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农业生物技术学报  2019, Vol. 27 Issue (3): 481-487    DOI: 10.3969/j.issn.1674-7968.2019.03.012
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
蛋鸡骨钙素基因启动子变异及其转录活性研究
王涵, 陈烨, 岳巧娴, 周荣艳*, 陈辉, 王德贺, 锡建中
河北农业大学 动物科技学院,保定 071001
Study on Variations and Transcriptional Activity of Osteocalcin Gene Promoter in laying hens (Gallus gallus domesticus)
WANG Han, CHEN Ye, YUE Qiao-Xian, ZHOU Rong-Yan*, CHEN Hui, WANG De-He, XI Jian-Zhong
College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China
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摘要 骨钙素(osteocalcin, OCN)是由成骨细胞分泌合成的一种非胶原骨基质蛋白,在骨重建、骨矿化等骨代谢过程中发挥重要作用。本研究旨在研究蛋鸡(Gallus gallus domesticus) OCN基因表达调控机制。选取20只50周龄大午粉蛋鸡,提取血液DNA;以Ensembl基因组数据库中OCN基因序列(ENSGALG00000029494)为模板设计引物,扩增OCN基因启动子;利用Sanger测序方法在OCN基因启动子区寻找潜在的遗传变异。采用JASPAR2018在线软件进行转录因子结合位点预测,并构建启动子不同单倍型荧光素酶报告基因重组载体,转染鸡成纤维细胞系DF-1后检测双荧光素酶活性。结果表明,所扩增蛋鸡OCN基因启动子区具有转录活性,含有维生素D受体(Vitamin D receptor, VDR)、RUNX (Runt related transcription factor)1、RUNX2和RUNX3等骨代谢相关转录因子结合位点,测序结果发现在启动子区存在插入片段CCGCACTCTGCACTTTGCGGCCG和缺失片段CCACA,以及C>G和A>G突变,组成4种单倍型;双荧光素酶检测结果表明,启动子区4种单倍型均具有转录活性,但不同单倍型间转录活性存在极显著差异(P<0.01),其中单倍型活性最高。本研究确定了OCN基因核心启动子区,且不同变异类型启动子的转录活性存在差异,为该基因在蛋鸡骨代谢中的转录调控提供了基础资料。
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王涵
陈烨
岳巧娴
周荣艳
陈辉
王德贺
锡建中
关键词 蛋鸡骨代谢骨钙素基因(OCN)启动子    
Abstract:Osteocalcin (OCN) is a kind of non-collagen bone matrix protein secreted and synthesized by osteoblasts, which plays an important role in bone metabolism such as bone remodeling and bone mineralization. This study aimed to investigate the mechanism of gene expression regulation of OCN in laying hens (Gallus gallus domesticus). Twenty 50-week-old DAWUFEN laying hens were selected and the blood was collected for DNA extraction. The primers for promoter region of OCN gene were designed according to the template sequence downloaded from Ensembl database (ENSGALG00000029494). Potential genetic variations in the promoter region of OCN gene were identified by Sanger sequencing. JASPAR2018 was used for transcription factors binding site prediction. The luciferase reporter gene recombinant vectors with different haplotypes were constructed and the dual-luciferase activity was detected by transfecting chicken fibroblast line DF-1. The results showed that the promoter region of the OCN gene in the amplified layer had the transcriptional activity and contained binding sites for bone metabolism-related transcription factors such as Vitamin D receptor (VDR), Runt related transcription factor (RUNX) 1, RUNX2, and RUNX3. Sequencing results revealed the presence of the insert fragment CCGCACTCTGCACTTTGCGGCCG and the deletion fragment CCACA in promoter region, as well as C>G and A>G variations. These variants constituted 4 haplotypes. The dual luciferase assay showed that all the 4 haplotypes in the promoter region had transcriptional activity, but the activity of different haplotypes had extremely significant difference (P<0.01), and the transcriptional activity of haplotype was the highest. In this study, the core promoter region of the OCN gene was identified, and there were differences in the promoter transcriptional activities of different haplotypes. A theoretical basis for the transcriptional regulation of OCN gene could be helpful for bone metabolism of laying hens.
Key wordsLaying hen    Bone metabolism    Osteocalcin (OCN)    Promoter
收稿日期: 2018-08-13     
ZTFLH:  S831.2  
基金资助:河北省硕士研究生创新项目(No. CXZZSS2018057)、河北首批青年拔尖人才支持计划(2016-2018)和河北农业大学青年学术带头人支持项目(2015-2017)
通讯作者: *rongyanzhou@126.com   
引用本文:   
王涵, 陈烨, 岳巧娴, 周荣艳, 陈辉, 王德贺, 锡建中. 蛋鸡骨钙素基因启动子变异及其转录活性研究[J]. 农业生物技术学报, 2019, 27(3): 481-487.
WANG Han, CHEN Ye, YUE Qiao-Xian, ZHOU Rong-Yan, CHEN Hui, WANG De-He, XI Jian-Zhong. Study on Variations and Transcriptional Activity of Osteocalcin Gene Promoter in laying hens (Gallus gallus domesticus). 农业生物技术学报, 2019, 27(3): 481-487.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.03.012     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I3/481
 
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