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基于 GFP 标记的新型番茄 Torrado 病毒载体的构建及其在植物病毒运动监测中的应用。

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants.

机构信息

Department of Molecular Biology and Biotechnology, Institute of Plant Protection-National Research Institute, Władysława Węgorka 20St, 60-318 Poznań, Poland.

出版信息

Viruses. 2020 Oct 20;12(10):1195. doi: 10.3390/v12101195.

Abstract

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTV-Kra) that could infect and . Importantly, a modified variant of the viral RNA2-with inserted sGFP (forming, together with virus RNA1, into ToTV-Kra)-was engineered as well. RNA2 of ToTV-Kra was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTV-Kra-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

摘要

绿色荧光蛋白 (GFP)-标记病毒是广泛应用于病毒感染过程中疾病分子决定因素研究的基础研究工具。在这里,我们描述了番茄 torrado 病毒分离株 Kra(ToTV-Kra)的新一代遗传稳定的感染性克隆,这些克隆能够感染 和 。重要的是,我们还构建了一个插入 sGFP 的病毒 RNA2 的改良变体(与病毒 RNA1 一起形成 ToTV-Kra)。通过引入额外的 sGFP 开放阅读框(ORF)并用对应于假定病毒蛋白酶识别位点的氨基酸编码序列进行侧翼修饰,对 ToTV-Kra 的 RNA2 进行了修饰。我们的进一步分析表明,通过机械接种和系统传播,sGFP 标记的 ToTV-Kra 成功地进行了传代。因此,该克隆可能应用于研究感染过程中 ToTV 在体内细胞、组织和器官水平的定位。通过进行全株成像,然后进行荧光和共聚焦显微镜分析,在感染的植物中证实了 ToTV-Kra 衍生荧光信号的存在。所有这些信息都通过 sGFP 特异性免疫沉淀和 Western blot 分析得到了验证。torradovirus-plant 相互作用的分子生物学仍未得到充分描述;因此,这里获得的结果为进一步研究开辟了新的可能性。sGFP 标记病毒感染性克隆的应用及其开发方法可用于在广泛的植物病理学背景下分析植物-病毒相互作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2bc9/7588970/e0d040978744/viruses-12-01195-g001.jpg

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