中文摘要：

【目的】克隆小鼠八聚体结合转录因子1（octamer-binding transcription factor 1,Oct-1）基因序列,探讨八聚体结合转录因子1在小鼠黑素细胞中过表达对毛色主效基因表达的影响及在毛色形成中的作用。【方法】使用实验室冻存的第5代小鼠黑素细胞,通过普通PCR方法用引物以小鼠黑素细胞c DNA为模板克隆Oct-1基因c DNA序列,构建小鼠Oct-1克隆载体和真核表达载体;通过KEGG PATHWAY、NCBI、Transfec等软件对获得的序列进行生物信息学分析;在细胞水平通过细胞转染技术过量表达小鼠Oct-1;转染后使用荧光显微镜观察细胞转染效率,采用分光光度计对小鼠黑素细胞中黑色素含量进行测定,并进行Real-time PCR实验检测转染后黑素细胞中毛色主效基因在m RNA水平表达量的变化,Western blot实验检测转染后细胞中MITF、TYR、TYRP-1和TYRP-2蛋白水平的变化。【结果】经测序和拼接最终获得长度为2 313 bp的小鼠Oct-1基因的c DNA序列;成功构建真核表达载体,载体上连有小鼠黑素细胞特异性TYRP-2基因启动子和一个启动报告基因绿色荧光蛋白;通过KEGG PATHWAY分析获得与毛色形成有关的34个候选基因,NCBI查找出34各个基因的启动子,由Transfec启动子分析软件找出Oct-1可以调节的毛色主效基因;细胞转染后,在荧光显微镜下可观察到黑素细胞带有绿色荧光说明转染效率明显;分光光度计检测显示,转染后小鼠黑素细胞中黑色素含量减少（P〈0.05）;荧光定量检测结果显示,小鼠黑素细胞中Oct-1 m RNA表达量显著增加（P〈0.001）,表明小鼠Oct-1转染效率显著,MITF m RNA显著降低至0.70倍（P〈0.01）,TCF m RNA显著降低至0.66倍（P〈0.01）,Ras、Frizzled、ERK2和TYRP-2 m RNA的表达未见变化,TYR m RNA显著增加至7.69倍（P〈0.01）,TYRP-1 m RNA升高至3.11倍（P〈0.01）,αMSH m RNA显著增加至18.49倍（P〈0.001）,AC m RNA显著增加至6.88倍（P〈0.01）,c-kit m RNA?

英文摘要：

【Objective】The aim of the study was to clone the octamer-binding transcription factor 1, to investigate whether over-expression of Oct-1 regulated the major genes of coat color in melanocytes of mouse at the transcriptional levels and to explore its influence on the formation of melanin. 【Method】 The CDS region in Oct-1 gene were cloned from melanocytes of mouse by primers and PCR to build a mouse Oct-1 cloning vector and eukaryotic expression vector. The KEGG PATHWAY, NCBI, and Transfec softwares were adopted to analyze the biological information of the obtained sequence. Over-expression of Oct-1 was conducted in the melanocytes of the 5th generation mouse through the cell transfection technique and transfer efficiency was observed by fluorescence microscope. The content of melanin in melanocytes was detected by spectrophotometer. The level of major genes were detected using real-time PCR and the proteins of MITF, TYR, TYRP-1 and TYRP-2 were detected using western blot.【Result】Results showed that the 2 313 bp c DNA sequence of Oct-1 gene was obtained by sequencing and splicing. Eukaryotic expression vector was successfully constructed with specific TYRP-2 gene promoter of mouse and a startup report gene of green fluorescent protein. KEGG PATHWAY analysis obtained 34 candidate genes related with coat color, and the promoters of these 34 candidate genes were found by NCBI. The major genes of coat color regulated by Oct 1 was determined by using Transfec software. Under the fluorescence microscope, green fluorescence could be identified in melanocytes of mouse. The contents of melanin in melanocytes were reduced（P〈0.05）. Real-time PCR results showed that Oct-1 m RNA was significantly increased（P〈0.001）,witch indicated Oct-1 high transfection efficiency. MITF m RNA was significantly reduced to 0.70 times（P〈0.01） and TCF m RNA was significantly reduced to 0.66 times（P〈0.01）. The expressions of Ras, Frizzled, ERK2 and TYRP-2 m RNA did not change. TYR m RNA was significantly increase