Two-step PCR mediated Red recombination technique for rapid deletion of Yersinia pestis sRNA and large chromosomal fragment

WANG Renxia 1 LIU Rongjiao 1 LI Ziwei 1 WANG Ruihuan 1 YANG Ruifu 2 HAN Yanping 2 DENG Zhongliang 1

(1.Department of Sanitary Inspection, School of Public Health, University of South China, Hengyang, Hunan Province, China 421001)
(2.State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China 100071)

【Abstract】[Objective] Based on the λ Red recombination system, a two-step PCR method was developed to delete small non-coding RNA (sRNA) and large chromosomal fragment in Yersinia pestis. [Methods] Two PCR procedures were done to amplify product formed of a kanamycin resistance gene flanked by long (600–1 000 bp) homology arms. The PCR fragment carrying a kanamycin resistance gene flanked by regions homologous to the target locus was electroporated into a recipient 201 strain of Yersinia pestis expressing the homologous recombination system encoded by plasmid pKD46, which promoted the replacement of the target gene with kanamycin resistance fragment. Finally, the recombinant clones were identified by PCR. [Results] The homologous extensions of 600–1 000 bp were constructed by two PCR method, which increased the efficiency of homologous recombination, the sRNA RyhB1 (108 bp) and RyhB2 (106 bp) and the large chromosomal fragments 47-2 (10.4 kb), 47-3 (21.6 kb), 47-3a (9.2 kb) and 47-3b (6.1 kb) were successfully deleted. [Conclusion] The two-step PCR mutation technique was a simple and efficient method for the precise modification of sRNA and large fragment chromosome of Yersinia pestis. This method was suitable for gene knockout of the whole genome of Yersinia pestis, which provided a powerful tool for gene expression and regulation, pathogenicity and virulence study of Yersinia pestis.

【Keywords】 Yersinia pestis; Red recombination system; sRNA; large chromosomal fragments; gene knockout;

【Funds】 Key Projects of Hunan Provincial Department of Education (15A165)

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(Translated by SONG GJ)

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This Article

ISSN:0001-6209

CN: 11-1995/Q

Vol 57, No. 07, Pages 1126-1137

July 2017

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Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • References