Figure 1
Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP4351 digested with XbaI (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with XbaI; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with XbaI (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative CT (2−∆∆CT) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (***p < 0.001).