烟草坏死病毒A外壳蛋白介导的寄主特异性长距离运输机制研究

Virus long-distance transport, also called systemic movement, usually causes severe damage on host plant, in which various factors are involved in the mechanism of interaction between virus and plant. According to the phenomena that virus systemic infection is normally restricted within a limited plant species, in this proposal, Tobacco necrosis virus A (TNV-A) will be used for study on the molecular mechanism of virus long-distance transport mediated by its coat protein, in which the special domains were found in our previous research to be essential for long-distance transport restrictively in different host plant species. On the bases of our obtained results, the functional domain or specific amino acids of TNV-A coat protein that are essential for long-distance transport in specific the plant species will be accurately identified by biological assays after site-directed mutagenesis and viral fragment recombination. In further, fluorescence-labeled microscopy would be carried out for localization of the mutant viruses on plant organell and phloem-related cells associated with virus long-distance movement. Also, the interaction between plant hosts and the mutated viruses will be assayed by the techniques of amino acid phosphorylation, co-immunoprecipitation and transcriptome sequencing. With the results of this project, the crucial domain or amino acid and their function model of TNV-A coat protein, that are essential for systemic infection restricted in soybean (Glycine max L.) or Nicotiana benthamiana, are expected to be understood, providing the new data for the mechanism of plant virus systemic infection.

植物病毒的长距离运输，或称作系统性运动，往往导致对寄主的危害加重，其机制涉及病毒和植物的多种因子，通过互作而实现。基于植物病毒系统侵染中存在的寄主种类特异性现象，本项目拟以烟草坏死病毒A为对象，在已发现其外壳蛋白局部区域可特异性决定该病毒系统侵染的寄主种类和其他相关工作积累的基础上，对其外壳蛋白介导的长距离运输的分子机制进行研究。 研究工作将利用氨基酸点突变及与近缘病毒的片段重组，通过生物测定对决定病毒寄主特异性长距离运输的关键区域或氨基酸位点进行精细定位。在此基础上，进一步采用荧光标记显微技术对相应病毒突变体的植物细胞器定位和输导情况进行研究，并通过磷酸化修饰检测、免疫共沉淀和感病寄主转录组测序等技术，对寄主与以上病毒突变体的互作情况进行分析。以期确认烟草坏死病毒A外壳蛋白中特异性影响系统侵染大豆或本生烟的关键功能区或氨基酸位点及其作用方式，并为病毒系统侵染寄主植物的机制提供新的资料。