Abstract: This study aimed to establish an antibiotic-resistance-free gene knockout method for Glaesserella parasuis based on the Flp-FRT site-specific recombination system, thus providing a genetic manipulation tool for the research on virulence factors, pathogenic mechanism, genetic engineering vaccines of G. parasuis. Firstly, the cI857/P_RM/P_R expression regulation system from λ bacteriophage was used to control the expression of Flp recombinase to realize resistance gene excision from the resistance-marker mutants of G. parasuis. Then, the Flp expression plasmid was introduced into the G. parasuis CF7066 by electroporation, and the obtained recombinant strain was naturally transformed with the suicide plasmid containing the homologous DNA sequence flanked with FRT sites to generate the marker mutants. Following the regulation expression of Flp recombinase, the markerless mutants were screened by colony PCR and the Flp expression plasmid was cured by improving the culturing temperature to 42 ℃. Furthermore, this knockout system was improved by mutation of unilateral FRT site. Initially，the Flp recombinase carried by temperature-sensitive shuttle plasmid pSHG5C-Flp was verified to be able to express under the control of λ repressor and promotor of cI857/P_RM/P_R and to excise the resistance gene in the mutant of ΔnanH::Kan^R. Subsequently, the G. parasuis CF7066 was electrotransformed with pSHG5C-Flp in advance, then it was naturally transformed with the suicide plasmids of pKF-ΔnanH and pKF-Δapd2 successively. Correspondingly, the markerless single gene and double gene deletion mutants of ΔnanH and ΔnanHΔapd were generated from the primary transformants of the primary generation, and the Flp recombinase expression plasmid pSHG5C-Flp also was cured. Although we achieved successive two gene knockout, the knockout efficiency for the second gene was obviously declined, which was probably due to the accumulating Flp recombinase that probably interfered the homologous recombination mediated by the plasmid DNA from natural transformation. Thus, we screened the mutated one FRT site and introduced it into the plasmid of pKFM-Δapd2 to conduct natural transformation. As a result, the introduction of mutated FRT site into the suicide plasmid guaranteed the occurrence of homologous recombination and deletion mutation, which increased the effectiveness and stability of this method. This study provided a novel markerless and double gene knockout system for G. parasuis and paved a way for the research on molecular pathogenetic mechanism and genetic engineering vaccines.

Key words: Glaesserella parasuis, expression regulation, Flp-FRT, gene knockout, markerless deletion