双报道基因系统的构建以及在N蛋白生物功能研究中的应用

The Construction of lacZ and galK Double Reporter System and Its Use in the Study of Lambda Phage N Gene Regulation

为研究 λ噬菌体调控因子 Ｎ 蛋白的生物功能，应用 λ噬菌体感染、 Ｐ１ 噬菌体转导、体内、体外重组方法，通过对遗传标记的筛选，将 ｌａｃ Ｚ 和 ｇａｌ Ｋ 报道基因、λ噬菌体 ｐ Ｌ 操纵子负责调控转录、翻译的 Ｄ Ｎ Ａ 序列以及转录终止子装配到大肠杆菌染色体上，构建成菌株 Ｚ Ｈ１０４１、 Ｚ Ｈ１０４２ 和 Ｚ Ｈ１１４２．应用 Ｚ Ｈ１１４２ 菌株模拟 λ噬菌体的自然状态对其调控蛋白 Ｎ 的生物功能进行了研究．研究证明， Ｎ 蛋白不仅在转录水平上正向调控基因表达，还具有一种新的在翻译水平上负向调节自身基因表达的生物功能．

The construction and use of lacZ and galK double reporter system that facilitates study of N mediated gene regulation is described.Bacterial genetics methods,including Lambda phage and P1 phage techniques were used to construct the strains.The lacZ gene was fused in frame to the first 33 condon of N gene and used as the reporter for N gene expression.Transcription terminators were placed between lacZ and galK ,making galK dependent upon the lambda pL promoter and N antitermination.In presence of N,β galactosidase(β Gal)level was reduced dramatically relative to no N,indicating that N represses its own expression.Repression of N gene expression was at the translational level and occurred at the same time in the same cell and on the same transcript as antitermination was occurring to allow galK expression.