| Hypoxia affects various aspects of physiological activities in aquatic animals,including oxygen transport,organismal metabolism,signal transduction,cell proliferation and apoptosis,and vascular formation,and other biochemical processes.Studies have found that the tolerance of aquatic animals to hypoxia varies among different species,with mollusks demonstrating greater tolerance to hypoxia compared to other aquatic organisms such as fish,crustaceans,echinoderms,and annelids.The Tegillarca granosa(blood clam),an important economically cultivated bivalve mollusk,mainly inhabits the intertidal zone or tidal flat,where it is susceptible to periodic hypoxia or desiccation stress.Our preliminary research has found that T.granosa was more tolerant of hypoxia compared to other bivalve mollusks.In order to explore the molecular mechanisms underlying T.granosa’s response to hypoxic stress,we have conducted the following study:1.Using transcriptome sequencing technology,we analyzed the enriched signaling pathways and biological processes in the hemocytes of blood clam under hypoxia stress,and explored the molecular regulatory mechanisms underlying its hypoxia tolerance.Hemocytes were subjected to hypoxia stress with dissolved oxygen(DO)concentration of 0.5 mg/L for 6,24,72,and 120 h,and differentially expressed genes(DEGs)were screened and subjected to Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis at 72 and 120 h after stress.The results showed that the number of DEGs increased from 6 h to 120 h under hypoxia stress.GO functional analysis mainly involved negative regulation of JUN kinase activity,negative regulation of protein hydrolysis,and immune system processes,indicating active antiapoptosis and stress resistance processes.KEGG pathway analysis revealed enrichment in signaling pathways related to insulin and pancreatic secretion,HIF-1 pathway,calcium signaling,and cell apoptosis,suggesting that these signaling pathways may play important roles in the hypoxia tolerance of blood clam.2.Based on the differential gene expression profiles and enriched pathways under low oxygen stress,7 differentially expressed genes were selected for q RT-PCR validation,including HIF-1α(hypoxia-inducible factor 1 alpha),JUN protein,DUSP10(dualspecificity protein phosphatase 10),CCKR(cholecystokinin receptor),TRIM(tripartite motif protein),EIF-4E(eukaryotic translation initiation factor 4E),and PTHR2(parathyroid hormone receptor).Through q RT-PCR analysis,it was found that HIF-1α,JUN,DUSP10,CCKR,TRIM,and EIF-4E were upregulated,while PTHR2 was downregulated,which was consistent with the transcriptome results.3.Due to the important role of the calcium ion channel protein CaV2 in the calcium signaling pathway and its significant response after low oxygen stress in blood clam,an in-depth study was conducted on the calcium channel protein CaV2 in the calcium signaling pathway of blood clam.The sequence,expression level and tertiary structure of its domains were analyzed and compared with other common bivalves.Specifically,sequence alignment and tertiary structure prediction analyses were conducted on the calcium ion transport domain sequences of blood clam and representative species of other bivalves.Additionally,experiments were performed to measure changes in intracellular and extracellular calcium ion concentration signals as well as CaV2,NFAT gene m RNA expression levels in blood clams under low oxygen stress.The experimental results showed that under low oxygen stress,the extracellular calcium ion concentration of blood clam decreased significantly,while the intracellular calcium ion concentration increased significantly;CaV2 expression was down-regulated and NFAT expression was upregulated,which was consistent with the results of transcriptome analysis.It is speculated that the increase in intracellular calcium ion concentration activates some negative transcription factors such as NFAT,which inhibits the transcription of CaV2 and leads to a decrease in its transcription level,this is preliminarily verified by the results of interference experiments on NFAT.Sequence alignment of the structural domain revealed that there were 4 conserved residue sites mutated in Arcidae shellfish compared to other bivalves,which are threonine residue at position 10 changed to isoleucine,alanine residue at position 198 changed to methionine,leucine residue at position 290 changed to alanine,and tyrosine residue at position 314 changed to phenylalanine.The three-dimensional structure prediction analysis showed that the conformation of the calcium ion transport domain of bivalves is highly conserved.However,in the region from Position 23 to 37,blood clam exhibits a folded structure in the conformation,while other bivalves exhibit irregularly coiled conformation.Therefore,this study suggests that specific differences in sequence and conformation of the calcium ion transport domain of blood clam relative to other bivalves may change the spatial structure and molecular stability of CaV2 protein,leading to a decrease in its activity or alteration in its physicochemical properties,which in turn weaken its calcium ion transport function.In summary,this study found that the calcium signaling pathway,pancreatic secretion pathway,and apoptosis pathway may play important roles in the adaptation of the blood clam to low oxygen conditions,and that the calcium signaling pathway played a crucial role.The expression of CaV2 in the calcium signaling pathway of blood clam hemocytes showed a decreasing trend after hypoxic stress,based on transcriptional site prediction and interference experiments,it is speculated that CaV2 is caused by negative regulation of transcription factor NFAT.The decrease in CaV2 expression on the cell membrane will slow down the excessive influx of calcium ions into Hemocytes after low oxygen stress,thereby avoiding downstream reactions that are too intense and detrimental to cell survival.It was also found that the specific differences in the sequence and spatial configuration of calcium ion transport domain in blood clam compared to other bivalves can cause differences in the physicochemical and functional properties of CaV2 in blood clam compared to other bivalves,which may be related to the blood clam’s tolerance to low oxygen.This study provides theoretical references for further elucidating the molecular mechanisms of blood clam’s tolerance to low oxygen and the cultivation of low oxygen-tolerant strains. |
PDF Full Text Request