【摘要】 旨在了解生长分化因子5(GDF5)可能的调控序列。本研究通过基因组比对,扩增了牛GDF5基因5′侧翼区,并通过产物纯化、连接、转化及测序比对,确定了2043bp的启动子序列。同时综合考虑已经证实的人GDF5基因启动子结构及应用启动子在线分析软件,对该序列进行分析。结果发现,牛GDF5基因5′侧翼区没有CpG岛,序列比对发现,牛和人GDF5基因启动子区域同源性为78%;牛GDF5基因启动子没有TATAbox或CAATbox结构,其转录起始位点位于翻译起始密码子ATG上游-359bp位置,其潜在的转录因子有AML-la,Ap-1,AmL-la,CdxA,SRY,CdxA,TATA,AmL-la,GTATA-1,MZF1,CdxA,Nkx-2,CdxA,S8和SRY,其中Ap-1,AmL-la,SRY,Nkx-2,S8和SRY高度保守,与人的序列完全一致;推测发现牛GDF5基因启动子-457至-423bp序列中的GT重复序列可以增强启动子的活性,且最小增强子处于-458和-377bp之间。研究结果推测判定了牛GDF5基因启动子的转录起始位置、转录结合位点、活性序列及最小增强子序列,为以后深入研究GDF5基因在牛软骨细胞中的表达机制提供了理论基础。更多还原

【Abstract】 This experiment was conducted to study the potential regulatory sequences of growth differentiate factor 5(GDF5).The 5’ flanking region of bovine GDF5 gene was amplified through comparing human GDF5 gene,and then a 2 043 bp promoter sequence was confirmed after product purification,ligation,transformation and sequence.In addition,considering the confirmed human GDF5 promoter structure and the analytic result of promoter online software,the promoter sequence was analyzed.It was found that there are no CpG island existing in 5’ flanking region of bovine GDF5 gene using CpG Island Searcher program,and comparing with the human GDF5 5’ flanking sequence,about 78% nucleotide identity was showed.Meanwhile,the result showed that no typical TATA or CAAT boxes were found in bovine GDF5 promoter,and the transcription start site was mapped to-359 bp of translation start site,and the potential transcription factor binding motifs were predicted,including AML-la,Ap-1,AmL-la,CdxA,CdxA,TATA,AmL-la,GTATA-1,MZF1,CdxA,Nkx-2,CdxA,S8 and SRY,in which only Ap-1,AmL-la,Nkx-2,S8 and SRY were located at the identical positions in human and bovine,both of which were conserved.Moreover,it also showed that around the promoter region from-457 to-423 bp the repeat of GT could increase the promoter activity and a minimal enhancer element reside from-458 to-377 bp in bovine GDF5 promoter.In this experiment,we inferred the transcription start site,transcription factor binding motifs,activity sequence and minimal enhancer element in bovine GDF5 promoter,which may be helpful for exploring the mechanism of gene expression in chondrocyte-restrictive.更多还原