The expression of three genes related to cell division was significantly higher, two for a 123 kD protein of cell division (Cdc123) and one encoding a suppressor of kinetochore, and one PIM1 gene was significantly less expressed in the Adriamycin molecular weight primordial stage. Cdc123 proteins are regulators of eIF2 in Saccharomyces cerevisiae and are regulated by nutrient availability . This simultaneous increase indicates the predominance of
phase G1 of cell division. As the formation of spores occurs in already differentiated primordia, it is likely that the collected phase contains a larger number of non-differentiated primordia. There was also a significant increase of six genes of unknown function, one of them showing no similarity Selleck PI3K Inhibitor Library with any sequence in the available public data banks. Expression analyses of genes involved in basidiomata development by RT-qPCR The gene expression profile obtained by the macroarray in two distinct phases suggested physiological changes in mycelia prior to basidiomata production. However, more detailed analyses should be performed to monitor the expression of key genes (previously described in the literature as involved in basidiomata development). Quantitative PCR is an accurate technique to analyze gene expression. It is 10,000 to 100,000 times more sensitive than RNase protection
assays and 1,000 times more sensitive than dot blot hybridization . Therefore, a more detailed RT-qPCR analysis was performed with 12 ESTs in selleck kinase inhibitor order to observe a possible relationship between transcript levels of all samples collected (Figure 6). RNA Adenosine was obtained from mycelium samples of all seven developmental stages: white, yellow and reddish pink phases, before and after stress, and during basidiomata formation.
Figure 6 RT-qPCR of genes expressed in different phases during the culture of M. perniciosa in basidiomata-inducing medium. A – MpPRIA1; B – MpPRIA2; C – MpPLYB; D – MpRHEB; E – MpGLU; F – MpADE; G – MpCPR; H – MpRHO1-GEF, I – MpMBF; J – MpRAB; K – MpCYP; L – MpRPL18. In Y axis values of RQ using primers constructed for each gene and in axis X corresponding samples of RNA originated from mycelia in the following stages: 1 = cDNA of mycelium white stage, 2 = cDNA of yellow mycelium stage, 3 = cDNA of reddish pink mycelium stage, 4 = cDNA of reddish pink mycelium before stress, 5 = cDNA of reddish pink mycelium after stress, 6 = cDNA of mycelium with primordia and 7 = cDNA of basidiomata. RQ = relative quantification measured by ddCt. (*) – significant at 5% probability, (**) – significant at 1% probability by the statistical t test. The hypothesis that nutrient depletion might act as a signal for fructification was confirmed since some genes related to primary metabolism and to nutrient uptake were down-regulated when primordia emerged.