Construction Of Random Transposon Mutant Library Of Riemerella Anatipestifer And Screening & Identification Of The Virulence-related Genes

Riemerella anatipestifer disease, also called duck infectious serositis and caused by Riemerella anatipestifer (RA), is an infectious disease that brings significant economic losses to the duck breeding industry. Currently, the whole-genome sequences of four strains of RA have been reported in GenBank. There are 2,000 encoding genes in RA genome, few of which have the known functions. It is a key method to construct a large random mutation library for studying the RA gene functions.In this study, taking the RA Yb2 as receptor strain and the escherichia coli (E.coli) BW19851 containing plasmid pEP4351 as donor strain, the transposon Tn4351 was randomly introduced into the genomes of Yb2 after introducing pEP4351 into Yb2 through the process of conjugation and transduction. Then, positive exconjugants were selected using the erythromycin resistance gene from Tn4351, based on which a random mutation library including approximately 3,100 mutants was established. The transduction efficiency was calculated to be 7.1 ×10-6.Sixty-nine mutants with attenuated virulence were preliminarily obtained based on the infection experiment of inoculated ducklings. The transposon insertion sites of 27 mutants were identified through determining the sequences of insertion sites by inverse-PCR method. Southern blot method was used to detect the frequency of introducing Tn4351 into the genomes of mutants, thus confirming that the virulence attenuation was attributed to the simple insertion mutation.The mutant Y2666 with obvious attenuation in virulence was selected from those re-checked, and primers were designed based on the transposon-insertional inactivation of genes. Then the mutant genes were cloned into the shuttle expression vector of RA and E.coli, and also introduced into Y2666 through the processes of conjugation and transduction. Further, the gene-complementary strain cY2666 was obtained by resistance selection and PCR identification. Meanwhile, the growth curves of the wild strain Yb2, Y2666 and cY2666 were mapped. To verify whether the mutant genes of Y2666 have a significant influence on the virulence of RA Yb2, the lethal dose 50 (LD50) of Yb2, Y2666 and cY2666 as well as the blood bacterial load of these three strains at 12h and 24h after the inoculation of ducklings were determined by animal experiments respectively. The results show that Yb2, Y2666 and cY2666 have similar growth curves; there exists a significant difference in the LD50 of ducklings; the LD50 of Y2666 has been increased by 209 times as compared with that of Yb2, while the LD50 of cY2666 has been decreased by approximately 3 times as against that of cY2666. In addition, the blood bacterial load of Y2666 at 12h and 24h after inoculation has been significantly reduced as compared with Yb2. Finally, the above results demonstrate that the gene insertion and deletion in Y2666 has a great impact on the virulence of RA Yb2.In conclusion, the transposon-based random mutation library of Riemerella anatipestifer was constructed successfully. Some potential virulence-related genes were identified, one of which was further verified. This study has laid a basis for identifying the virulence-relate genes of Riemerella anatipestifer.