Harpin_(Xooc)蛋白在毕赤酵母中的分泌表达及其生物活性检测  

Secreted Expression of HarpinXooc in Pichia pastoris and Its Bioactivity Detection

作　　者：何丹[1] 刘俊[1] 高学文[1] 沈波[1] 

机构地区：[1]南京农业大学农业部病虫监测与治理重点实验室,南京210095

出　　处：《中国生物防治》2008年第1期46-52,共7页Chinese Journal of Biological Control

基　　金：国家自然科学基金(30570041);国家863计划(2006AA10A203);高等学校博士学科点专项科研基金(20060307012)

摘　　要：采用PCR方法从水稻细菌性条斑病菌RS105菌株中扩增harpinXooc编码基因hrf2,将其克隆到酵母表达载体pPICZαA的分泌信号肽基因下游,获得重组表达质粒pPICZαA-hrf2。重组表达质粒线性化后电击转化至毕赤酵母宿主菌X-33,经抗生素Zeocin筛选和PCR鉴定后,得到重组酵母菌X-33/pPICZαA-hrf2。用甲醇诱导重组酵母菌表达目标蛋白,发酵上清液经浓缩后进行SDS-PAGE电泳分析,在约18kD处有特异目标条带出现。Western blot检测表明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harpinXooc能够诱导烟草产生过敏反应和促进烟草生长,活性高于在大肠杆菌中表达的harpinXooc。The hrf2 gene was amplified from genome of Xanthomonas oryzae pv. oryzicola RS105 by PCR and inserted into the downstream of the α-factor signal sequence of the Pichia pastoris expression vector pPICZαA. The linearized recombinant plasmid was transformed into P. pastoris strain X-33 by electroporation. The recombinant strains were chosen by YPDS plates containing Zeocin and identified by PCR. SDS-PAGE analysis proved a special band about 18 kD and Western blot demonstrated good antigenicity of the expressed protein. The results of bioactivity detection showed that the recombinant harpinxooc had higher activity to induce hypersensitive response and promote tobacco growth than harpinXooc expressed in Escherichia coli.

关 键 词：毕赤酵母 HarpinXooc蛋白 生物活性检测 

分 类 号：Q785.6[生物学—分子生物学] S435.72[农业科学—农业昆虫与害虫防治]