Schematic representation of the T-DNA and molecular analysis of <i>ZoCDPK1-</i>expressing tobacco.

<p>(A) Gateway recombination reaction between pCR^®8/GW/TOPO-ZoCDPK1 entry vector and pMDC85 destination vector and resultant T-DNA region of the vector pMDC85-ZoCDPK1-GFP. RB, right T-DNA border; LB, left T-DNA border; 35S, cauliflower mosaic virus 35S promoter; nos T, NOS terminator; Hyg^r, hygromycin phosphotransferase II gene. (B) RT-PCR analysis using <i>ZoCDPK1</i> specific primers and <i>EF1α</i> specific primers of wild type and five independent T3 transgenic lines. (C) Western blot analysis of wild type and and transgenic tobacco (CD-1 and CD-6) lines. Proteins (20 µg per lane) were fractionated by 12.5% SDS-PAGE and immunobloted with GFP-specific antibody (D) Quantitative real time PCR analysis of <i>ZoCDPK1</i> transcripts in wild type and transgenic tobacco lines (CD-1 and CD-6) under normal, salinity and drought conditions. The <i>EF1α</i> gene was used as endogenous control in all experiments. Relative gene expression was given as the fold expression change using the equation 2^-ddCt. Data are presented as mean <u>+</u> SE (n=3) and error bars represent SE.