par DE基因对重组质粒pLM2在Enterobacter gergoviae 57-7中稳定性的影响

EFFECT OF parDE GENE ON THE STABILITY OF RECOMBINANT PLASMID pLM2 IN ENTEROBACTER GERGOVIAE 57-7

将 parDE基因克隆到载有组成型表达nifA基因的质粒 pST10 2 1上 ,得到重组质粒pLM2 .将 pLM2 ,pST10 2 1分别转到玉米联合固氮菌Enteorbactergergoviae 5 7 7(E5 7 7) ,获得转化子E5 7 7(pLM2 ) ,E5 7 7(pST10 2 1) ,二者在c(NH+ 4 ) =5 0mmolL-1的Kp培养基中表达的固氮活性一致 .将pLM2 ,pST10 2 1分别转到E .coliDH5α(pMGFP2 .1) ,培养物用 4 88nm光激发后在 5 10nm均有相同的的荧光特征发射峰 .上述实验表明 ,重组质粒pLM2仍保留了原质粒pST10 2 1中nifA的功能 .但质粒 pLM2和 pST10 2 1在E5 7 7中的稳定性试验表明 ,E5 7 7(pST10 2 1)在无抗生素的LB中继代培养 70代后 ,质粒几乎全部丢失 (仅存 1% ) ;而E5 7 7(pLM2 )继代培养 10 0代后 ,质粒全部存在 (10 0 % ) .图 4表 1参

The par DE gene was cloned to the plasmid pST1021, which contained and expressed the nif A gene constitutively, and a recombinant plasmid pLM2 was obtained. The pLM2 and pST1021 were transformed to maize associative nitrogen fixation bacteria Enteobacter gergoviae 57 7(E57 7), and E57 7(pLM2)and E57 7(pST1021) were obtained, respectively. Both of them expressed the Nase activity in Kp medium with c (NH + 4)=50 mmol L -1 . The pLM2 and pST1021 were transformed to E.coli DH5α(pMGFP2.1), respectively, and both cultures had same emission fluorescent peak at 510 nm when they were excited by 488 nm. The above experiments showed that the recombinant plasmid pLM2 kept the nif A function. Furthermore, pLM2 maintained in E57 7(pLM2) 100% after 100 generation in antibiotic free LB medium, but pST1021 only maintained in E57 7(pST1021) 1% after 70 generation. Fig 4, Tab 5, Ref 12