中文摘要：

【目的】Sall4是一种锌指结构转录因子,对建立和维持动物多能干细胞具有重要意义。为探索猪Sall4的表达和转录调控机制,克隆了猪Sall4启动子,对其进行了功能验证和核心调控区的筛选。【方法】从猪基因组中克隆获得2.1 kb Sall4启动子片段,并构建相应的报告载体;将报告载体p E2.1转染不同种类的细胞,进行细胞特异性表达检测;采用实时荧光定量PCR方法,检测Sall4在猪不同组织和细胞中的表达变化;通过生物信息学方法,分析Sall4启动子上各种关键顺式作用元件和潜在的转录结合位点;将多能转录因子Oct4、Sox2、Klf4、Myc、Esrra/b和Smad与Sall4启动子共转染293T细胞,利用双萤光素酶报告系统检测Sall4启动子活性;采用DNA片段缺失的方法,构建一系列Sall4启动子片段缺失报告载体,将缺失片段载体分别转入293T细胞中,并利用双萤光素酶报告系统检测启动子的活性。【结果】Sall4在多能干细胞、生殖细胞和癌化肿瘤细胞中：包括P19、293T、CHO和Hela等细胞中特异性高表达。另外,通过实时荧光定量PCR检测结果显示,Sall4的表达具有明显的组织特异性,其在猪i PS细胞和生殖相关组织,如睾丸和卵巢组织中表达最高,而在脑、心、肝和肌肉等组织中的表达较低,表明该基因与细胞多能性维持具有互作关系。应用JASPAR和GPMiner软件分析发现,Sall4启动子上有TATA box、GC box、CAAT box等顺式作用元件,以及Oct4、Sox2、Klf4、Myc、Esrra/b、Stat3和Smad等多能转录因子的预测结合位点。其中,Oct4、Sox2和TGF-beta信号通路因子对Sall4启动子活性有较显著的激活作用,与对照组比较Sall4启动子活性提高了3倍;而Klf4因子对Sall4启动子活性有一定的抑制作用。采用分段PCR的方法,对2.1 kb启动子进行了片段缺失,分别构建了p L2.1、p L1.0和p L0.5等3个报告载体,检测结果发现,在p L2.1与p L1.0之间相差1 kb左右,但是启动子的?

英文摘要：

【Objective】Sall4（spalt-like transcription factor 4）, as a zinc finger transcription factor, plays an important role in establishing and maintaining the pluripotency of embryonic stem cells. To investigate the expression pattern and regulatorymechanism of Sall4, we cloned porcine Sall4 promoter and functionally analyzed the core regulatory domains of this promoter. 【Method】A 2.1 kb fragment of porcine Sall4 promoter was cloned from pig genomic DNA by PCR and was used to construct reporter vectors. The pE 2.1 vector was transfected into different kinds of cells to determine the expression of fluorescent protein for cellular specificity. The Sall4 expression in different tissues was detected by real-time RT-PCR. The core regulatory elements of Sall4 promoter, which share homology to known transcription factor binding sites, were analyzed by GPminer, Methprimer and JASPER program. Sall4 promoter and pluripotent factors, including Oct4, Sox2, Klf4, c-Myc, Esrra/b and Smad were co-transfected into 293 T cells to determine the activation of Sall4 promoter. A series of deletion fragments with different sizes of Sall4 promoter were produced by PCR and inserted into p GL3-Basic vector. After transient transfection into 293 T, dual luciferase assay was used to measure promoter activities. 【Result】 Sall4 has specific expression in pluripotent cell type P19, reproductive cell type CHO and cancerization cell types 293 T and Hela. The real-time fluorescent quantitative RT-PCR results showed that the expression of porcine Sall4 was significantly higher in ovary and testis tissues and porcine i PS cells, but there was low expression in brain, heart, liver and muscle tissues, suggesting that Sall4 is a key factor maintaining pluripotency of stem cells. Bioinformatics analysis of Sall4 promoter by JASPER and GPminer programs indicated that cis-acting elements like TATA box, GC box and CAAT box, and the potential binding sites for Oct4, Sox2, Klf4, Myc, Esrra/b, Stat3 and Smad were found within the promoter sequence.