Abstract Mass mortality caused by pathogens has severely affected the oyster cultivation industry, but our knowledge about the innate immune system of oysters is still limited. The nuclear factor-κB (NF-κB) signaling pathway plays a vital role in many cellular, developmental, and biological processes. And inhibitor of NF-κB (IκB) proteins can prevent NF-κB nuclear localization and DNA binding, thereby inhibiting NF-κB functions. However, little is known about their existence and function in invertebrates. In this study, a novel homolog of the IκB gene was identified in the Pacific oyster Crassostrea gigas (designated as CgIκB4). The open reading frame of CgIκB4 is 1029 bp and encodes a putative protein of 342 amino acids. Comparative and phylogenetic analyses revealed that the CgIκB4 belongs to the invertebrate IκB family. The results of quantitative real-time PCR showed that CgIκB4 transcripts are expressed in all test tissues, and with the highest expression level in the stomach and labial palpus, and that the expression is significantly induced after LPS, PGN, and poly(I:C) challenges. Importantly, Co-IP assays showed that CgIκB4 can interact with oyster NF-κB family proteins (i.e., CgRel1 and CgRel2) directly. The results of dual-luciferase reporter assays showed an inhibition of CgIκB4 on CgRel1-dependent NF-κB and TNFα activation. These findings demonstrate that CgIκB4 may play an important role in the innate immunity of C. gigas through regulation of NF-κB activity. And the research results will be helpful for deciphering the mechanisms of innate immunity in invertebrates and provide theoretical basis for molecular breeding of disease-resistance oysters.

中文翻译：

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NF-κB信号通路中一个牡蛎IκB基因的分子克隆及免疫功能研究

摘要 病原体引起的大规模死亡严重影响了牡蛎养殖业，但我们对牡蛎先天免疫系统的了解仍然有限。核因子-κB (NF-κB) 信号通路在许多细胞、发育和生物过程中起着至关重要的作用。NF-κB (IκB) 蛋白抑制剂可以阻止 NF-κB 核定位和 DNA 结合，从而抑制 NF-κB 功能。然而，人们对它们在无脊椎动物中的存在和功能知之甚少。在这项研究中，在太平洋牡蛎 Crassostrea gigas（指定为 CgIκB4）中发现了 IκB 基因的新型同源物。CgIκB4 的开放阅读框是 1029 bp，编码 342 个氨基酸的推定蛋白质。比较和系统发育分析表明 CgIκB4 属于无脊椎动物 IκB 家族。实时定量 PCR 结果显示 CgIκB4 转录物在所有受试组织中均有表达，以胃和唇睑的表达水平最高，LPS、PGN 和 poly(I:C ) 挑战。重要的是，Co-IP 分析表明 CgIκB4 可以直接与牡蛎 NF-κB 家族蛋白（即 CgRel1 和 CgRel2）相互作用。双荧光素酶报告基因检测的结果显示 CgIκB4 对 CgRel1 依赖性 NF-κB 和 TNFα 激活有抑制作用。这些发现表明 CgIκB4 可能通过调节 NF-κB 活性在 C. gigas 的先天免疫中发挥重要作用。研究结果将有助于破译无脊椎动物先天免疫机制，为抗病牡蛎分子育种提供理论依据。