抗植物病毒剂抑制烟草花叶病毒外壳蛋白复制的作用机制研究

Tobacco mosaic virus (TMV) coat protein (CP) is addressed in relation to its role in host resistance and disease development, the main function of TMV-CP is not only to protect of viral RNA from degradation, but also take an active part in virus and host responses, including the replication of virus and the long-distance movement. At present, a commercial drug of Ningnanmycin was found that it has inhibited the replication of TMV by biological test. However it is not clear how Ningnamycin inhibits the infection activity. In this study, the recombinant proteins of TMV-CP was cloned and expressed in vitro, the crystal structure of TMV-CP was salved, and the assembly function of TMV-CP was studied. The affinity between TMV-CP and Ningnanmycin was analyzed by interaction test in vitro. The binding-sites between TMV-CP and Ningnanmycin were found by molecular dynamics simulation. Meanwhile, the binding-sites between TMV-CP and Ningnanmycin were determined by co-crystallization. In vivo, the regulation of CP genes in TMV-infected tobacco and its coded proteins dealt with Ningnanmycin was studied using qPCR and Weston Blot methods and Protein Proteomics. Collectively, both in vitro and in vivo assay were used to explain the replication mechanism of TMV-CP by Ningnanmycin.

烟草花叶病毒 (TMV) 外壳蛋白 (CP) 与宿主的致病性和病毒的扩增密切相关，除了保护病毒RNA不被降解外，还参与病毒与宿主之间的应答反应，包括病毒的复制、长距离运动等。目前通过生物测试发现药剂宁南霉素对CP的复制具有抑制作用。然而，抗植物病毒剂抑制CP复制的作用机制尚未研究清楚？针对这一现象，本研究以宁南霉素为例，通过克隆表达CP的重组蛋白，培养并解析其晶体结构，验证CP的组装功能。采用体外结合实验研究宁南霉素与CP的亲和力。采用分子计算寻找宁南霉素与CP发生相互作用的位点，并结合基因突变分析验证分子模拟结果，同时，通过共结晶进一步确定关键结合位点。在体内，通过qPCR和Weston Blot 研究抗病毒剂对感染TMV的烟草中CP gene和其编码蛋白的影响。同时，运用蛋白组学进行药剂对感染TMV烟草体内差异蛋白的分析。通过离体和活体实验共同解释抗植物病毒剂抑制CP复制的作用机制。

项目的背景.烟草花叶病毒外壳蛋白与宿主的致病性和病毒的扩增密切相关，除了保护病毒RNA不被降解外，还参与病毒与宿主之间的应答反应，包括病毒的复制、长距离运动等。目前通过生物测试发现药剂宁南霉素对CP的复制具有抑制作用。然而，抗植物病毒剂抑制CP复制的作用机制尚未研究清楚。.主要研究内容.本研究以宁南霉素为例，通过克隆表达CP的重组蛋白，培养并解析其晶体结构，验证CP的组装功能。采用体外结合实验研究宁南霉素与CP的亲和力。采用分子计算寻找宁南霉素与CP发生相互作用的位点，并结合基因突变分析验证分子模拟结果。在体内，通过qPCR和WB 研究抗病毒剂对感染TMV的烟草中CP gene和其编码蛋白的影响。同时，运用组学技术进行药剂对感染TMV烟草体内差异蛋白的分析。通过离体和活体实验共同解释抗植物病毒剂抑制CP复制的作用机制。.重要结果.发现了宁南霉素的剂量与TMV-CP disk的解组装有关。分析了宁南霉素的作用位点位于TMV-CP 的亚基与亚基结合的界面之间，验证了宁南霉素阻碍了TMV-CP disk的正常组装，促使TMV-CP disk解组装成为单体TMV-CP monomers的分子机制。明确了宁南霉素增加了感病烟草中的苯丙氨酸氨裂解酶、过氧化物酶，聚氧磷酸酶和超氧化物等防御酶的表达，激活了氧化还原和代谢过程，抑制了 CP 亚基因的表达，增强了宿主烟草的抗病性。.科学意义.构建了宁南霉素与烟草花叶病毒外壳蛋白的互作模型。初步解释了宁南霉素抑制烟草花叶病毒外壳蛋白解组装的分子机理，对抗烟草花叶病毒药剂的创制和田间应用提供技术支撑。