Molecular cloning of plant virus gene from RF-dsRNA and the production of antisera against proteins encoded by the gene.

在过去的十年里，植物病毒的分子克隆已经进行了。克隆植物病毒的目的之一是提高病毒的检测和诊断水平。作为合成DNA的模板，RNA或DNA通常是从纯化的病毒制剂中提取的，形式相对纯度较高，而且数量相当大。这些策略依赖于从受感染植物中提纯病毒颗粒。然而，有许多顽固的病毒或病毒分离株不能用现有的方法进行纯化，因此，标准的核酸模板无法用于它们的克隆。正是那些没有可用的抗血清的病毒，需要替代的检测和诊断方法。对于其中几种病毒，从草本或木本宿主中提取dsRNA技术的应用使病毒复制核酸(RF-dsRNA)的检测成为可能。本研究的目的是从受病毒感染的植物中提纯dsRNA，以获得克隆。以柑橘衰退病毒侵染组织中提取的dsRNA为模板，完成了柑橘衰退病毒的分子克隆，并进行了DNA合成和聚合酶链式反应。该方法对其他无法获得纯化病毒制剂的植物病毒具有普遍的适用性。进一步将经PCR扩增的cDNA与A蛋白基因融合到表达载体中，获得融合蛋白免疫兔。产生的抗血清与CTV感染植物中表达的非结构蛋白发生反应。

Molecular cloning of plant viruses has been carried out during the past decade. One objective of cloning plant viruses has been the improvement of virus detection and diagnosis. As templates for cDNA synthesis, RNA or DNA are usually extracted from purified virus preparations in relatively pure form and in rather large amounts. These strategies rely on the purification of virus particles from infected plants. However, There are many recalcitrant viruses or virus isolates that can not be purified by current methods and, therefore, the standard nucleic acid templates are not accessible for their cloning. It is the viruses for which there are no available antisera that alternate methods of detection and diagnosis are needed. For several of these viruses, the application of dsRNA extraction techniques from herbaceous or woody hosts has permitted the detection of virus replicative nucleic acids (RF-dsRNA). The objective of this study were the production of cDNA clones generated from dsRNA purified from virus-infected plants. The molecular cloning of citrus tristeza virus by using dsRNA that were extracted from virus-infected tissue as the template for cDNA synthesis and PCR was sccomplished. The method should have general utility for other plant viruses where purified virus preparations can not be obtained. Furthermore, the cDNA amplified by PCR was fused to the Protein A gene in an expression vector and the fusion protein was obtained to immunize a rabbit.. The resulting antiserum reacted with non-structural protein expressed in CTV-infected plants.