人野生型p53肿瘤抑制基因在烟草中的遗传转化.doc

人野生型p53肿瘤抑制基因在烟草中的遗传转化
【摘要】目的: 构建人野生型p53肿瘤抑制基因的植物表达载体,并建立p53转基因烟草植株. 方法: 将人野生型p53肿瘤抑制基因编码区克隆于pUC19, pBI426和pCAMBIA2301载体,构建含人野生型p53基因的植物表达载体pCAMBIA2301/p53,p53基因由2×CaMV 35S启动子控制表达. 利用叶盘共培养法经根瘤农杆菌EHA105介导转化烟草,获得转基因烟草植株;用组织化学法,PCR、Southern杂交检测转基因烟草植株. 结果: 经组织化学法,PCR, Southern杂交检测表明人野生型p53基因已整合到转基因烟草植株的基因组中,并获得了的转p53基因烟草植株. 结论: 成功建立了含人野生型p53基因的转基因烟草植株,为进一步检测p53基因的生物活性和开辟生产药用蛋白的新途径奠定了基础.
【关键词】人野生型p53基因;转基因烟草;根瘤农杆菌;基因转化;抗菌药
【Abstract】AIM: To construct the plant transformation vector containing human wildtype p53 tumor suppressor gene and establish human wildtype p53 transgenic o plants. METHODS: The human wildtype p53 coding sequence was subcloned into vectors pUC19, pBI426 and pCAMBIA2301 to obtain plant expression vector pCAMBIA2301/p53. TDNA regions of the pCAMBIA2301/p53 binary vector contained constitutive 2
×Cauliflower mosaic virus (2×CaMV) 35S promoter, nopaline synthase terminator, and neomycin phosphotransferase Ⅱ(npt Ⅱ)gene, which allowed the selection of transformed plants against kanamycin. The o (Nicotiana tobacum Xanthi) plants were transformed by cocultivating leaf discs method via Agrobacterium tumefaciens EHA105 harboring the plant expression vector. The generated transgenic o plants were selected by kanamycin, and identified by histochemical assay, PCR, Southern blot. RESULTS: Histochemical assay, PCR and Southern blot analyses demonstrated