马铃薯块茎特异性启动子驱动的人白细胞介素-12的遗传转化研究

Genetic Transformation of Human IL-12 Driven by Tuber-specific Promoter from Potato(Solanum tuberosum)

为获得高效表达人白细胞介素-12(h IL-12)蛋白的转基因马铃薯植株,利用根瘤农杆菌侵染法将前期构建的马铃薯块茎特异性启动子Ppatatin驱动的h IL-12植物表达载体导入马铃薯,通过共培养、筛选、分化等过程获得转基因植株,并结合PCR、RT-PCR、GUS染色及ELISA分析对转基因植株进行鉴定。结果显示,获得12个马铃薯转基因株系,PCR检测表明其中的10个株系目的基因已导入马铃薯基因组,转基因阳性率为83%;RT-PCR分析表明其中的7个株系外源基因在转录水平成功获得表达,ELISA分析表明其中的5个株系有明显的蛋白水平表达。对这7个株系后代的检测表明外源基因成功表达且具有良好的遗传稳定性。

To obtain transgenic potato plants that express human interleukin 12（h IL-12）protein,the early-constructed plant expression vector driven by Ppatatin of potato tuber-specific promotor was transformed into potato by Agrobacterium-mediated transfection,followed by co-cultivation,screening and differentiation,and the transgenic plants were acquired. Techniques of PCR,RT-PCR,GUS staining,and ELISA analysis were performed for the identification of transgenic plants. The results showed that 12 transgenic lines were obtained,and in the 10 transgenic lines the target gene was in the potato genome,i.e.,positive rate was 83%. RT-PCR analysis illustrated that exogenous genes expressed successfully in 7 transgenic lines at m RNA level,moreover,ELISA data showed that 5 transgenic lines presented the high level of h IL-12 protein expression. In addition,the expression of h IL-12 was genetically stable in these 7 transgenic potato lines.