豌豆来源的毒性抗虫白蛋白cDNA在毕赤酵母中异源表达及抗虫毒性分析

Heterologous Expression of Insecticide Pea Albumin in Pichia pastoris and Toxicity to Weevils Sitophilus oryzae

一种来源于豌豆的白蛋白 (PA)对多种昆虫具有明显的毒性作用 .为了进一步研究其抗虫机理 ,采用基因工程的方法富集蛋白质 .将该白蛋白基因克隆到毕赤酵母分泌型表达载体pPICZαA .并导入毕赤酵母菌GS115中表达 .通过PCR和Northern印迹法分析基因的整合及转录 ,对工程菌进行发酵 ,采用两步培养 :第一步富集菌体 ,第二步甲醇诱导工程菌表达重组蛋白 ,并分泌到培养基中 .经过超滤和HPLC分离 ,重组抗虫白蛋白得率约 10mg L .重组蛋白对象鼻虫敏感株表现出很强的毒力 ,半致死剂量LC50 为 4 7,与直接纯化于豌豆种子的白蛋白毒力一致 ,而对象鼻虫抗性株无明显毒力 .

A kind of pea albumin protein shows high toxicity to several kinds of insect. In order to obtain more protein to investigate the insecticidal mechanism, the pea albumin protein (PA) gene obtained from plasmid pET-DP by PCR amplification was cloned into Pichia pastoris expression vector pPICZαA. With transformation, PA protein can be expressed as secretory protein in the Pichia pastoris strain GS115. The yield of purified recombinant protein was estimated to be 10 mg/L. This expressed protein shows a high toxicity to sensitive strain of weevils (Sitophilus oryzae) Benin, whereas it has no significant effect on the resistant strain Chine. The LC 50 of PA expressed protein is 4.7, equals to that of PA extracted from pea seeds.