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

作　　者：张云华[1] 杨秀芬[1] 刘延锋[1] 玉山江[1] 邱德文[1] 

机构地区：[1]中国农业科学院植物保护研究所农业部作物有害生物综合治理综合性重点实验室,北京100081

出　　处：《生物工程学报》2011年第11期1631-1636,共6页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2006AA10A210)资助~~

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BglⅡ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。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.

关 键 词：激发子 pebC1基因 毕赤酵母 生物活性 

分 类 号：Q78[生物学—分子生物学]