Figure 1
Analysis of PiCypA overexpressing transgenic tobacco plants (T1 generation) under osmotic stress.
(a) Structure of T-DNA region of pCAMBIA1301 containing PiCypA gene (495 bp) in XhoI and EcoRI restriction enzyme sites of MCS region with CaMV35S promoter and poly A terminator (pCAMBIA1301-PiCypA). (b) Visualization of β-glucuronidase (GUS) activity in the leaf tissue of WT and vector control (VC) controls and the transgenic lines (L12.1, L12.2 and L12.3) by using X-gluc. The hygromycin resistant putative tobacco transformants were used for GUS assay. (c) PCR analysis of the transgenic lines by the use of PiCypA gene specific forward and reverse primers. Lane 1 (M) is DNA marker, lane 2 is WT, lane 3 is VC and rests of lanes (4–6) are transgenic lines showing the required amplification (495 bp). (d) The PiCypA transgenic lines and control plants (WT and VC) were analyzed by the Southern blot hybridization to show the integration of the PiCypA gene along with CaMV35S promoter and poly A terminator (1.1 kb) in the transgenic lines. The genomic DNAs were digested with HindIII and probed with radiolabelled ORF of PiCypA gene. The gel is cropped for clarity. (e) Relative mRNA expression in all the three transgenic lines checked by Real Time PCR. (f) Western blot analysis of the WT and VC control plants and T1 transgenic lines (12.1, 12.2 and 12.3) using PiCypA polyclonal antibodies. The gel is cropped for clarity. Full gel figures for (d) and (f) are shown in supplementary figure S4.
