罗非鱼是世界上最重要的养殖鱼类之一。它是一种热带和亚热带淡水鱼，适应温暖水域但对寒冷天气敏感。极冷天气可能导致罗非鱼严重压力和大量死亡。本研究旨在调查寒冷压力对尼罗罗非鱼（Oreochromis niloticus）抗冻蛋白（AFP）基因上调的影响。共研究了两组鱼（每组15尾，每组均有5个重复，在玻璃纤维水槽中，每槽容积为70升）：1）对照组；鱼经过两周的实验室条件适应，并且整个实验期间水温保持在25℃，饲喂商用饲料（粗蛋白质含量30%）；2）寒冷压力组；与对照组相同的条件，唯一不同在于温度。最初，温度每12小时降低1摄氏度。当水温降到6-8摄氏度时，鱼开始出现死亡症状。在这个阶段，分别从两个组中取得了组织（肌肉）样品。采用差异显示PCR（DD-PCR）检测和表征了暴露于寒冷应激的鱼类的免疫反应。结果表明，与对照组相比，寒冷应激组中检测到了九种不同的上调基因。这些基因分别为：整合素α-2（ITGA-2）、间隙连接蛋白γ-1类似蛋白（GJC1）、WD重复含蛋白59异构体X2（WDRP59）、NUAK家族SNF1样激酶、G蛋白偶联受体-176（GPR-176）、肌动蛋白细胞骨架调控复合物蛋白pan1样蛋白（PAN-1）、旋螺蛋白（WHRN）、抑癌性 7蛋白异构体X2（ST7P）和ABC超家族A蛋白 1 异构体 X2（ABCA1）。使用600bp的特定PCR产物进行抗冻基因II型扩增，并进行克隆和测序分析，结果显示检测到的基因为抗冻基因II型，相似性范围为70％至95％。体外转录基因诱导了一个分子量为22kDa的抗冻蛋白。抗冻基因ITGA-2和WD重复蛋白属于凝集素家族（糖蛋白）。总之，在寒冷应激下，尼罗罗非鱼表达了许多防御基因，其中包括一个约0.6kbp的开放阅读框。© 2023 BioMed Central Ltd. ，Springer Nature的一部分。

Tilapia is one of the most essential farmed fishes in the world. It is a tropical and subtropical freshwater fish well adapted to warm water but sensitive to cold weather. Extreme cold weather could cause severe stress and mass mortalities in tilapia. The present study was carried out to investigate the effects of cold stress on the up-regulation of antifreeze protein (AFP) genes in Nile tilapia (Oreochromis niloticus). Two treatment groups of fish were investigated (5 replicates of 15 fish for each group in fibreglass tanks/70 L each): 1) a control group; the fish were acclimated to lab conditions for two weeks and the water temperature was maintained at 25 °C during the whole experimental period with feeding on a commercial diet (30% crude protein). 2) Cold stress group; the same conditions as the control group except for the temperature. Initially, the temperature was decreased by one degree every 12 h. The fish started showing death symptoms when the water temperature reached 6-8 °C. In this stage the tissue (muscle) samples were taken from both groups. The immune response of fish exposed to cold stress was detected and characterized using Differential Display-PCR (DD-PCR).The results indicated that nine different up-regulation genes were detected in the cold-stressed fish compared to the control group. These genes are Integrin-alpha-2 (ITGA-2), Gap junction gamma-1 protein-like (GJC1), WD repeat-containing protein 59 isoform X2 (WDRP59), NUAK family SNF1-like kinase, G-protein coupled receptor-176 (GPR-176), Actin cytoskeleton-regulatory complex protein pan1-like (PAN-1), Whirlin protein (WHRN), Suppressor of tumorigenicity 7 protein isoform X2 (ST7P) and ATP-binding cassette sub-family A member 1-like isoform X2 (ABCA1). The antifreeze gene type-II amplification using a specific PCR product of 600 bp, followed by cloning and sequencing analysis revealed that the identified gene is antifreeze type-II, with similarity ranging from 70 to 95%. The in-vitro transcribed gene induced an antifreeze protein with a molecular size of 22 kDa. The antifreeze gene, ITGA-2 and the WD repeat protein belong to the lectin family (sugar-protein).In conclusion, under cold stress, Nile tilapia express many defence genes, an antifreeze gene consisting of one open reading frame of approximately 0.6 kbp.© 2023. BioMed Central Ltd., part of Springer Nature.