ABSTRACT: ADP-ribosylation factors (Arfs) are guanosine triphosphate (GTP)-binding proteins that play essential roles in membrane trafficking, and they have been recently reported to be involved in innate immunity in crustaceans. However, little information is available on Arfs in the oriental river prawn Macrobrachium nipponense and their response to ammonia nitrogen stress. In this study, we identified a novel M. nipponense Arfn gene (MnArfn). The full-length cDNA of MnArfn was 1076 bp. It contained a 537 bp open reading frame (ORF) and encoded a 178 amino acid protein with a predicted molecular weight of 19.85 kDa. Sequence and phylogenetic analyses showed that MnArfn was an unidentified Arf, sharing 55-61% identity with other known Arfs. Quantitative real-time PCR (qPCR) indicated that all examined tissues (hepatopancreas, stomach, gill, heart, muscle, and eyestalk) expressed MnArfn. Hepatopancreas and gills, 2 organs involved in environmental stress management, had the highest expression. Under conditions of ammonia nitrogen stress, MnArfn expression in hepatopancreas and gills was significantly up-regulated at 6, 12, and 24 h. Western blotting experiments also revealed that MnArfn was distributed in all examined tissues, with the highest expression in hepatopancreas and gills, consistent with qPCR results. The findings from this study indicate that MnArfn may play an important role in the response of M. nipponense to ammonia nitrogen stress, which provides a new avenue to study the resistance mechanism(s) of crustaceans to ammonia nitrogen and to screen for individuals with resistance to unfavorable environments.

中文翻译：

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日本沼虾新的ADP-核糖基化因子基因的表征及其对氨氮胁迫的响应

摘要：ADP-核糖基化因子（Arfs）是三磷酸鸟苷（GTP）结合蛋白，在膜运输中起重要作用，最近已报道它们参与甲壳类动物的先天免疫。但是，关于东方河虾Macrobrachium nipponense中的Alfs及其对氨氮胁迫的响应的信息很少。在这项研究中，我们确定了新的日本沼虾Arfn基因（MnArfn）。MnArfn的全长cDNA为1076 bp。它包含一个537 bp的开放阅读框（ORF），并编码一个178个氨基酸的蛋白质，预测分子量为19.85 kDa。序列和系统发育分析表明，MnArfn是一个未知的Arf，与其他已知的Arfs具有55-61％的同一性。实时定量PCR（qPCR）表明，所有检查的组织（肝胰腺，胃、,、心脏，肌肉和眼球）均表达MnArfn。肝胰腺和腮是参与环境压力管理的两个器官，其表达最高。在氨氮胁迫下，MnArfn在6、12和24小时，肝胰腺和g中的表达显着上调。蛋白质印迹实验还显示，MnArfn分布在所有检查的组织中，在肝胰脏和g中的表达最高，与qPCR结果一致。从本研究的结果表明，MnArfn可以在响应中发挥重要作用日本沼虾为氨的氮胁迫，其提供了新的途径，以甲壳类的抗性机制（一个或多个）研究为氨的氮和筛选具有抗性的个体不利的环境。