Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.)
Figure 2
Expression analyses of transgenic plants containing the chimeric nat-T-phylloplanin-GFP gene. (a) Gene integration and expression analysis was determined by reverse transcriptase-polymerase chain reaction (RT-PCR); amplification products from independent transgenic lines (R1 progeny, 2nd generation), representative samples, after electrophoresis, are displayed on ethidium bromide stained 1% agarose gel. RT-PCR products for T-phylloplanin-GFP with the expected 1304 bp band for independent line numbers T1.1 to T1.4 and T2.1 to T2.2; untransformed tobacco control KY14 (W), positive controls by taking pBS-nat-T-phylloplanin-GFP (P, a clone of the chimeric nat-T-phylloplanin-GFP gene in Bluescript) used as template are shown. (b) Expression analysis (mRNA levels) of independent transgenic tobacco lines (R1 progeny, 2nd generation) containing the native T-phylloplanin-GFP construct by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in stably transformed tobacco. Independent plant lines with : = 3 : 1 were selected for analysis. The relative expression levels of nat-T-phyllo-GFP-specific mRNA in independent transgenic tobacco lines were analyzed using the comparative threshold cycle (Ct) method and were presented as fold-changes compared with the reference transgenic line #R. Independent tobacco lines (T1.1 to T1.4 and T2.1 to T2.2) are indicated on the histogram. Wild-type plants were used as the reference (R) line. Data are expressed as mean ± S.D. of 5 observations. The amplified RT-PCR products of the nat-T-phylloplanin-GFP gene in each independent line showed similar melting curves with a Tm of 79.9°C, indicating product homogeneity, whereas the internal control tubulin gene had a Tm of 81.7°C (data not shown). (c) Western blot analysis of transgenic lines expressing the chimeric nat-T-phylloplanin-GFP gene probed with GFP-specific polyclonal antibodies showed the expected bands as marked (145 kD). The nat-T-phylloplanin-GFP band was detected with polyclonal GFP-antibodies in the independent transgenic lines (T1.1 to T1.4 and T2.1 to T2.2); no band was detected in wild-type tobacco (N. tabacum cv. KY14) plants (W).
