摘要： SKP2A是调控细胞周期转录因子降解的F-box蛋白。为研究该蛋白在小麦-叶锈菌互作过程的作用,首先克隆获得中国春小麦Ta-SKP2A全长序列,然后将其与pGBKT7载体连接,转入酵母Y187感受态细胞中,构建酵母诱饵表达载体,并检测其表达产物对酵母细胞有无毒性和自激活效应。结果表明,Ta-SKP2A编码区序列长度为1146 bp,其ORF区编码一条由382个氨基酸组成的多肽。在ORF区两侧连接酶切位点(EcoR Ⅰ和BamH Ⅰ),并将其与载体pGBKT7连接,成功构建了包含目的基因的诱饵载体pGBKT7-Ta-SKP2A,且含重组诱饵质粒的酵母菌株在SD/-Trp营养缺陷平板上生长良好,其过夜培养物OD₆₀₀ > 0.8,表明重组诱饵质粒表达产物对酵母细胞无毒性。含重组诱饵质粒的酵母菌株在二缺、三缺及四缺营养缺陷平板上不能生长,表明诱饵质粒的表达产物不能自激活报告基因。成功构建了一个包含Ta-SKP2A的重组诱饵质粒,为深入筛选与其互作的靶蛋白,研究其在小麦-叶锈菌互作体系中的作用机制奠定了基础。

关键词: 小麦, F-box基因SKP2A, 诱饵载体构建, 毒性检测, 自激活效应

Abstract: SKP2A is an F-box protein that regulates the proteolysis of cell cycle transcription factors.In order to understand the role of SKP2A in the interaction between wheat and leaf rust pathogen, a full-length sequence of Ta-SKP2A was firstly obtained from wheat cv.China Spring, and ligated with pGBKT7 vector.Recombinant vector was transformed into yeast Y187 competent cell, further detected its self-activated activity and toxicity effect for yeast cells.Results showed that Ta-SKP2A was 1 146 bp and encoded a polypeptide of 382 amino acids.The ORF with restriction enzyme sites of BamH I and EcoR I was inserted into expression vector pGBKT7.The results showed that the bait vector pGBKT7-Ta-SKP2A was successfully constructed by sequencing.The recombinant bait plasmid grew well on SD/-Trp plate, which showed that the bait plasmid was not toxic to yeast cell.The yeast strains containing bait plasmid couldn't grow on SD/-Leu/-Trp, SD/-Leu/-Trp/-His and SD/-Trp/-Leu/-His/-Ade plate, so the bait plasmid didn't have transcriptional activation.A recombinant bait plasmid with Ta-SKP2A was successfully constructed, which would lay foundation for screening for targeted protein interacted with Ta-SKP2A, and would help to understand its function in the interaction of wheat and leaf rust pathogen.

Key words: Wheat, F-box gene Ta-SKP2A, Construction of bait vector, Toxicity detection, Transcriptional activation

中图分类号: 

• Q784