| Immune response is a process in which the immune system regulates various physiological activities of the organism to eliminate pathogens and maintain the dynamic balance and relative stability of internal environment after the organism is invaded by pathogens.During immune response,the number and function of immune cells,especially haemocytes,will change dynamically to maintain immune homeostasis.In the early stage of immune response,haemocytes experience rapid proliferation to effectively resist the pathogen,and once the pathogen threat conrtolled,haemocytes will reduced to prevent the damage caused by over-immunity.Transcription factor B lymphocyte induced mature protein 1(Blimp-1)plays an important regulatory role in the genesis and development of various immune cells.The dynamic changes of haemocytes are also indispensable in the regulation of immune response.At present,the dynamic change characteristics and regulatory mechanism of haemocytes during immune response in invertebrates are still unclear,and the function and role of Blimp-1 in invertebrate immune defense are unexplored,either.In this study,the oyster Crassostrea gigas was studied using bioinformatics,molecular biology and cell biology techniques.Blimp-1(CgBlimp-1)and its key downstream gene CgMyc-A were identified,and the regulatory effects of CgBlimp-1 and CgMyc-A on the proliferation of haemocytes were investigated.The conclusions were listed as follows:1.CgBlimp-1 and CgMyc-A were evolutionarily conserved.Transcription factors CgBlimp-1 and CgMyc-A were identified from the genome of Crassostrea gigas.The full-length c DNA sequence of CgBlimp-1 was of 2616 bp with an open reading frame(ORF)of 2502 bp encoding a peptide of 834 amino acid residues.There was a SET domain(residues 77-201)at the N-terminal and five Zinc finger C2H2(Zn F_C2H2)domains(residues 558-580,586-608,614-636,642-664,and 670-692)at the C-terminal of CgBlimp-1.The similarity of amino acid sequence to that of Crassostrea virginica was 86.83%,and 32.43% to that of Danio rerio.The full-length c DNA sequence of CgMyc-A was of 2223 bp with an ORF of 1149 bp encoding a peptide of 382 amino acid residues.Its protein domain composition was relatively conservative,including Myc domain(5-118 aa)at the N-terminal and HLH domain(302-354 aa)at the C-terminal.The similarity of amino acid sequence to that of Crassostrea angulate was 98.43%,and 26.86% to that of Mus musculus.By constructing the evolutionary tree of the two proteins,they were all clustered together with invertebrate Blimp-1 and C-Myc,respectively.2.CgBlimp-1 and CgMyc-A were constitutively expressed in different tissues,and increased significantly after Vibrio splendidus stimulation.qRT-PCR was used to detect the expression of CgBlimp-1 and CgMyc-A m RNA in different tissues.The result showed that the m RNA of CgBlimp-1 and CgMyc-A expressed constitutively in tested tissues and haemocyte.The m RNA expression level of CgBlimp-1 was the highest in gill,and higher in hepatopancreas,labial flap and mantle,which was 20.07 fold(p < 0.001),13.19 fold(p < 0.01),6.10 fold(p < 0.01)and 5.21 fold(p < 0.01)of that in haemocyte,respectively.The m RNA expression level of CgMyc-A was the highest in gill,and higher in haemocyte,which was 2.33 fold(p < 0.01)and 1.89 fold(p < 0.05)of that in mantle,and in other tissues did not show significant differences.Further detection showed that the expression level of CgBlimp-1 and CgMyc-A were significantly different in three subpopulations of haemocyte,and both of them were the highest in granulocyte.After V.splendidus stimulation,the m RNA expression level of CgMyc-A in haemocytes up-regulated significantly at 3,12,and 48 h,which was 2.19-fold(p < 0.01),2.05-fold(p < 0.001)and 1.63-fold(p < 0.01)of that in control group,respectively.The m RNA expression level of CgBlimp-1 in haemocytes up-regulated significantly at 24,48,and 96 h,which was 4.16-fold(p < 0.05),7.68-fold(p < 0.01)and 2.65-fold(p < 0.05)of that in control group,respectively.3.The proliferation of haemocytes was reduced after inhibiting the activity or expression of CgMyc-A.After injecting 10058-F4,a small molecule inhibitor of C-Myc protein,the proliferation activity of haemocyte was detected after stimulation with V.splendidus.The proportion of Ed U+ haemocytes significantly reduced(0.70-fold,p < 0.001)in experimental group(10058-F4).In CgMyc-A-RNAi oysters,at 24 h after V.splendidus stimulation,the proportion of Ed U+ haemocytes notably reduced,which was 0.82-fold(p < 0.01)of that in EGFP-RNAi.4.The expression of CgBlimp-1 inhibited proliferation of haemocytes after arresting cell cycle in G1/G0 phase.When the expression of CgBlimp-1 was knocked-down in vivo by RNAi,the m RNA expressions of downstream transcription factor CgMyc-A(1.63-fold of that in control group,p < 0.05),hematopoietic related factor CgWnt-1(2.04-fold,p < 0.001),CgRunx-1(1.62-fold,p < 0.05),cell cycle related gene CgCDK2(1.70-fold,p < 0.05)increased significantly at 24 h after V.splendidus stimulation.Western blot result was consistent with the nucleic acid level detection.Granulocytes were sorted out by flow cytometry.At 24 h after V.splendidus stimulation,the proportion of granulocytes in total haemocytes significantly upregulated,which was 1.29-fold(p < 0.01)of that in Seawater,after the expression of CgBlimp-1 was knocked-down,the proportion of granulocytes in total haemocytes continued to increase,which was 1.14-fold(p < 0.05)of that in EGFP-RNAi.In CgBlimp-1-RNAi oysters,at 24 h after V.splendidus stimulation,the proportion of haemocytes and granuloctes in G0/G1 phase decreased dramatically,which was 0.81-fold(p < 0.001)and 0.67-fold(p < 0.001)of that in control group,respectively.And the proportion of haemocytes and granuloctes in G2/M phase increased significantly,which was 2.77-fold(p < 0.01)and 1.97-fold(p < 0.001)of that in control group,respectively.Concomitantly,the ratio of Ed U+ haemocytes(1.56-fold,p < 0.01)and granuloctes(1.82-fold,p < 0.05)upregulated notably.Furthermore,the m RNA expression levels of CgIL17-1,CgIL17-2 and CgIL17-4 in haemocytes increased significantly in CgBlimp-1-RNAi oysters,which was 1.71-fold(p < 0.05),144.70-fold(p < 0.01)and 1.93-fold(p < 0.05)of that in control group,respectively.Aforementioned results suggested that CgBlimp-1 could reduce the proliferation of granulocytes by arresting cell cycle in G1/G0 phase and avoid over-expression of interleukin to maintain homeostasis in the immune response of oyster.These studies provided important reference for further analysis of immune regulation of invertebrates. |
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