Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128–133 aa in length, and mature peptides of 109–111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2–3 fold) of five (nkl 2–4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.

Nk-lysin (Nkl) 是哺乳动物中自然杀伤细胞和细胞毒性 T 细胞的抗菌肽 (AMP) 产物，最近已在许多有鳍鱼类中进行了表征。在这项研究中，我们鉴定了六个与 Nkl 序列同源的基因，并描述了它们在虹鳟鱼 ( Oncorhynchus mykiss ) 中的 mRNA 表达模式和丰度。六个 Nkls 的 cDNA 序列编码长度为 128-133 个氨基酸的前体肽和长度为 109-111 个氨基酸的成熟肽。nkl1-4基因的基因组DNA由5个外显子和4个内含子组成，而类似nkl的a和b基因由4个外显子和3个内含子组成。这些肽的染色体位置表明nkl1位于染色体臂 25q 上，而其他五个nkl基因聚集在染色体臂 19q 上。系统发育分析揭示了硬骨鱼中 Nkl 的保守结构，进一步的蛋白质序列分析表明所有六个nkl基因都属于 Saposin 蛋白质家族的 Nkl 亚家族。六种鳟鱼 Nkl 同源物的组织特异性 mRNA 表达模式不对称，其中nkl1、nkl3和nkl 样 a 和 b出现在免疫活性器官如脾、鳃、肠和肾以及松果体、脑中和卵母细胞。但是，nkl2和nkl4，显示脑、松果体和卵母细胞组织中的主要丰度。使用 mRNA 测序，在暴露于嗜冷黄杆菌感染的幼鳟鱼苗（1 g bw）的全身池中，我们观察到 5 个（ nkl 2-4 和nkl- like a & b ）的适度上调（2-3 倍） )在五天的攻击后时间过程中的六个nkl mRNA。然而，在幼鳟鱼（270 g bw）中通过 qPCR 测量的脾组织中没有记录到上调。再次使用 mRNA 测序，在暴露于各种水产养殖压力源的幼鳟鱼 (~57.7 g bw) 的鳃中测定了 mRNA 丰度。结果表明，所有六个nkl s ( nkl1-4 和 nkl-like a 和 nkl-like b) 在暴露于高温时被下调，并且nkl1在盐度挑战后显着下调。总体而言，这些新表征的 AMP 可能有助于宿主先天免疫，因为它们在病原体攻击和生理应激源后受到调节。