| Orange-spotted grouper(Epinephelus coioides)is one of the dominant aquaculture species in China.However,diseases are an important factor restricting its aquaculture production.As a new natural immunopotentiator,?-glucan has a good application prospect in aquaculture industry.In this paper,transcriptome property and m RNA expression of immune-related genes in orange-spotted grouper under the stimulation by ?-glucan were analyzed.Then,the influence of ?-glucan on expression of immune-related genes was explored.Orange-spotted grouper were intraperitoneally injected with 0.2 m L ?-glucan(0.05 g/m L concentration)and the thymus,spleen,head-kidney and peripheral lymphocytes tissues were respectively transcriptome sequenced after stimulation at 6 h and 72 h.As the result of the research,123677 unigene were assembled and the average length was 1711.30 bp.A total of 96355 unigene were functional annotated by using Blast alignment database.The number of unigene annotated in six bioinformatics databases were 91955(Nt),66089(Nr),14126(GO),24018(COG),9673(KEGG)and 54148(Swiss Prot),respectively.This data revealed that the assemble result was reliable.Based on this result,we further analyzed and found that:(1)According to the analysis of DEGs,74 DEGs involved in six immune-related categories were screened,which were related with antigen processing and presentation,cell adhesion molecules,complement system,cytokine,pattern recognition receptor and phagocytosis.(2)The DEGs were analyzed by Gene Ontology(GO)and seven immune-related terms were selected,including the immune response,B cell receptor signaling pathway,chemokine receptor activity,leukocyte chemotaxis,chemokine-mediated signaling pathway,interleukin-1 receptor binding and lysosomal membrane.(3)Through KEGG pathway enrichment analysis,we got 19 category immune-related and significantly enriched pathways.Toll-like receptor signaling pathway and RIG-I-like receptor signaling pathway were both selected from peripheral lymphocytes,spleen,head-kidney,thymus tissues.T cell receptor signaling pathway,NOD-like receptor signaling pathway,Natural killer cell mediated cytotoxicity and Hematopoietic cell lineage were all selected from peripheral lymphocytes,spleen,head-kidney and tissues.B cell receptor signaling pathway and Intestinal immune network for Ig production were selected from peripheral lymphocytes and head-kidney tissues.(4)Seven immune-related DEGs,which differences were significant,were randomly selected to validate by q RT-PCR,and the result of q RT-PCR was the same as that of the RNA-Seq.We further studied the temporal expression of DEGs in immune related tissues of orange-spotted grouper stimulated by different ?-glucan concentration.The groups were injected 0.2m L b-glucan at five different concentrations(0.015g/m L,0.03g/m L,0.06g/m L,0.12g/m L,0.15g/m L)and the control group were injected with the 0.2 m L PBS.Then the thymus,spleen,head-kidney and peripheral lymphocytes tissues were respectively obtained after stimulation at 1d,2d,3d,4d,5d,6d and 7d.Finally seven kinds of immune-related genes(IL-1??TLR13?IFN??MHC-I?TNF??C3-1and CD2)were studied in the present tissue samples by the Real-time PCR technique.The results were:(1)The expression of IL-1?,TNF? and TLR13 genes were highly depends on injection dose.All of them reached the highest level when injected with a concentration of 0.06 g/m L;when injected with higher concentration,the gene expressions were significantly down-regulation.(2)The expression of C3-1 and MHC-I genes were influenced by b-glucan: both of them reached the highest level at concentration of 0.06g/m L;when injected with a concentration higher than 0.12g/m L,the gene expressions remained at a high level.(3)IFN? gene showed a concentration-dependent expression patterns and reached highest level at a concentration of 0.12g/m L and up-regulated with concentration increase.(4)CD2 gene expression was related to the injection of b-glucan concentration and days after stimulated.The gene relative expression was significantly up-regulated five days after simulated with the concentration higher than 0.12g/m L.The results of this study will provide reference data for research on the mechanism of fish immune system which was modulated by ?-glucan. |
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