Journal: Frontiers in Plant Science
Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses
Figure Lengend Snippet: Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).
Article Snippet: The transformed calli were selected on Chu-N6 medium supplemented with 50 mg/L Hygromycin (Invitrogen, USA) and 1 mg/L PPT (Duchefa, Germany).
Techniques: Transgenic Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Quantitative RT-PCR, Over Expression, Recombinant, Staining, Activity Assay, Produced