Screening and identification of miRNAs regulating Tbx4/5 genes of Pampus argenteus.
Screening and identification of miRNAs regulating Tbx4/5 genes of Pampus argenteus.
复制标题
银鲳Tbx4/5基因调控miRNA的筛选与鉴定
作者：
Zhang C;Zhang S;Liu M;Wang Y;Wang D;Xu S
银（Pampus argenteus）是分布最广、经济价值最高的中上层鱼类之一。然而，银鲳的一个独特的形态特征是失去了腹鳍，这在一定程度上会增加食物捕获过程中的能量需求，因此不利于人工养殖。Tbx 4/5基因是高度保守的调节因子，在脊椎动物中调节肢体发育，并且反过来由microRNAs（miRNAs）调节。然而，直接调控P. argenteus中Tbx 4/5基因的miRNAs仍有待阐明。首先克隆了银鲳Tbx 4/5基因，并利用高通量测序技术对银鲳鳍发育关键期（孵化后第1、7和13天）的小RNA转录组进行了测序。随后通过生物信息学分析预测了调控银鲫Tbx 4/5基因的miRNAs，并利用双荧光报告系统对相关miRNAs进行了体外验证。共鉴定出662个miRNAs，其中257个为已知miRNAs，405个为新发现的miRNAs。与第1天相比，在第7天有182个miRNA差异表达（DE），其中77个和105个miRNA分别下调和上调，而在第13天有278个miRNA差异表达，其中136个和142个miRNA分别下调和上调。与第13天相比，第7天有4种miRNA被DE，其中3种miRNA下调，1种miRNA上调。聚类分析结果显示，DE基因在幼虫发育的第1 ~ 7天和第1 ~ 13天呈负表达，表明幼虫处于分化的高峰期。然而，在幼虫发育的第7天和第13天之间，DE基因的数量相对较少，这表明发育的开始。随后预测DE miRNAs的潜在靶基因，并进行靶基因的基因本体论（GO）和京都基因和基因组百科全书（KEGG）富集分析。结果表明，DE miRNAs参与了鱼类的生长、发育和信号转导途径，其中Wnt和Fgfs信号通路在鱼类的生长发育中起重要作用。双荧光报告基因检测结果表明，miR-102、miR-301 c和miR-589对Tbx 4基因的3′-UTR具有显著的负调控作用，而miR-187、miR-201、miR-219和miR-460对Tbx 5基因的3′-UTR具有显著的负调控作用。总之，研究结果表明miRNAs在调节银鲳腹鳍的生长和发育中起着重要作用。本研究为进一步阐明银鲫miRNAs与靶基因的相互作用提供了参考。
Silver pomfret (Pampus argenteus) is one of the most widely distributed and economically important pelagic fish species. However, an unique morphological feature of P. argenteus is the loss of pelvic fins, which can increase the energy requirement during food capture to some extent and is therefore not conducive to artificial culture. Tbx4/5 genes are highly conserved regulatory factors that regulate limb development in vertebrates and are in turn regulated by microRNAs (miRNAs). However, the miRNAs that directly regulate the Tbx4/5 genes in P. argenteus remain to be elucidated. The Tbx4/5 genes of P. argenteus were first cloned, and the small RNA transcriptomes were sequenced by high-throughput sequencing during the critical period of the fin development at days 1, 7, and 13 of hatching. The miRNAs regulating the Tbx4/5 genes of P. argenteus were subsequently predicted by bioinformatics analysis, and the related miRNAs were verified in vitro using a dual fluorescence reporter system. A total of 662 miRNAs were identified, of which 257 were known miRNAs and 405 were novel miRNAs were identified. Compared to day 1, 182 miRNAs were differentially expressed (DE) on day 7, of which 77 and 105 miRNAs were downregulated and upregulated, respectively, while 278 miRNAs were DE on day 13, of which 136 and 142 miRNAs were downregulated and upregulated, respectively. Compared to day 13, four miRNAs were DE on day 7, of which three miRNAs were downregulated and one miRNA was upregulated. The results of hierarchical clustering of the miRNAs revealed that the DE genes were inversely expressed between days 1 and 7, and between days 1 and 13 of larval development, indicating that the larvae were in the peak stage of differentiation. However, the number of DE genes between days 7 and 13 of larval development was relatively small, suggesting the initiation of development. The potential target genes of the DE miRNAs were subsequently predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of target genes were performed. The results suggested that the DE miRNAs were involved in growth, development, and signal transduction pathways, of which the Wnt and Fgfs signaling pathways are known to play important roles in the growth and development of fins. The results of dual fluorescence reporter assays demonstrated that miR-102, miR-301c, and miR-589 had a significant negative regulatory effect on the 3′-UTR of the Tbx4 gene, while miR-187, miR-201, miR-219, and miR-460 had a significant negative regulatory effect on the 3′-UTR of the Tbx5 gene. Altogether, the findings indicated that miRNAs play an important role in regulating the growth and development of pelvic fins in P. argenteus. This study provides a reference for elucidating the interactions between the miRNAs and target genes of P. argenteus in future studies.
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