基于TMT技术对高温胁迫条件下大菱鲆幼鱼心脏的差异蛋白筛选分析

TMT-Based Proteomic Analysis of Scophthalmus maximus Hearts Under High Temperature Stress

水温是影响鱼类生存生长的重要因素,而心脏对于维持脊椎动物机体稳态有关键作用。因此,为探究与大菱鲆(Scophthalmus maximus)耐高温性状相关的关键蛋白或代谢通路,本研究利用TMT联合LC-MS/MS技术开展高温胁迫下大菱鲆心脏差异表达蛋白谱分析。将大菱鲆幼鱼分为3组进行实验:control组(14.0±0.5)℃(适宜温度),HT1组(20.0±0.5)℃(高温),HT2组(28.0±0.5)℃(极高温)。定量分析鉴定到的肽段数量为35434个,其中特有肽段数量为31704个;鉴定到的蛋白质总数4765个。3个实验组间两两比较形成3个比较组,并进行差异蛋白筛选,结果显示,HT1/control共筛选到87个差异表达蛋白(DEPs),HT2/control共筛选到171个DEPs,HT2/HT1共筛选到188个DEPs。KEGG富集结果显示,HT2组与control组和HT2组与HT1组的差异蛋白均主要富集在与脂肪酸代谢、炎症以及免疫防御相关的通路;热休克蛋白家族中多个蛋白(HSP90B、HSP70等)在HT2组相较于control组均显著上调。蛋白互作网络结果显示,关联度最高的是HSP90B。首次发现,高温胁迫下心脏组织纤维蛋白原相关蛋白(FGA、FGB和FGG)表达水平呈上升趋势,其在热响应中的作用仍需进一步研究。结果表明,极高温可能引起大菱鲆的脂肪酸代谢紊乱,引发炎症反应,而热休克蛋白家族蛋白的上调可能对缓解热胁迫下的应激症状有积极作用。本研究为进一步阐明大菱鲆热应激的分子机制提供了理论依据,并为大菱鲆的耐高温选育提供候选蛋白标记。

Water temperature is one of the most important ecological environmental factors affecting fish survival and growth.When the water temperature is higher than the optimal temperature for fish survival,fish homeostasis may be disrupted.The heart plays a crucial role in maintaining vertebrate homeostasis.In this study,TMT and LC-MS/MS were performed to analyze differentially expressed protein(DEP)profiles in the hearts of turbot under high-temperature stress to explore the key proteins and metabolic pathways related to the high-temperature tolerance traits of turbot.Ninety turbot(mean weight 23.01±3.63 g,mean length 10.34±0.76 cm)were used in experiments.Three temperature points for heat stress experiments were designed:control=(14.0±0.5)℃,HT1=(20.0±0.5)℃,and HT2=(28.0±0.5)℃.Three parallel groups were established for each temperature group,with 10 turbots placed in each group.Fold Change≥1.2 or<0.83,P<0.05 was used as a criterion to screen for DEPs in turbot.Through mass spectrometry analysis of heart tissues from turbot,85,731 spectra were identified,with 35,434 peptides,of which 31,704 were unique,and 4,765 proteins were identified.The HT1/control group in the heart of turbot had a total of 87 differentially expressed proteins,including 77 upregulated and 10 down-regulated proteins;the HT2/control group in the heart of turbot had 171 DEPs,including 149 upregulated and 22 down-regulated proteins;the HT2/HT1 group in the heart of turbot had a total of 188 DEPs,including 130 upregulated and 58 downregulated proteins.Through bioinformatics analysis,the DEPs between the 28℃group and the 14℃group,as well as the 28℃group and the 20℃group,were mainly enriched in KEGG pathways related to fatty acid metabolism,inflammation,and immune defense.Multiple proteins in the heat shock protein family(HSP90B,HSP70,etc.)were significantly upregulated in the 28℃group compared to the 14℃group.The protein interaction network revealed that HSP90B exhibited the highest correlation.A new discovery is that the expression levels of fibrinogen-related proteins(FGA,FGB,and FGG)increased,and their role in the thermal response requires further research.These results indicate that extremely high temperatures may cause fatty acid metabolic disorders in turbot,leading to inflammatory reactions.The upregulation of heat shock protein family proteins may have a positive effect on alleviating stress symptoms under heat stress.This study identified DEPs,such as HSP90B,HSPA5,and HYOU1,which were significantly upregulated under heat stress,as candidate proteins for high-temperature tolerance breeding in turbot and provided a theoretical basis for further elucidating the molecular mechanism of heat stress in turbot.