Functional Analysis Of Chemokines And Their Receptors Under Acute Hypoxic Conditions In Takifugu Rubripes

Takifugu rubripes,as an important aquatic organism in northern China,has its economic yield deeply influenced by the oxygen content of seawater.With the significant expansion of T.rubripes aquaculture in recent years,the effect of different dissolved oxygen levels in seawater on its culture has become a hot topic of research.In this study,chemokines and receptors were identified and analyzed based on the genome of T.rubripes.Three tissues(brain,blood and gill)of T.rubripes were processed and transcriptome sequencing analysis was performed under different dissolved oxygen conditions,and the expression levels of chemokines and their receptors under different dissolved oxygen conditions were explored on the basis of transcriptome data.The DNA pull down technique was further used to analyze the chemokines and their receptors that showed significant differential expression under hypoxic stress at the level of gene transcriptional regulation,with the aim of dissecting the gene regulatory networks associated with these chemokines and their receptors,and thus revealing the relationship between the relevant chemokines and their receptors and the molecular mechanisms underlying their functions under hypoxic stress.The main results are as follows:1.Bioinformatics methods were used to data mine the genome of T.rubripes,using reported chemokine sequences of three species,Ictalurus punctatus,Danio rerio and Homo sapiens,aligned to the T.rubripes genome.9 CXC chemokine ligand(CXCL)genes,11 CXC chemokine receptor(CXCR)genes,22 CC chemokine ligand(CCL)genes,and 25 CC chemokine receptor(CCR)genes were systematically identified from the genome of T.rubripes.The phylogenetic tree shows that scleractinians fish are very closely related to each other evolutionarily compared to mammals,amphibians and birds.Covariance analysis showed that the genes upstream and downstream of the chromosomal region where the chemokine and its receptor are located in T.rubripes are conserved with the gene distribution structure of the corresponding region in D.rerio.Protein structural domain analysis revealed that all of the above 21 CC chemokine members contained conserved CC structural domains except for CCL17,which was a non-coding RNA,and all of the 9 CXC chemokine members contained conserved CXC structural domains.Except for CCR3.2 and CCR3.3,which are non-coding RNAs,all of the 34 chemokine receptors mentioned above contain a conserved 7-transmembrane GPCR structural domain specific to chemokine receptors.2.By processing and transcriptome sequencing analysis of three tissues(brain,blood and gill)of T.rubripes under different dissolved oxygen conditions,and then uniformly homogenizing the counts of the three tissues,the results showed that different tissues showed their own characteristics in response to hypoxia.In the brain,the transcriptome changed the least with 14 differentially expressed genes.In the blood the transcriptome changed the most,with 745 differentially expressed genes.It can be speculated that the tolerance of T.rubripes to hypoxia is prominent in the brain.In addition,by comparing the differential genes screened in each tissue,we found that each tissue showed a large number of differential genes each different from other tissues,while the proportion of differential genes shared between tissues was low,which was closely related to the functions of these tissues and organs.The results of GO function and KEGG signaling pathway enrichment analysis showed that most of the differentially expressed genes were enriched in biological processes such as regulation of muscle contraction,glutamatergic synapse,synaptic membrane,and the signaling pathways regulating HIF-1,AMPK,chemokines and amino acid metabolism might play an important role in the hypoxic response of T.rubripes.In addition,genes such as hif1,vegfa,gck,pfkfb and agr play a key role in the response to hypoxia.In the brain it may provide brain cells with abundant cellular structure and reduction substrates by enhancing glycolysis,while reducing oxidative phosphorylation and reactive oxygen levels to avoid oxidative stress death of brain cells.In gill tissues,it may remove abnormal cells by promoting apoptosis to maintain the stability of the internal environment of gill tissues under hypoxic stress.In blood,it may play a pleiotropic immunomodulatory role by catalyzing the conversion of the semi-essential amino acid L-arginine to ornithine and urea,thereby limiting the activation and proliferation of T cells to enhance hypoxic tolerance in the blood.3.After hypoxic stress,Gene set enrichment analysis(GSEA)showed that chemokine signaling pathway showed a down-regulated expression trend in brain,blood and gill of T.rubripes,but only in gill(adjusted P value<0.05).Heatmap clustering analysis of the expression of chemokines and their receptors in three tissues showed that the expression patterns of chemokines and their receptors were more different among tissues,while the expression patterns were similar within tissues,indicating that the expression of chemokines was tissue-specific.We selected chemokine CCL25 b and its receptor CCR9 b,which showed differential intra-tissue and inter-tissue expression changes under hypoxic stress,for DNA pull down experiments,and the mass spectrometry identification data and GO enrichment analysis showed that six transcription factors(C1QBP,PURA,ARHGAP35,NME2,RAP2 C,and DRG1)were enriched to the core promoter regions of CCL25 b and CCR9 b,and these preliminary results will help advance the understanding of the underlying biological processes related to the regulatory mechanisms of CCL25 b and CCR9 b expression.Although the actual function of those proteins in regulating CCL25 b and CCR9 b gene expression is unknown,we provide new ideas for the search of DNA-interacting proteins of CCL25 b and CCR9 b.