| 韦 吉1#, 栾宏瑛1#, 王荟洁1, 刘 晶1, 王洪洋1*, 陈爱娥2*.致病疫霉诱导的马铃薯酵母双杂交文库构建及无毒蛋白PiAVR3b寄主靶标筛选[J].植物保护,2022,48(4):114-122. |
| 致病疫霉诱导的马铃薯酵母双杂交文库构建及无毒蛋白PiAVR3b寄主靶标筛选 |
| Construction of yeast two-hybrid cDNA library induced by Phytophthora infestans and screening host targets for avirulence protein PiAVR3b in potato |
| 投稿时间：2021-06-14 修订日期：2021-07-09 |
| DOI：10.16688/j.zwbh.2021340 |
| 中文关键词: 马铃薯 致病疫霉 酵母双杂交 PiAVR3b蛋白 靶标蛋白 |
| 英文关键词:potato Phytophthora infestans yeast two-hybrid PiAVR3b protein target protein |
| 基金项目:国家自然科学基金（31800134，32060499）；云南省基础研究计划（2019FB023） |
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| 中文摘要: |
| 致病疫霉是引起番茄、马铃薯晚疫病的植物病原卵菌, 其分泌的效应蛋白在抑制寄主免疫方面发挥重要功能。鉴定效应蛋白的寄主靶标对研究植物与病原菌互作具有重要意义。本研究构建了高效马铃薯酵母双杂交cDNA文库, 并通过筛选致病疫霉无毒蛋白PiAVR3b的互作蛋白来评价构建文库质量。分别提取接种致病疫霉后24 h和48 h的马铃薯栽培种‘合作88’叶片RNA, 等量混合后构建cDNA文库。次级文库容量为1.6×107 cfu/mL, 重组率为100%, 插入片段平均在1 000 bp以上。以PiAVR3b为诱饵, 通过酵母双杂交筛选初步获得10个候选靶标蛋白。经点对点验证, 进一步确定了4个与PiAVR3b互作的靶标蛋白, 分别是马铃薯MYB-like蛋白、线粒体外膜孔蛋白、碳分解代谢抑制蛋白、肽基脯氨酰异构酶。本研究构建的酵母双杂交文库为致病疫霉效应蛋白靶标筛选及植物与病原菌互作研究奠定了基础。 |
| 英文摘要: |
| Phytophthora infestans is a phytopathogenic oomycete that causes the late blight disease on potato and tomato. Generally, P.infestans secretes an arsenal of effector molecules that suppress host innate immunity. Identification of the host target proteins of effectors is greatly significant for the study on plant-pathogen interaction. Here, a yeast two-hybrid cDNA library induced by P.infestans was constructed. The library quality was evaluated by screening the target proteins of avirulence protein PiAVR3b. RNA was extracted from leaves of potato cultivar ‘Hezuo 88’ infected by P.infestans 24 h and 48 h post inoculation, respectively. Then RNAs mixed in equal amount was used for cDNA library construction. The titer of the secondary yeast two-hybrid cDNA library was 1.6×107 cfu/mL with a recombination rate of 100%. The average length of the inserted cDNA was more than 1 000 bp. Ten candidate interaction proteins were obtained by yeast two-hybrid using PiAVR3b as a bait, of which four target proteins were identified through yeast two-hybrid peer-to-peer verification, including MYB-like DNA-binding domain protein, mitochondrial outer membrane protein porin, carbon catabolite repressor protein, and peptidylprolyl isomerase. In this study, a high-quality yeast two-hybrid library for identifying the host target proteins of P.infestans effectors was constructed. Our results lay a foundation for further study on the interaction mechanisms of P.infestans and potato. |
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