Transcriptome Response Of Sillago Sihama Exposed To Hypoxia Stress And Molecular Evolution Analysis Of Major Differentially Expressed Genes

Sillago sihama,commonly known as silver sillago,is a marine fish species belonging to the family Sillaginidae and widely distributed in the Indo-West Pacific.The silver sillago has a delicious taste,high nutritional value,and it is an emerging commercial marine aquaculture species in China.It is sensitive to hypoxia and variations exist among various populations and strains,while the genetic basis is still unclear.To date,no gene expression profiling for hypoxia tolerance trait in S.sihama has been reported;hence it’s essential to explore the genes involved in hypoxia stress.In this study,healthy fish were divided into four treatment groups,including 1 hour of hypoxia（hypoxia1h,DO = 1.5mg/L）,4 hours of hypoxia（hypoxia4h,DO = 1.5 mg/L）,4 hours of reoxygen（reoxygen4h,DO = 8.0 mg/L）after 4 hours of hypoxia（DO = 1.5 mg/L）,and normoxia or control group（DO = 8.0 mg/L）.We sequenced the transcriptome of gill,heart and liver tissues of four treatments from S.sihama by RNA-seq.Differentially expressed genes（DEGs）and their associated pathways were examined under hypoxia.We identified the major differentially expressed genes and their adaptation to hypoxia in S.sihama.The results will enrich the transcriptional expression patterns of different tissues in response to hypoxia stress,and lay a foundation for further revealing the genetic mechanism of hypoxia tolerance traits.The main findings are as below:（1）A comparative transcriptional profiles of gill tissue in response to hypoxia were conducted in S.sihama.In this study,we compared the transcriptome response to hypoxia stress in the gill tissue of S.sihama.A total of 3550 genes were identified as differentially expressed genes（DEGs）（log₂foldchange > 1 and P-value < 0.05）,including 1103,1451 and 996 genes in hypoxia1 h,hypoxia4h and reoxygen4 h groups,respectively.Only 247 DEGs were differentially co-expressed in all treatment groups.According to Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis,DEGs were significantly enriched in steroid biosynthesis,biosynthesis of amino acids,glutathione metabolism and metabolism of xenobiotics by cytochrome P450,ferroptosis and drug metabolism—cytochrome P450 pathways.Of these,the cytochrome P450（CYP）and glutathione Stransferase（GST）gene families were widely expressed.（2）A total of 3068 genes were significantly differentially expressed（og₂FC > 1.0,P <0.05）in transcriptome heart tissue under hypoxia stress.776,1141 and 1151 DEGs were identified at different hypoxic times 1h,4h,and reoxygen 4h in the heart tissue of S.sihama.The enrichment pathway analysis showed that the DEGs were significantly enriched in ribosome biogenesis in eukaryotes,retinol metabolism,DNA replication and the oxidative phosphorylation（OXPHOS）pathways.Thirteen DEGs from the RNA-seq results were validated by qRT-PCR.（3）A total of 3457 DEGs were identified in the transcriptome of liver tissue under hypoxia stress（og₂FC>1.0,P<0.05）.Of these,506,1721,and 1230 DEGs were identified at hypoxia 1 hour,hypoxia 4 hours,and reoxygenation 4 hours in the liver tissue,respectively.The enrichment analysis showed that the DEGs were significantly enriched in metabolic and translation changes pathways such as mapk signaling pathway,p53 signaling pathway,fatty acid metabolism,protein export,ribosome biogenesis in eukaryotes.The DEGs of seventeen genes validated the RNA-seq results by qRT-PCR.（4）We compared co-expressed DEGs from gill,heart and liver tissue under hypoxia stress.The results showed that liver tissue had a greater effect on hypoxia stress.Metabolic-related genes such as Slc2A1,Slc16A3,Ldha,and Ddit4 were highly expressed in three tissues after hypoxia treatment.Particularly,three genes,Egln3,Ddit4 and uncharacterized gene（novel.1595）,were differentially expressed in gill,heart and liver tissue and could be considered candidate hypoxia-specific biomarkers in all duration time of hypoxia.（5）Seventeen GST genes were identified and annotated in S.sihama.Phylogenetic analysis showed that the GST gene family contained two subgroups（cytosolic and MAPEGs）,and lacked three subgroups（i.e.Pi,Kappa,and MGST2）.Phylogenetic and syntenic analysis revealed that GST genes were conserved during evolution.The c DNA lengths of Ss GST ranged from 423 to 729 bp,with the number of amino acids from 140 to242 aa.The Ss GSTZ1 and Ss GSTM3 had a large number of exons,which contained 9 and6 exons,whereas Ss PTGES contained 3 exons,which is the smallest number of exons.8Ss GSTs were significantly differentially expressed under hypoxia stress in S.sihama by RNA-seq and qRT-PCR analysis.The expression results of Ss MGST3 b,Ss GSTO1,Ss GSTT1 b,and Ss GSTR2 in gill tissue were significantly up-regulated after 4 hours of reoxygenation.Ss GSTR3 was up-regulated expression in hypoxia 1hr group,while Ss GSTT1 b and Ss FLAP were significantly down-regulated in hypoxia 4hr group of heart tissue.ConclusionTaking together,three tissues（gill,heart and liver）exposure to hypoxia challenge were surveyed in this study.Several genes related to hypoxia were identified,including GSTs,CYPs and metabolism related genes（such as Slc2A1,Slc16A3,Idh A,Hsp and Ddit4）.It was found that apoptosis,stress and inflammation related pathways were significantly enriched under hypoxia stress.Moreover,we also identified the Ss GST,which shared common expression in three tissues.The results provide a foundation for further research on the molecular mechanism of hypoxia tolerance in S.sihama,and enrich the understanding of the mechanism of hypoxia adaptation in teleost fishes.