谷氨酸棒杆菌质粒pXZ10145自发缺失突变体的特性及其重组质粒的构建

Characterization of the Natural Deletion Mutant of Plasmid pXZ10145 in Corynebacterium glutamicum and Construction of Its Recombinant Plasmid

用谷氨酸棒杆菌质粒pXZ10145转化钝齿棒杆菌T6-13原生质体,得到自发缺失突变体pNAT65,该质粒为2.4kb,仍带有氯霉素抗性,经物理图谱分析表明,质粒pXZ10145Sma I到Cla I位点之间的片段已缺失,仍保留EcoR I、Xba I、Bcl I三个酶的单切点。质粒pNAT65与pBR322用EcoR I酶切连接得到重组质粒pNAR67,这一质粒在E.coli中复制并表现Ap,Tc抗性,但氯霉素抗性能力大大降低,只能抗2μg/ml。

Plasmid pNAT65 carrying the Chloramphenicol resistance marker waschosen from a number of natural deletion mutants of pXZ10145 whenCorynebacterium crenatum T6-13 protoplast was transformed with pXZ10145 DNA from Corynebacterium glutamicum 1014-6T. The size ofpNAT65 is 2.4kb determined by electrophoresis on 0.7% agarose gel.Thephysical map of plasmid pNAT65 was determined with EcoRⅠ, XbaⅠ,BclⅠ and PvuⅡ. One recombinant plasmid, pNAR67, was constructed from DNA fra-gments of pNAT65 and pBR322 digested with EcoRⅠ respectively. Thisplasmid was capable of replication in E.coli expressed ampicillin resistan-ce in E.coli and lower chloframphenicol resistance about 2μg/ml.