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Studies on the multiplication mechanism of plant viruses by electron microscopic in situ hybridization

电镜原位杂交研究植物病毒增殖机制

基本信息

批准号：
07660054
负责人：
HOSOKAWA Daijiro
金额：
$ 1.47万
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 Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

In recent years, remarkable progress has been made in the understanding of viral genome structures and their expression strategy. However, information concerning the replication of viral genome in host cells is still fragmentary. In this study, we thus examine the intracellular distribution of RNA molecules of tobacco mosaic virus (TMV) and potato virus X (PVX) in tobacco protoplasts using in situ hybridization at the electron microscopic level to gain the beter approach to define the mechanism of repllication of viral genome.In situ hybridization at the electron microscopic level can be carried out using two different methohs : Pre-embedding method and post-embedding method. However, these methods were not established in plant cells infected with viruses. Therefore, we first determind the optical technical conditions and obtained the best result as follow. The protoplasts infected with TMV or PVX were fixed in 4% paraformaldehyde plus 0.5% glutaraldehyde for 2 hr at 4ﾟC and embedded in Lowicryl K4M.Polymerization of Lowicryl K4M was carried out with irradiation by UV light. Ultrathin sections were prepared and hybridized with digoxigenin-labelled RNA probes for over night at 45ﾟC.The detection of hybrids was carried out using successively sheep anti-digoxigenin antibody and rabbit anti-sheep IgG antibody conjugated to colloidal gold. In protoplasts infected with TMV,hybridization signals were localized over the slightly electron-dense small areas of the cytoplasm. No labelling was observed on other organelles. In protoplasts infected with PVX,gold labelling were also localized over the small areas in the cytoplasm. However, specific structures were not found in these areas.In future work, it should be possible to obtain simultanous in situ, hybridization of viral RNA and immunogold detection of viral proteins and to define a site of virus replication.

近年来，对病毒基因组结构及其表达策略的研究取得了显著进展。然而，有关病毒基因组在宿主细胞中复制的信息仍然是零碎的。本研究利用原位杂交技术研究了烟草花叶病毒（TMV）和马铃薯X病毒（PVX）的RNA分子在烟草原生质体中的分布，为进一步研究病毒基因组的复制机制提供了一种新的方法。预埋法和后埋法。然而，这些方法在感染病毒的植物细胞中没有建立。为此，我们首先确定了最佳的工艺条件，得到了如下的最佳结果。将感染TMV或PVX的原生质体用4%多聚甲醛+0.5%戊二醛在4 ℃下固定2小时，包埋在Lowicryl K4M中，用紫外光照射进行聚合。制备超薄切片，与地高辛标记的RNA探针在45 ℃杂交过夜，用羊抗地高辛抗体和兔抗羊IgG抗体与胶体金偶联进行杂交检测。在感染TMV的原生质体中，杂交信号定位于细胞质中电子密度稍高的小区域。在其他细胞器上未观察到标记。在PVX感染的原生质体中，金标记也定位于细胞质中的小区域。在今后的工作中，应该有可能获得原位杂交、病毒RNA的杂交和病毒蛋白的免疫金检测，并确定病毒复制的位点。