热敏感的生精作用特异表现基因Esx1的启动子作用研究

发布时间：2018-04-07 17:13

本文选题：热敏感　切入点：生精作用　出处：《大连医科大学》2016年硕士论文

【摘要】：Esx1(Extra-embryonic tissue-spermatogenesis-homeobox gene 1)系一X性联同源箱(X-linked homeobox)蛋白基因,具有三个mRNA的异构型,分别是a型、b型及x型。序列比对发现,彼此差异主要在5'端的非转译区(5'-untranslated regions)与部分下游序列。这些不同异构型的表现明显地是受到不同的启动子区域所调节,而此调节又高度地对热敏感。为了了解Esx1的组织特异性表现,分别抽取小鼠wj丸及胎盘的总RNA进行定量反转录聚合酶连锁反应(quantitative RT-PCR analyses)。结果显示在wj丸中主要表现a型及x型mRNA,而胎盘中主要表现x型。为了解存在于启动子区域(promoter region)内的近端作用因子(cis-acting elements),首先透过短暂性转染(transient transfection)分析小鼠Esx1基因的整个5'端邻近区域(5'-flanking sequences)(核苷酸序列从-1493到+464),其后再利用泳动迟滞试验(electrophoretic mobility shift assay,EMSA)及DNase I足迹试验(DNase I footprinting analysis,DFA)作佐证。报导基因分析结果显示核苷酸序列-1015到-459可在分离的初级精母细胞(primary spermatocytes)及圆形精细胞(round spermatids)内驱动下游的荧光酵素基因大量表现;核苷酸序列从+14到+189可在分离的精原细胞(spermatogonia)内启动报导基因的基础表现。DFA及EMSA则进一步地证实序列-901到-882能够专一地与wj丸及圆形精细胞总核蛋白萃取液结合,唯与胎盘、脑及肝脏总核蛋白质萃取液则否。利用TESS(transcription element search system)数据库比对此20个硷基发现,CACCC结合蛋白可能为调控wj丸中Esx1基因选择性转录的重要转录因子之一。删除此20 bp序列可使启动子活性降低23%。在未来,则可利用RNA干扰技术(RNA interference)进一步地分析CACCC结合蛋白在Esx1表现时的角色。
[Abstract]:Esx1(Extra-embryonic tissue-spermatogenesis-homeobox gene 1 is a X-linked homeobox) protein gene with three mRNA isomorphisms, type a and type x, respectively.Sequence alignment showed that the differences were mainly in the 5'-terminal non-translated regions (5'-untranslated regions) and some downstream sequences.The performance of these different isomerism is obviously regulated by different promoter regions and this regulation is highly sensitive to heat.In order to understand the tissue specificity of Esx1, the total RNA of mouse WJ pill and placenta were extracted for quantitative reverse transcription polymerase chain reaction (QRT) and quantitative RT-PCR analysis.The results showed that type a and type x mRNAs were mainly present in WJ pills, while those in placenta were mainly of type x.In order to understand the proximal action factor cis-acting elements in promoter region, the whole 5'terminal adjacent region of mouse Esx1 gene was analyzed by transient transfection.Then the nucleotide sequence ranged from -1493 to 464N.Electrophoretic mobility shift assayma (EMSA) and DNase I footprint test (DNase I footprinting analysis) were used as evidence.The results of gene analysis showed that nucleotide sequences -1015 to -459 could drive a large number of fluorescein genes downstream in isolated primary spermatocytes (primary spermatocytes) and round spermatocytes.Nucleotide sequences ranging from 14 to 189 could initiate the basic expression of the reporter gene in isolated spermatogonia. DFA and EMSA further confirmed that sequences -901 to -882 could specifically bind to WJ pill and circular spermatocyte total nuclear protein extract, but only to placenta.Brain and liver total nuclear protein extracts were not.Using the TESS(transcription element search system database, it was found that the CACCC-binding protein might be one of the important transcription factors regulating the selective transcription of Esx1 gene in WJ pills.Deletion of the 20 BP sequence reduced the promoter activity by 23%.In the future, RNA interference technique can be used to further analyze the role of CACCC binding proteins in the expression of Esx1.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R339.21

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