| Rainbow trout(Oncorhynchus mykiss)is regarded as a typical cold-water fish with poor heat tolerance.Given the growing threat of global warming,high temperature in summer has become one of the biggest threats to rainbow trout culture.The rainbow trout farming,especially in intensive aquaculture in cage,is severely restricted by heat stress which determined by an excessively high temperature in summer.Understanding of the molecular mechanisms underlying heat stress in the rainbow trout is of great theoretical significance for us to elucidate its adaptation mechanism to thermal and will contribute to promote the development of heat-resistant breeding and practical techniques for the trout.However,the molecular mechanisms of the heat-stress response in rainbow trout is incompletely understood.At present work,the rainbow trout with consistent genetic background were treated with acute(rapidly increase the temperature from 18℃ to 25℃ at 2.5℃/h speed)and chronic(continuously increase the temperature from 18℃ to 25℃ at 1℃/d speed)heat stress.And then,quantitative proteomics and metabolomics were used to systematically analyze the variety and abundance of proteins and small molecule metabolites in rainbow trout liver samples.Subsequently metabolic pathways involved in differential molecules were analyzed using bioinformatics methods.In addition,the preliminary correlation analysis of proteomic and metabolomic data under chronic heat stress was also carried out.The main results are summarized as follows:1.Lable label-free quntitative proteomic analysis of rainbow trout liver responses to acute heat stress.We identified 16746 peptides,3362 proteins and 152 differentially expressed proteins.Of these,37 were uniquely expressed in the heat-stress group and 35 were uniquely expressed in the control group.In addition,42 proteins were significantly upregulated(fold-change>2)and 38 proteins were significantly downregulated(fold-change>2).GO(Gene Ontology)analysis indicated that these differentially expressed proteins were primarily expressed in the nucleus,extracellular matrix,and cytoplasm,and were associated with a variety of functions,including protein binding/bridging and enzyme facilitation.KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis of the differentially expressed proteins showed that,during high temperature stress,12 KEGG pathways including estrogen signaling pathway,fat digestion and absorption,complement and coagulation cascades,and platelet activation pathway,were significantly enriched.2.DIA quntitative proteomic analysis of rainbow trout liver responses to chronic heat stress.We identified 67028 peptides,12196 proteins and a total of 390 differentially expressed proteins were screened in the liver under heat stress and control conditions.Of these differentially expressed proteins,175 proteins were significantly upregulated and 215 proteins were significantly downregulated.GO function annotation and enrichment analysis shown that the differentially expressed proteins are mainly located in the organelle envelope and extracellular regions and are mainly involved in the biological processes of cellular fatty acid and lipid metabolism.Of particular concern is that the several members of HSP family such as HSP70,HSPA4,HSPA5(GRP78/BiP),HSPA12 A,HSPA13,HSP90 α,DNAJB1,DNAJC3,HSP30 were up-regulated while some proteins involved in lipid metabolism,fatty acid metabolism and carboxylic acid metabolism including ACOX1,ACOX3,FADS2,SCP2 were down-regulated after heat stress.KEGG pathway enrichment analysis were also performed and the results indicated that differentially expressed proteins were highly associated with 20 KEGG pathways especially the protein processing in endoplasmic reticulum,endoplasmic reticulum associated protein degradation and PPAR(Peroxisome proliferators-activated receptor)signaling pathway.3.Metabolomics quntitative proteomic analysis of rainbow trout liver responses to chronic heat stress.A UHPLC-Q-TOF-MS based untargeted metabolomics analysis was carried out to compare the difference of metabolites composition and abundance between heat stress group and control group.The significantly different metabolites were identified based on the combination of a statistically significant threshold of variable influence on the projection(VIP)values obtained from the OPLS-DA model and Student’s t-test on the raw data,and the metabolites with a VIP>1.0 and p-value < 0.05 were considered statistically.We identified 152 different metabolites,80 were significantly upregulated and 72 were downregulated.Furhter,KEGG enrichment analysis revealed L-proline,L-histidine,L-phenylalanine,L-tryptophan,L-methionine,L-tyrosine,L-serine,L-aspartate were significantly mapped to several important pathways related to aminoacyl-tRNA biosynthesis,protein digestion and absorption,amino acid metabolism.And palmitic acid,oleic acid,linoleic acid,eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)were significantly mapped to glycerophospholipid metabolism,biosynthesis of unsaturated fatty acids and linoleic acid metabolism pathways.4.Correlation analysis of proteomic and metabonomics data from rainbow trout liver responses to chronic heat stress.An integrated analysis of the two kinds of-omics data(proteomic and metabonomics)was performed by calculating Pearson correlation coefficient of abundance of differential expression protein and different metabolite and searching the Co-pathway that shared between the two-omics data.Finally,it was found that the metabolites such as linoleic acid,oliolenic acid,eicosapentaenoic acid,palmioleic acid,palmitic acid and oleic acid had significant up-regulation in the abundance of rainbow trout liver in the chronic heat stress group.Co-pathway study has shown that pathways such as unsaturated fatty acid synthesis,α-linolenic acid metabolism,fatty acid degradation,and primary bile acid biosynthesis have been significantly enriched in the two types of omics studies.These results suggest that acute heat stress may have adverse effects on the reproductive performance and immune function of rainbow trout,and it may affect the fat metabolism and absorption in rainbow trout liver and lead to liver function impairment.While the protein synthesis ability of rainbow trout liver is decreased after heat stress.The chronic heat stress may cause the liver injury through endoplasmic reticulum stress and affect liver lipid metabolism of the trout.Under chronic heat stress conditions,rainbow trout use amino acids rather than glucose as preferential energy source.Metabolites such as linoleic acid,alpha-linolenic acid,eicosapentaenoic acid,palmioleic acid,palmitic acid and oleic acid in rainbow trout liver can be expected to be markers of chronic heat stress in rainbow trout.The present study provides novel insights into the molecular mechanisms regulating the thermal response of rainbow trout.The results could be used as important reference on breeding of new heat-resistant strains,genetic improvement and developing techniques related to early warning and mitigation of heat stress for rainow trout in aquaculture. |
PDF Full Text Request