| In recent years,due to the comprehensive influence of natural and human factors such as natural wind and tide,water temperature,seasonal change,sewage discharge,coastline development,etc,there are frequent periodic and continuous low oxygen states in the sea area of the cultivation of the sea cobia.Cobia cultured waters often exhibit periodic and persistent hypoxic conditions.The high density of aquaculture areas exacerbated the occurrence of hypoxia,which directly caused economic losses to the cobia fish breeding industry relatively.At present,the research on cobia in hypoxia is mainly focused on physiological and biochemical mechanisms,and the molecular regulation mechanism of cobia’s adaptationto hypoxia has not been reported.In view of this,in this study,cobia juvenile fish were used as experimental materials to study the changes of liver metabolism under acute hypoxia,the changes of serum of serum biochemical indexes,gill and liver injury,liver transcriptome sequencing and comparative analysis of cobia under hypoxia-reoxygenation.The Rapid c DNA End Amplification(RACE)and Real-time Quantitative Polymerase Chain Reaction(RQ-PCR)were used to clone the hypoxia-related gene Heme oxygenase(HO)of cobia and explored the expression changes of HO gene in different tissues under hypoxia-reoxygenation.The research results obtained are as follows:1.Effect of acute hypoxia on the changes of liver metabolic function of cobia.After acute hypoxic stress,the Superoxide dismutase(SOD)activity and Glutathione peroxidase(GSH-Px)activity of cobia liver decreased,lipid peroxide(LPO)activity and Malondialdehyde(MDA)content increased,and the difference was very significant from before stress;Catalase(CAT)activity decreased slightly,and without significant difference from before hypoxia stress.After acute hypoxia stress,Glycogen(LG)content and ATPase activity of cobia decreased to varying degrees,and the differences were significantly different from those before hypoxia stress.After acute hypoxia stress,LDH activity,PK activity and HK activity in liver of cobia were increased to varying degrees,and the differences were significantly different from those before hypoxia stress.2.The effects of hypoxia reoxygenation on serum biochemical indexes,gill and liver tissue structure of juvenile cobia.Under hypoxic stress,serum lipid metabolism enzymes Triglyceride(TC),serum liver function related indicators Alkaline phosphatase(ALP),Serum total protein(TP),Seralbumin(ALB),and serum antioxidant enzyme SOD indicators all showed a downward trend,and there was no significant difference.After 8 hours of reoxygenation,AST and cat were higher than those in the control group,ALT and MDA were lower than those in the control group,there was significant difference;after 24 hours of reoxygenation,AST was lower than that in the control group,there was significant difference;After 48 hours of reoxygenation,ALT,MDA and AST were significantly lower than those in the control group,and the other indicators were not significantly different from the control group.After hypoxia treatment,the blood vessels of the gill segments contracted and slightly curved,the number of red cells in the gill segments increased,the number of mucinous cells and mitochondria rich cells in the bottom part of the gill segments increased,and the liver tissues showed disorder of tissue arrangement and many vacuoles.After reoxygenation,gill and liver tissues of juvenile fish returned to normal level with reoxygenation time.3.Transcriptomic Analysis of juvenile cobia liver submitted to hypoxic.Analyzed the hypoxic-reoxygenated cobia larvae liver by transcriptome sequencing to obtain 100,743,276,600 and 335,810,922 transcripts,and then118,176 unigenes sequences were assembled with a total data volume of 74,119,587 bp and an average length of 1921.Annotated the five major database functions of ungenes and finded that the most annotated is the NR database(62142,accounting for52.58%),and the least annotated is the GO database(23108,approximately19.55%).Further comparative analysis of the transcriptome found that in the hypoxicstress group,the reoxygenated 8h group,the reoxygenated 24 h group and the reoxygenated 48 h group,there were 1,689,651,236,and 1150 differential genes in the hypoxic stress group,the reoxygenated 8h group,the reoxygenated 24 h group,and the reoxygenated 48 h group,respectively.Compared the differentially expressed genes to the GO database,the main enriched genes are related to the GO functional items such as the structural components of ribosomes,matrix-dependent cell migration,hormone activity,and oxidoreductase activity.By comparing the differentially expressed genes to the KEGG database,a total of 43,054 differentially expressed genes were enriched into 212 KEGG pathways including PPAR signaling pathway,Ribosome signaling pathway,AMPK signaling pathway,HIF-1 signaling pathway,and fatty acid metabolism.Through the interaction of various metabolic pathways,changes in the transcription level of the juvenile fish body had occurred,which controled the response to oxygen and regulates other functions of the cell and in response to hypoxic stress,also determined the hypoxic molecular adaptation strategy of juveniles.4.Cloning and expression analysis of hypoxia-related gene HO in cobia.The full-length c DNA of the cobia HO gene was cloned by using RACE technology.The full-length c DNA is 1639 bp,the open reading frame(ORF)is 840 bp,encods 279 amino acids,and 5 ’-UTR 518 bp,3 ’-UTR 281 bp.Phylogenetic analysis showed that cobia HO had the highest homology with Lates calcarifer and Seriola dumerili,which were 92.80% and 90.91%,respectively.The results of real-time quantitative PCR showed that cobia fish HO was expressed in 9 tissues,with the highest expression in gills.In addition,there was no significant change in the expression of HO in hypoxic-reoxygenated m RNA in the liver.The m RNA expression of gill HO increased after hypoxia stress,and the m RNA expression increased andthen decreased before and after reoxygenation,and remained low expression until the end.In conclusion,this study used cobia juveniles as experimental objects.On the one hand,the study explored the effects of acute hypoxia stress on changes in liver metabolic function and hypoxia stress and reoxygenation on serum biochemicalindicators,gills and liver tissue structure of juvenile cobia from the biochemical and tissue levels;From the molecular level,the transcription and regulation of hypoxia-related genes in the juvenile cobia liver transcriptome and the gene structure and expression level of cobia HO were discussed.The results of this study are intend to explain the biological process and molecular regulation mechanism of hypoxia adaptation in cobia,and provide theoretical basis for the study of adaptation to hypoxia in fish. |
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