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不带抗药性基因的nifA质粒的构建
Construction of a nifA containing Plasmid Without Antibotics resistant Genes
【摘要】 本研究克隆了萤光假单胞菌AS1.55(PseudomonasfluorescensAS1.55)的亮氨酸基因(leu+)EcoRI片段(~6.6kb),并获得含有该片段的重组质粒pBR322-LEU。从pBR322-LEU质粒中分离出leu+EcoRⅠ片段,将其插入到nifA质粒pMC71A的EcoRⅠ位点使氯霉素抗性基因(Cmr)失活,从而构建了不带抗药性基因的nifA质粒pMC71A-LEU。用pMC71A-LEU转化30株leu-水稻根际固氮菌阴沟肠杆菌E26(EnterobactercloacaeE26),其中一株E26-1能够互补,转化频率为1×103转化子/μg质粒DNA。pMC71A-LEU在E26-1菌株中不够稳定,在LB培养基中连续培养30代后,丢失率为60%~80%。E26-1(pMC71A-LEU)与亲株E26(pMC71A)相比,耐铵固氮能力没有变化。
【Abstract】 A 6.6kb Eco RⅠ fragment of the gene encoding for leucine biosynthetase from Pseudomonas fluorescens strain AS1.55 was cloned using plasmid pBR322 as the vector. The leu + Eco RⅠ fragment was isolated from the plasmid pBR322 LEU and was inserted into the Eco RⅠ site of chloramphenicol resistant gene in the nif A plasmid pMC71A. A resultant nif A plasmid pMC71A LEU was obtained and resulted in the inactivation of the resistance to chloramphenicol. Among thirty strains of the leu diazotroph Enterobacter cloacae E26 in the rice rhizosphere transformed with the plasmid pMC71A LEU, only one strain E26 1 can be complemented with a transformation frequency of 1×10 3 transformants/μg plasmid DNA. The plasmid pMC71A LEU was not stable in the strain E26 1, and the plasmid could be lost at a rate of 60%~80% after thirty generations cultivated in LB medium. As compared to its parental strain E26(pMC71A), the E26 1(pMC71A LEU) strain has no much changed in the capability of nitrogen fixation and the resistance to ammonia.
【Key words】 leu + gene; gene cloning; plasmid pMC71A LEU; Enterobacter cloacae;
- 【文献出处】 农业生物技术学报 ,JOURNAL OF AGRICULTURAL BIOTECHNOLOGY , 编辑部邮箱 ,1998年04期
- 【分类号】Q74,
- 【被引频次】1
- 【下载频次】54