| Takifugu obscurus is the main species of puffer cultured in China,a typical anadromous fish,and one of the "Three Fresh Foods of Yangtze River".It is often active in the lower and middle water bodies,while its gills and gill pores are relatively small,and its respiratory capacity is weak,so it is more susceptible to low dissolved oxygen than other fish.In the process of breeding,due to the high density of breeding,baiting and the relative closure of the water is more prone to oxygen deficiency.Therefore,for the sustainable development of river herring aquaculture industry,it is important and urgent to strengthen the research on the response of T.obscurus herring to hypoxic stress.The subject of this study is the T.obscurus,three environmental conditions were set:normoxia(DO: 7.0 ± 0.2 mg/L),hypoxia stress(DO: 0.9 ± 0.2 mg/L)and normoxia recovery(DO: 7.0 ± 0.2 mg/L,4 h,8 h,12 h,24 h),the transcriptomic and metabolomic analyses of gill and muscle tissues of T.obscurus were performed by high-throughput sequencing and liquid chromatography-mass spectrometry,and the differential expression genes(DEGs)and metabolites(DEMs)associated with hypoxia were screened out,and then the pathways significantly enriched in genes and metabolites were analyzed to preliminarily analyze the mechanism of hypoxia tolerance of T.obscurus from the molecular level,and the following results were obtained:1.Transcriptome analysis was performed on gill tissues under acute hypoxic stress,992,877,1 561,1 412 and 679 differentially expressed genes were identified in the hypoxic group versus the normoxic,reoxygenated 4 h,reoxygenated 8 h,reoxygenated 12 h and reoxygenated24 h groups,respectively,and 56 genes were significantly expressed in all five comparison groups.The results showed that the main GO entries obtained with significant differential gene enrichment were the transmembrane receptor protein serine/threonine kinase signaling pathway,the signaling pathway of the transforming growth factor β receptor(TGF-β),immune response,negative regulation of cellular processes,collagen trimerization,extracellular regions,DNA-binding transcription factor activity,sequence-specific DNA binding and transcriptional regulator activity,and these GO entries are associated with growth and development,immune response,and apoptosis.The KEGG pathways that were significantly enriched in differential genes were mainly cell adhesion molecules(CAMs),cytokine-cytokine receptor interactions,VEGF signaling pathway,MAPK signaling pathway and calcium signaling pathway,etc.The differential genes on these pathways were mainly involved in stress response,immune function,angiogenesis and apoptosis processes.Among them,ccn1、ccn2、il-8、ccl20、ackr3 were screened as immune-related genes.Bmp10、bambi、smad6、smad7 were growth and development-related genes.And dusp1,ddit4 are related genes involved in apoptotic process.2.Transcriptome analysis of muscle tissue under acute hypoxic stress,1 287,941,1 261,841 and 1 453 differentially expressed genes were identified in the hypoxic group versus the normoxic,reoxygenated 4h,reoxygenated 8 h,reoxygenated 12 h and reoxygenated 24 h groups,respectively,and 57 genes were significantly expressed in all five comparison groups.The results showed that the GO entries significantly enriched in differentially expressed genes were cytoskeleton,catalytic activity,DNA-binding transcription factor activity,and immune response,which are mainly involved in cell growth and development,lipid accumulation,and inflammatory response processes.The KEGG pathways with significant enrichment of differential genes are mainly ECM receptor interaction,tight junctions,cytokine-cytokine receptor interaction,linoleic acid metabolism,MAPK signaling pathway,etc.The differential genes on these pathways mainly play a role in the process of cell injury and energy metabolism.In addition,some genes that were significantly affected by hypoxic stress and related to growth or immunity were screened,such as bhlhe40、mybph、sik1、ltb4r、ccl20、ccl28.These genes were associated with muscle development of T.obscurus or regulated various immune responses.3.Metabolomic analysis was performed on gill tissues under acute hypoxic stress.36,18,24 and 34 different metabolites were identified in the positive ion mode and 6,5,4 and 6different metabolites were identified in the negative ion mode between the hypoxia group and the normal-oxygen,4h,12 h and 24 h reoxygenated groups,respectively.The results revealed that the relevant metabolic pathways in which the differential metabolites were mainly enriched were biosynthesis of phenylalanine,tyrosine and tryptophan,phenylalanine metabolism,purine metabolism,Fox O signaling pathway,and m TOR signaling pathway,which are related to lipid metabolism,amino acid metabolism,oxidative stress,and apoptosis in cells.4.Metabolomic analysis was performed on muscle tissue under acute hypoxic stress.44,17,64 and 79 different metabolites were identified in the positive ion mode and 9,5,10 and 28 different metabolites were identified in the negative ion mode between the hypoxia group and the normal-oxygen,4h,12 h and 24 h reoxygenated groups,respectively.The results revealed that the metabolites were enriched in the KEGG pathways of arginine and proline metabolism,glutathione metabolism,glycine,serine and threonine metabolism,glycerophospholipid metabolism,glyoxylate and dicarboxylic acid metabolism,and TCA cycle,and were mainly involved in lipid metabolism,amino acid metabolism,and energy metabolism.This study enriched the transcriptome and metabolome data of T.obscurus under acute hypoxic stress,screened the differential genes and differential metabolites related to hypoxia,and also provided a new research idea for the regulatory mechanism of the organism under hypoxic stress,further clarified the molecular mechanism of hypoxia tolerance of T.obscurus,and provided some theoretical basis for the selection and breeding of new hypoxia-tolerant species of fish at the molecular level in the future,as well as a reference for the study of gill and muscle genes and metabolites of other organisms. |
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