Zhang Yunhua, Yang Xiufen, Liu Yanfeng, Yu Shanjiang, Qiu Dewen
Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Nov;27(11):1631-6.
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.
为了在毕赤酵母中表达PebC1，通过PCR从灰葡萄孢菌BC-4-2-2-1的基因组中扩增pebC1序列，并亚克隆到毕赤酵母表达载体pPIC9K中，构建pPIC9K-pebC1。重组质粒经Bgl II线性化后，通过电穿孔法转化到毕赤酵母GS115中。通过MD和G418-YPD平板筛选重组毕赤酵母GS115/pPIC9K-pebC1，并通过PCR进一步确认。用甲醇诱导蛋白表达，并用SDS-PAGE进行分析。SDS-PAGE分析显示有一条约39 kDa的特异条带，Western blotting表明表达的蛋白具有良好的抗原性。生物测定结果表明，重组蛋白PebC1可诱导黄瓜和拟南芥对灰霉病产生抗性。
