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Studies on transcription and replication of viral genomes in infected plant cells by situ hybridization

原位杂交研究受感染植物细胞中病毒基因组的转录和复制

基本信息

批准号：
61560049
负责人：
HOSOKAWA Daijiro
金额：
$ 1.09万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

In situ hybridization is potentially a very powerful technique for detecting the localization of viral gemones in infected cells. Unfortunatelly until now, it have no wide application to plant virus-infected cells. We have developed convenient method of in situ hybridization for tobacco mosaic virus (TMV)-infected cells using biotinylated RNA probes.To prepare sense-strand RNA probes, TMV-RNA were fragmented (200-600 nucleoties long) by alkali treatment in the presence of Mg^<2+>. The RNA fragments were labeled with photobiotin by mercury vapour lamp irradiation. To prepare antisense-strand RNA probes, the cloned cDNA of TMV coat protein gene was inserted into a transcription vector, pGITC, that carries the SP 6 promoter at the transcription initiation site. The transcription vector was linearized just downstream of the cDNA insert with Bam H I, and was transcribed to obtain biotin labeled antisense RNA probe, using the SP 6 polymerase in the presence of biotin-11-UTP.When these probes were hybridized with TMV-RNA isolated from virions or doublestrand TMV-RNA extracted from TMV-infected tobacco plant on nylon membrane, hybridization reactions were detected by tagging avidin-al^kaline phosphatase followed by incubation in nitro blue tetrazolium.By in situ hybridization with these probes, viral nucleic acids were detected in the TMV-infected tobacco protoplasts spread on slides.In situ hybridization at electron microscope levels are in progress.

原位杂交可能是一种非常强大的技术，用于检测受感染细胞中病毒基因组的定位。不幸的是，到目前为止，它还没有广泛应用于植物病毒感染的细胞。我们开发了使用生物素化RNA探针对烟草花叶病毒(TMV)感染的细胞进行原位杂交的便捷方法。为了制备正义链RNA探针，在Mg^2+存在下通过碱处理将TMV-RNA片段化(200-600个核长)。通过汞蒸气灯照射，RNA 片段被光生物素标记。为了制备反义链 RNA 探针，将 TMV 外壳蛋白基因的克隆 cDNA 插入转录载体 pGITC，该载体在转录起始位点携带 SP 6 启动子。转录载体在 cDNA 插入片段的下游用 Bam H I 线性化，并在生物素-11-UTP 存在的情况下使用 SP 6 聚合酶转录以获得生物素标记的反义 RNA 探针。当这些探针与从病毒体分离的 TMV-RNA 或从 TMV 感染的烟草植物提取的双链 TMV-RNA 杂交时，在尼龙上膜上，通过标记亲和素碱性磷酸酶，然后在硝基蓝四唑中孵育来检测杂交反应。通过与这些探针的原位杂交，在涂在载玻片上的TMV感染的烟草原生质体中检测到病毒核酸。电子显微镜水平的原位杂交正在进行中。