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Expression of soybean-embryo lipoxygenase 2 in transgenic tobacco tissue
Authors:Wenlian Deng  W Scott Grayburn  Thomas R Hamilton-Kemp  Glenn B Collins  David F Hildebrand
Institution:(1) Department of Agronomy, University of Kentucky, 40546-0091 Lexington, KY, USA;(2) Departments of Horticulture and Landscape Architecture, University of Kentucky, 40546-0091 Lexington, KY, USA
Abstract:To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. lsquoKY 14rsquo by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5prime-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3prime to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1–0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50–529% increase in C6-aldehyde production. The impact on C6-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C6-aldehydes than that of other well-characterized LOX isozymes.Abbreviations AMV alfalfa mosaic virus - CaMV cauliflower mosaic virus - IEF isoelectric focusing - kDa kilodalton - LOX lipoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank Bernard Axelrod (Purdue University) for supplying the lipoxygenase 2 cDNA, and Arthur G. Hunt (University of Kentucky) for supplying the pKYLX712 and pBS/AMV. The advice of Arthur G. Hunt, Chris L. Schardl, Sadik Tuzun and Dwight Tomes is greatly appreciated, as is the technical assistance of Udaya Chand and Robert Versluys.
Keywords:Gene expression (lipoxygenase)  Fatty acid peroxidation  Lipoxygenase  Lipid metabolism  Nicotiana (transgenic)
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