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本文旨在通过酵母双杂交技术研究杨扇舟蛾颗粒体病毒(ClanGV)的经口感染因子PIF0和其他经口感染因子PIF1~PIF6之间的互作关系。首先根据ClanGV的基因组序列信息,利用在线软件TMHMM Server v.2.0对PIF0~PIF6进行跨膜区分析,确定需要扩增的片段。然后以诱饵载体pGBKT7和捕获载体pGADT为出发载体,构建了PIF0的重组诱饵载体(B0)和PIF1~PIF6的重组捕获载体(A1~A6)。自激活实验证明,重组诱饵载体B0没有自激活特性,可以进一步开展蛋白的互作研究。酵母双杂交结果证明,PIF0作为诱饵载体时,可以与PIF3和PIF5的重组捕获载体发生蛋白相互作用,与PIF1、PIF2、PIF4和PIF6都没有互作现象。本研究结果将有助于揭示PIF0在ClanGV经口感染过程中的功能,并可为阐明ClanGV的经口感染机制以及开发新型病毒杀虫剂和增效剂奠定基础。
The aim of this study was to investigate the interaction between oral infection factor PIF0 and other oral infection factors PIF1 ~ PIF6 in Claire's cockroach granulosis virus (ClanGV) by yeast two-hybrid technique. First of all, based on the genome sequence information of ClanGV, the transmembrane region of PIF0 ~ PIF6 was analyzed by on-line software TMHMM Server v.2.0 to determine the fragment to be amplified. Then, the bait vector pGBKT7 and the capture vector pGADT were used as the starting vectors to construct the recombinant bait vector (P10) and the PIF1 ~ PIF6 recombinant capture vector (A1 ~ A6). The self-activation experiment proves that the recombinant bait vector B0 has no self-activating property and can further carry out protein interaction research. The results of yeast two-hybrid assay demonstrated that PIF0 could interact with the recombinant capture vector of PIF3 and PIF5 and had no interaction with PIF1, PIF2, PIF4 and PIF6 when it was used as bait vector. The results of this study will help to reveal the function of PIF0 in oral infection of ClanGV and lay a foundation for elucidating the oral infection mechanism of ClanGV and developing new virus insecticides and synergists.