Abstract: This study aims to establish a practical method for an unmarked gene knockout of Pseudomonas plecoglossicida NyZ12 that degrades cyclohexylamine，which is significant for further studying the molecular mechanism of it degrading cyclohexylamine. The upstream and downstream flanking DNA fragments of the target gene were fused by overlapping PCR，and then cloned into the suicide vector pEx18km. The recombinant plasmid was introduced into Escherichia coli strain S17 pir and then transferred into NyZ12 by conjugation. The mutants by inverse screening sacB gene in pEx18km was identified by PCR and sequencing. The mutant NyZ12Δ4637 from NyZ12 strain orf4637 were successfully constructed. In conclusion，the unmarked gene-knockout mutant was acquired using homologous recombination of suicide vector，and there was no resistance residual of screening marker on the genome of the mutant. This study provides a reliable gene-knockout technology for investigating the genetic functions of NyZ12.

Key words: gene knockout, suicide vector, homologous recombination, conjugative transfer