灰葡萄孢菌激发子pebC1在毕赤酵母中的分泌表达及其生物活性分析

Expression of a protein elicitor pebC1 from Botrytis cinerea in Pichia pastoris

摘要:

为了实现激发子PebC1编码基因在毕赤酵母中的分泌表达，采用PCR方法从灰葡萄孢菌BC-4-2-2-1菌株中扩增获得激发子PebC1的编码序列，将其亚克隆至酵母分泌型表达载体pPIC9K中，以此片段构建了pPIC9K-pebC1重组表达质粒。重组表达质粒经Bgl Ⅱ线性化处理，电击转化至毕赤酵母宿主菌GS115，经MD、G418-YPD平板和PCR法筛选，获得了重组毕赤酵母菌GS115/pPIC9K-pebC1。用甲醇诱导重组酵母菌表达目标蛋白，发酵液经SDS-PAGE电泳分析，在约39 kDa处出现特异目标条带。Western blotting检测结果说明，重组表达产物具有良好的抗原性。生物活性检测表明，酵母重组表达蛋白PebC1能够诱导拟南芥和黄瓜幼苗对灰霉病的抗性。

Abstract:

In order to express PebC1 in Pichia pastoris, the pebC1 sequence was amplified from genome Botrytis cinerea BC-4-2-2-1 by PCR and subcloned into the Pichua pastoris expression vector pPIC9K to generate pPIC9K-pebC1. The recombinant plasmid was linearized by Bgl II and transformed into Pichia pastoris GS115 by electroporation. Recombinant Pichia pastoris GS115/pPIC9K-pebC1 was screened by MD and G418-YPD plates and further confirmed by PCR. The protein expression was induced by methanol and analyzed by SDS-PAGE. SDS-PAGE analysis showed a special band about 39 kDa and western blotting indicated a good antigenicity of the expressed protein. Bioassay results showed that the recombinant protein PebC1 can induce resistance to gray mould disease of cucumber and Arabidopsi thaliana.