摘要：Boule基因是小鼠精子发生的必需因子，为了解小鼠Boule基因5’启动子区特征，本文鉴定和分析了小鼠Boule基因核心启动子区。利用克隆测序获得小鼠Boule基因5’调控区序列，采用生物信息学方法分析其序列特征；采用双荧光酶检测系统鉴定小鼠Boule基因缺失表达载体荧光素酶活性；采用生物信息学网站预测其核心启动子区的潜在转录因子结合位点等。结果发现在获得的2034bp的小鼠Boule基因5’调控区序列存在两个CpG岛；双荧光素酶活性分析发现，-562nt~-337nt区域是小鼠Boule基因的核心启动子区；转录因子结合位点预测发现，小鼠Boule基因核心启动区存在与精子发生相关的转录因子，如Sp1、SOX10和HOXA5等。小鼠Boule基因核心启动子的鉴定以及其上与精子发生相关的转录因子结合位点的发现，为今后进一步的研究小鼠睾丸组织中Boule基因的表达调控奠定了基础。关键字：小鼠；Boule基因；核心启动子；
Identification and analysis of core promoter region in mouse Boule gene
Abstract：Boule gene is a necessary factor for spermatogenesis in mice, the aim of this study was to understand
5’ promoter region features of Boule gene. The sequence of 5’ regulatory region of mouse Boule gene was obtained by cloning and sequencing, and was analyzed by bioinformatics methods. Luciferase activity of the different length of Boule gene expression vector was examined using Dual-Luciferase assay system; Potential transcription factor binding sites in core promoter region were predicted with bioinformatics website. The results indicated that 5’ regulatory region of Boule gene is 2034 bp in length and contains two CpG island; the core promoter region of Boule gene was located in -562 nt ~ -337 nt by Dual-Luciferase assay system. It contained many transcription factor binding sites related to spermatogenesis, such as Sp1,SOX10 and HOXA5. The identification of the Boule gene core promoter and transcription factor binding sites provide the foundation for further research on the expression of Boule gene in mouse testes.
Key words: mouse; Boule gene; core promoter
目录
小鼠Boule基因核心启动子区的鉴定与分析.4引言.4
1材料和方法5
1.1实验材料.5
1.1.1实验动物和睾丸组织收集..5
1.1.2载体及感受态细胞..5
1.1.3工具酶及试剂盒.5
1.1.4主要仪器和设备.5
1.1.5主要试剂和耗材.5
1.1.6主要分子生物学软件6
1.2实验方法.6
1.2.1小鼠Boule基因全序列的获取和分析.6
1.2.2睾丸组织基因组DNA提取..6
1.2.3PCR扩增..6
1.2.4引物设计.7
1.2.5DNA凝胶电泳.7
1.2.6DNA回收..7
1.2.7测序..8
1.2.8载体构建.8
1.2.8.1酶切..8
1.2.8.2回收..8
1.2.8.3连接..8
1.2.8.4转化..8
1.2.8.5提取质粒.8
1.2.9细胞培养.9
1.2.10转染.9
1.2.11荧光素酶活性检测.9
2结果与分析9
2.1小鼠Boule基因5’调控区克隆与序列分析..9
2.1.1PCR扩增结果.9
2.1.2Boule基因5’调控区序列分析..10
2.1.3转录起始位点预测.10
2.1.4启动子区预测和分析11
2.1.5调控元件预测和分析11
2.1.6小鼠Boule基因CpG岛的预测和分析.11
2.2小鼠Boule基因核心启动子鉴定.12
2.2.1片段的扩增.12
2.2.2缺失表达载体构建与鉴定.13
2.2.3荧光素酶活性分析.13
2.2.4核心启动子区域转录因子预测.14
3讨论与展望..14
3.1小鼠Boule基因5’调控区克隆与序列分析.14
3.2小鼠Boule基因核心启动子区的特征15致谢：.15 小鼠Boule基因核心启动子区的鉴定与分析:http://www.youerw.com/shengwu/lunwen_205218.html