极细链格孢菌peaT1基因在毕赤酵母中的表达与功能 分析

Expression H113of peaT1 gene from Alternaria tenuissima in Pichia pastoris and its function

摘要:

建立了在毕赤酵母(Pichia pastoris)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Sal I或Bgl II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a-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35 kD。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16.13 mg, 该纯化蛋白能显著地促进小麦幼苗的生长。

Abstract:

In this study, peaT1 gene was subcloned into the Pichia pastoris 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 α-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.