摘要： 旨在对猪 SIM1 基因的转录调控机制及表达模式进行初步探讨。采用PCR法扩增猪 SIM1 基因5'端上游的启动子区,应用生物信息学方法对这一区域内潜在的转录因子结合位点进行预测,采用5'端侧翼序列缺失获得8段长度不等的启动子片段,并分别克隆至荧光素酶报告基因表达质粒(pGL3-Enhancer)中,利用双荧光素酶报告活性检测分析 SIM1 基因启动子区不同长度片段在293T、MGC803和Bel7402细胞中瞬时转染后的活性。同时,采用Western Blot实验检测SIM1蛋白在猪7个不同组织的表达模式。结果表明,各 SIM1 启动子片段在293T、MGC803和Bel7402细胞中均有活性,在293T细胞活性最低;-699～-489 bp存在关键顺式调控元件,这一区域发现Smad、CEBPα、PAX6等关键转录因子结合位点;Western Blot实验首次在中枢神经系统外发现 SIM1 的表达,SIM1蛋白在脑和下丘脑表达量最高,在睾丸的表达量次之,在肝脏、皮脂和甲状腺表达量较高,在肌肉表达量最低。研究结果提示, SIM1 基因在神经细胞发育、脂肪代谢和性腺发育过程都可能起到重要作用。

关键词: 猪, SIM1 基因, 启动子活性, 表达模式

Abstract: To investigate the transcriptional regulation mechanism and expression pattern of porcine Single-minded 1 (SIM1) gene.5' promoter fragment was amplified with PCR method and the potential transcription factor binding sites were predicted with bioinformatics method;Eight promoter segments with different length were obtained by promoter deletion analysis and cloned into pGL3-Enhancer vector, then the promoter activities were determined in transiently transfected 293T, MGC803 and Bel7402 cells by the dual-luciferase assay system, respectively.Meanwhile, SIM1 protein expression pattern was detected in seven tissues using Western Blot (WB) method.The results showed that all these SIM1 promoters presented activities in 293T, MGC803 and Bel7402 cells, and the lowest in 293T cells.The region of -699 — -489 bp contained the key cis-regulatory elements and further bioinformatics analysis found some key transcription factor binding sits, such as Smad, CEBPα and PAX6.WB experiments revealed that, besides its known expression in central nervous system, SIM1 protein could be detected in all examined tissues.Grayscale analysis showed a strong SIM1 protein expression in brain, hypothalamus and testes, a weak to moderate expression in muscle, thyroid gland, subcutaneous fat and liver.These results indicated that SIM1 gene may play important role in nerve cell development, fatness metabolism and gonads development.

Key words: Pig, SIM1 gene, Promoter activity, Expression pattern

中图分类号: 

• Q78