极细链格孢菌peaT1基因在毕赤酵母中的表达与功能分析  

Expression of peaT1 gene from Alternaria tenuissima in Pichia pastoris and its function

作　　者：刘延锋[1] 曾洪梅[1] 玉山江[1] 杨秀芬[1] 毛建军[1] 邱德文[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100081

出　　处：《生物工程学报》2009年第3期413-417,共5页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展规划项目(973项目)(No.2003CB114204);国家高技术研究发展计划(863计划)(No.2006AA10A210);国家自然科学基金项目(No.30571252)资助~~

摘　　要：建立了在毕赤酵母(Pichiapastoris)中分泌表达PeaT1蛋白的技术。将来源于极细链格孢菌(Alternaria tenuissima)的基因peaT1亚克隆至酵母分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K-peaT1,分别用SalI或BglII酶切线性化后电击转入毕赤酵母GS115菌株中,经MD平板筛选、PCR鉴定获得整合有外源基因的重组菌株。在α-Factor及AOX1基因启动子和终止信号的调控下,PeaT1在酵母中大量表达并分泌到胞外,SDS-PAGE检测表明表达蛋白的表观分子量约为35kD。表达蛋白上清稀释液能诱导烟草产生对TMV的抗性,其枯斑数抑制率可达到30.37%。每升表达上清液经超滤浓缩和离子交换层析可纯化目的蛋白16.13mg,该纯化蛋白能显著地促进小麦幼苗的生长。In this study, peaT1 gene was subcloned into the Pichia pasWris expression vector pPIC9K, which contained both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pPIC9K-peaT1. The recombinant plasmid was linearized by Sal Ⅰ or Bgl Ⅱ and transformed into P. pastoris GS115 by electroporation method. Recombinant strain was screened by Minimal Dextrose Medium and further confirmed by PCR. The gene was in frame integrated into the Pichia genome through homologous recombination, resulting the recombinant strain. Regulated by the a-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant protein was expressed and secreted into the supernatant. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 35 kD. Bioassay results showed that the inhibition rate of the expressed protein against TMV was 30.37%. The supernatant was collected and then purified by anion exchange chromatography. This protein can promote seedling growth of wheat obviously.

关 键 词：peaT1 极细链格孢菌 毕赤酵母 

分 类 号：Q93[生物学—微生物学] Q78