摘要：

旨在从cDNA文库中筛选潜在与番茄褪绿病毒(ToCV)外壳蛋白(CP)互作的蛋白,探究ToCV的侵染机制及CP在侵染过程中的功能,以应对当前越夏、秋延后番茄生产中因番茄褪绿病毒病大肆传播而致使番茄产量和品质严重受影响的状况。以感染ToCV的Moneymaker番茄品种作为试验材料,采用Gateway技术构建感染ToCV的番茄核体系酵母cDNA文库,并构建酵母双杂交诱饵载体pGBKT7-CP(CP-BD)。以CP为诱饵蛋白,通过核体系酵母文库筛选出上百种参与多种生理过程的潜在互作蛋白,并进一步通过一对一的酵母双杂交验证以及NCBI BLAST比对确定与ToCV CP互作的相关蛋白。构建了核体系酵母文库,初级文库总克隆数为1.60×10⁷,重组率达100%,平均插入片段长度超过1 000 bp;次级文库总克隆数同样为1.60×10⁷,重组率为100%,平均插入片段长度大于1 000 bp,达到质量标准,满足后续酵母杂交试验的条件。由文库筛选得到的ToCV CP互作蛋白涵盖了细胞过程、生物调节、细胞物质运输等类别,其中在病毒复制和运输、侵染宿主细胞以及调节宿主细胞的代谢和细胞周期等生物过程相关的蛋白数量较多。此外,还涉及蛋白质结合、核酸结合、水解酶活性等相关蛋白,其中RNA酶最为丰富,在病毒侵染过程中发挥重要作用。最终确定了HSPs、DnaJ与TCPs等30种与ToCV CP互作的蛋白,为后续深入研究ToCV的侵染机制及CP在侵染过程中的功能奠定了良好基础。

关键词: 番茄褪绿病毒, 外壳蛋白, Gateway技术, 酵母双杂交, 核体系

Abstract:

This study aims to identify potential proteins that interact with the coat protein(CP)of Tomato chlorosis virus (ToCV)from a cDNA library,to explore the infection mechanism of ToCV and the role of CP in the infection process.The research highlights the significant impact of Tomato chlorosis virus disease on tomato yield and quality during summer and late-autumn production.The Moneymaker tomato variety infected with ToCV was used as the experimental material.Using Gateway technology,a nuclear yeast cDNA library was constructed from ToCV-infected tomatoes,and a yeast two-hybrid bait vector,pGBKT7-CP(CP-BD),was developed.CP was employed as the bait protein to screen the nuclear yeast cDNA library,identifying hundreds of potential interacting proteins involved in various physiological processes.Further verification was performed using one-on-one yeast two-hybrid assays and NCBI BLAST analysis to confirm the proteins interacting with ToCV CP.The constructed nuclear yeast cDNA library had a primary capacity of 1.60×10⁷,with a 100% recombination rate and an average insert size exceeding 1 000 bp.The secondary library also achieved a capacity of 1.60×10⁷,with a 100% recombination rate and an average insert size greater than 1 000 bp,meeting the quality standards for subsequent yeast hybridization experiments.Proteins interacting with ToCV CP,identified through library screening,were categorized into cellular processes,biological regulation,and intracellular material transport.Notably,many of these proteins were associated with processes such as viral replication and transport,host cell infection,and the regulation of host cell metabolism and the cell cycle.Additionally,the identified proteins included those with functions such as protein binding,nucleic acid binding,and hydrolase activity.Among these,ribonucleases were the most abundant,playing a critical role in the viral infection process.Ultimately,30 proteins,including HSPs,DnaJ,and TCPs,were confirmed to interact with ToCV CP.These findings provide a strong foundation for further research into the infection mechanism of ToCV and the functional role of CP in the infection process.

Key words: Tomato chlorosis virus, Coat protein, Gateway technology, Yeast two-hybrid, Nuclear system

中图分类号: 

• S432.4侵(传)染性病害