番茄脂氧合酶基因的表达和酶活性分析

Expression and Activity Analysis of Lipoxygenase Gene in Tomato

将番茄脂氧合酶基因Tom loxD克隆至酵母表达载体pPIC9K中,转化毕赤酵母GS115菌株,构建组成型表达Tom loxD的酵母工程菌株。SDS-PAGE和W estern blot分析表明,经甲醇诱导,Tom loxD蛋白在酵母中得到表达,表达的融合蛋白分子量约为103.56 kD,与预测的分子量一致。酶活性分析表明,酵母中表达的Tom loxD蛋白具有脂氧合酶活性。半定量RT-PCR分析表明,Tom loxD基因在番茄中表达受机械损伤的诱导,损伤处理的番茄叶片中脂氧合酶的活性明显增高。说明Tom loxD基因在番茄防御信号中发挥作用。

The TomloxD gene was cloned into pPIC9K vector and was transformed into Pichia pastoris expression host strain GS115.A transformant strain was selected.The molecular weighs of the expression fusion protein were about 103.56 kD.SDS-PAGE and Western blot analysis of the concentrated supernatant confirmed the exogenous TomloxD gene was expressed efficiently in transformed yeast and a novel protein band about 100 kD was the fusion protein.The analysis of lipoxygenase activity showed the TomloxD transgenic yeast had high levels of lipoxygenase activities,which demonstrate the TomloxD protein has lipoxygenase activity.The semi-quantitative RT-PCR analysis suggested that the TomloxD expression was response to mechanical injury,and the change of lipoxygenase activities exhibited the same as that of TomloxD gene transcript after mechanical injury.The findings showed TomloxD may play a role as a component of the octadecanoid defense-signaling pathway.