本研究旨在了解牦牛FKBP6基因5′调控区和启动子区特征,为探讨牦牛和犏牛睾丸组织中FKBP6基因差异表达机制提供依据。利用克隆测序获得牦牛FKBP6基因5′调控区序列,采用生物信息学方法分析其序列特征;采用双荧光素酶报告基因系统鉴定牦牛FKBP6基因核心启动子区,利用生物信息学软件预测与精子发生有关的转录因子结合位点。通过克隆测序和序列拼接获得了1 354bp的牦牛FKBP6基因5′调控区序列,与普通牛的一致性为99.71%;牦牛FKBP6基因5′调控区序列含有潜在的启动子区、典型的CAAT-Box和CpG岛,但未见TATA-Box;荧光素酶活性分析发现,牦牛FKBP6基因的核心启动子区位于5′调控区的-263--167nt区域,含有CAAT-Box、E-Box、CTCF和CREB等与精子发生相关的转录因子结合位点。牦牛FKBP6基因核心启动子的鉴定和精子发生相关转录因子结合位点的发现为进一步研究牦牛睾丸组织中FKBP6基因的表达调控奠定了基础。
The aim of this study was to understand 5′regulatory region and promoter region features of FKBP6 gene,to provide evidence for exploring the mechanisms of differential expression of FKBP6 gene in yak and yak-cattle testicular tissue.The sequence of 5′regulatory region of yak FKBP6 gene was obtained by cloning and sequencing,and was analyzed by bioinformatics methods.The core promoter region of FKBP6 gene was identified using Dual-luciferase assay system and the transcription factor binding sites related to spermatogenesis were predicted using bioinformatics software.The 5′regulatory region of yak FKBP6 gene was 1 354 bp in length by cloning,sequencing and splicing,which shared the similarity of 99.71% with cattle.The 5′regulatory region sequence of yak FKBP6 gene contained several potential promoter regions,typical CAAT-Box and CpG island,but no TATA-Box.The core promoter region of FKBP6 gene was located in-263--167 nt of 5′regulatory region of FKBP6 gene by luciferase activity analysis and contained transcription factor binding sites related to spermatogenesis including CAAT-Box,E-Box,CTCF and CREB.The identification of the FKBP6 gene core promoter and the transcription factor binding sites provide a basis for further research on the regulation of FKBP6 gene expression in yak testis.