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. 2014 Oct 10;9(10):e109962. doi: 10.1371/journal.pone.0109962

Figure 1. Construction and characterization of R. anatipestifer mutant strain RA2640 and complementation strain cRA2640.

Figure 1

(A) Slide agglutination test: Mutant strain RA2640 lacked serum agglutination ability to serotype 2 positive sera. (B) Southern blot analysis. Lane M: DM15000 DNA Marker (CWBIO, Beijing, China). Lane 1: A single insertion of Tn4351-transposon was identified in mutant strain RA2640. Lane 2: No hybridization band was detected in wild-type strain Yb2. Lane 3: XbaI fragment (10.4 kb) of pEP4351 was identified with the ermF gene probe. (C) Real-time RT-PCR analysis. The expression of upstream AS87_04040 gene and downstream AS87_04055 gene were measured. The changes of mRNAs were expressed as fold expression and calculated using the comparative CT (2-△△CT) method. The expression of AS87_04050 in mutant strain RA2640 couldn't be detected. Error bars represent SD from three replicates. (D) PCR analysis. Lane M: DM2000 DNA Marker (CWBIO, Beijing, China). Lane 1: The Erm and R. anatipestifer 16S rRNA were amplified from the mutant strain RA2640 using primer pairs Erm-F/Erm-R and RA 16S rRNA-F/RA 16S rRNA-R, showing an 833-bp fragment of Erm or a 496-bp fragment of RA 16S rRNA. Lane 2: A 496-bp fragment was amplified from wild-type strain Yb2 with primers RA 16S rRNA-F/RA 16S rRNA-R. Lane 3: The AS87_04050 gene and R. anatipestifer 16S rRNA were amplified from the complementation strain cRA2640 using primer pairs AS87_04050 comp-F/AS87_04050 comp-R and RA 16S rRNA-F/RA 16S rRNA-R, showing a 972-bp fragment of AS87_04050 or a 496-bp fragment of RA 16S rRNA.. Lane 4: The AS87_04050 gene and R. anatipestifer 16S rRNA were amplified from wild-type strain Yb2 using primer pairs AS87_04050 comp-F/AS87_04050 comp-R and RA 16S rRNA-F/RA 16S rRNA-R, showing a 972-bp fragment of AS87_04050 or a 496-bp fragment of RA 16S rRNA.