Changes Of Transcriptome Of Takifugu Rubripes Swim Bladder And Functional Analysis Of Key Differentially Expressed Genes Under Chronic Hypoxia Stress

Takifugu rubripes is deeply loved by people because of its delicious taste,rapid growth and high nutritional value.Now it has developed into an important industrial culture processing fish in northern China,and is one of the main export fish to earn foreign exchange.Hypoxia is considered to be one of the most serious factors threatening the stability of aquatic ecosystem and the quality and safety of aquaculture production because of its serious impact on the growth,breeding,behavior and physiology of fish.With the rapid development of puffer aquaculture in China,the research on dissolved oxygen in the process of puffer fish culture is becoming more and more urgent.In this study,based on transcriptome sequencing(RNA-seq)technique,the response mechanism of swim bladder tissue of T.rubripes under long-term hypoxia was analyzed for the first time,and based on the swim bladder itself,the difference in function of two closely related oriental swim bladders was revealed for the first time.Furthermore,the potential interaction of hypoxia-related chemokines was explored by fusion expression in vitro and GSTpull down technology,in order to analyze the relationship between hypoxia and hypoxia and the molecular mechanism of its function as much as possible.The main results are as follows:1.The transcriptome changes of swim bladder of T.rubripes after continuous hypoxia for10 days were studied by RNA-seq technique.The results of functional enrichment showed that under long-term hypoxia stress,swim bladder tissue could maintain the normal excitation of cells by regulating the ion concentration in the body,and activate the activity of downstream related proteins through calcium signal transduction pathway.to regulate the body to cope with the adverse effects of hypoxia.At the same time,insulin secretion is activated in a long-term hypoxic environment,which plays a certain role in reducing the level of blood glucose and reducing the disorder of lipid metabolism in the hypoxic environment.In addition,cell cycle,DNA replication,two pathways related to cell growth,are down-regulated in long-term hypoxia environment,indicating that the T.rubripes can reduce energy consumption by inhibiting the normal division and growth of swim bladder tissue cells.2.The swim bladder tissues of T.rubripes and Takifugu obscurus were compared and analyzed by RNA-seq technique.Significant differentially expressed genes were enriched and analyzed based on GO and KEGG.The enrichment results showed that there were significant differences in neuroactive ligand receptor signal transduction between the two species of Takifugu swim bladder,and different types of ligand-receptor interactions often mediate different signal transduction and play different functions.In addition,we also found that a large number of genes related to auditory function have been significantly enriched.Therefore,we believe that the swim bladder is functionally species-specific,even between two closely related species of Takifugu.The difference of this function may be caused by the mechanism of adaptive evolution.3.According to the results of transcriptome and based on p ET-41b(+)expression vector,we successfully constructed five fusion expression vectors of chemokine mature peptide with GST tag,and all of them were induced by IPTG to obtain the target fusion protein.At the same time,after further optimization of the induced expression conditions,a sufficient amount of fusion protein GST-CCL19l1 was successfully purified in the supernatant.Furthermore,a number of proteins with potential interaction with CCL19l1 were obtained by GST-pulldown combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).Through GO and KEGG enrichment analysis,we believe that chemokines can mediate glycolysis through HIF1 signaling pathway,and then participate in fish adaptation to hypoxia.Although the specific mechanism of this effect is not clear and needs to be further revealed through relevant studies,it is sufficient to confirm that chemokines are involved in hypoxia stress and play a key role.