Tn7转座子在大肠杆菌、迟钝爱德华氏菌及鳗弧菌中的应用

Tn7转座子是一种定点插入型转座子,固定地插在细菌的glmS基因下游的attTn7位点,而glmS基因与细胞壁的合成有关,存在于所有的细菌中,所以Tn7转座子可以插入到所有细菌的染色体,因此Tn7转座系统已被开发为细菌基因组改造的重要工具。为了方便对大肠杆菌、迟钝爱德华氏菌和鳗弧菌的改造和研究,探讨应用Tn7转座子对其基因组进行改造的操作步骤,并利用阿拉伯糖启动子pBAD诱导的Flp/FRT系统建立了高效、快捷的抗性消除方法。

Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses

lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

运用生色基因标记黄瓜根围促生菌(PGPR)筛选菌株

采用三亲交配方法 ,通过Tn7转座系统将lacZY标记基因导入黄瓜根围促生菌 (PG PR)筛选菌株PseudomonasfluorescensCN1 1 6和PseudomonascorrugataCN31的利福平抗性突变株中 ;标记假单胞菌菌株则被赋予了利用乳糖作为唯一碳源的能力 ,在只有乳糖的M9培养基上生长能分解X Gal,菌落显出特有的蓝色 ;经Southern杂交分析 ,证明标记基因lacZY存在于转化菌株的染色体上 ;经验证标记菌株标记性状稳定 ,与对应的野生菌株比较其它性状如培养性状、形态特征、生防效果等基本不变 ;PGPR菌株利福平抗性和生色基因标记的结合 (双标记 )能最大限度地将土壤中引入的PGPR菌株与土著细菌分开 ,检测下限可达 1 0CFU mL ,为PGPR在根围的分子生态学研究提供了一个较好的工具。