## 3 Results  
### Effect of tri5 Gene Overexpression on the Expression of Trichodermin Biosynthetic Genes  
The expression of seven genes (_tri4_, _tri3_, _tri11_, _tri6_, _tri10_, _tri12_ and _tri14_) of the trichodermin biosynthetic pathway was analyzed from RNAs extracted from mycelia of the wild-type strain IBT40841 and three selected transformants overexpressing the _tri5_ gene: Tb38 tri5, Tb40 tri5 and Tb41 tri5, incubated 3 days in MM [24] supplemented with 20 g/L glucose as described above.  
The highest levels of expression were observed in the Tb41 tri5 transformant, a strain that produces 2.8-folds more trichodermin than the wild type strain [13]. Expression of _tri14_ was slightly lower in this transformant that in the wild-type strain (Figure 1).  
The expression values of the _tri3_, _tri4_, _tri11_ and _tri12_ genes in the Tb38 tri5 transformant were similar to or lower than those of the wild-type strain. No large differences were observed in the expression levels of the _tri3_ and _tri12_ genes between the wild-type and the Tb40 tri5 strains. However, the _tri11_ and _tri14_ genes showed a lower level of expression, and the _tri4_ gene showed a considerably higher level of expression in Tb40 tri5 than in the wild-type strain. In the case of the regulatory genes, _tri6_ and _tri10_, the level of expression was higher in all the transformants than in the wild-type strain, the greatest differences being seen in the values of _tri10_ gene expression between the wild-type and the Tb40 tri5 and Tb41 tri5 transformants. It was previously observed under identical growth conditions that the transformants Tb38 tri5, Tb40 tri5 and Tb41 tri5 showed higher _tri5_ transcript levels than the  
Figure 1: RT-PCR quantification of expression of seven genes from the trichothecene biosynthetic pathway (_tri_ genes) in the wild-type strain IBT40841 and three _tri5_-overexpressing transformants Tb38 tri5, tb40 tri5 and Tb41 tri5. The basal condition used for relative measurements was MM without glucose (\(2^{-\Delta\Delta\Gamma}=1\)). IBT40841 \(\beta\)-_tubulin_ was used as an internal reference gene.  
wild-type strain [13], and although the transformation cassette had been inserted twice into the genomes of the three _tri5_-overexpressing transformantsants, they were inserted in different genome locations in the different transformantsants.  
Effect of Tyrosol on the Expression of the tri4, tri6 and tri10 Genes in the tri5 Overexpression Transformants  
The different strains were grown for 1 or 3 days in MM [24], with 20 g/L glucose, supplemented with 0.25 or 5 mM of the antioxidant tyrosol. The pattern of expression of the _tri4_, _tri6_ and _tri10_ genes was similar for the two tyrosol concentrations (Figure 2).  
In general, all the genes were repressed by the addition of tyrosol in comparison with the use of MM lacking tyrosol. All the genes showed a higher level of expression after 1 day of growth. In addition, the expression of the genes was higher when the lower concentration of tyrosol was used. The greatest differences in the expression levels were observed between the wild-type strain and the transformantsants Tb40 tri5 and Tb41 tri5 after 1 day of growth in the presence of 0.25 mM tyrosol.  
Figure 2: RT-PCR quantification of (**A**) _tri4_, (**B**) _tri6_ and (**C**) _tri10_ gene expression in the wild-type strain and three _tri5_ overexpressing transformantsants. The experiment was carried out with mycelia grown for 1 or 3 days in MM [24], with 0.25 or 5 mM tyrosol. The basal condition and the internal reference genes used were those described in Figure 1.