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牙鲆精氨酸酶Ⅱ基因的克隆以及免疫应答表达分析
马慧鑫1,2, 王 磊1,3, 汪林庆1,4, 周 茜1,3, 陈松林1,3
1.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.上海海洋大学水产与生命学院 上海 201306;3.青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛 266071;4.福州大学生物科学与工程学院 福州 350116
摘要:
本研究克隆得到牙鲆(Paralichthys olivaceus)精氨酸酶Ⅱ基因(ArginaseⅡ, Arg-Ⅱ)全长cDNA序列,并检测了Arg-Ⅱ在牙鲆免疫组织和细菌感染过程中的时空表达模式。结果显示,Arg-Ⅱ基因cDNA全长1882 bp,包含1050 bp开放阅读框,编码349个氨基酸(预测分子量为38.02 kDa,等电点为6.42)。Arg-Ⅱ基因编码的氨基酸序列具有典型的精氨酸酶结构特征,推测与哺乳动物中的功能类似。系统进化分析显示,鱼类Arg-Ⅱ基因合成一簇,其中,Arg-Ⅱ基因与鲈鱼(Lates calcarifer)相关度最高(93%)。实时荧光定量结果显示,Arg-Ⅱ基因在健康牙鲆的肝、脾和鳃等免疫组织中有较高表达。进一步研究发现,经迟缓爱德华氏菌(Edwardsiella tarda)感染的牙鲆成鱼中,主要免疫组织中Arg-Ⅱ mRNA的表达量在细菌感染后6~12 h显著上调,随后表达量恢复正常。离体培养的牙鲆巨噬细胞经迟缓爱德华氏菌感染后,Arg-Ⅱ基因也呈显著上调模式。因此,本研究结果表明,Arg-Ⅱ基因参与牙鲆响应迟缓爱德华氏菌感染的免疫过程,揭示Arg-Ⅱ基因可能在牙鲆抗病免疫中发挥重要作用。
关键词:  牙鲆  精氨酸酶Ⅱ;迟缓爱德华氏菌;免疫表达;巨噬细胞
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The Different Expression Patterns of the Gene Arginase Ⅱ in Paralichthys olivaceus and the Correlation Between its Expression and Edwardsiella tarda Infection
MA Huixin1,2, WANG Lei1,3, WANG Linqing1,4, ZHOU Qian1,3, CHEN Songlin1,3
1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306;3.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071;4.College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116
Abstract:
Arginase Ⅱ(Arg-Ⅱ) is a type of arginase. It mainly participates in the urea cycle, and has been found to play an important role in the pathological process. However, its role in fish is not well reported. In this milieu, we obtained the full cDNA of Arg-Ⅱ of Japanese flounder (Paralichthys olivaceus) and detected its space-time expression profile in the immune tissue and bacterial infected tissue of Japanese flounder. The results showed that the Arg-Ⅱ gene consists of 1882 nucleotides, including an open reading frame (1150 bp) that encodes a protein of 349 amino acids (with deduced molecular mass of 38.02 kDa and theoretical isoelectric point of 6.42). The amino acid of Arg-Ⅱ protein in Japanese flounder displayed the typical structure of arginase, therefore we assumed that it might have functions similar to that in mammals. Phylogenetic tree analysis also confirmed that fish Arg-Ⅱ formed a cluster, and Japanese flounder Arg-Ⅱ was most closely related to that of Lates calcarifer (93%). Quantitative real-time PCR (q-RT PCR) analysis showed that the mRNA of Arg-Ⅱ is mainly expressed in immune tissues, such as the liver, spleen, gills, and kidney. Intensive study showed that the mRNA expression of gene Arg-Ⅱ was significantly upregulated after 6~12 h of Edwardsiella tarda challenge, and then returned to normal condition in the immune tissues of Japanese flounder. The expression of Arg-Ⅱ was also significantly upregulated in the in vitro cultured E. tarda challenged Japanese flounder phagocyte. The present study demonstrates that the gene Arg-Ⅱ might participate in the immune response of Japanese flounder to E. tarda infection, thus indicating that the gene Arg-Ⅱ might play an important role in the antibacterial immunity of Japanese flounder.
Key words:  Paralichthys olivaceus  ArginaseⅡ  Edwardsiella tarda  Expression  Macrophage