羊驼MC1R在绵羊黑色素细胞内特异性表达的转基因载体的构建

发布时间：2018-03-01 07:30

  本文关键词： MC1R 羊驼 绵羊黑色素细胞 荧光定量PCR Western blot　出处：《山西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：经过多项研究和实验得出,黑色素皮质素受体-1 (Melanocortin-1 Receptor, MC1R)在控制哺乳动物毛色基因中是关键性的主要控制基因。本试验将羊驼MC1R基因和慢病毒载体连接,构建一个转基因表达载体。将载体转入绵羊黑色素细胞中,使其在绵羊黑色素细胞中特异性表达,从基因和蛋白水平鉴定转基因表达载体的活性和效果,为生产表达羊驼MC1R基因的转基因绵羊奠定基础。(1)根据NCBI上羊驼MC1R (GenBank:FJ517582.2)的基因序列,设计带有酶切位点的特异性引物,扩增到大小为1069bp的完整的羊驼MC1R CDS区,该序列与其他物种MC1R的相似度达99%,可用于下一步连接载体。(2) MC1R基因经限制性内切酶Sal I和Xba Ⅰ酶切后,与同样经Sal I和Xba Ⅰ酶切后的慢病毒载体通过T4 DNA连接酶连接,测序鉴定正确。(3)重组质粒转染绵羊黑色素细胞,36h后荧光显微镜下观察到绿色荧光,48h提取细胞RNA和总蛋白质。(4)运用荧光定量PCR分析MC1R、MITF、TYR和YRP2在实验组(转染重组质粒)、Negtive Control组(转染慢病毒空载体质粒)和空白细胞组(不进行转染)的mRNA表达量。结果显示实验组MC1R, MITF, TYR和YRP2基因的mRNA表达量分别为空白对照组的3.193倍,2.021倍,3.218倍,3.140倍,并呈差异显著。(5)运用蛋白免疫印迹(Western blot)技术检测MITF、TYR和YYRP2在实验组,转染Negtive Control组和空白细胞组的平均蛋白含量。结果为：转染表达载体的实验组的MITF、TYR、TYRP2蛋白表达量是KB组的1.852倍、3.537倍,2.045倍,且差异均显著。综上所述,绵羊黑色素细胞转入羊驼MC1R转基因表达载体后,调控黑色素细胞的活性和黑素生成的主效基因MC1R,MITF,TYR和TYRP2的基因和蛋白表达量均提高。羊驼MC1R转基因表达载体构建成功,效果良好。
[Abstract]:After many studies and experiments, it was found that Melanocortin-1 Receptor-1 (MC1R) is the key gene in the control of mammalian coat color. In this study, the MC1R gene of alpaca was linked with lentivirus vector. A transgenic expression vector was constructed. The vector was transferred into sheep melanocytes and specifically expressed in sheep melanocytes. The activity and effect of the transgenic expression vector were evaluated at the gene and protein levels. To lay the foundation for the production of transgenic sheep expressing the MC1R gene of alpaca. According to the gene sequence of MC1R GenBank: FJ517582.2) on NCBI, a specific primer with enzyme cutting site was designed, and the complete MC1R CDS region of alpaca was amplified. The sequence is similar to MC1R of other species and can be used to ligate the MC1R gene by restriction endonuclease Sal I and Xba 鈪，

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