STRUCTURAL CHARACTERISTICS OF IΚBΑ AND C-REL AND EXPRESSION ANALYSIS UNDER LPS STIMULATION IN HYRIOPSIS SCHLEGELII

The functions of nuclear factor-κappaB (NF-κB) and inhibitors of NF-κB (IκB) in freshwater shellfish are still unclear. In this study, we analyzed the sequence structure and expression characteristics of IκBα and c-Rel (HsIκBα and Hsc-Rel) and the correlation between HsIκBα and Hsc-Rel in Hyriopsis schlegelii. The results showed that the full-length cDNA of HsIκBα was 1783 bp with an 1083 bp open reading frame that encoded a total of 360 amino acids. The cDNA of Hsc-Rel gene was 2414 bp with the ORF of 2298 bp that encoded a total of 765 amino acids. The GST-pull down experiment was completed by constructing HsIκBα-ORF-GST and Hsc-Rel-RHD-HIS recombinant plasmids, prokaryotic expression and purification of two fusion proteins. The direct interaction between HsIκBα and Hsc-Rel-RHD was found. HsIκBα and Hsc-Rel mRNA level was the highest in hepatopancreas tissues and the lowest in hemocytes. At 6h after LPS stimulation, the expression of HsIκBα in 8 tissues and Hsc-Rel in 7 tissues were down-regulated; At 12h after LPS stimulation, HsIκBα was upregulated in 10 tissues, and all reached the peak. Hsc-Rel was only upregulated in hemocytes, heart, hepatopancreas and intestine. At 24h after LPS stimulation, HsIκBα was down-regulated in 10 tissues, but Hsc-Rel was up-regulated in all tissues except kidney, where the expression of hemocytes, foot, and intestine reached its peak. Between 36h—72h after LPS stimulation, the expression of HsIκBα and Hsc-Rel in some tissues gradually returned to basic levels, the results showed that the expression of HsIκBα and Hsc-Rel significantly changed with LPS stimulation, indicating that they were involved in the immune response, and there may be a NF-κB pathway in response to LPS.