人野生型p53肿瘤抑制基因在烟草中的遗传转化

Transformation of human wild-type p53 tumor suppressor gene in tobacco

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pCAM-BIA2301/p53,p53基因由2×CaMV35S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.

AIM： To construct the plant transformation vector containing human wild-type p53 tumor suppressor gene and establish human wild-type p53 transgenic tobacco 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 tobacco （Nicotiana tobacum L. Cuttivar Xanthi ） plants were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens EHA105 harboring the plant expression vector. The generated transgenic tobacco plants were selected by kanamycin, and identified by histochemical assay, PCR, Southern blot. RESULTS ： Histochemical assay, PCR and Southern blot analyses demonstrated the stable integration of the human wild-type p53 gene into the tobacco genome. Transgenic tobacco plant with p53 gene was obtained successfully. CONCLUSION： Transgenic wild-type p53 tobacco plants have been established, which can serve as a base for further studies on detecting the biological activities of p53 gene and exploiting the new way of producing pharmaceutical proteins.