用酵母双杂交筛选与小麦黄花叶病毒CP互作的寄主因子

为筛选与小麦黄花叶病毒外壳蛋白(wheat yellow mosaic virus coat protein,WYMV-CP)互作的小麦蛋白,构建了小麦茎叶酵母双杂交cDNA文库,并从中筛选获得了与WYMV-CP互作的小麦蛋白基因片段。从小麦茎叶中提取小麦总RNA,分离mRNA后,利用GATE-WAY技术将反转录得到的小麦cDNA片段定向转移到目的载体,同源重组反应后成功构建小麦cDNA文库。经检测,小麦cDNA文库的滴度为5.86×106 CFU·mL-1,库容量为2.34×107 CFU,重组率为95.8%。根据NCBI上WYMV-CP开放阅读框信息,构建了诱饵载体pGBK-CP,通过酵母双杂交筛选获得了若干阳性克隆,共转化试验初步验证了WYMV-CP与PEPCK、PAL等蛋白的互作关系。本研究利用酵母双杂交技术成功获得与WYMV-CP互作的小麦蛋白基因片段,为明确WYMV的致病机制提供了科学的依据。

Screening of Host Factors Interacting with Wheat Yellow Mosaic Virus CP by Yeast Two-hybrid System

In order to screen the wheat proteins which interact with the coat protein of wheat yellow mosaic virus, wheat cDNA library was constructed. And some wheat genes were screened from the cDNA library. After the separation of mRNA from wheat leaf and stem, the wheat cDNA fragments were directional transferred to the purpose vector using the GATE-WAY technology. Following the homologous recombination reaction, the wheat yeast two hybrid cDNA library was constructed. According to the open reading frame information of wheat yellow mosaic virus coat protein in NCBI, the bait vector pGBK-CP was constructed, and wheat protein genes interacting with wheat yellow mosaic virus coat protein were screened by yeast two-hybrid system, which were tested using co-transformation technology. After the detection, the titer of yeast two-hybrid cDNA library is 5.86×10⁶ CFU·mL^-1, and the capacity is 2.34×10⁷ CFU, and recombination rate is 95.8%, which can be used in the yeast two-hybrid system. Several positive clones were obtained by the yeast two-hybrid screening, and the interaction relationship between wheat yellow mosaic virus coat protein and PEPCK, PAL proteins were confirmed through co-transformation test. This study provides more theory for explore WYMV pathogenic mechanism on the molecular level.