| Takifugu obscurus is an important economic fish in China.With the increasing consumption market in recent years,the culture scale is expanding.But high-density culture and rising temperatures often lead to anoxia in the water,resulting in death from lack of oxygen.Therefore,hypoxia has become an important limiting factor in the culture of T.obscurus.Therefore,it is particularly important to study the hypoxia tolerance of T.obscurus.At present,studies on hypoxia of T.obscurus mainly focus on physiological and biochemical aspects,and the molecular regulatory mechanism to deal with hypoxia is rarely reported.Therefore,in this study,transcriptome and metabolome techniques were used to analyze liver and brain tissues of T.obscurus under acute hypoxia stress,to explore the molecular regulatory mechanism of hypoxia and reoxygenation of T.obscurus from the perspective of transcription and metabolism levels,and to analyze the important signal pathways involved in regulation,so as to provide certain theoretical basis for the study of hypoxia tolerance of T.obscurus.It can lay a foundation for the breeding of hypoxic tolerance varieties.The main research results of this paper are as follows:1.Transcriptomic analysis of the molecular regulatory mechanism of liver response to hypoxia in T.obscurusIn transcriptome analysis of liver tissue,1141 differential genes were identified under hypoxia condition,and 798,1480,761 and 1484 differential genes were found in 4h,8h,12h and24h reoxygenation groups,respectively.The differentially expressed genes were compared into the GO database,and the mainly enriched genes were related to GO functional items such as molecular functional regulatory factors,extracellular regions,activity of signal transducers,and activity of cross-mode signal receptors.By comparing the differentially expressed genes in KEGG database,MAPK,AGE-RAGE and apoptosis signaling pathways play important roles in liver tissue of T.obscurus exposed to hypoxia stress.The expression of HSP72 in MAPK pathway was significantly up-regulated,and the up-regulation of HSP72 inhibited the activation of JNK signaling pathway and prevented cell apoptosis.Egr-1 is up-regulated in AGE-RAGE signaling pathway,and Egr-1 plays an active role in cell growth,proliferation,differentiation,and development.In addition,PI3K is also significantly upregulated,and the activation of PI3K can affect the expressions of fibroblast growth factor（FGF）,vascular endothelial growth factor（VEGF）and human growth factor（HGF）.As a result,VEGF expression is indeed upregulated.VEGF plays an important role in promoting angiogenesis,inhibiting endothelial cell apoptosis and regulating vascular permeability.The apoptosis signaling pathway was significantly enriched at both hypoxia and reoxygenation stages,but was no longer enriched at 12h after reoxygenation.FADD and c-jun were up-regulated at hypoxia and down-regulated at 4h after reoxygenation.These two genes play an important role in the inhibition of apoptosis.2.Transcriptomic analysis of the molecular regulatory mechanism of brain response to hypoxia in T.obscurusIn the transcriptome analysis of brain tissue,1723 differential genes were identified under hypoxia condition,and 1731,2165,1299 and 666 differential genes were detected in the 4h,8h,12h and 24h reoxygenation groups,respectively.Through GO enrichment analysis,it was found that differential genes were mainly concentrated in GO functional items such as signal receptor binding,extracellular regions,ion transport,and hormone activity.A comparison of differ-expressed genes in KEGG database showed that Apelin and MAPK signaling pathways played an important role in the response to acute hypoxia in the brain tissue of T.obscurus,and were enriched in both hypoxia and reoxygenation,until 24h after reoxygenation.After hypoxia,AMPK in Apelin signaling pathway showed a downward trend.Studies have shown that AMPK plays an important role in regulating growth and remodeling metabolism.When activated,AMPK promotes the catabolic pathway to produce more ATP and inhibits the anabolic pathway.At 8h after reoxygenation,AMPK began to be up-regulated.In brain tissue,the role of AMPK is believed to be in the pathway of energy sensing neurons and energy balance in the body.In order to maintain neural activity and energy balance under hypoxia state,brain tissue may not produce more ATP like normal catabolic pathways,but to reduce the metabolic rate and process for sustained energy balance and neuronal activity.When recovering from hypoxia,the body has enough oxygen to deliver,so AMPK activity is increased again.By 24h,this pathway is no longer enriched,which may indicate that the body has recovered from the process of hypoxia stress.In the MAPK signaling pathway,ERK and PKC are down-regulated,and the activation of these two genes can promote cell differentiation and growth.Therefore,down-regulated after hypoxia may inhibit cell growth and differentiation,and these two genes are up-regulated after reoxygenation.In addition,c-fos gene in this pathway is upregulated after hypoxia,and c-fos gene is believed to be related to traumatic stimulation of neurons in brain tissue studies.Therefore,its upregulation indicates that hypoxia stimulates the brain tissue of T.obscurus,and the gene is downregulated after reoxygenation.3.Metabolomics analysis of the molecular regulatory mechanism of liver response to hypoxia in T.obscurusIn the analysis of liver metabolites,88 metabolites were identified under the positive ion mode,among which 81 were up-regulated and 7 were down-regulated.13 metabolites were identified under the negative ion model,of which 6 were up-regulated and 7 were down-regulated.Through metabolite screening and KEGG enrichment pathway analysis,it was found that the arachidonic acid metabolic pathway,Fox O signaling pathway and m TOR signaling pathway were significantly enriched during hypoxia,and these pathways were no longer enriched after 12h reoxygenation.AMP is accumulated in Fox O signaling pathway,and AMP activates AMPK to play its role,which is a key molecule in energy metabolism,such as regulating glucose uptake,glycolysis and fatty acid oxidation,and promoting ATP production.Therefore,the up-regulation of AMP may be related to the maintenance of metabolic balance.When cells are stimulated by hypoxia,m TOR is inhibited.However,it is worth noting that the significant enrichment of this pathway is also due to the significant up-regulation of AMP.As mentioned above,up-regulation of AMP will activate AMPK,and activated AMPK will lead to the inhibition of m TOR activity.In addition to the above signaling pathways,the arachidonic acid metabolic pathway was also significantly enriched,and LTE4was up-regulated in the pathway.LTE4is produced by inflammatory white blood cells and is closely associated with respiratory inflammation.Therefore,the upregulation of LTE4may indicate the effect of hypoxia on the respiratory system of T.obscurus.4.Metabolomics analysis of the molecular regulatory mechanism of brain response to hypoxia in T.obscurusIn brain metabolome analysis,41 different metabolites were identified by positive ion model,of which 24 were up-regulated and 17 were down-regulated.The negative ion model identified 22 differential metabolites,of which 14 were up-regulated and 8 down-regulated.Through metabolite screening and KEGG enrichment pathway analysis,it was found that6-Gingerol,a key metabolite,was down-regulated after hypoxia stress.This metabolite has antioxidant and anti-inflammatory effects,and plays a positive role in cell apoptosis.Therefore,the down-regulation of this metabolite may inhibit cell apoptosis.After reoxygenation,sphinolipid metabolic pathway is significantly enriched,and phosphoethanolamine is down-regulated in this pathway.This metabolite has the function of inhibiting angiogenesis,indicating that after reoxygenation,it begins to promote the growth and differentiation of related cells,while under hypoxia,it plays an inhibitory role in cell growth and cell differentiation. |
