但在96 h显著下降(p<0.05);肝脏溶菌酶活性8-96 h显著上升(p<0.05);超氧化物歧化酶活性48-96 h显著上升(p<0.05);过氧化氢酶活性在24-96 h显著上升(p<0.05);丙二醛含量在整个胁迫期间都显著上升(p<0.05)。热休克蛋白相关基因表达结果显示,HSP70基因总体呈现出先升后降再上升的趋势,分别以12 h和48 h表达量最高(p<0.05);HSP90α基因总体呈现出先升后降的趋势,于24 h表达量最高(p<0.05),而后随即下降,于96 h显著降低(p<0.05)。高温下,高体鰤肝脏组织结构发生明显变化,血清和肝脏中的抗氧化和免疫相关指标发生了显著的变化,同时热休克蛋白基因的表达也出现了明显的调整。这些变化表明,高温增强了高体鰤的免疫反应。具体来说,高体鰤通过调节抗氧化和免疫相关指标,以及通过基因表达的调节,来应对高温带来的免疫系统压力。4.肝脏转录组结果显示,剔除假阳性差异基因后,C48h组与H48h组之间有430个差异表达基因(DEGs),C96h组与H96h组之间有1612个DEGs,两组共有的DEGs有141个。这141个DEGs共筛选出了51个GO term,如代谢过程、生物调节、细胞部分、细胞器部分、催化活性、转运蛋白活性等;共富集到25条KEGG通路中,如丁酸代谢、淀粉和蔗糖代谢、类固醇激素生物合成途径等。对转录组测序结果中的hoga1、l2hgdh、LOC111236694、gnmt、tapt1、pter、utp4、prlh和glyctk基因进行了qRT-PCR验证,其表达水平与转录组分析中发现的差异基因是一致的,从而验证了我们的测序数据的准确性。其中关键基因glyctk、l2hgdh、gnmt、hoga1的表达量在高温下显著下调,影响了甘氨酸、丝氨酸和苏氨酸代谢通路、丁酸代谢通路、甘氧酸和二羧酸代谢通路、经由细胞色素P450代谢异生物质通路的功能,导致高体鰤在能量代谢、氧化应激、脂肪变性等方面受阻。更多还原

【Abstract】 The Greater Amberjack(Seriola dumerili.),is a precious economic fish species in China.It has advantages such as fast growth rate,short breeding cycle,high profit,tender meat,and comprehensive nutrition.It is mainly produced in the South China Sea islands,Hainan Island,and along the coast of Guangdong and Fujian.The suitable water temperature range for the growth of Greater Amberjack is 20-31℃,and it has low tolerance to high temperatures.However,with the global warming trend,continuous high temperatures and extreme weather conditions have caused further increase in water temperature in the South China Sea,making the breeding of Greater Amberjack vulnerable to high mortality due to the high temperature stress.Currently,high temperature has become an important environmental stress factor affecting the healthy breeding of Greater Amberjack.Therefore,this study set up a high temperature group(32℃)and a control group(28℃),to investigate the physiological responses and molecular regulation mechanisms of Greater Amberjack under high temperature stress,in order to provide basic data for understanding the high temperature response mechanism of Greater Amberjack.The main results are as follows:The results show that the digestive tissue structure of Greater Amberjack is significantly damaged as the stress time increases under high temperature stress.Especially at 48 h of stress,the intestinal tissue structure experiences significant pathological damage,with widened gaps between intestinal villi,shortened villi,and presence of an empty intestinal cavity.Furthermore,the activities of amylase,trypsin,and lipase in the intestinal tract of the high temperature group significantly increased at different time points during stress.The 16 S sequencing results of the intestinal tract showed that high temperature did not significantly affect the diversity of the intestinal microbial community of Greater Amberjack.At the phylum level,the predominant bacterial groups were Deferribacteres,Bacteroidetes,Firmicutes,Actinobacteria,and Verrucomicrobia.At the genus level,the predominant bacterial groups were Acinetobacter,Bacillus,Pseudoalteromonas,Pseudomonas,and Psychromonas.High temperature stress altered the intestinal tissue structure of Greater Amberjack,leading to significant changes in digestive enzyme activity and intestinal microbial composition.These changes worsened with increasing stress time,indicating that high temperature stress disrupted the digestive system function of Greater Amberjack,impacting their intestinal health.The results on metabolic-related indicators of Greater Amberjack under high temperature stress show that the serum glucose content significantly increased at 24 h and 48 h,then decreased at 72-96 h in the high temperature group.The liver lactate content remained unchanged,while the liver triglyceride content significantly increased at 18-72 h of stress.The activities of hexokinase,pyruvate kinase,carnitine palmitoyltransferase,and phosphoenolpyruvate carboxykinase in the liver increased significantly at different time points during stress.Nontargeted metabolomics sequencing identified differential metabolites,with enrichment in pathways such as lipid metabolism and amino acid metabolism,indicating that high temperature increased the metabolic burden on the liver of Greater Amberjack,thereby disrupting normal liver metabolism function.The results on changes in the liver tissue structure of Greater Amberjack under high temperature stress show that the liver exhibited varying degrees and forms of damage as stress time increased.At 24 h of stress,the liver tissue showed mild fat accumulation and vacuolar degeneration,with more extensive fat accumulation and vacuolar degeneration observed at 48 h.Severe liver tissue damage,including cell necrosis and apoptosis,was observed at 96 h of stress.The results of serum and liver tissue immune indicators show that the complement C3 content in the serum significantly increased at 18-96 h of stress,while the complement C4 content decreased at 8-96 h.The immunoglobulin M content in the serum significantly increased at 0-72 h but decreased at 96 h.The lysozyme activity in the liver increased at 8-96 h of stress,along with increased activities of superoxide dismutase and catalase,and an overall increase in malondialdehyde content during the entire stress period.The expression levels of heat shock proteinrelated genes,HSP70 and HSP90α,fluctuated under high temperature stress,with the highest expression levels observed at 12 h and 48 h for HSP70,and at 24 h for HSP90α.High temperature stress caused significant changes in the liver tissue structure of Greater Amberjack,as well as significant alterations in antioxidant and immunerelated indicators in the serum and liver.The expression of heat shock protein genes was also notably adjusted,indicating enhanced immune response of Greater Amberjack under high temperature stress.The transcriptome results of the liver showed 430 differentially expressed genes(DEGs)between the C48 h group and H48 h group,and 1612 DEGs between the C96 h group and H96 h group,with 141 common DEGs identified between the two groups.These141 DEGs were associated with 51 GO terms,such as metabolic processes,biological regulation,cellular components,catalytic activity,and transporter activity.They also enriched in 25 KEGG pathways,including butanoate metabolism,starch and sucrose metabolism,and steroid hormone biosynthesis.The qRT-PCR validation of key genes,such as hoga1,l2 hgdh,gnmt,tapt1,pter,utp4,prlh,and glyctk,confirmed the accuracy of the sequencing data.The downregulated expression of key genes(glyctk,l2 hgdh,gnmt,hoga1)affected pathways related to metabolism,oxidative stress,and lipid degeneration,hindering the energy metabolism,oxidative stress response,and lipid metabolism in Greater Amberjack under high temperature stress.更多还原