| In aquatic ecosystems,dissolved oxygen(DO)has been the main source of oxygen for aquatic organisms.Factors such as climate change,water pollution and intensive fish farming often expose fish to hypoxic environments,which negatively affects their behavior,growth,physiological metabolism,and immune response.And in severe cases,hypoxia could even affect fish reproduction and survival,causing huge economic losses to the fish farming industry.Takifugu rubripes mainly distributed in the coastal areas of China,Korean Peninsula and Japan.Due to its delicious meat and high nutritional value,T.rubripes has become an important marine economic fish in the coastal areas of China.However,with the significant expansion of T.rubripes aquaculture,its economic yield is deeply influenced by the DO concentration in seawater.Therefore,understand the molecular regulatory mechanism of T.rubripes in response to hypoxic stress is of great significance for the breeding of hypoxia-tolerant species.The adaptive mechanisms of T.rubripes in response to acute hypoxia was explored before.Therefore,based on our previous study,we further analyzed the transcriptomic changes in the blood of T.rubripes under chronic hypoxia using RNA-Seq technology in the present study.In addition,we also compared the transcriptomic data of chronic hypoxic blood with acute hypoxic blood and chronic hypoxic brain to investigate the transcriptomic changes of blood under different hypoxic levels(acute and chronic hypoxia),and the transcriptomic changes of different tissues(brain and blood)under the same hypoxic level,and then comprehensively understood the expression patterns and differential expression levels of genes in the hypoxic environment of T.rubripes.At the same time,available transcriptome data was used to screen the differentially express FOXO3 transcription factor under hypoxia.On the one hand,molecular biological experiments were conducted to explore the interaction between FOXO3 transcription factor and their target genes.On the other hand,bioinformatics was used to predict the transcription factor binding sites(Motif)of FOXO3 transcription factor.The results of this study not only reveal the mechanism of hypoxia tolerance and the tissue-specific molecular characteristics of T.rubripes in response to hypoxia,but also provide new insight for studying the regulation of gene transcription.The results are as follows:1.Changes of transcriptome in blood of T.rubripes under chronic hypoxia.To understand the molecular responses of T.rubripes to chronic hypoxia stress,RNA-Seq technology was used to study transcriptomic changes in the blood of T.rubripes under normoxia and hypoxia.Combined with bioinformatics,a large number of differentially expressed genes(DEGs)were identified,and gene function enrichment analysis were also performed.The results were as follows: 1)a total of 710 DEGs were identified,including 370 up-regulated and 340 downregulated genes;2)the functional enrichment results showed that chronic hypoxia could promote anaerobic metabolism by up-regulating glycolysis/gluconeogenesis,fructose and mannose metabolism to provide energy supply under hypoxic environment;3)blood could inhibit energyconsuming biological processes such as immune regulation and cell proliferation to reduce energy consumption under chronic hypoxia;however,the suppression of innate immune processes might make fish more susceptible to diseases.2.Tissue differences in response to hypoxic stress in T.rubripesTo understand the tissue-specific molecular characteristics of T.rubripes in response to hypoxia,we analyzed the transcriptomic data of blood and brain of T.rubripes under chronic hypoxia.The results were as follows: 1)The brain of T.rubripes had a prominent tolerance to chronic hypoxia,and the tissue specificity of the same species was strong;2)In contrast to the immune response mechanism in blood,the brain could significantly up-regulate the expression of immune-related pathways and genes to enhance immune defense under hypoxia;3)The brain was able to promote substance synthesis and energy supply under hypoxic environments by actively regulating ribosomes,glycolysis,arachidonic acid(ARA)metabolic pathways and other metabolism-related pathways;4)Compared with blood,which responded to hypoxic stress by inhibiting cell cycle and other energy-consuming biological processes,the brain preferred to promote apoptosis to remove useless cells to maintain the homeostasis of the body’s internal environment and thus adapt to hypoxic environments.3.Adaptation mechanisms of T.rubripes in response to different DOTo understand the molecular mechanisms of T.rubripes when they coping with different DO levels,we analyzed the transcriptomic data of blood of T.rubripes under acute and chronic hypoxia.The results were as follows: 1)As same as the regulatory mechanism of chronic hypoxic blood,under acute hypoxic stress,blood could also reduce energy expenditure in hypoxic environments by suppressing innate immune responses,which reflected the functional properties of blood tissues in the organism;2)Signal transduction was the main functional class involved in DEGs under acute hypoxic blood.Blood could play an essential role in enhancing myocardial protection,angiogenesis,and rapid activation of anaerobic metabolism to coordinate intracellular energy metabolism and homeostasis through upregulating the expression of HIF-1 and AMPK signaling pathways and downstream genes.4.Functional exploration of FOXO3 transcription factor under hypoxiaBased on the transcriptome data,FOXO3 transcription factor was screened to be differentially expressed under hypoxic stress.On the one hand,the DNA binding domain of FOXO3 was obtained by the prokaryotic expression,and the fragmented DNA was used to bind to the purified GST-FOXO3 protein for screening the DNA fragments that could bind to the GST-FOXO3 protein.The results identified four downstream target genes might be directly regulated by FOXO3 transcription factor,namely STIP1,Tbx16,RIPOR1,and CPSF4.These results laid the foundation for the role of FOXO3 transcription factor in the development,cell proliferation,and apoptosis of the organism.On the other hand,a DNA Motif of FOXO3 transcription factor,namely "AAAACAAGA",was predicted by bioinformatics analysis as a binding site(Motif)of FOXO3 transcription factor,which was involved in the regulation of gene transcription under hypoxic environment. |
PDF Full Text Request