| MA Qian,FAN Yanjun,ZHUANG Zhimeng,LIU Shufang. 2018. Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis). Acta Oceanologica Sinica, 37(2):76－84 |
| Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis) |
| 半滑舌鳎骨形态发生蛋白2基因的克隆、表达分析及启动子活性分析 |
| Received:March 03, 2017 Revised:November 07, 2011 |
| DOI：10.1007/s13131-018-1164-x |
| Key words:cloning gene expression pattern promoter transcriptional activity bone morphogenetic protein Cynoglossus semilaevis early developmental stages |
| 中文关键词: 基因克隆 表达模式 启动子活性 骨形态发生蛋白 半滑舌鳎 早期发育 |
| 基金项目:The Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences under contract No. 2016RC-LX02; the National Natural Science Foundation of China under contract No. 31201981. |
| Author Name | Affiliation | E-mail | | MA Qian | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China | | | FAN Yanjun | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China | | | ZHUANG Zhimeng | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
Function Laboratory for Marine Biology and Biotechnolgy, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China | zhuangzm@ysfri.ac.cn | | LIU Shufang | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China | |
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| Abstract: |
| BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp, which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR (qRT-PCR). The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch (dph); then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5'-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of -179 to +109. The predicted transcription factor binding sites (E-box binding factors, zinc finger transcription factor, etc.) in this region might participate in the transcriptional regulation of the bmp2 gene. |
| 中文摘要: |
| 骨形态发生蛋白2（BMP2）在脊椎动物的骨形成、尤其是诱导成骨方面具有至关重要的作用。本研究克隆了半滑舌鳎（Cynoglossussemilaevis）BMP2基因的cDNA序列，分析其在半滑舌鳎成鱼的组织表达差异及其在早期发育阶段的定位和表达变化趋势，并检测了其启动子活性。研究结果表明，半滑舌鳎BMP2基因cDNA全长为2048bp，编码422个氨基酸。BMP2mRNA在半滑舌鳎成鱼的14个组织中具有广泛的分布，并在脊髓中表达量最高。此外，BMP2基因在早期胚胎发育阶段（原肠期）具有极高水平的表达，其表达量约为其他发育阶段表达量的10倍。整体原位杂交检测结果表明，BMP2mRNA在1日龄仔鱼主要在冠状幼鳍、心和肝表达，在脊索有微量表达；随后，3日龄仔鱼的BMP2mRNA主要表达于脊索。通过对BMP2基因5'上游调控区片段进行启动子活性分析，发现了-179～+109片段可能包含启动子活性增强原件，生物信息学预测该片段包含的锌指转录因子等可能参与了BMP2的转录调控。 |
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