| The mud crab(Scylla paramamosain)is an economically significant crab species cultured in southeastern coastal areas of China.However,due to the crabs’ habitat in sediment rich in organic matter,the spoilage of this sediment consumes oxygen and leads to hypoxia stress.The proline hydroxylase(PHD)/ hypoxia-inducible factor(HIF)pathway is a crucial oxygen-sensing signal pathway found in most metazoans that plays a critical role in maintaining cell oxygen homeostasis.Despite its importance,there is currently no systematic study on the PHD/HIF oxygen-sensing signal pathway and its regulatory mechanism in mud crabs.In this study,the anti-oxidation level and gene expression changes of mud crabs under different dissolved oxygen concentrations were analyzed.Additionally,the transcriptional regulation mechanism of the key gene HIF-1in the PHD/HIF signal pathway of mud crabs under hypoxia stress and its interaction with PHD2 was elucidated.The main findings indicate that:1.The impact of hypoxia stress on the gill tissue of mud crabs and its relationship with oxidative stress.The results revealed that hypoxia stress-induced oxidative stress in the gill tissue.Specifically,this study found that the total antioxidant capacity(T-AOC)of the gill tissue in the moderately hypoxic group(dissolved oxygen concentration 3.0 ±0.2 mg/L,named "DO3")was significantly higher than that of the normoxic control group(dissolved oxygen concentration 7.5 ± 0.2mg/L,named "CK")after 6,12,and 24 h of hypoxia stress.Similarly,the T-AOC of the gill tissue in the acute hypoxia group(dissolved oxygen concentration 1.0 ± 0.2 mg/L,named "DO1")was also significantly higher than that of the control group after 12 and 24 h of hypoxia stress.Additionally,the content of malondialdehyde(MDA)in the gill tissue increased significantly after hypoxia stress.Specifically,the MDA content of the DO3 group was significantly higher than that of the control group after 6 h of hypoxia stress,but gradually decreased and returned to normal levels.Conversely,the MDA content of the DO1 group was significantly higher than that of the control group after 6,12,and 24 h of hypoxia stress,showing a continuous upward trend.These results suggest that mud crabs can effectively mitigate oxidative damage caused by hypoxia stress by enhancing their antioxidant capacity,thus preventing cellular damage.2.The transcriptome analysis of gill tissue in mud crabs under hypoxia stress.In this study,the gill tissue of mud crabs after 24 h of hypoxia stress at different dissolved oxygen concentrations was used as the experimental material for transcriptome analysis.A total of 94267 Unigenes were obtained by transcriptome sequencing,with an average length of 993 bp and N50 of 1844 bp,which were annotated in the public database to identify 53,687 genes.Among these genes,394 differentially expressed genes(DEGs)were found between DO1 and DO3 groups,481 DEGs were found between DO1 and CK groups,and 177 DEGs were found between DO3 and CK groups.Furthermore,10 DEGs were randomly selected to verify the accuracy of transcriptome data using fluorescence quantitative analysis.KEGG enrichment analysis showed that 230,229,and 177 signal pathways were significantly enriched between DO1 and CK groups,DO1 and DO3 groups,and between DO3 and CK groups,respectively.These pathways were related to energy metabolism,immunity,ion transport,and antioxidant stress.Notably,the expression of genes related to the antioxidant stress pathway was significantly up-regulated,indicating that mud crabs activate antioxidant system to cope with oxidative stress under hypoxia stress.In the PHD/HIF signal pathway,we found that the expression of the Elongin B gene involved in HIF-1α degradation was down-regulated,and the expression of the p300/CBP gene involved in HIF-1αtranscription was up-regulated.However,there was no significant change in the expression of the HIF-1α gene,indicating that the PHD/HIF signal pathway of mud crabs may have a more complex regulatory mechanism.3.The regulation of the anti-oxidation system and HRE-dependent transcription activity of HIF-1α in mud crabs(SpHIF-1α).HIF-1α is a key gene involved in the regulation of cell hypoxia adaptation.The results showed that T-AOC of gill tissue in mud crabs significantly decreased when SpHIF-1α was disturbed after 6 and 12 h of hypoxia stress,while MDA content significantly increased after 24 h of hypoxia stress(dissolved oxygen concentration was 1.0 ± 0.2 mg/L).SpHIF-1α interference also led to a significant decrease in the expression level of antioxidant-related genes.Notably,the expression level of the superoxide dismutase gene decreased significantly after 12 and24 h of hypoxia stress,the expression level of the catalase gene decreased significantly after 6 and 12 h of hypoxia stress,and the expression level of glutathione-S-transferase decreased significantly only after 24 h of hypoxia stress.In addition,the expression level of glutaredoxin(Grx)2,3,and 5 subtypes were significantly lower than that of the control group after 6,12,and 24 h of hypoxia stress.In order to further explore the regulatory mechanism of SpHIF-1α,we successfully cloned the promoter region sequence of Sp Grx3 and identified the existence of three hypoxia response elements(HRE).Promoter activity analysis demonstrated that SpHIF-1α could activate the hypoxic reporter gene containing HRE,while this activation was lost after HRE site mutation,indicating that the transcriptional activity of SpHIF-1α was dependent on HRE.4.Cloning and functional study of PHD2(Sp PHD2)from mud crab.In this study,we successfully cloned the full-length c DNA sequence of Sp PHD2 and found another variable splicing form of Sp PHD2-iso2.The c DNA length of Sp PHD2 is 1926 bp,including 333 bp of 5’ untranslated region,354 bp of 3’ untranslated region,and 1239 bp of the open reading frame(ORF).Sp PHD2 ORF encodes 412 amino acids,and the predicted molecular weight of the protein is about 46.336 k Da.In contrast,the amino acid encoded by Sp PHD2-iso2 ORF lacks the aa33-133 region.Conservative domain analysis showed that the Sp PHD2 protein contained a P4 Hc domain at the C-terminal end and a zf-MYND domain at the N-terminal,while the zf-MYND domain of Sp PHD2-iso2 protein was missing.Phylogenetic analysis showed that Sp PHD2 was highly homologous with the PHD2 gene of other crustaceans.Sp PHD2 was expressed in all tissues tested,and the highest expression was found in the heart and hemolymph.Sp PHD2 protein and Sp PHD2-iso2 protein are mainly present in the cytoplasm,and hypoxia stress cannot change their subcellular localization.When Sp PHD2 is disturbed,the expression level of SpHIF-1α was significantly increased,thus activating the expression of the downstream target gene.In contrast,when Sp PHD2 is overexpressed,the transcriptional activity of SpHIF-1α was significantly inhibited.In addition,compared with the complete Sp PHD2 protein,Sp PHD2-iso2 protein has a stronger inhibitory effect on SpHIF-1α transcriptional activity.5.The negative feedback regulatory mechanism of SpHIF-1α on Sp PHD2 under hypoxic stress.The expression level of Sp PHD2 was found to increase significantly after 12 h of hypoxic stress and then decreased but remained significantly higher than the control group at all times.However,disturbing SpHIF-1α significantly lowered the expression level of Sp PHD2 compared to the control group at 6,12,and 24 h of hypoxic stress,indicating that Sp PHD2 was also regulated by SpHIF-1α under hypoxia.Further analysis revealed that there were four HRE sites in the promoter region of Sp PHD2.Dual luciferase activity analysis showed that all of these HRE sites could respond to SpHIF-1α.The study suggests that SpHIF-1α may negatively regulate Sp PHD2 via HRE during hypoxia,highlighting the complexity of the PHD/HIF signaling pathway in mud crabs.In summary,this study first reveals the molecular mechanism of the PHD/HIF signal pathway in response to hypoxia in mud crab and provides a theoretical reference for exploring the mechanism of hypoxia tolerance in crustaceans. |
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