密码激酶（Cry）作为一种重要的黄素蛋白，在调节先天免疫反应中发挥关键作用，例如炎症细胞因子的释放。在本研究中，从中国杂色梭子蟹中鉴定出一个密码激酶同源物（EsCry），它含有典型的DNA光酶结构域和FAD结合结构域。EsCry的转录本在一天中的11点高度表达，并在凌晨3点达到最低水平，而白细胞介素增强子结合因子（EsILF）、脂多糖诱导的TNF-α因子（EsLITAF）、肿瘤坏死因子（EsTNF）和白细胞介素-16（EsIL-16）的转录本则呈相反的节律表达模式。通过dsEsCry注射使EsCry表达抑制后，时间蛋白（EsTim）、周期蛋白（EsCyc）、昼夜节律调节蛋白（EsClock）、周期蛋白（EsPer）以及EsLITAF、EsTNF、EsILF、EsIL-16的mRNA转录本明显上调，腹背星状神经元活动调节因子（Dorsal）的磷酸化水平也显著上调。在NF-κB抑制剂注射后，EsCry-RNAi梭子蟹中的EsLITAF、EsTNF、EsILF和EsIL-16的转录本显著下调。体外观察到EsCyc与EsCry、EsCyc与Dorsal之间的相互作用。这些结果表明，EsCry通过抑制它们的转录因子LITAF和ILF，通过NF-κB信号传导途径，负调控细胞因子TNF和IL-16的表达，从而提供了更好地理解蟹类细胞因子产生的昼夜节律调节机制的证据。版权所有 © 2023 Elsevier B.V. 发表。

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.Copyright © 2023. Published by Elsevier B.V.