Expression, molecular characterization and detection of lipoxygenase activity of tomloxD from tomato

TomloxD is a lipoxygenase from tomato (Lycopersicon esculentum Mill.), which encodes a protein of 908 amino acid residues with a calculated molecular mass of 102.31 kDa. The predicted three-dimensional structure of TomloxD showed a typical lipoxygenase fold: a beta-barrel structure in the N-terminal region and the catalytic site in the C-terminal domain. The semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggested that, the TomloxD transcript was higher in roots, flowers, younger leaves and fruits of breaker+4 days stage and lower in mature leaves, fruits from the mature green and breaker stages. TomloxD expression response to mechanical injury and the change of lipoxygenase activities exhibited is the same as that of TomloxD transcript after mechanical injury. The TomloxD gene was cloned into pPIC9K vector and was transformed into Pichia pastoris expression host strain GS115. A transformant strain was selected and expressed efficiently. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the concentrated supernatant showed that exogenous TomloxD was expressed in transformed yeast. Lipoxygenase activity of the supernatant showed that, TomloxD transgenic yeast had high levels of lipoxygenase activities, which demonstrated that the TomloxD has lipoxygenase activity. TomloxD may play a role as a component of the octadecanoid defense-signaling pathway.