Transcriptome And BSR-Seq Integrated Analysis Of Differential Hypoxia Tolerance In Trachinotus Blochii

Trachinotus blochii has rapidly developed into the third-largest species of marine aquaculture in China within ten years.However,due to its rapid growth and exuberant metabolism,oxygen consumption of fish is high,and it is extremely vulnerable to hypoxia stress and death,leading to serious economic losses.In this study,we simulated hypoxia stress conditions and constructed the hypoxia tolerance group(Tol group)and intolerance group(Intol group).On the one hand,we used high-throughput sequencing to analyze the m RNA expression profiles of the brain and liver under hypoxia conditions to clarify the main pathways and key genes involved in hypoxia.On the other hand,we used BSR-Seq analysis to identify and screen SNPs closely linked to differential genes of hypoxic tolerance.Based on the data of genome and genetic linkage groups,we accurately located functional genes to systematically reveal the reasons for differences in hypoxia tolerance among different groups.These provided theoretical support for genetic breeding and scientific aquaculture of Trachinotus blochii.1.the transcriptome data showed that the pathways involved in high expression genes in the brain are mainly related to signal transduction,endocrine,and environmental information processing,while the pathways involved in high expression genes in the liver are mainly related to metabolism.The results of gene differential expression analysis showed that a total of 31 genes in the brain showed significant differences in expression between the Intol group and the Tol group,and in the liver,a total of 710 differentially expressed genes were screened between the Intol group and the Tol group.During acute hypoxia,the differentially expressed genes in the brain are significantly enriched in amino acid metabolism(histidine metabolism and β-alanine metabolism).The differentially expressed genes in the liver are significantly enriched in metabolic processes and signal transduction pathways.In the process of adapting to hypoxia,the fish first used anaerobic glycolysis to adapt to the energy of hypoxia.However,as stress continues,glycolysis is inhibited,and the glycogen degradation rate in the liver is significantly decreased in the Tol group,making lipid metabolism becomes the preferred mode of energy metabolism.Besides,acetyl-Co A may be produced through lipid metabolism in the Tol group,which provides metabolic substrates for other tissues to enter the tricarboxylic acid cycle and energy for the body.The Intol group may be due to the consumption of a large amount of glucose and lipids and glycogen,leading to the disorder of glucose and lipids metabolism and death.Although more in-depth research is needed to get accurate conclusions,we speculate that lipid metabolism in the Trachinotus blochii liver plays an important role in adaptation to hypoxia.2.Through the BSR-Seq(Bulked Segregant RNA-Seq)analysis of the brain and liver of differential hypoxia tolerance groups in Trachinotus blochii,it was determined that the candidate regions in the brain were located in linkage groups 8,18,and 22,and the candidate regions in the liver were located in linkage groups 10,16,18,and 22.It is speculated that the hypoxia tolerance of Trachinotus blochii may be jointly determined by multiple linkage groups.768 and 348 candidate genes were located in the candidate regions of the brain and liver,respectively.GO enrichment analysis showed that the Trachinotus blochii is closely related to cell structure,migration,energy metabolism and cell apoptosis in the process of hypoxia tolerance.The enrichment analysis of the KEGG pathway showed that this experiment screened the candidate genes of the significantly enriched pathway under hypoxic stress that were involved in anaerobic energy metabolism,stress response,immune response,waste discharge from the body,and cell death.The specific pathway of arachidonic acid metabolism in the brain of the Tol group may improve the ability to adapt to hypoxia by regulating lipid decomposition.There are 5important genes involved in the arachidonic acid metabolism pathway among the candidate genes,which are Trachinotus＿GLEAN＿10010123(PTGS2),Trachinotus＿GLEAN＿10010124(pla2g4a),Trachinotus＿GLEAN＿10015446(ALOX5),Trachinotus＿GLEAN＿10015447(ALOX5)and Trachinotus＿GLEAN＿10017022(PLA2G12B).Among these important genes,the PTGS2 gene located on LG8 has a G/A nonsynonymous mutation at position20641628,and the encoded amino acid was changed from hydrophobic aspartic acid to asparaginate.The specific pathway of RIG-I-like receptor signaling pathway in the liver may mediate the metabolic system and the immune system,linking glucose metabolism with immune regulation.The death of the Intol group may be due to the accumulation of lactic acid caused by the activation of early anaerobic glycolysis at the early stage of hypoxic stress,and the activation of type I interferon was inhibited,which resulted in the decreased immunity of Trachinotus blochiii.There are 4 important genes involved in the RIG-I-like receptor signaling pathway among the candidate genes,which are Trachinotus＿GLEAN＿10002391(CYLD),Trachinotus＿GLEAN＿10017046(trim72),Trachinotus＿GLEAN＿10018773(Ifih1)and Trachinotus＿GLEAN＿10018778(TANK).Among these important genes,the CYLD gene located on LG16 has a G/T nonsynonymous mutation at position 13629651,and the encoded amino acid was changed from alanine acid to valine.The Ifih1 gene located on LG18 has a G/C nonsynonymous mutation at position16153700,the encoded amino acid was changed from hydrophilic glycine to hydrophobic alanine.By predicting the gene domains of PTGS2,CYLD and Ifih1,it was found that the region of 20641628 G/A variant site of PTGS2 is specifically matched with the protein domain of prostaglandin endoperoxide synthase,and the region of 13629651 C/T variant site of CYLD is specifically matched with the CAP-Gly domain.In conclusion,this study found that under the acute hypoxia stress,there is a close relationship between the hypoxic tolerance and metabolism of Trachinotus blochii through the combined analysis of RNA-Seq and BSR-Seq,and the the balance of glucose and lipid metabolism plays a key role in the hypoxic tolerance of Trachinotus blochii.