小鼠黑素细胞中过表达Oct-1对毛色主效基因的影响

发布时间：2018-07-03 05:27

本文选题：八聚体结合转录因子 + 黑色素　；参考：《中国农业科学》2017年04期

【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(P0.05);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(P0.001),表明小鼠Oct-1转染效率显著,MITF m RNA显著降低至0.70倍(P0.01),TCF m RNA显著降低至0.66倍(P0.01),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(P0.01),TYRP-1 m RNA升高至3.11倍(P0.01),αMSH m RNA显著增加至18.49倍(P0.001),AC m RNA显著增加至6.88倍(P0.01),c-kit m RNA显著增加至18.75倍(P0.001),ET1 m RNA增加至1.50倍(P0.05),ETB-R m RNA显著增加至13.47倍(P0.001),CAM m RNA增加至1.46倍(P0.05);蛋白免疫印迹结果显示,小鼠黑素细胞中Oct-1转染组MITF蛋白显著降低至0.67倍(P0.01),TYR蛋白增加至1.16倍(P0.05),TYRP-1蛋白升高至1.15倍(P0.05),TYRP-2蛋白未见变化。【结论】通过PCR和克隆技术及核酸测序技术获得了小鼠Oct-1基因全长2 313 bp的CDS区,经生物信息学分析找出Oct-1作用的毛色主效基因,过表达Oct-1后使黑素细胞中MITF和TCF的表达降低,TYR、TYRP-1、αMSH、AC、c-kit、ET1、ETB-R和CAM的表达增加。证实Oct-1可调节毛色主效基因的表达,参与黑色素合成的调节,改变毛色。
[Abstract]:[objective] to clone mouse octamer binding transcription factor 1 (octamer-binding transcription factor 1) gene sequence. To investigate the effect of octamer binding transcription factor 1 (Octamer binding transcription factor 1) on the expression of major coat color genes in mouse melanocytes and the role of octamer binding transcription factor 1 in the formation of coat color. [methods] the fifth generation of mouse melanocytes, which were frozen in laboratory, was used. The cDNA sequence of Oct-1 gene was cloned by using mouse melanocyte c DNA as template by ordinary PCR method, and the cloned vector and eukaryotic expression vector of mouse Oct-1 were constructed, and the obtained sequences were analyzed by bioinformatics using KEGG PATHWAYA NCBITransfec software. After transfection, the transfection efficiency of mouse Oct-1 was observed by fluorescence microscope, and the melanin content in mouse melanocytes was measured by spectrophotometer. Real-time polymerase chain reaction was used to detect the expression of coat color major genes in melanocytes after transfection. Western blot assay was used to detect the changes of the protein levels of MITFTYROTYRP-1 and TYRP-2 in transfected melanocytes. [results] the results were obtained by sequencing and splicing. The cDNA sequence of mouse Oct-1 gene was obtained with the length of 2 313 BP. The eukaryotic expression vector was successfully constructed with mouse melanocyte specific TYRP-2 gene promoter and a priming reporter gene green fluorescent protein. By KEGG PATHWAY analysis, 34 candidate genes related to the formation of coat color were identified by NCBI, and the major color genes of Oct-1 were identified by Transfec promoter analysis software. Under fluorescence microscope, melanocytes with green fluorescence showed significant transfection efficiency; spectrophotometer showed that melanin content in mouse melanocytes decreased after transfection (P0.05); fluorescence quantitative analysis showed that, The expression of Oct-1 mRNA in mouse melanocytes was significantly increased (P0.001), indicating that the transfection efficiency of mouse Oct-1 was significantly reduced to 0.70 times (P0.01) and the expression of TCF mRNA significantly decreased to 0.66 times (P0.01). TYRP-1 mRNA increased to 3.11 fold (P0.01), 伪 MSH mRNA significantly increased to 18.49 fold (P0.001) to 6.88 times (P0.01) c-kit mRNA significantly increased to 18.75 times (P0.001) and 1.50 times (P0.05) to 1.50 fold (P0.05). In mouse melanocytes, the MITF protein of Oct-1 transfection group decreased significantly to 0.67 fold (P0.01) and increased to 1.16 fold (P0.05), the protein of TYRP-1 increased to 1.15 times (P0.05). [conclusion] Oct-1 was obtained by PCR, cloning and nucleic acid sequencing. The full length of the gene was 2 313 BP CDS region. By bioinformatics analysis, Oct-1 was found to be the dominant gene of coat color. After over-expression of Oct-1, the expression of MITF and TCF in melanocytes decreased, and the expression of ETB-R and CAM increased in 伪 MSHG / ACC-kit1ET1 and TYRP-1, respectively. Oct-1 can regulate the expression of major genes, regulate melanin synthesis and change the color of coat.
【作者单位】：山西农业大学动物科技学院;山西医科大学基础医学院;
【基金】：国家高科技研究发展计划(“863”计划,2013AA102506) 公益性行业(农业)科研专项(201303119) 山西农业大学创新团队建设计划(CXTD201201)
【分类号】：Q953

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