髓样分化因子88(MyD88)是Toll样受体(TLR)信号通路的关键衔接子,在动物先天免疫信号转导中起着至关重要的作用。然而,MyD88介导的贝类信号转导机制尚未得到很好的研究。在这项研究中,一个新的MyD88基因,CfMyD88-2,在志孔扇贝中被鉴定,ChlamysFarreri.1779bp长的开放阅读框编码592个氨基酸。CfMyD88-2的N端包含一个保守的死亡域(DD),随后是TIR(TLR/白细胞介素-1受体)结构域。多序列比较结果表明,TIR结构域序列高度保守。系统发育分析显示,CfMyD88-2首先与紫菜MyD88-4和紫菜MyD88-4相关。CfMyD88-2mRNA在所有扇贝组织中均有表达,通过qRT-PCR检测,在地幔和肝胰腺中表达水平最高。此外,CfMyD88-2mRNA表达在病原体相关分子模式后显著增加(PAMPs,如脂多糖,肽聚糖,或聚肌苷酸-聚胞嘧啶酸)刺激。HEK293T细胞免疫共沉淀实验结果表明,CfMyD88-1和CfMyD88-2均与扇贝的TLR蛋白相互作用,提示扇贝中存在多个功能性TLR-MyD88信号轴。双荧光素酶报告基因检测表明,在HEK293T细胞中过表达的CfMyD88-2激活了干扰素(IFN)α,IFN-β,IFN-γ,和NF-κB报告基因,表明蛋白质具有多种功能。亚细胞定位实验结果表明,CfMyD88-2主要定位于人细胞的细胞质中。总之,新鉴定的CfMyD88-2可以应对PAMP的挑战,参与TLR免疫信号,并可能激活下游效应基因,如NF-κB基因。这些研究结果将有助于推进无脊椎动物先天免疫理论,为今后扇贝抗病选育提供参考。
Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-β, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.