Molecular cloning of recalcitrant plant viruses genes by a modified RAPD method using RF-dsRNA

在过去的十年里，人们对植物病毒基因进行了分子克隆和测序。植物病毒分子分析的目标之一是改进检测和诊断病原病毒的方法。作为合成DNA的模板，核酸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，有许多顽固的病毒不能用现有的方法纯化，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，从草本或木本寄主植物中提取dsRNA技术的应用使病毒复制核酸(RF-dsRNA)的检测成为可能。本研究的主要目的是从受病毒感染的植物组织中提取RF-dsRNA，以获得克隆。本研究建立了检测这些病毒的简易单管RT-PCR方法。此外，还测定了柑橘衰退病毒和黄瓜黄化病毒弱分离物和强分离物的全核苷酸序列。番茄侵染性黄化病毒是近年来在日本新发现的一种病毒。

Molecular cloning and sequencing of plant virus genes has been carried out during the past decade. One objective of molecular analysis of plant viruses has been the improvement of the methods to detect and diagnose pathogenic viruses. As templates for Cdna synthesis, nucleic acids are usually extracted from purified virus preparations in relatively pure form and in rather large amounts. Theses strategies rely on the purification of virus particles from infected plants. However, there are many recalcitrant viruses 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 host plants has permitted the detection of viral replicative nucleic acids (RF-dsRNA). The main objective of this research was the production of cDNA clones generated from RF-dsRNA extracted from virus-infected plant tissues. In this research the simple one tube RT-PCR method to detect these viruses was established. Furthermore, full nucleotide sequences of mild and severe isolates of citrus tristeza vireus and Cucumber yellows virus were determined. Tomato infectious chlorosis virus, a member of Criniviruses, was newly recognized in Japan.