點帶石斑魚 CD9 基因啟動子之活性與特性及添加 poly(I:C) 與感染石斑魚虹彩病毒的表現分析

Abstract

High economic benefits make groupers important aquacultural species in Taiwan. However, the disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Therefore we take Epinephelus coioides which easily raised as our model to study the interactions between groupers and GIV. In 2012, Liu and Wu injected poly(I:C), GIV and LPS into orange-spotted groupers and discovered the expression pattern of CD9 resembling to those genes associated with interferon (IFN). IFN influencing antiviral effects are important bridges between innate immunity and adaptive immunity, but the role of CD9 remains unknown. We gained the total length of CD9 cDNA from orange-spotted grouper (Yang, 2012), finding that CD9 mainly expressed in immune organs. Thus, we hope to get CD9 promoter for further analysis to realize its relationship to GIV. According to the total length of CD9 cDNA gained, we obtained 1.2 Kb and 2.6 Kb CD9 promoters from genome walking. Through analysis of sequence and transcription factor binding sites, we predicted that CD9 promoters might contain binding sites of AP-1, ApoD, C/EBP, NF-κB, TNF-α-Y-BOX, IRF1 and IRF2. By luciferase assay, we found that 1.2 Kb CD9 prmoter but not 2.6 Kb CD9 promoter regularly expressed in GK cells and GB cells, and their activities weren't affected by different concentration of poly(I:C). In deletion analysis of CD9 promoter in GK cells, 2.6 Kb CD9 promoter might have negative regulatory factor binding sites. Some binding sites located in 124 bp near the side close to transcription initiation point and the part between 400 bp and 679 bp of CD9 promoter might play a key role to affect expression. CD9 promoters with different lengths were also not affected by various concentration of poly(I:C), but the infection of GIV inhibit CD9 promoters, causing extremely low activity. Cotransfection with subunit of NF-κB, c-rel in GK cells up-regulated CD9 promoter. Measuring endogenious CD9 expressions in response to poly(I:C) treatment in GK cells by RT-PCR showed no differences. Although the percoll purified white blood cells from head kidney and spleen of orange-spotted grouper showed no CD9 transcriptional difference after poly(I:C) treatment, IFNa and Mx expression showed significant upregulation. In summary, the CD9 transcriptional regulation might associate with NF-κB, and might be inhibited by the infection of GIV. However, the transcription factors involving in CD9 promoter regulation during GIV infection remain further study.