For visualization of EGFP expression, spores of pHxk1-EGFP transformed strains were inoculated in MM and grown for 10 h at 28 °C. These cultures were used directly for microscopic observation. Fluorescence and light microscopy were performed using a Nikon Eclipse 80i fluorescence microscope and images were captured under control of nis-elements ar software. Previous work showed that H. jecorina hexokinase-negative strains were not able to grow on MM containing d-fructose as the sole carbon source (Hartl & Seiboth 2005).
To test the utility of the two polyols d-mannitol and d-sorbitol as osmotic stabilizer and as a selective carbon source we tested the growth of H. jecorina TU-6H and the parent strain on MM agar plates with different carbon sources. Growth tests showed that TU-6H strains were find more not able to grow on MM containing 10 g L−1d-mannitol and d-sorbitol, whereas the parent strain was able to use both polyols as sole carbon source. This indicates that, in H. jecorina, the two polyols d-mannitol and d-sorbitol are catabolized via d-fructose as intermediate (Elorza & Arst, 1971; Solomon et al., 2007). To demonstrate the utility of this transformation system, H. jecorina TU-6H was transformed
with the reporter plasmid pHxk1-EGFP. For determination of the optimal selective carbon source and osmotic stabilizer, we compared the effect of 1 M d-sorbitol and 1 M d-mannitol in the regeneration medium on transformation efficiency. Results from three independent parallel transformation experiments showed that d-mannitol leads to higher transformation efficiencies than d-sorbitol. Using d-mannitol, approximately 500–1000 colonies μg−1
Selleck PI3K Inhibitor Library plasmid DNA were obtained, whereas using d-sorbitol, only 100–200 colonies were found. Adenosine triphosphate In control transformation assays with the EGFP expression, plasmid pIG1783 alone or without plasmid DNA, no colonies were found on the selective plates. As shown in Fig. 1a, transformed colonies of variable sizes were seen on selective plates. After purification of the transformants, their growth on different carbon sources was compared with growth on the parental strains (Fig. 1b). The growth of the transformants was fully restored on MM with d-mannitol or d-glucose as carbon source. The presence of the reporter plasmid pHxk1-EGFP in d-mannitol-utilizing transformants was confirmed by the presence of both hxk1 and egfp in the isolated gDNA of different transformants as detected by PCR (Fig. 2a). Three randomly picked transformants were further analyzed by Southern blot to determine the pattern of integration. Figure 2b shows that pHxk1-EGFP integrated into the genome of all analyzed transformants at the hxk1 locus; in all cases, the plasmid integrated next to the promoter region of hxk1. Some of the transformants showed additional hybridizing bands, most likely due to tandem integration or insertion of the plasmid by ectopic integration as well as targeted integration.