牛Sry启动子调控序列的鉴定

Identification of the Regulation Sequences of Bovine Sry Promoter

【目的】Sry是大多数哺乳动物雄性性别发育的决定基因,但人们仍未找到其表达的调控规律,本试验对牛Sry5′端调控序列作了初步的研究,为深入研究牛Sry的表达调控奠定了基础。【方法】克隆牛Sry5′端侧翼1056bp长的DNA序列,利用生物信息学方法对这一区域内潜在的转录起始位点进行了预测,并构建了10个不同长度的缺失牛Sry5′部分侧翼序列的报告基因载体;进一步分离了胎牛生殖嵴,进行生殖嵴细胞的原代培养,并对胎牛生殖嵴细胞进行了性别和特征鉴定;最后利用荧光素酶双报告基因分析系统,在生殖嵴细胞内检测了牛Sry核心启动子区域的位置。【结果】体外培养的生殖嵴细胞可以表达雄性生殖嵴细胞的特征基因Sry、Sox9、Sf-1和Dax1,牛Sry5′端-93、-419和-722处存在3个潜在的转录起始位点(TSS),-599—-565区域35bp内存在控制Sry基础转录活性的顺式调控元件,其中存在多个潜在的转录因子结合位点。【结论】牛Sry5′端UTR区-599—-565bp区域35bp存在部分调控序列。

【Objective】 Sry is the pivotal gene initiating male sex determination in most mammals,but how its expression is regulated is still not clear.The study was to identify the regulation sequences of bovine Sry promoter,and to understand the transcriptional regulation of the Sry.【Method】 A 1056 bp long bovine Sry 5＇-flanking sequences,and the potential transcriptional start sites in this region were predicted by bioinformatics method as well as reporter gene analysis was isolated,Bovine genital ridge cells were isolated and deletion recombinant plasmids with different lengths of Sry 5＇ flanking regions were constructed.Sry 5＇-flanking promoter activity analysis was performed in bovine genital ridge cells by the dual-luciferase reporter assay system.【Result】 The results indicated that the genital ridge cells cultured in vitro could express the characteristic genes of male genital ridge,including Sry,Sox9,Sf-1 and Dax1.Through the prediction analysis,three potential transcription start sites for bovine Sry were identified at-93,-419 and-722 upstream from the translation initiation site.Further analysis demonstrated that a 35 bp long fragment（-599 to-565） in the 5＇-flanking sequence was important for the basic transcriptional activity.Prediction of the 35 bp long fragment demonstrated that there were several potential transcription factor binding sites in this region.【Conclusion】 There were several potential regulation sequences existed in the 35 bp long fragment（-599 to-565） of bovine Sry 5＇UTR。