Computational protocol: Overexpression of AtOxR gene improves abiotic stresses tolerance and vitamin C content in Arabidopsis thaliana

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Protocol publication

[…] Full-length amino acids sequences were aligned using ClustalX, and then imported into the Molecular Evolutionary Genetics Analysis (MEGA) package version 3.1 []. Phylogenetic analyses were conducted using the neighbor joining method in MEGA. The following accession numbers were used: AtOxR (GenBank accession number: NP_568854), Oryza sativa (NP_001058445), Glycine max (NP_001235783), Zea mays (ACG39093), Setaria italica (XP_004966149), Medicago truncatula (XP_003610078), Eutrema salsugineum (ESQ42667), Capsella rubella (EOA14055), Theobroma cacao (EOY31908), and Ricinus communis (XP_002532085).Yeast transformations were performed using a lithium acetate-based method []. The empty vector plasmids pYES2-AtOxR and pYES2 were used as controls, and were introduced into the yeast strain InVSCI. The transformed yeast strains were grown in synthetic defined (SD) medium lacking the appropriate amino acids for the selective growth for the expression plasmids. For the response assays, yeast transformants were cultured in liquid yeast extract peptone dextrose (YPD) medium (1 % yeast extract, 2 % peptone, and 2 % glucose) until they reached an optical density at 600 nm of ≈ 0.6, and were then diluted 10−1-, 10−2-, 10−3-,10−4-, and 10−5-fold using double distilled H2O. Then, aliquots of each dilution were spotted onto solid yeast YPD and YPG medium (1 % yeast extract, 2 % peptone, and 2 % galactose) supplemented with different concentrations of NaCl (0.7 M, 0.9 M, 1 M), Mannitol (1 M, 1.2 M, 1.5 M), H2O2 (4 mM, 4.5 mM, 4.8 mM), MnCl2 (1 mM, 1.5 mM), MgCl2 (0.8 M, 1 M), CdCl2 (160 μM, 180 μM), BaCl2 (2 mM, 4 mM), CuCl2 (7 mM, 8 mM), FeCl3 (7 mM, 10 mM), AlCl3 (5.5 mM, 6 mM, 6.5 mM, 7 mM). A yeast transformant of the pYES2 empty vector was used as a control, and growth was monitored for 3–7 days at 30 °C. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Arabidopsis thaliana
Chemicals Ascorbic Acid, Aspirin, Hydrogen Peroxide, Oxygen