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MB Sample ID: SA343187
| Local Sample ID: | Young Leaf_Malus domestica_YLR3 |
| Subject ID: | SU003292 |
| Subject Type: | Plant |
| Subject Species: | Malus domestica/Pyrus communis/Apple-pear intergeneric hybrid |
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Collection:
| Collection ID: | CO003285 |
| Collection Summary: | For metabolite profiling, ripe fruit and young and old leaves of apple, pear and hybrid were collected from each individual maintained in the germplasm collection of Fondazione Edmund Mach. 100 mg of fresh tissue (FW) was extracted in 4 mL 80% v·v-1 methanol, sonicated for 20 min at 60 Hz in a water bath at 25ºC, agitated for further 20 min and kept in dark for 48 h, filtered through a 0.22 µm PTFE filter and stored at 4 ºC. For enzyme assays, E. coli strains harbouring pGEX-4T-1 with putative AS were grown in Terrific Broth (12 g·L-1 tryptone, 24 g·L-1 yeast extract, 9.4 g·L-1 K2HPO4, 2.2 g·L-1 KH2PO4, 4 mL·L-1 glycerol) at 37 °C and recombinant proteins were induced by supplementation of 0.5 mM IPTG at optical density OD600 of 0.5 – 0.6 and incubation at 20 ºC with agitation at 200 rpm for 16 h. Protein extraction was carried out by resuspending cells with B-PER™ Complete reagent supplemented with cOmplete™ protease inhibitor cocktail (Roche) followed by protein purification by Pierce™ GST spin purification kit, according to manufacter’s instructions. Quantitation of proteins was carried out by Pierce™ BCA protein assay kit and Bradford reagent (Sigma) after crude extraction and Glutathione S-Transferase (GST) - fusion protein purification, respectively. Enzyme activity was assayed in 200 µL reactions using 1 mM hydroquinone, 2 mM UDP-glucose, 5 µg purified protein in 200 mM Tris HCl, pH 7.5 buffer, incubated at 50 °C for 1 h and terminated by adding 300 µL methanol, as previously described35. |
| Sample Type: | Plant tissue/Enzyme assay |
| Storage Conditions: | -80℃ |
