Castiglia, Daniela (2011) Glucosio-6P deidrogenasi citosolica di orzo: overespressione, caratterizzazione e regolazione da stress biotico e da sugar sensing. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Blumeria gramins,G6PDH, sugar sensing|
|Date Deposited:||06 Dec 2011 09:49|
|Last Modified:||17 Jun 2014 06:04|
A Glucose-6 phosphate dehydrogenase (G6PDH) encoding sequence was obtained from barley (Hordeum vulgare cv. Nure) roots and leaves. This sequence was 1767 bp long, and encoded for a 509 aminoacids long protein. The derived amino acid sequence was compared with known G6PDH sequences from various sources. The phylogenetic analisys suggests that this sequence would produce a cytosolic G6PDH isoform (cyt- G6PDH). This coding sequence was cloned in the expression vector pET-3d and recombinant cyt-G6PDH protein overexpressed in E.coli BL21 (DE3). The recombinant protein was purified and characterized. The enzyme specifically reacts vs potato cyt-G6PDH antibodies and exhibits kinetic parameters in agreement with those measured on cyt-G6PDH previously purified from barley roots. In vivo, the cyt-G6PDH expression level increased in the leaves upon infection with Blumeria graminis, more intensively in resistant plants with respect to susceptible plants, suggesting a main role of this isoform in the defence mechanisms developed in plants in both in specific and aspecific response to pathogens. Moreover, the regulation of cyt-G6PDH appears to be triggered by sugar sensing mechanisms: enzyme activity increased in hydroponic-grown plants upon 50mM fructose supply, sharper in roots than in leaves.
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