Enhancement of Vitamin A and E Level In Soybean Via Expressing Multiple Genes In a Single Open Reading Frame.

Enhancement of vitamin A and vitamin E levels in Soybean (Glycine max Merr.) via expressing multiple genes in a single open reading frame

Pham, Thu Dung and Zhang, Zhanyuan

Laboratory of Plant and Growth Facility, Division of Plant Science. University of Missouri, Columbia.

 Using unique “cleavage” activity of Foot – and – mouth – disease virus (FMDV) 2A protein at its own carboxyl – terminus, we process artificial polyproteins produced in transgenic Soybean to increase level both of vitamin A and vitamin E. To enhance level of vitamin E, we express both the Arabidopsis VTE3 and VTE4 genes encoding 2 - methyl – 6 – phytylbenzoquinol methyltransferase and γ - tocopherol methyltransferase), respectively (these enzyme catalyze in committed steps in totopherol synthesis to enhance α- tocopherol level of the total tocopherol content in Soybean seed). Besides, we use only crtB encoding bacterial phytoene synthase gene to enhance β – carotenoid (pro – vitamin A). In our system, VTE3, VTE4 and crtB were fused into a single open reading frame (ORF) with 2 copies FMDV 2A protein placed between them under control of a single set of promoter (Soybean glycinin gene promoter) and terminator sequence (Soybean vegetative storage protein), so transgenic express could be more stabile. We also design various constructs (VTE3-2A-crtB-2A; VTE3-2A-VTE4-2A-crtB, crtB-2A-VTE3-2A-VTE4...) in transgenic Soybean to identify the function of FMDA 2A protein in Soybean cells. Various expressions of these genes in each construct show the “cleavage” efficiency of FMDV 2A suggest that the context flanking of 2A might affect its activities. Our data demonstrate that using 2A protease as a linker, VTE3, VTE4 and crtB could be express in a single open reading frame to increase level of tocopherol and β-carotenoid in Soybean.

Keywords: VTE3 (2 - methyl – 6 – phytylbenzoquinol methyltransferase), VTE4 (γ - tocopherol methyltransferase), crtB (phytoene synthase), FMDV 2A (Foot – and – mouth – disease virus), single open reading frame.

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