The expected 806-bp hxk1 fragments were obtained after PCR using reverse-transcribed RNAs from five different transformants as templates, indicating transgenic hxk1 expression in these transformants (Fig. 2c). To visualize expression of the EGFP reporter, positive transformants were investigated by microscopy. Most of the investigated transformants showed the typical green fluorescence (Fig. 3). The fluorescence could be detected easily by fluorescence microscopy, suggesting the EGFP expression levels were high in transformants. About 20% of a total of 50 randomly selected colonies from the transformation plates were regarded as abortive Selleck PI3K inhibitor transformants without
further growth after transfer to fresh selective medium, which was in accordance with a previous report using H. jecorina pyr4 as a homologous marker (Gruber et al., 1990). Individual colonies from a single spore isolated from nonabortive transformants were tested for phenotypic stability. Subcultivation of the transformants RAD001 nmr on MM without selective pressure followed by a growth test for d-mannitol utilization demonstrated that the
HXK+ phenotype remained stable for at least five successive generations. Complementation of an auxotrophic mutation in a recipient strain to prototrophy is probably the most successful strategy for genetic transformation and selection of transformants. Auxotrophic markers are often preferred over dominant markers due to the high cost of the antibiotics used as selective agents and the detrimental effects of such PRKACG agents on the cell during the transformation and selection procedures. In this study, we investigated whether an hxk1-negative strain can be used as a recipient for the development of a carbon source-dependent genetic transformation system. An analysis of phenotypic differences in carbon utilization between the parental strain TU-6 and the hxk1 deletion strain TU-6H showed that the latter strain was not able to metabolize d-mannitol or d-sorbitol, which are both commonly used as effective osmotic stabilizers for fungal transformation (Ruiz-Díez 2002; Li et al., 2006). These physiological characteristics
were the prerequisites for the development of the transformation system in which these polyols could be used as both selective agent and osmotic stabilizer. Transformation of TU-6H with the selectable marker hxk1 showed that this gene is responsible for d-mannitol and d-sorbitol utilization via d-fructose. A comparison of the effect of the two polyols on transformation efficiencies showed that the efficiency was around five times higher for d-mannitol than for d-sorbitol. As d-sorbitol is usually applied as osmotic stabilizer in conventional H. jecorina transformation (Penttiläet al., 1987; Gruber et al., 1990; Mach et al., 1994), the replacement of it by d-mannitol might provide a useful alternative for transformation assays where high transformation efficiencies are required.