Cardi, Manuela (2010) Cloning, expression and characterization of P2-glucose-6P dehydrogenase from barley (Hordeum vulgare) and poplar (Populus trichocarpa. Effects of Abscissic acid (ABA) on activity, occurrence and expression of different isoforms of glucose-6P dehydrogenase in barley. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||G6PDH, OPPP, ABA, Populus trichocarpa, Hordeum vulgare|
|Date Deposited:||02 Dec 2010 10:59|
|Last Modified:||30 Apr 2014 19:45|
Glucose 6-phosphate dehydrogenase (G6PDH, EC 184.108.40.206) is the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), an important provider of NADPH. The effect of exogenous ABA on the activity, expression level and protein amount of different G6PDH isoforms has been studied in barley plants. G6PDH activity increased by 50% in ABA treated roots for 12h; this increase resembles a similar (+35%) effect observed in P2-G6PDH transcript levels .The transcript Cy-G6PDH levels did not change appreciably upon ABA supply. Similar variations were observed in protein levels in immunoblotting analyses. In leaves, ABA did not affect the cytosolic protein levels, while a sudden decrease was observed for chloroplastic P1-G6PDH protein; therefore the two-fold increase in G6PDH activity observed after ABA treatment is likely related to an increase in mRNA level (+ 50%) and protein amount (+ 85%) of leaf P2-G6PDH. Altogether, these results suggest a specific role for the plastidial P2-isoform in ABA treated barley plants, encouraging a better characterization of this plastidic isoform. Therefore, genes of the plastidic isoform of G6PDH (P2-G6PDH) from two different organisms, barley (Hordeum vulgare) and poplar (Populus trichocarpa) have been cloned in two different expression vectors, pET3d and pET15b (His-Tag), and overexpressed in E.coli. The purified enzyme was checked by mass spectrometry analysis, and for the reactivity against specific P2—G6PDH antibodies; the main kinetic parameters, and differential sensitivity to reduction by DTT were determined.The recombinant P2-G6PDHs exhibits molecular weights of 55.5 kDa and 61kDa for barley and poplar, respectively. The main kinetic parameters measured for both HvP2-G6DPH and PtP2G6PDH are in agreement (e.g. high KiNADPH) with the values known for the most of other P2-type G6PDHs. The recombinant barley protein is moderately sensitive to reductants (DTT); moreover the Populus trichocarpa enzyme presents a redox potential (-280 mV) favourable for control by either thioredoxins m or f.
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