【摘要】 以临床分离的一株铜绿假单胞菌 (Pseudomonasaeruginosa)PA68作受体菌 ,将具有卡那霉素抗性标记的质粒pSMC2 8通过电转化导入到受体菌中 ,研究细胞生长状态、电击电压、细胞浓度、感受态细胞的贮备方式对转化效率的影响。结果表明 ,在细胞生长至OD5 40 =0 7～ 0 8时收集菌体 ,在低温 (2℃ )条件下 ,制备浓度为 10 11个细胞 mL的感受态细胞 ,在较高的电压 (2 6kV)电击下 ,能获得较高的转化效率。最高可达 1 68× 10 8个转化子 μgDNA(CFU μgDNA)。用此优化的转化条件 ,在国际上首次成功地将Mu转座复合物导入到P .aeruginosa中 ,并获得 2 4× 10 4 CFU μgDNA的高转化效率。由于Mu转座重组技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点 ,保证了表型的改变与转座子插入位点所在的基因突变的一一对应关系 ,为进一步研究P .aerugi nosa的基因组功能奠定基础更多还原

【Abstract】 A P.aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain.Optimum conditions including growth stage of the strain,electroshock voltage,concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28.It was showed that the highest transformation efficiency was up to 1.68×108 CFU/μg DNA under the optimium conditions in which the competent cells were collected at logarithmic growth phase （OD₅40=0.7～0.8） and concentrated to about 1011 cells/ml,the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV.With this optimal condition,Mu transponson complexes have been successfully transformed into P.aeruginosa strain PA68 and the obtained efficiency was 2.47×104 CFU/μg DNA.This is the first time to electroporate Mu trsnsposon complexes into Pseudomonas spp.The artificial Mu transposons could integrate into bacterial genomes at a single site randomly.Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion.That will be very helpful for the study of genomic function of Pseudomonas spp.更多还原