中国科学院海洋研究所机构知识库

牙鲆（Paralichthys olivaceus）是一种重要的水产养殖鱼类，在亚洲东部等地区大规模养殖。肿大细胞病毒（megalocytivirus）和迟缓爱德华氏菌（Edwardsiella tarda）是牙鲆等许多养殖鱼类的严重病原。本研究对肿大细胞病毒感染后的牙鲆脾脏进行全转录组测序，通过生物学信息分析并结合实验对病原诱导的编码RNA和非编码RNA进行了系统鉴定、免疫功能分析以及抗感染免疫作用研究。另外，本研究还对迟缓爱德华氏菌感染后的牙鲆进行了脾脏全转录组测序，分析了迟缓爱德华氏菌以及肿大细胞病毒共诱导的miRNA。

通过对肿大细胞病毒感染的牙鲆脾脏转录组数据分析，我们在感染后2、6和8天分别鉴定到225、275和877个差异表达基因（DEG）。其中，728个DEGs的表达量显著上调，659个DEGs的表达量显著下调。大多数DEGs的表达具有时间特异性，并形成四种不同的表达模式。Gene Ontology（GO）和Kyoto Encyclopedia of Genes and Genomes（KEGG）功能富集分析显示DEGs参与多个信号通路，其中约三分之一与免疫相关。通过加权共表达网络分析（WGCNA）筛选到了16个关键免疫DEGs，其共参与7个免疫通路，这些通路之间存在广泛的相互作用，形成复杂的调控网络。

随后，通过对肿大细胞病毒感染的牙鲆脾脏小RNA组数据进行分析，共鉴定了1,327个microRNAs（miRNAs），其中171个miRNA（命名为DEmiRs）在病毒感染期间以时间依赖性的方式表现出明显的差异性表达。基于靶基因预测软件分析结果，共有805个基因（命名为DETmRs）被预测为DEmiRs的靶基因，其表达量不仅在病毒感染后发生显著性变化，且与目标DEmiRs的表达量呈负相关。通过对免疫相关的DETmRs及其目标DEmiRs的综合分析，确定了12个关键DEmiRs，它们与相应的DETmRs一起形成了包含84个DEmiR-DETmR关系对的调控网络。此外，19个DEmiRs靶向上述转录组分析结果中的6个关键免疫DEGs，共同形成了由21个miRNA-mRNA关系对组成的调控网络。通过对测序数据进一步分析，鉴定了9,434个环状RNA（circRNA），其中169个circRNA（命名为DEcircRs）在肿大细胞病毒感染期间表现出显著差异表达以及时间依赖性。通过对DETmR-DEmiR和DEcircR-DEmiR相互作用进行综合分析，构建了竞争性内源RNA（ceRNAs）调控网络，该网络由参与抗病毒免疫的circRNA、miRNA和mRNA三联体构成。

MiRNAs在抗感染免疫中发挥重要作用，其通过调控靶基因的转录后表达，广泛参与鱼类多种免疫过程。基于全转录组数据分析结果，我们鉴定了肿大细胞病毒和迟缓爱德华氏菌共诱导的miRNA，并且针对其中一个miRNA（miR-29-x）开展了免疫功能研究。结果表明，miR-29-x的靶基因为claudin 4（CLDN4）。qRT-PCR检测发现miR-29-x和CLDN4的表达量在病原感染后均发生显著变化，且变化趋势呈负相关。CLDN4是五次跨膜蛋白，通过激光共聚焦显微镜观察发现CLDN4在细胞质膜上表达。CLDN4蛋白有三段序列暴露于细胞膜外，分别命名为E1、E2和E3，其中E2对于CLDN4的胞内定位具有重要影响。CLDN4胞外段E3对应的小肽具有与多种细菌结合的能力以及对Vibrio anguillarum的杀伤能力。另外发现，过量表达CLDN4能够增加迟缓爱德华氏菌对细胞的粘附作用。

综上所述，我们从mRNA、miRNA及circRNA等多层次对肿大细胞病毒诱导的牙鲆RNA进行了鉴定分析和研究，发现编码RNA和非编码RNA深度参与牙鲆抗感染免疫，揭示了一个肿大细胞病毒和迟缓爱德华氏菌共诱导的miRNA的靶基因的抗感染免疫作用。这些研究结果加深了我们对鱼类RNA免疫功能和免疫机制的理解，也为鱼类病毒和细菌病的免疫防控提供了理论指导。R

Japanese flounder (Paralichthys olivaceus) is an important aquaculture fish that is farmed on a large scale in eastern Asia and other regions.Megalocytivirus and Edwardsiella tardaare serious pathogens of many cultured fish including flounder. In this study, transcriptome sequencing was performed to examinethe expression profiles ofcodingRNAs and non-coding RNAs inthe spleen of flounderinfected with megalocytivirus. The RNAs were systematically identified, analyzed for immune function, and studied for anti-infective immunity.Inaddition, we also analyzed the spleen transcriptomeof E. tarda-infected flounder, and examined the miRNAs co-induced byE. tardaand megalocytivirus.

By analyzing the transcriptomic data of flounder infected with megalocytivirus, we identified 225, 275 and 877 differentially expressed genes (DEGs) at 2, 6 and 8 days post infection, respectively. Of these, 728 DEGs were significantly up-regulated and 659 DEGs were significantly down-regulated in expression. The expression of most DEGs was time-specific and formed four distinct expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis revealed that DEGs are involved in multiple signaling pathways, about one-third of which were related to immunity. A total of 16 key immune DEGs were screened by weighted co-expression network analysis (WGCNA), which are involved in seven immune pathways with extensive interactions between them, forming a complex regulatory network.

A total of 1,327 microRNAs (miRNAs) were identified by analyzing data from the small RNAs of flounder infected with megalocytivirus. Of these miRNAs, 171 miRNAs (named DEmiRs) showed significant differential expression in a time-dependent manner during virus infection. A total of 805 genes (named DETmRs) were predicted to be the target genes of these DEmiRs, and their expression not only changed significantly after viral infection but also correlated negatively with the expression of their target DEmiRs. Integrative analysis of immune-related DETmRs and their target DEmiRs identified 12 key DEmiRs. These DEmiRs together with their target DETmRs formed an interaction network containing 84 pairs of DEmiR and DETmR. In addition, 19 DEmiRs targeted six key immune DEGs from the above regulatory transcriptome analysis results, which together formed a regulatory network consisting of 21 miRNA-mRNA relationship pairs. Further analysis of the sequencing data led to the identification of 9,434 circular RNAs (circRNAs), of which 169 circRNAs (named DEcircRs) exhibited significant and time-dependent differential expressions during megalocytivirus infection. Through comprehensive analysis of DETmR-DEmiR and DEcircR-DEmiR interactions, a regulatory network of competing endogenous RNAs (ceRNAs) was constructed, consisting of a triad of circRNAs, miRNAs and mRNAs involved in antiviral immunity.

MiRNAs play an important role in anti-infection immunity, and are widely involved in a variety of immune processes in fish by regulating the post-transcriptional expression of theirtarget genes. Based on whole-transcriptome data analysis, we screened miRNAs co-induced by megalocytivirusand Edwardsiella tarda,and examined the immune function of one of these miRNAs (miR-29-x). The results showed that claudin 4 (CLDN4) was the target gene of miR-29-x. qRT-PCR assay revealed significant changes in the expression of both miR-29-x and CLDN4 after pathogen infection, and their changing trends were negatively correlated. CLDN4 was found to localize in the cytoplasmic membrane by confocal microscopy. CLDN4 is a five-time transmembrane protein, and has three extracellular regions named E1, E2 and E3, respectively. E2 has a marked effect on the subcellular localization of CLDN4. The small peptide corresponding to E3 was able to bind to a variety of bacteria and kill Vibrio anguillarum. Furthermore, overexpression of CLDN4 was able to increase the adhesion of Edwardsiella tardato fish cells.

In conclusion, we identified, analyzed and studied flounder RNAs induced by megalocytivirus from multiple levels, including mRNA, miRNA and circRNA. We found that these coding RNA and non-coding RNA were deeply involved in flounder immunity against pathogen infection, and that the target gene of one of the virus-bacteria-coinduced miRNAs possessed possessed antibacterial activity. These findings deepen our understanding of RNA immune functions and immune mechanisms in fish, and also provide theoretical guidance for immune prevention and control of viral and bacterial diseases in fish.