刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析

畅孟阳; 李彬; 荣小军; 王锦锦; 于永翔; 王印庚; 廖梅杰; 张正; 范瑞用; 刘清兵

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刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析
畅孟阳,李彬,荣小军,王锦锦,于永翔,王印庚,廖梅杰,张正,范瑞用,刘清兵
1.上海海洋大学水产与生命学院上海 201306;2.中国水产科学研究院黄海水产研究所农业农村部海洋渔业可持续发展重点实验室山东青岛 26607;3.中国水产科学研究院黄海水产研究所农业农村部海洋渔业可持续发展重点实验室山东青岛 266071;4.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266071;5.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266072;6.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266073;7.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266074;8.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266075;9.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266076;10.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室山东青岛 266077;11.青岛瑞滋集团有限公司山东青岛 266409

摘要:

microRNA参与基因的转录后调控，在真核生物的生长发育、细胞分化和免疫防御等过程中发挥重要作用。刺参(Apostichopus japonicus)病害问题已成为产业发展的主要限制因素之一，而其病害发生的分子机制尚待进一步完善。本研究以刺参重大疾病“腐皮综合征”的重要致病原灿烂弧菌(Vibrios splendidus)为侵染菌株，通过人工侵染实验制备患病刺参样本，采用miRNA-seq技术对侵染组(PT16S)和对照组(PT10H)各3头刺参的体壁组织进行miRNA测序，通过相关生物信息学软件对miRNAs进行鉴定和分析，筛选差异表达miRNAs (DEmiRNAs)并预测其靶基因，构建关键调控途径的miRNA-mRNA调控网络。结果显示，PT10H组平均得到5 902 588条有效序列，194个已知miRNA和19个新的miRNA；PT16S组平均得到5 053 529条有效序列，182个已知miRNA和42个新的miRNA。对2组鉴定到的miRNA进行差异表达分析，共筛选到2个上调和11个下调的具有显著差异的DEmiRNAs (P≤0.05)，上调的DEmiRNAs靶基因预测结合到3010个靶基因，注释到585个GO terms及24条信号通路(P≤0.05)，下调的DEmiRNAs靶基因预测到19 072个靶基因，注释到514个GO terms以及22条信号通路(P≤0.05)。对筛选到的DEmiRNAs进行实时荧光定量PCR (qRT-PCR)验证，显示miRNA-seq与qRT-PCR的一致率达到70%。根据KEGG分析结果构建泛素介导的蛋白水解途径和Notch信号通路的miRNA-mRNA调控网络，结果显示，13个DEmiRNAs分别靶向结合134个与泛素介导的蛋白水解相关的mRNAs和109个与Notch信号通路相关的mRNAs，Aja-miR-184、Aja-miR-2478和Aja-miR-9277p等DEmiRNAs可能参与对Notch信号通路和对泛素介导的蛋白水解的调控。相关研究结果将为刺参疾病发生调控网络建立和机制解析提供依据。

关键词: 刺参 microRNA 灿烂弧菌胁迫应答靶基因 miRNA-mRNA调控网络

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Identification of differential expression microRNAs and target genes analysis of sea cucumber (Apostichopus japonicus) in response to Vibrio splendidus infection

CHANG Mengyang^1,2, LI Bin^2,3, RONG Xiaojun^4,5, WANG Jinjin^6,7, YU Yongxiang^8,9, WANG Yingeng^10,11, LIAO Meijie^12,11, ZHANG Zheng^13,11, FAN Ruiyong¹⁴, LIU Qingbing¹⁴

1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;2.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, China;3.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, Shandong 266071, China;4.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266072, China;5.Laboratory for Marine Fisheries SNNN OO称ᘚ异QR ¦ĀĀT풐ᖂK异na;6.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266073, China;7.Laboratory for Marine Fisheries ST\\UU称ᘚ弲Y[¦ĀĀKᢀᖂL弲I;8.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266074, China;9.Laboratory for Marine Fisheries Sew RomanႠᕖ饀ᶝ￬Ɛ䀀✀Times New Roman⮐࿎￬Ɛ蘀䀀ᜀ宋体es New Roman殀࿍I饀ᶝ;10.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266075, China;11.Laboratory for Marine Fisheries Sæêîî瘀眀砀礀稀笀츀簀紀耀脀舀茀케萀蔀蘀蜀퀀턀蠀褀言謀谀툀贀踀팀퐀픀輀退鄀鈀錀鐀销阀需頀餀騀鬀鰀鴀鸀鼀혀ꀀꄀꈀꌀꐀꔀ휀ꘀ꜀ꠀ꤀ꨀ��꬀가관글꼀뀀넀눀대됀딀똀뜀렀뤀먀묀�밀봀븀뼀쀀섀숀쌀쐀씀였윀󐀀저樀琀쨀찀쬀촀欀氀戀洀渀搀昀最漀椀猀I饀ᶝ;12.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266076, China;13.Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266077, China;14.Qingdao Ruizi Company, Qingdao, Shandong 266409, China

Abstract:

microRNAs (miRNAs), involved in post-transcriptional gene regulation, play important roles in the growth, development, cell differentiation, and immune defense of eukaryotes. Apostichopus japonicus has become an economically important species for marine aquaculture in China; however, the outbreak of diseases, such as skin ulcer syndrome (SUS), has led to great losses in aquaculture development. Therefore, molecular mechanisms underlying disease occurrence must be further elucidated. In the present study, Vibrio splendidus, an important causative pathogen of SUS, was used as the stress strain (1×106 CFU/mL) in an artificial infection experiment. Body wall of the diseased (PT16S) and healthy (PT10H) samples was subjected to miRNA-Seq. Differentially expressed miRNAs (DEmiRNAs) were screened using bioinformatics. The target genes of DEmiRNAs were predicted and used for constructing miRNA-mRNA regulatory networks. Through miRNA-Seq, respectively 5 902 588 and 5 053 529 valid reads were generated for the PT10H and PT16S samples. A total of 13 DEmiRNAs (P≤0.05) were screened between PT10H and PT16S, of which two were upregulated and 11 downregulated. Target gene prediction revealed that the two upregulated DEmiRNAs bound to 3010 target genes, which were annotated to 585 GO terms and 24 signaling pathways (P≤0.05), while the 11 downregulated DEmiRNAs bound to 19 072 target genes, which were annotated to 514 GO terms and 22 signaling pathways (P≤0.05). In the validation test, the consistency rate of the sequencing and qRT-PCR data reached 70%. Two immune-related pathways (ubiquitin-mediated proteolysis and Notch signaling) were selected and used to construct the miRNA-mRNA regulatory networks. The 13 DEmiRNAs could bind 134 ubiquitin-mediated proteolytic mRNAs and 109 Notch signaling mRNAs. Specifically, Aja-miR-1559-3p, Aja-miR-7550-5p, Aja-miR-2478, and Aja-miR-9277-3p may be involved in the regulation of ubiquitin-mediated proteolysis and Notch signaling. Our results provide primary data for understanding the post-transcriptional regulatory mechanisms of diseases in sea cucumber.

Key words: Apostichopus japonicus microRNA Vibrio splendidus Stress response Target genes miRNA-mRNA network