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DHAC-induced transgene overexpression in 35S-gshI GMO poplar (Populus × canescens)
Published November 15, 2007
78-83

Relative gene expression levels of transgene gshI (γ-glutamylcysteine synthetase cloned from E. coli) were analyzed by qRT-PCR in two transgenic poplar (Populus × canescens) clones (11ggs and 6lgl) and wild type (WT). An extremely high expression level of transgene gshI was observed in the 6lgl clone (13.5-fold) compared to 11ggs (1.0) sample...s, which level was doubled (1.8-fold) in the DHAC (5.6-dihydro-5'-azacytidine hydrochloride) treated (at 10-4 M for 7 days) 6lgl samples but not in the 11ggs clone (0.4-fold). Contrary to this result, relative copy number of transgene gshI in the 6lgl clone was found to be less 60% less (1.0) then in the 11ggs samples (1.6). Relative expression levels of proper poplar gene gsh1-poplar showed significantly higher responsiveness to DHAC treatment than transgene gshI with the highest expression level in the untransformed (WT) poplar
clone (19.7-fold) compared to transformed 6lgl (8.7-fold) and 11ggs (2.5-fold) clones. For internal controls constitutively expressed housekeeping genes a-tubulin were applied. For data analysis, the 2−ΔΔCt method was used. DHAC applied in long-term cultures (27 days) at low concentrations (10-8 – 10-6 M) showed morphogenetic activity by initiating de novo root development of leaf discs.

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