体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kan^r)基因对成团肠杆菌固氮质粒pEA9进行活体遗传标记。将来自质粒pEA9的3.0kb片段(nif ENX)克隆到pBR322载体中，再将卡那霉素抗性(Kan^r)基因插入到3.0kb的片段中，构建成供体质粒pST5。将该质粒转化到含有待标记质粒pEA9的E.a.339菌株中，然后在AP培养基中消除供体质粒，筛选得到40个失去了pST5并保持卡那霉素抗性的克隆，分析表明它们不是质粒pEA9和pST5的共整合体，而是卡那霉素抗性基因通过两个质粒在nifENX区域内的DNA间的同源重组整合到了质粒pEA9上。

in vivo Labelling of nif-plasmid pEA9 from Enterobacter agglomerans 339

The authors describe the in vivo labelling of the plasmid pEA9 in Enterobacter agglomerans 339 with a ka-namycin resistance gene. For labelling purposes the donor plasmid pST5 was constructed. This plasmid contains the nif ENX region from pEA9,in which a kanamycin resistance gene is cloned. pST5 was transformed into E. a. 339 and subsequently cured from the host. Curing was achieved with AP medium. Fourty strains that had lost pST5,but retained the kanamycin resistance,could be isolated. It showed that none of these clones contained co-ntegrates of pST5 and pEA9. This is evident that in all clones the kanamycin resistance gene was integrated into pEA9 by homologous recombination.