2016, 23(3): 500-512. doi: 10.3724/SP.J.1118.2016.15346
关键词： 半滑舌鳎 , BMP4 , 克隆 , 基因表达 , 整体原位杂交 , 骨骼发育
Keywords： Cynoglossus semilaevis , BMP4 , molecular cloning , gene expression , whole-mount in situ hybridization , skeletal development
本研究克隆了半滑舌鳎(Cynoglossus semilaevis)骨形态发生蛋白4(BMP4)基因的cDNA序列,并分析其在半滑舌鳎成鱼的组织表达分布及其在早期发育阶段的定位和表达变化趋势。结果表明,半滑舌鳎BMP4基因cDNA全长为1680 bp,包括了419 bp的5'非翻译区(5'UTR)、编码403个氨基酸的1212 bp的开放阅读框(ORF)以及49 bp的3'非翻译区(3'UTR)。同源性分析和分子进化聚类分析结果显示,半滑舌鳎与已报道慈鲷科(Cichlidae)各鱼种的同源性最高,并与之共同聚为鱼类BMP4分支。BMP4 mRNA在半滑舌鳎成鱼的13个组织中具有广泛的分布,并在鳃组织中表达量最高,其次在背鳍、软骨等组织具有中等水平的表达。此外, BMP4基因在早期胚胎发育阶段(卵裂期和原肠期)具有极高水平的表达,此后其表达水平逐渐降低;但在后期仔鱼期(孵化后3日龄和4日龄),其表达水平再次升高。整体原位杂交检测结果表明, BMP4 mRNA在早期仔鱼期主要在头部及躯干前部表达;后期仔鱼BMP4基因主要在冠状幼鳍、上下颌及胸鳍处表达;进入稚鱼期后在鳃盖骨及各鳍均有表达。上述研究结果揭示了BMP4在半滑舌鳎早期骨骼发育中的重要作用,为揭示海水硬骨鱼类骨骼发生、发育的调控机制奠定了理论基础。
The growth and development of the vertebrate skeleton is regulated by the bone morphogenetic proteins (BMPs), among which BMP4 is one of the most active osteoinduction factors. In this study, we cloned the BMP4 gene of the tongue sole(Cynoglossus semilaevis) and described its expression profiling. The BMP4 cDNA sequence was 1680 bp, and contained a 5'-untranslated region (5'-UTR) of 419 bp, an open reading frame (ORF) of 1212 bp encoding a protein of 403 amino acids, and a 3'-UTR of 49 bp. Homology and phylogenetic analyses revealed that the deduced amino acid sequence of the C. semilaevis BMP4 protein shares highest identity with those of Cichlidae species, and fish BMP4 forms a single lineage distinct from the BMP4 proteins of other vertebrates. The tissue expression distribution of the BMP4 gene was examined in 13 adult tissues. BMP4 mRNA was predominantly expressed in the gill, and less in the dorsal fin, cartilage, etc. In the early developmental stages, BMP4 mRNA was also detected at the egg, larval, juvenile, and fingerling stages, with the highest expression in the egg stage (cleavage and gastrula periods), and the second highest in the larval stage (3-4 days old). Whole-mount in situ hybridization showed that BMP4 mRNA was firstly detected in head and anterior region in the early larval stage, and then appeared in the crown-like larval fin, jaw, and pectoral fin. BMP4 mRNA was then predominantly expressed in all the fins and the operculum during the early juvenile stage. These results demonstrate the important role of BMP4 in the fin development of C. semilaevis, and provide a theoretical foundation for investigating the mechanisms regulating fish bone development.
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