| 许艳1#, 李冉2#, 宋健2, 王丹2, 周雷2, 李俊娇2, 张丹丹2, 陈捷胤2, 戴小枫2*, 杨星勇1*.大丽轮枝菌及激素处理后棉花酵母双杂交文库的构建[J].植物保护,2021,47(2):46-55. |
| 大丽轮枝菌及激素处理后棉花酵母双杂交文库的构建 |
| Construction of a sea-island cotton yeast two-hybrid library under the conditions of infection with Verticillium dahliae and treatment with hormones |
| 投稿时间：2019-12-15 修订日期：2020-02-20 |
| DOI：10.16688/j.zwbh.2019691 |
| 中文关键词: 大丽轮枝菌 植物激素 酵母双杂交 cDNA文库 互作蛋白 |
| 英文关键词:Verticillium dahliae plant hormones yeast two-hybrid system cDNA library interaction proteins |
| 基金项目:国家重点研发计划（SQ2018YFE011242，2017YFD0201904）；国家自然科学基金（31772245，31970142） |
| 作者 | 单位 | E-mail | | 许艳1#, 李冉2#, 宋健2, 王丹2, 周雷2, 李俊娇2, 张丹丹2, 陈捷胤2, 戴小枫2*, 杨星勇1* | 1. 重庆师范大学生命科学学院, 重庆401331
2. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京100193 | 戴小枫daixiaofeng_caas@126.com；杨星勇yangxy94@swu.edu.cn |
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| 中文摘要: |
| 大丽轮枝菌是引起棉花、马铃薯等重要作物黄萎病的土传病原真菌, 其分泌的胞外蛋白是侵染寄主的重要毒力因子, 因此研究胞外蛋白与寄主的相互作用具有重要意义。本研究旨在构建高效的棉花酵母双杂交cDNA文库, 并通过已知大丽轮枝菌效应子VCR1互作蛋白的筛选来评价构建文库的质量。分别采用大丽轮枝菌及茉莉酸甲酯、水杨酸和乙烯处理海岛棉‘海7124’以诱导其抗病基因表达, 混合RNA样品构建酵母双杂交cDNA文库, 文库滴度达1.18×108 cfu/mL, 检测重组率100%, 插入片段在300~2 000 bp之间。以效应子VCR1为诱饵筛选获得27个潜在互作蛋白, 并进一步确证了1个与VCR1互作的靶标蛋白。本研究构建的酵母双杂交文库将为筛选与大丽轮枝菌效应子互作的蛋白及后续互作机理研究提供重要的研究基础。 |
| 英文摘要: |
| Verticillium dahliae is a soil-borne fungal plant pathogen that causes the destructive vascular wilt on many hosts, including economically important cotton and potato crops. Generally, the extracellular proteins secreted by V.dahliae play a crucial role in its virulence on the host. The objective of this study was to construct a yeast two-hybrid library from cotton infected with V.dahliae and treated with hormones, and evaluate the library quality by screening the interaction proteins of a known V.dahliae effector VCR1. To construct the library, the RNA was extracted from the cultivar Gossypium barbadense ‘Hai 7124’ inoculated with V.dahliae or treated with hormones. The high quality of library was assessed by the high cDNA library titer (1.18×108 cfu/mL), the distribution length of insertion size is 3002 000 bp, and the absolute recombination efficiency. Subsequent screening of this library for the known V.dahliae effector VCR1 yielded 27 candidate interaction proteins, and one of them was proven to interact with VCR1 when validated using the yeast two-hybrid system. In conclusion, a high-quality sea-island cotton yeast two-hybrid library with good efficiency for identifying the interaction proteins of V.dahliae effectors was constructed in this study, which would be helpful for revealing molecular mechanisms underlying the interactions between V.dahliae and cotton. |
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