Akirins are highly conserved, small, nuclear proteins that have been recognized as key regulators of multiple gene expression patterns and play important roles in immune responses. Previous studies on Akirins have mainly focused on the model species Drosophila and mice, while there have been few studies on fish. In the present study, two Akirin genes, Akirin1 and Akirin2, were identified from Megalobrama amblycephala. The open reading frames (ORFs) of M. amblycephala Akirin1 and Akirin2 were 570 bp and 558 bp in length, encoding 189 and 185 amino acids, respectively. Quantitative real-time PCR analysis indicated that both Akirin1 and Akirin2 mRNA were ubiquitously expressed in all 10 examined tissues with different expression patterns. After Aeromonas hydrophila infection, the mRNA levels of Akirin1, Akirin2, nuclear factor κB subunit p50 (NF-κB p50) and nuclear factor κB subunit p65 (NF-κB p65) were significantly induced in the liver and spleen. Knockdown and overexpression of Akirins in L8824 cells showed that Akirin2 positively regulated the expression of NF-κB p50, NF-κB p65, liver-expressed antimicrobial peptide 1 (LEAP-1) and liver-expressed antimicrobial peptide 1 (LEAP-2), while Akirin1 had no significant effect on the expression of these genes above. In addition, the recombinant M. amblycephala Akirin1 (rAkirin1) and Akirin2 (rAkirin2) proteins were successfully obtained and intraperitoneal injection of them could help protect M. amblycephala from A. hydrophila infection, with rAkirin2 having a better protective effect than rAkirin1. Furthermore, in vitro glutathione S-Transferase (GST) pull-down assays showed that M. amblycephala Akirin2 could bind to the IPT domain of NF-κB p50 and luciferase assays showed that NF-κB p65 could promote the activities of LEAP-1 and LEAP-2 promoters, which suggested that Akirin2, rather than Akirin1, could interact with the IPT domain of NF-κB p50 and induce the transcription of downstream antimicrobial peptides LEAP-1 and LEAP-2 though NF-κB p65 to enhance the immunity of M. amblycephala to A. hydrophila infection. All the above results indicated that M. amblycephala Akirin2 plays an essential role in the defense against A. hydrophila via the NF–κB signaling pathway, providing a theoretical basis for in-depth study of the innate immune mechanism of fish and promotion of healthy aquaculture.

Akirins 是高度保守的小型核蛋白，已被公认为多种基因表达模式的关键调节因子，并在免疫反应中发挥重要作用。以往对Akirins的研究主要集中在模式物种果蝇和小鼠上，而对鱼类的研究较少。在本研究中，两个Akirin基因，Akirin1和Akirin2 ，是从Megalobrama amblycephala中鉴定出来的。M. amblycephala Akirin1和Akirin2的开放阅读框 (ORF)长度分别为 570 bp 和 558 bp，分别编码 189 和 185 个氨基酸。定量实时 PCR 分析表明Akirin1和Akirin2 mRNA 在所有 10 个具有不同表达模式的检查组织中普遍表达。嗜水气单胞菌感染后，肝脏和脾脏中Akirin1、Akirin2、核因子κB亚基p50（NF-κB p50）和核因子κB亚基p65（NF-κB p65）的mRNA水平明显升高。在 L8824 细胞中敲除和过表达Akirins表明Akirin2正调控NF-κB p50、NF-κB p65、肝脏表达的抗菌肽 1 ( LEAP-1 ) 和肝脏表达的抗菌肽 1 (LEAP-2），而Akirin1对上述这些基因的表达没有显着影响。此外，成功获得了重组的M. amblycephala Akirin1 (rAkirin1) 和Akirin2 (rAkirin2) 蛋白，腹腔注射它们可以帮助保护M. amblycepha免受A. hydrophila感染，其中rAkirin2 比rAkirin1 具有更好的保护作用。此外，体外谷胱甘肽 S-转移酶 (GST) pull-down 试验表明M. amblycephala Akirin2 可以与 NF-κB p50 的 IPT 结构域结合，荧光素酶试验表明 NF-κB p65 可以促进LEAP-1和飞跃2启动子，这表明 Akirin2，而不是 Akirin1，可以与 NF-κB p50 的 IPT 结构域相互作用，并通过 NF-κB p65 诱导下游抗菌肽LEAP-1和LEAP-2的转录，以增强M. amblycephala的免疫力到A.hydrophila感染。以上结果表明，M. amblycephala Akirin2通过NF-κB信号通路在防御嗜水A.hydrophila中发挥重要作用，为深入研究鱼类的先天免疫机制和促进健康水产养殖提供理论依据。 .