利用转座子将parDE与pCPP430体内重组提高生防工程菌的遗传稳定性

带有梨火疫欧氏杆菌 (Erwiniaamylovora)完整的hrp基因簇的重组粘粒pCPP43 0转化到植物的附生菌成团泛菌 (Pantoeaagglomerans) 3 0 8R中后可以使成团泛菌获得在烟草等植物上引发过敏反应和诱导植物抗病性的能力。pCPP43 0携带着约 40kb的梨火疫欧氏杆菌的染色体DNA ,在成团泛菌 3 0 8R中不能稳定遗传。本文首先把广宿主范围质粒RK2的控制质粒稳定性的parDE片段插入到转座载体pUT mini Tn5Km的唯一克隆位点NotI上 ,然后利用Tn5的转座特性 ,将parDE体内重组到pCPP43 0上 ,经过在无抗生素选择压下反复传代和过敏反应活性测定 ,筛选到了遗传稳定性显著提高的重组生防工程菌。

Use Transposon and parDe Through in Vivo Cloning to Promote the Genetic Stability of Plasmid pCPP430

Plasmid pCPP430 carrying the hrp gene cluster after transformated into 308R (Pantoea agglomerans) can cause the hypersensitive response, and simultaneously induce the plant resistance to disease. It was genetically unstable in P. agglomerans. 0.8 kb parDE region of the broad-host range plasmid RK2 is responsible for plasmid partition. It can mediate plasmid maitenance of many kinds in Rhizobium meliloti, and also can promote the genetic stability of recombinant plasmid in the biocontrol bacteria P. agglomerans. In this paper, we cloned parDE into pCPP430 in vivo through transposition to promote its genetic stability. parDE was amplified by PCR, inserted into pGEM-T vector and cut out and religated to NotI-cut transposon vector pUT/mini-Tn5 Km to get a parDE containing mini-Tn5, pTnp. After conjugation between S17-1/lambda pir (pTnp) and 308R (pCPP430), parDE was cloned in vivo into plasmid pCPP430 to obtain pRTnp. It was demonstrated that the insertion of parDE in pCPP430 increased significantly the plasmid's stability in P. agglomerans.