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

原位杂交是一种潜在的非常强大的技术，用于检测感染细胞中病毒gemones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细胞。利用生物素化RNA探针，建立了烟草花叶病毒（TMV）感染细胞的原位杂交方法。在Mg^<2+>存在下，用碱处理将TMV-RNA片段化（长度为200-600个核苷酸），制备意义链RNA探针。在汞灯照射下用光生物素标记RNA片段。将克隆的TMV外壳蛋白基因cDNA插入到转录起始位点携带sp6启动子的转录载体pGITC中，制备反义链RNA探针。转录载体在cDNA插入物的下游用Bam H I线性化，在生物素-11- utp存在的情况下，使用SP 6聚合酶转录得到生物素标记的反义RNA探针。将这些探针与从病毒粒子中分离的TMV-RNA或从感染tmv的烟草植株中提取的双链TMV-RNA在尼龙膜上杂交，标记亲和素-碱性磷酸酶，然后在硝基蓝四氮唑中孵育，检测杂交反应。利用这些探针进行原位杂交，检测了传播于载玻片上的tmv感染的烟草原生质体中的病毒核酸。电镜水平的原位杂交正在进行中。

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.