Zhang Pengjuan, Li Chenghua, Zhang Peng, Jin Chunhua, Pan Daodong, Bao Yongbo
Department of aquaculture, Ningbo University, Ningbo, Zhejiang Province, P.R China.
Department of Aquatic Germplasm Resources, Zhejiang Wanli University, Ningbo, Zhejiang Province, P.R China.
PLoS One. 2014 Jun 20;9(6):e100492. doi: 10.1371/journal.pone.0100492. eCollection 2014.
Skin ulceration syndrome (SUS) is considered to be a major constraint for the stable development of Apostichopus japonicus culture industries. In this study, we investigated protein changes in the coelomocytes of A. japonicus challenged by Vibrio splendidus using isobaric tags for relative and absolute quantification (iTRAQ) over a 96 h time course. Consequently, 228 differentially expressed proteins were identified in two iTRAQs. A comparison of the protein expression profiles among different time points detected 125 proteins primarily involved in response to endogenous stimuli at 24 h. At 48 h, the number of differentially expressed proteins decreased to 67, with their primary function being oxidation reduction. At the end of pathogen infection, proteins responsive to amino acid stimuli and some metabolic processes were classified as the predominant group. Fifteen proteins were differentially expressed at all time points, among which eight proteins related to pathologies in higher animals were shown to be down-regulated after V. splendidus infection: paxillin, fascin-2, aggrecan, ololfactomedin-1, nesprin-3, a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (Adamts7), C-type lectin domain family 4 (Clec4g) and n-myc downstream regulated gene 1 (Ndrg1). To gain more insight into two SUS-related miRNA (miR-31 and miR-2008) targets at the protein level, all 129 down-regulated proteins were further analyzed in combination with RNA-seq. Twelve and eight proteins were identified as putative targets for miR-31 and miR-2008, respectively, in which six proteins (5 for miR-31 and 1 for miR-2008) displayed higher possibilities to be regulated at the level of translation. Overall, the present work enhances our understanding of the process of V. splendidus-challenged sea cucumber and provides a new method for screening miRNAs targets at the translation level.
皮肤溃疡综合征（SUS）被认为是刺参养殖业稳定发展的主要制约因素。在本研究中，我们使用相对和绝对定量的等压标签（iTRAQ）在96小时的时间进程中研究了受灿烂弧菌攻击的刺参体腔细胞中的蛋白质变化。结果，在两个iTRAQ中鉴定出228种差异表达蛋白。不同时间点蛋白质表达谱的比较检测到125种主要在24小时参与对内源刺激反应的蛋白质。在48小时，差异表达蛋白的数量减少到67种，其主要功能是氧化还原。在病原体感染结束时，对氨基酸刺激有反应的蛋白质和一些代谢过程被归类为主要类别。在所有时间点有15种蛋白质差异表达，其中8种与高等动物病理学相关的蛋白质在灿烂弧菌感染后被下调：桩蛋白、成束蛋白-2、聚集蛋白聚糖、嗅觉介素-1、核膜蛋白-3、具有血小板反应蛋白1型基序的解整合素样金属蛋白酶（Adamts7）、C型凝集素结构域家族4（Clec4g）和N-myc下游调控基因1（Ndrg1）。为了在蛋白质水平上更深入地了解两个与SUS相关的miRNA（miR-31和miR-2008）的靶标，结合RNA测序对所有129种下调蛋白进行了进一步分析。分别鉴定出12种和8种蛋白质作为miR-31和miR-2008的假定靶标，其中6种蛋白质（miR-31为5种，miR-2008为1种）在翻译水平受到调控的可能性更高。总体而言，本研究工作增进了我们对灿烂弧菌攻击海参过程的理解，并提供了一种在翻译水平筛选miRNA靶标的新方法。
