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目的构建能在大肠埃希菌(E.coli)和铜绿假单胞菌(PA)间高效接合转移的质粒。方法通过NCBI blastn程序分析质粒pCVD442接合反应起始序列oriT,PCR扩增oriT后插入pMD18-T,再将pMD18-oriT转化E.coli DH5α,经酶切、测序验证后用SmaⅠ和HindIIII双酶切亚克隆至质粒pUCP24,获得质粒pUCP24T,将pUCP24T转化E.coli后研究pUCP24T从E.coli到PA的接合效率和质粒稳定性等。结果成功构建pUCP24T重组质粒,在E.coli和PA的接合实验中,E.coli(pUCP24T)-PA共培养2 h的接合效率平均为3.588×10-2,按12 h一代连续9代继代培养108 h,抗生素压力下质粒保存率为98.29%,无抗生素的培养基中质粒保存率为21.76%。结论成功构建高接合效率的接合转移质粒pUCP24T。
Objective To construct a plasmid capable of efficiently binding and transferring between Escherichia coli and Pseudomonas aeruginosa (PA). Methods The ORF of ORF of pCVD442 was amplified by NCBI blastn and inserted into pMD18-T after PCR amplification of oriT. The recombinant plasmid pMD18-oriT was transformed into E. coli DH5α. After digestion with restriction endonucleases and digestion with SmaI and HindIIII Subcloned into plasmid pUCP24 to obtain plasmid pUCP24T pUCP24T transformation of E.coli pUCP24T from E. coli to PA binding efficiency and plasmid stability and so on. Results The recombinant plasmid pUCP24T was successfully constructed. The conjugation efficiency of E.coli (pUCP24T) -PA for 2 h was 3.588 × 10-2 in the co-culture of E. coli and PA, After 108 h cultivation, the plasmid preservation rate under antibiotic stress was 98.29%, and the plasmid preservation rate in the medium without antibiotics was 21.76%. Conclusion The high efficiency of junctional transfer plasmid pUCP24T was successfully constructed.