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        Screening Carica papaya native promoters driving stilbene synthase expression in Arabidopsis thaliana for resveratrol glucoside (piceid) synthesis

        Luzminda Carlos-Hilario,Richard Shimshock,Cherie Ng,Jon-Paul Bingham,David A. Christopher 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.5

        The use of promoters for downstream metabolic engineering in plants requires efficiently screening their transcriptional strength and the resulting levels of the desired metabolite. Carica papaya is a complex slowgrowing tropical tree, which is difficult to rapidly screen for promoter activity. Therefore, we used the simple model system, Arabidopsis thaliana (Columbia), to compare the effectiveness of two native papaya promoters (Cp9 and Cp29) with a positive control (CaMV35S promoter), in driving transcription of the stilbene synthase gene (Vst1) and production of the phytoalexin trans-resveratrol glucoside (piceid). Single transgene copy numbers were verified via Southern analysis of at least three independent transgenic lines for each promoter construct. The Cp29 and control CaMV35S promoters consistently produced high Vst1 mRNA levels, whereas Vst1 mRNA was 50–60 % less in the Cp9:Vst1 lines. The evolutionarily related chalcone synthase (CHS) gene produced 20 and 40 % less mRNAs relative to WT in the Cp29:Vst1 and 35S:Vst1 lines, respectively, but was unchanged in the Cp9:Vst1 lines. The transgenic lines accumulated piceid as identified through RP-HPLC and tandem mass spectrometry. Piceid levels were the highest in the 35S:Vst1 followed by Cp29:Vst1 and Cp9:Vst1 in the studied tissues. No overt deleterious phenotypes were observed in the Cp9:Vst1 and Cp29:Vst1 lines; however, 35S:Vst1 produced smaller plants. The levels of secondary metabolites (anthocyanins) and seed pigments (tannins) decreased in the CaMV35S:Vst1 and Cp29:Vst1 lines, likely due to competition between CHS and stilbene synthase for precursors p-coumaroyl- and malonyl-CoA. The Cp29:Vst1 expression in Arabidopsis produced adequate levels of piceid for future disease-resistance studies.

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