Gene Reactivation Induced by DNA Demethylation in Wild Type and 35S-gshI-rbcS Transgenic Poplars (Populus x canescens) Novel plant sources for phytoremediation

For gene reactivation DNA demethylating agent DHAC (5,6-dihydro-5'-azacytidine hydrochloride) (10 -4 M for 7 days) was applied in aseptic leaf cultures of wild type (WT) and 35S- gshI-rbcS GM (genetically modified) transgenic (TR) poplar (Populus x canescens) clone lgl6. Gene expression levels were determined by RT-qPCR (reverse transcriptase quantitative PCR) measuring the mRNA levels of the prokaryotic gshI-rbcS- mRNA (γ-glutamylcysteine synthetase) cloned from E. coli, and two endogenous poplar genes of gsh1-mRNA and gst-mRNA (glutathione S-transferase). For internal control, the constitutively expressed housekeeping poplar genes α-tubulin and actin were used, and the 2 −ΔΔCt method was applied for data analysis. After DHAC treatment the expression levels of 35S- gshI-rbcS transgene showed a double (1.8 - times) increment. The endogenous poplar gene gsh1 increased by 19.7-fold in the WT, and by 8.7-fold in the TR clone. The endogenous gst gene showed a 4.9 - times (in WT) and a 2.9-times (in TR) increment. Sequence analysis of DNA methylating enzymes were analyzed in silico and significant distinction was found among the three main plant DNA methylases (METases) of METs (maintenance methyltranferase), CMTs (chromomethylases) and DRMs (de novo domains rearranged DNA methylases). The DHAC-treated WT poplars with increased gene expressions of gsh1 and gst might provide novel plant sources for application for detoxification and soil remediation concerning general public frightened by GM poplars.