白粉菌侵染后的拟南芥酵母双杂交cDNA文库构建及其利用

 拟南芥广谱抗病基因RPW8对拟南芥白粉病菌、霜霉病菌和烟草花叶病毒等均具有抗性。为了深入研究其广谱抗病机制,筛选鉴定与RPW8具有直接相互作用的蛋白,我们以含有RPW8的拟南芥纯合转基因系S5为材料,接种拟南芥白粉菌系UCSC1,36 h后取样,构建了白粉菌侵染初期的拟南芥cDNA文库。为了提高文库对长片段基因5′端的覆盖率,分别使用含有oligo(dT)和oligo(dN)的接头引物反转录cDNA第一链,PCR扩增双链cDNA。将纯化后的双链cDNA与线性化载体pGADT7-Rec混合,利用同源重组技术在酵母菌株Y187中构建cDNA文库。经检测,文库转化效率为5.0×10⁶/3 μg pGADT7-Rec,滴度为2.5×10⁸ CFU·mL^-1,插入片段长度在350～2 000 bp之间,平均插入片段大小为750 bp。用RPW8.1和RPW8.2构建诱饵载体,分别获得11和12个候选互作蛋白。结果表明此cDNA文库质量较好,适用于互作蛋白的筛选。

Construction and utilization of a yeast two-hybrid cDNA library from Arabidopsis thaliana infected by powdery mildew

RPW8, a broad-spectrum resistance locus in Arabidopsis thaliana confers enhanced resistance to Golovinomyces cichoracearum, Hyaloperonospora parasitica, and Tobacco mosaic virus. To identify its interacting candidates that may be helpful for understanding the mechanisms of RPW8-mediated broad-spectrum resistance, an Arabidopsis thaliana yeast two-hybrid cDNA library was constructed by using plant seedlings at an early stage of powdery mildew infection. Total RNA was isolated from the homozygous line S5 with a single copy of RPW8 at 36 h after inoculation of the powdery mildew strain UCSC1. mRNA was purified and the first-strand cDNA was reverse transcribed by oligo(dT) and oligo(dN) primers, respectively, in order to improve the 5’ terminus coverage of genes with large size. The cDNA library was constructed by homologous recombination between ds cDNA and linearized pGADT7-Rec vector in yeast strain Y187. The transformation efficiency of the constructed cDNA library was 5.0×10⁶/3 μg pGADT7-Rec and the titer was 2.5×10⁸ CFU·mL^-1. The length of the insertion fragments ranged from 350 bp to 2 000 bp with an average length of 750 bp. The cDNA library was then applied to screen for RPW8.1- and RPW8.2-interacting proteins, and 11 and 12 candidates were obtained, respectively. These data suggest that the cDNA library is in high quality and suitable for screening the interacting proteins of an interested protein in the Arabidopsis thaliana-powdery mildew pathosystem.