Transgenic analysis of promoter constructs.

<p>(<b>a</b>) <b>Comparative stable expression analysis of parent and hybrid promoter-GUS constructs in transgenic tobacco plants.</b> Promoter activities of parent and hybrid promoters were monitored in 21-days-old tobacco (<i>Nicotiana tabacum</i> cv. Samsun NN) seedlings (R1 progeny, 2nd generation, Kan^R) grown aseptically on an MS-agar medium in presence of kanamycin (300 µg/ml) and 3% sucrose. Soluble protein extracts (5 µg) from whole seedlings were used for the GUS assay. The data presented in the histogram as an average of three independent experiments for each construct with respective standard deviation (SD). The statistical analysis revealed a <i>P</i> value of 0.001 implying highly significant. In the histogram, GUS constructs: (1) Untransformed control tissue extract from the wild type <i>Nicotiana tabacum</i> cv Samsun NN (2) pKYLXGUS, with CaMV35S promoter, (3) pKFScpGUS, with FScp promoter; (4) pKFSGUS, with FS promoter; (5) pKFGUS, with F promoter; (6) pKFcpGUS, with Fcp promoter; (7) pKFSuasGUS, with FSuas promoter; (8) pKFuasGUS, with Fuas promoter; (9) pKFuasFScpGUS, with FuasFScp promoter; (10) pKFSuasFcpGUS, with FSuasFcp promoter; were shown. (<b>b</b>) <b>Comparative expression analysis of transgenic plants expressing GUS constructs of parent and hybrid promoters by qRT-PCR assay.</b> For each construct, 21-days-old seedlings (R1 progeny, 2nd generation, Kan^R) from independent transgenic lines were selected. Estimation of relative <i>GUS</i> transcript accumulation in transgenic plants developed using GUS constructs driven by CaMV35S, FScp, FS, F, Fcp, FSuas, Fuas, FuasFScp and FSuasFcp promoters was performed by qRT-RCR as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The data presented in the histogram were average fold difference of <i>GUS</i> transcript ± SD of two independent experiments carried out using cDNA derived from two RNA samples extracted from two different plants expressing individual promoter constructs. In the histogram, each bar represents number of fold increase in transcript level of <i>GUS</i> gene in plants compared to CaMV35S (taken as 1.0). Histograms (2) pKYLXGUS; (3) pKFScpGUS; (4) pKFSGUS; (5) pKFGUS; (6) pKFcpGUS; (7) pKFSuasGUS; 8: pKFuasGUS; (9) pKFuasFScpGUS; (10) pKFSuasFcpGUS were shown.