| Hemocytes,the core and basis of the innate immune system,are important executor of immune defense in invertebrates.The morphological structure,cellular and molecular characteristics,as well as immune functions of different hemocyte subtypes are significantly different.Based on the cell size,nucleo-cytoplasmic(N:C)ratio,and the granularity,the hemocytes of shellfish were generally divided into three subtypes:Agranulocyte,Semi-granulocyte and Granulocyte.However,less was known in the molecular characteristics of different sub-hemocytes in shellfish.In the present study,potential molecular markers AATase(designed as CgAATase)and SOX11(designed as CgSOX11)of granulocyte,and CD9 antigen(designed as CgCD9 antigen)of agranulocyte were screened based on the analysis of the single cell transcriptome data of agranulocyte,semi-granulocyte and granulocyte in Pacific oyster Crassostrea gigas.Their molecular characteristics,expression patterns and potential value in hemocytes sorting were identified through molecular biological and cytobiology techniques.In the present study,an alcohol acyltransferase(designed as CgAATase)with specific expression pattern was identified from oyster C.gigas,and it could be employed as a potential marker for the isolation of oyster granulocytes.The open reading frame(ORF)of CgAATase was of 1431 bp,encoding a peptide of 476 amino acids with a typically conserved AATase domain.The mRNA transcripts of CgAATase were highest expressed in hemocytes.The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level(27.40-fold compared to control group,p<0.05)at 6 h after Vibrio splendidus stimulation.The total hemocytes were sorted as granulocytes,semi-granulocytes and agranulocytes by Percoll~?density gradient centrifugation.CgAATase transcripts were dominantly observed in granulocytes,which was 8.26-fold(p<0.05)and 2.80-fold(p<0.05)of that in agranulocytes and semi-granulocytes,respectively.The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic beads.CgAATase protein was mainly detected on the cytomembrane of granulocytes.About 85.7±4.60%of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic beads coated with anti-CgAATase monoclonal antibody,and 97.7±1.01%of the rest hemocytes(agranulocytes and semi-granulocytes)were negative for CgAATase.The isolated primary granulocytes could maintain cell activity for more than one-week in vitro culture that exhibited numerous filopodia.These results collectively suggested that CgAATase was a molecular marker of oyster granulocytes,and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.In the present study,a transcription factor SOX11 homologue(designated as CgSOX11)was obtained as the potential molecular marker of granulocytes,and it with specific expression pattern was identified.The full-length cDNA of CgSOX11 was of 1515bp with an ORF of 1020bp,encoding a 339-amino acid protein with a predicted molecular mass of 38kDa.SMART predicted that CgSOX11 contained a conservative HMG box domain.The results of multiple alignments revealed that the similarity of CgSOX11 was mainly located on the conserved high-mobility group(HMG)domain and C-terminal unknown domain,and the similarity was up to 80%.Phylogenetic tree analysis show that CgSOX11 was clustered with MySOX11 from the Mizuhopecten yessoensis,and joined the invertebrate group.The qRT-PCR was performed to verify the results from the single cell transcriptome.The qRT-PCR results revealed that CgSOX11 transcripts were highly expressed in hemocytes and gill,and relatively low levels of the labial palp,mantle,gonad,mantle,adductor muscle and hepatopancreas.The mRNA expression level in hemocytes and gill was 2.78-fold and 1.95-fold(p<0.05)of that in labial palp,and significant high expression in granulocytes,which was 19.14-fold(p<0.05)and 17.26-fold(p<0.05)of that in agranulocytes and semi-granulocytes,respectively.The expression level of CgSOX11 mRNA increased significantly at 3 h(8.03-fold compared to control group,p<0.05)after V.splendidus stimulation.The recombinant protein re-CgSOX11 was obtained by the prokaryotic expression system of E.coli,and was used to immune the Balb/C mouse for preparing the polyclonal antibody.The western blotting assay revealed two distinct bands with molecular weight 38kDa and 50kDa.The 38kDa band was consistent with the predicted molecular mass of CgSOX11.With the SUMO1,2,3 monoclonal antibodies,a distinct and unique band was revealed at the same site with 50kDa.Immunofluorescence assay revealed that CgSOX11and SUMO were located both on the nucleus of hemocytes.And thus it was proposed that CgSOX11 might be in SUMOylation in oyster.Combined with immunomagnetic beads and flow cytometry,the granulocytes were positive for CgSOX11 and could be successfully separated by immunomagnetic beads coated with anti-CgSOX11 polyclonal antibody,and the separation purity is 81.2%.These results collectively suggested that CgSOX11 was a molecular marker of oyster granulocytes.Based on the single-cell transcriptome sequencing of the three sub-hemocytes,in the present study,we obtained the potential molecular marker CD9 antigen(designed as CgCD9 antigen)by analyzing the data of transcriptome,and it with specific expression pattern was identified.The transcriptome data suggest that CgCD9 antigen transcript was dominantly in agranulocyte,and it could be employed as a potential marker for agranulocytes.The ORF of CgCD9 antigen was of693bp,encoding a peptide of 230 amino acids with a predicted molecular mass of 25kDa.SMART predicted that CgCD9 antigen contained a typically conserved tetraspanin domain.The mRNA transcripts of CgCD9 antigen were highest expressed in hemocytes,lower expressed in hepatopancreas,mantle,gonad,gill,ganglion,adductor muscle,and labial palp.CgCD9 antigen transcripts were dominantly observed in agranulocytes,which was 6.51-fold(p<0.05)and 2.19-fold(p<0.05)of that in semi-granulocytes and granulocytes,respectively.The specificity of CgCD9 antigen depend on the extracellular segment of the macrocyclic antigen.Therefore,a peptide fragment(11 aa)of the extracellular segment was synthesized,and was used to immune the Balb/C mouse for preparing the monoclonal antibody.The monoclonal antibody 3D8 against CgCD9 antigen were produced with the higher specificity 1.596.Immunofluorescence assay revealed that CgCD9 antigen protein was mainly detected on the cytomembrane of agranulocytes.The monoclonal antibody 3D8 was employed for the isolation of agranulocytes with the immunomagnetic beads.About 86.9±2.90%of the agranulocytes were positive for CgCD9 antigen and they could be successfully separated by immunomagnetic beads coated with anti-CgCD9 antigen monoclonal antibody.The isolated primary agranulocytes could maintain cell activity for more than one-week in vitro culture.These results collectively suggested that CgCD9 antigen was a molecular marker of oyster agranulocytes.In conclusion,our study firstly identified that the molecular marker CgAATase of granulocytes that can isolated the granulocytes with high purity and activity,the molecular marker CgSOX11 of granulocytes that is a transcription factor that regulate the occurrence,development and cell fate determination of hemocytes,the molecular marker CgCD9 antigen of agranulocytes that can isolated the agranulocytes with high purity and activity.These results further deepen the current understanding of granulocytes and agranulocytes,which provide a useful tool for the biological analysis of granulocytes and agranulocytes,and provide a molecular basis for the study of the occurrence,development,immune function and regulatory mechanism of hemocytes,and provide new idea for the prevention and treatment of diseases of important aquatic invertebrates. |
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