CAJ | 학술논문

阴沟肠杆菌(Enterobacter cloacae)E26(pMC73A)是带有耐氨质粒的固氮工程菌。本研究通过三亲杂交的方法将带有梨火疫病菌hrp基因簇的重组粘粒pCPP430导入E26(pMC73A)。接合子在番茄叶片上出现与对照DH5(pCPP430)一样典型的过敏反应症状，而出发菌E26(pMC73A)则没有过敏反应；从接合子中抽提到2条质粒DNA带，一条与pCPP430位置相同，大小约50 kb，另一条与pMC73A位置相同，大小约7 kb；接合子质粒DNA的酶切图谱正好是pCPP430和 pMC73A酶切图谱的叠加；用地高辛标记的hrpN DNA 探针进行Southern hybridization分析，在接合子的质粒酶切产物中得到预期的杂交带，而出发菌E26(pMC73A)无任何杂交信号。通过质粒抽提、酶切验证、Southern hybridization 分析以及在番茄叶片上的过敏反应测定，均表明生产植物抗性诱导蛋白的耐氨固氮工程菌构建成功。
음구장간균(Enterobacter cloacae)E26(pMC73A)시대유내안질립적고담공정균。본연구통과삼친잡교적방법장대유리화역병균hrp기인족적중조점립pCPP430도입E26(pMC73A)。접합자재번가협편상출현여대조DH5(pCPP430)일양전형적과민반응증상，이출발균E26(pMC73A)칙몰유과민반응；종접합자중추제도2조질립DNA대，일조여pCPP430위치상동，대소약50 kb，령일조여pMC73A위치상동，대소약7 kb；접합자질립DNA적매절도보정호시pCPP430화 pMC73A매절도보적첩가；용지고신표기적hrpN DNA 탐침진행Southern hybridization분석，재접합자적질립매절산물중득도예기적잡교대，이출발균E26(pMC73A)무임하잡교신호。통과질립추제、매절험증、Southern hybridization 분석이급재번가협편상적과민반응측정，균표명생산식물항성유도단백적내안고담공정균구건성공。
The plasmid pCPP430 harboring the functional hrp gene clusterof Erwinia amylovora was transformed by tri-parental mating into Enterobacter cloacae E26(pMC73A)which can fix nitrogen under relatively high ammonia concentrations.The transconjugants induced hypersensitive response when infiltrated into leaves of tomato.Two plasmids were isolated from the transconjugants and shown to have same physical map as that of pCPP430 plus pMC73A.Southern hybridization of this plasmid DNA with Dig-labeled hrpN DNA as probe revealed the presence of hrpN in the transconjugants.Thus,bioassay and molecular evidences demonstrated the successful construction of a bi-plasmid recombinant strain,which was capable of prouducing harpin.