基于BYL in vitro体系的抗病毒生物药剂分子作用机理研究

Plant viruses cause great losses to agriculture worldwide every year . BYL (evacuolated tobacco BY-2 protoplast Lysate) in vitro virus translation/replication system is widely applied to study the mechanisms of plant viral protein translation, replication and the host proteins involved in pathogenicity. Cytosinpeptidemycin is a biological antiviral agent developed by our research group of ShenYang agricultural University, which has good inhibitory effect on Tobacco mosaic virus (TMV). In this project, two biological antiviral agents of Ningnanmycin and Cytosinpeptidemycin together with TMV are used to further study their molecular antiviral mechanisms. In this study, Effect of antiviral agents on TMV accumulation in the single cell will be studied using BY-2 tobacco protoplast experiment. BYL in vitro system will be used to study the effect of the biological antiviral agents on TMV translation and negative RNA synthesis in separate experiments. Meanwhile, BYL system will also be used to study the effect of the biological antiviral agents on host proteins that is interacted with TMV RNA and P126 using Strepto-Tag and Co-IP method. The related host proteins will be identified through LC-MS/MS method. The studies of this project could provide important theoretical basis for the development of more specific and effective antiviral agents

植物病毒在世界范围内对农业生产造成严重危害。BYL in vitro病毒翻译复制体系（BYL体系）是脱去液泡的BY-2烟草原生质体裂解液，适用于植物RNA病毒翻译、复制机制以及相关宿主蛋白质的研究。嘧肽霉素是由项目组所在沈阳农业大学自主开发，可有效抑制烟草花叶病毒（TMV）所引起的病害的生物药剂。本项目通过米台霉素和宁南霉素两种抗病毒生物药剂和TMV来详细研究生物药剂的抗病毒分子机理。本研究拟采用BY-2原生质体法研究抗病毒生物药剂对TMV在单细胞内的作用；拟采用BYL 体系明确生物药剂对TMV的翻译以及RNA负义链合成的影响；拟基于BYL体系下采用Strepto-Tag法和免疫共沉淀法明确上述药剂对与TMV相互作的致病相关宿主蛋白质的影响，并通过液相色谱串联质谱法对目标宿主蛋白进行鉴定，进而明确生物药剂对病毒相关宿主蛋白质的影响。本研究将为更有效专抗病毒药剂的开发提供理论依据和重要参考。

植物病毒在世界范围内对农业生产造成严重危害。为了研究抗病毒生物制剂对于病毒的作用，以及对寄主植物产生的影响，本项目采用悬浮细胞BY-2原生质体系统和BYL体外系统研究抗病毒生物制剂嘧肽霉素和宁南霉素对模式病毒烟草花叶病毒（TMV）的翻译和复制方面的分子作用机理。明确了两种生物制剂嘧肽霉素和宁南霉素均可有效的抑制TMV的细胞内的核酸积累，在BYL体外系统中，嘧肽霉素可抑制TMV的蛋白质翻译和负义链RNA的合成。通过转录组测序和实时荧光定量PCR（qPCR）研究药剂对BY-2寄主细胞基因调控的作用。结果揭示嘧肽霉素处理后寄主热休克蛋白（HSP）家族多个基因以及NPK1等激酶的基因表达显著上调；宁南霉素处理后寄主先天免疫反应中的RLK1激酶以及RNA聚合酶RDR1等多个基因的表达量显著上调。上述结果表明两种抗病毒制剂诱导寄主抗性相关基因调控方面具有不同的作用。本项目的完成可为更有效专抗病毒药剂的开发提供理论依据和重要参考。

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