School of Agriculture, Ludong University, Yantai, 264025, China.
Laizhou Marine Development and Fishery Service Center, Yantai, 261499, China.
Fish Shellfish Immunol. 2023 Dec;143:109188. doi: 10.1016/j.fsi.2023.109188. Epub 2023 Oct 27.
Members of the nuclear factor-kappa B (NF-κB) family are crucial regulators of physiological processes such as apoptosis, inflammation, and the immune response, acting as vital transcription factors to perform their function. In this study, we identified a NF-κB homologous gene (CfRel1) in Zhikong scallops. The 3006-bp-long open reading frame encodes 1001 amino acids. The N-terminus of the CfRel1 protein harbors a conserved Rel homology domain (RHD) that contains a DNA-binding domain and a dimerization domain. According to the multiple sequence alignment results, both the DNA-binding and dimerization domains are highly conserved. Phylogenetic analysis indicated that CfRel1 is closely related to both the Dorsal protein of Pinctada fucata and the Rel2 protein of Crassostrea gigas. CfRel1 mRNA was expressed in all tissues tested in the quantitative reverse transcription PCR experiments, with hepatopancreatic tissue expressing the highest levels. Furthermore, after stimulation with lipopolysaccharide, peptidoglycan, or polyinosinic:polycytidylic acid, the mRNA expression level of CfRel1 was markedly increased. The co-immunoprecipitation test results showed that CfRel1 interacted with scallop IκB protein through its RHD DNA-binding domain, suggesting that IκB may regulate the activity of Rel1 by binding to this domain. Dual-luciferase reporter gene assays revealed that CfRel1 overexpression in HEK293T cells activated the activator protein 1 (AP-1), NF-κB, interferon (IFN)α, IFNβ, and IFNγ reporter genes, indicating the diverse functions of the protein. In summary, CfRel1 is capable of responding to attacks from pathogen-associated molecular patterns, participating in immune signaling, and activating NF-κB and IFN reporter genes. Our findings contribute to the advancement of invertebrate innate immunity theory, enrich the theory of comparative immunology, and serve as a reference for the future screening of disease-resistant strains in scallops.
贝类 NF-κB 家族成员是细胞凋亡、炎症和免疫反应等生理过程的关键调节因子，作为重要的转录因子发挥作用。在本研究中，我们在栉孔扇贝中鉴定出一个 NF-κB 同源基因（CfRel1）。该基因长 3006bp，开放阅读框编码 1001 个氨基酸。CfRel1 蛋白的 N 端含有一个保守的 Rel 同源结构域（RHD），该结构域包含一个 DNA 结合域和一个二聚化结构域。根据多序列比对结果，DNA 结合域和二聚化结构域均高度保守。系统进化分析表明，CfRel1 与菲律宾蛤仔 Dorsal 蛋白和中国蛤蜊 Rel2 蛋白亲缘关系较近。定量 RT-PCR 实验结果显示，CfRel1 在所有检测组织中均有表达，肝胰腺组织表达量最高。此外，经脂多糖、肽聚糖或多聚肌苷酸：多聚胞苷酸刺激后，CfRel1 的 mRNA 表达水平显著升高。免疫共沉淀试验结果表明，CfRel1 通过其 RHD DNA 结合域与扇贝 IκB 蛋白相互作用，提示 IκB 可能通过与该结构域结合来调节 Rel1 的活性。双荧光素酶报告基因检测结果显示，CfRel1 在 HEK293T 细胞中的过表达可激活激活蛋白 1（AP-1）、NF-κB、干扰素（IFN）α、IFNβ 和 IFNγ 报告基因，表明该蛋白具有多种功能。综上所述，CfRel1 能够对病原体相关分子模式的攻击作出反应，参与免疫信号通路，并激活 NF-κB 和 IFN 报告基因。本研究结果丰富了无脊椎动物先天免疫理论，为比较免疫学理论提供了补充，并为今后筛选扇贝抗病品系提供了参考。
