基于酵母双杂交系统的小麦cDNA文库构建及小麦矮缩病毒复制蛋白互作寄主因子的筛选
文献类型: 中文期刊
关键词: 小麦矮缩病毒;; 复制蛋白;; 酵母双杂;; cDNA文库;; 互作蛋白
期刊名称:植物保护
ISSN: 0529-1542
年卷期: 2025 年
页码:
收录情况: 北大核心(2023版) ; ; CSCD(2025-2026年度) ; ; 科技核心(2024版) ; ; 农林核心(2024版)
摘要: 小麦矮缩病毒(wheat dwarf virus, WDV)引起的小麦矮缩病是一种危害严重的小麦病害。为研究小麦与WDV的互作机制,本研究首先以小麦为供试材料,提取小麦幼苗叶片和根组织的总RNA,采用SMART (switching mechanism at 5' end of RNA transcript)技术构建了小麦全长cDNA文库。该文库滴度大于 4x10⁹ cfu/mL、初级文库库容量超过3.6x106 cfu,插入片段长度在500~2000 bp之间,平均长度大于1000 bp,重组率约100%,成功获得了高质量的小麦cDNA 文库。为筛选与WDV病毒复制蛋白(replication protein, Rep)互作的寄主因子,构建了酵母双杂交诱饵载体pGBKT7-Rep,验证其无毒性和自激活现象后,利用构建的文库初步筛选出32个与WDV-Rep蛋白发生相互作用的候选靶标蛋白。在此基础上,采用酵母双杂交系统对筛选获得的10个候选蛋白进行一对一互作验证,最终确定其中8个蛋白与WDV-Rep存在相互作用。生物信息学分析表明,这些阳性互作蛋白包括锌指转录因子TFIIIA、去SUMO化酶DeSI、E3 泛素连接酶 BRE1等,其功能涉及基因转录调控、蛋白质泛素化修饰、植物抗逆防御应答、光合作用能量转换和RNA加工等多个关键生物学过程。该研究为深入解析WDV致病机理和寄主抗病机制提供了材料和理论基础。
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