番茄脂氧合酶基因的表达和酶活性分析.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 20 11 2 胡廷章 肖国生 陈再刚 石汝杰 吴应梅 (, 404100) : 将番茄脂氧合酶基因 Tom loxD 克隆至酵母表达 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和W ester 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 : 脂氧合酶 番茄 脂氧合酶活性 毕赤酵母 Tom loxD Expression and Activity Analysis of L ipoxygenase Gene in Tomato Hu T i gzha g X iao Guoshe g Che Zaiga g ShiRujie W u Y i gm ei (Departm ent of Biology, Chongqing Three Gorges University, Chongqing 404100) Abstrac:t The Tom loxD ge e w as clo ed i to pPIC9K vector a d w as tra sform ed i to P ichia pastoris expressio host strai GS115. A tra sform a t strai w as selected. The m olecular w eighs of the express io fusio protei w ere about 103. 56 kD. SDS a d W ester blot a alysis of the co ce trated super ata t co firm ed the exoge ous Tom loxD ge e was expressed efficie tly i tra s- form ed yeast a d a ovel protei ba d about 100 kD w as the fusio protei . The a alysis of lipoxyge ase activity show ed the Tom loxD tra sge ic yeast had h igh levels of lipoxyge ase activities, wh ich demo strate the Tom loxD protei has lipoxyge ase activity. The sem -i qua titative RT-PCR a alysis suggested that theTom loxD expressio w as respo se tom echa ical i jury, a d the cha ge of lipoxyge ase activities exh ib ited the same as that of Tom loxD ge e tra script after m echa ical i jury. The fi d i gs show edTom loxD m ay play a role as a com po e t of the octadeca oid defe se-sig ali g pathway. K ey w ords: Lipoxyge ase Tom ato Lipoxyge ase activity P ichiapastoris Tom loxD