首页 >  渔业科学进展 >  刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析

2023, 44(2): 107-117. doi: 10.19663/j.issn2095-9869.20211228003

刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析

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 266071;

3. 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;

4. 青岛瑞滋集团有限公司 山东 青岛 266409

收稿日期:2021-12-28
修回日期:2022-01-19

基金项目:   国家重点研发计划(2018YFD0900305)、山东省农业良种工程课题(2020LZGC015)、中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2020TD40;2021GH05)共同资助。 

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

Identification of Differential Expression microRNAs and Target Genes Analysis of Sea Cucumber (Apostichopus japonicus) in Response to Vibrio splendidus Infection

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. Qingdao Ruizi Company, Qingdao, Shandong 266409, China

Received Date:2021-12-28
Accepted Date:2022-01-19

Keywords: Apostichopus japonicus , microRNA , Vibrio splendidus , Stress response , Target genes , miRNA-mRNA network

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。

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.

参考文献

[1] AGGARWAL K, SILVERMAN N.Positive and negative regulation of the Drosophila immune response.BMB Reports, 2008, 41(4):267-277
[2] BETEL D, WILSON M, GABOW A, et al.The microRNA.org resource:Targets and expression.Nucleic Acids Research, 2008, 36(suppl 1):D149-D153
[3] BOONCHUEN P, MARALIT B A, JAREE P, et al.MicroRNA and mRNA interactions coordinate the immune response in non-lethal heat stressed Litopenaeus vannamei against AHPND-causing Vibrio parahaemolyticus.Scientific Reports, 2020, 10(1):787
[4] CHEN H, XIN L, SONG X, et al.A norepinephrine-responsive miRNA directly promotes CgHSP90AA1 expression in oyster haemocytes during desiccation.Fish and Shellfish Immunology, 2017, 64:297-307
[5] 陈科, 程汉华, 周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能.遗传, 2012, 34(1):7-20CHEN K, CHENG H H, ZHOU R J.Molecular mechanisms and functions of autophagy and the ubiq-uitin-proteasome pathway.Hereditas, 2012, 34(1):7-20
[6] 郭睿, 杜宇, 周倪虹, 等.意大利蜜蜂幼虫肠道在球囊菌胁迫后期的差异表达微小RNA及其靶基因分析.昆虫学报, 2019, 62(1):49-60GUO R, DU Y, ZHOU N H, et al.Comprehensive analysis of differentially expressed microRNAs and their target genes in the larval gut of Apis mellifera ligustica during the late stage of Ascosphaera apis stress.Acta Entomologica Sinica 2019, 62(1):49-60
[7] HE X B, JING Z Z, CHENG G F.MicroRNAs:New regulators of toll-like receptor signalling pathways.BioMed Research International, 2014:945169
[8] KROL J, LOEDIGE I, FILIPOWICZ W.The widespread regulation of microRNA biogenesis, function and decay.Nature Reviews Genetics, 2010, 11(9):597-610
[9] LAN J F, ZHOU J, ZHANG X W, et al.Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii).Developmental and Comparative Immunology, 2013, 41(4):608-617
[10] LI C H, FENG W, QIU L, et al.Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.Fish and Shellfish Immunology, 2012, 33(2):436-441
[11] LI C, XU D X.Understanding microRNAs regulation in heat shock response in the sea cucumber Apostichopus japonicus.Fish and Shellfish Immunology, 2018, 81:214-220
[12] LI C, ZHAO M, ZHANG C, et al.miR210 modulates respiratory burst in Apostichopus japonicus coelomocytes via targeting toll-like receptor.Developmental and Comparative Immunology, 2016, 65:377-381
[13] 李席席.非O1霍乱弧菌对青虾的致病性、宿主的免疫反应及拮抗菌的益生效果研究.扬州大学博士研究生学位论文, 2020LI X X.Study on pathogenicity of non-O1 Vibrio cholerae to Macrobrachium nipponensis, host immune response and probiotic effect of antagonistic bacteria.Doctoral Dissertation of Yangzhou University, 2020
[14] 梁金荣.凡纳滨对虾免疫相关microRNA的克隆与功能分析.广东海洋大学博士学位研究生学位论文, 2020LIANG J R.Cloning and functional analysis of immune related microRNAs in Litopenaeus vannamei.Doctoral Dissertation of Guangdong Ocean University, 2020
[15] 林德麟.乳源miRNAs在动物体内的吸收规律及乳exosome对小鼠免疫功能的影响.华南农业大学博士研究生学位论文, 2017LIN D L.Absorption of milk-derived miRNAs in animals and effects of milk-derived exosomes on mice immune function.Doctoral Dissertation of Southern China Agriculture University, 2017
[16] 林锋.罗氏沼虾野田村病毒致病性及其快速检测技术研究.江苏大学博士研究生学位论文, 2018LIN F.Study on the rapid detection technology and characterization of Macrobrachium rosenbergii nodavirus and extra small virus isolated in China.Doctoral Dissertation of Jiangsu University, 2018
[17] LU M, ZHANG P J, LI C H, et al.MiR-31 modulates coelomocytes ROS production via targeting p105 in Vibrio splendidus challenged sea cucumber Apostichopus japonicus in vitro and in vivo.Fish and Shellfish Immunology, 2015a, 45(2):293-299
[18] LU M, ZHANG P J, LI C H, et al.miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.Scientific Reports, 2015b, 5:12608
[19] LV M, CHEN H H, SHAO Y N, et al.miR-137 modulates coelomocyte apoptosis by targeting 14-3-3ζ in the sea cucumber Apostichopus japonicus.Developmental and Comparative Immunology, 2017, 67:86-96
[20] LV Z, LI C H, ZHANG P J, et al.miR-200 modulates coelo-mocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus.Fish and Shellfish Immunology, 2015, 45(2):431-436
[21] NAM J W, RISSLAND O S, KOPPSTEIN D, et al.Global analyses of the effect of different cellular contexts on microRNA targeting.Molecular Cell, 2014, 53(6):1031-1043
[22] OU J, LI Y, DING Z F, et al.Transcriptome-wide identification and characterization of the Procambarus clarkii microRNAs potentially related to immunity against Spiroplasma eriocheiris infection.Fish and Shellfish Immunology, 2013:35(2):607-617
[23] SHAO Y N, LI C H, XU W, et al.miR-31 links lipid metabolism and cell apoptosis in bacteria-challenged Apostichopus japonicus via targeting CTRP9.Front Immunology, 2017, 8:263
[24] SHIMIZU T, TANAKA T, ISO T, et al.Notch signaling pathway enhances bone morphogenetic protein 2 (BMP2) responsiveness of Msx2 gene to induce osteogenic differentiation and mineralization of vascular smooth muscle cells.Journal of Biological Chemistry, 2011, 286(21):19138-19148
[25] SIGOILLOT F D, EVANS DR, GUY H I.Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades.Journal of Biological Chemistry, 2002, 277(18):15745-15751
[26] VALLET-GELY I, LEMAITRE B, BOCCARD F.Bacterial strategies to overcome insect defences.Nature Reviews Microbiology, 2008, 6(4):302-313
[27] 谢淑媚.缢蛏类胰岛素生长因子系统3个基因表达特征及功能初步分析.上海海洋大学博士研究生学位论文, 2018XIE S M.Expression characteristics and preliminary functional analysis of three genes in the lin-like growth factor system of razor clam Sinonovacula constricta.Doctoral Dissertation of Shanghai Ocean University, 2018
[28] XU P, GUO H, WANG H, et al.Identification and profiling of microRNAs responsive to cadmium toxicity in hepatopancreas of the freshwater crab Sinopotamon henanense.Hereditas, 2019, 156(1):34
[29] ZHANG C F, TONG C, TIAN F, et al.Integrated mRNA and microRNA transcriptome analyses reveal regulation of thermal acclimation in Gymnocypris przewalskii:A case study in Tibetan Schizothoracine fish.PLoS One, 2017, 12(10):e0186433
[30] 张春云, 王印庚, 荣小军.养殖刺参腐皮综合征病原菌的分离与鉴定.水产学报, 2006, 30(1):118-123ZHANG C Y, WANG Y G, RONG X J.Isolation and identification of causative pathogen for skin ulcerative syndrome in Apostichopus japonicus.Journal of Fisheries of China, 2006, 30(1):118-123
[31] 卓梅琴.黄颡鱼PI3Ks功能解析及其通路在胰岛素调控脂类代谢中的作用机制.华中农业大学博士学位研究生学位论文, 2018ZHUO M Q.Functional analysis of PI3Ks and its related signal pathway in insulin regulating lipid metabolism in yellow catfish Pelteobagrus fulvidraco.Doctoral Dissertation of Huazhong Agricultural University, 2018

相关文章

[1] 陈晶, 聂青, 刘妍. 《WHO基本药物示范目录》与我国《国家基本药物目录》动态调整程序比较与借鉴.水产学报,2015(3): 289-293.doi:10.3866/PKU.WHXB201503022
  • 导出引用
  • 下载XML
  • 收藏文章
计量
  • 文章下载量()
  • 文章访问量()

目录

刺参响应灿烂弧菌侵染差异microRNAs鉴定及靶基因分析