Figure 3.

Kanamycin‐resistant R. marina cells after long‐term incubation. (a) Kanamycin‐resistant colonies of R. marina after 30 days of incubation. Kanamycin‐resistant colonies appeared after 30 days of incubation, and they are shown in white arrows. (b) RT‐PCR analysis of the npt II gene. Transformations were carried out in the presence or absence of CPP and heat shock treatment. RNA was extracted from short‐term and long‐term incubated kanamycin‐resistant colonies. RT‐PCR was carried out using the npt II gene‐specific primer and the 16S rRNA gene (internal standard)‐specific primer sets. (c) Southern blotting analysis of the npt II gene. Transformations were carried out in the presence or absence of CPP and heat shock treatment. Genomic DNA was extracted from long‐term incubated kanamycin‐resistant colonies of R. marina. Southern blotting analysis was carried out using npt II‐ and the 16S rRNA gene (internal standard)‐specific probes. The PCR product of the npt II gene was used as a positive control. (d) Alignment of the 16S rRNA sequences between WT and long‐term incubated kanamycin‐resistant colonies of R. marina. 16S rRNA sequences of WT and eight long‐term incubated kanamycin‐resistant colonies of R. marina were determined. Sequences show a 3’ part of the 16S rRNA. The red triangle shows position 1,325 of the 16S rRNA