Molecular cloning of plant viruses genes using RF-dsRNA and production of antisera against viral proteins expressed in E.coli

植物病毒基因的分子克隆和测序在过去十年中已经开展。植物病毒分子分析的目的之一是改进病原病毒的检测和诊断方法。作为cDNA合成的模板，核酸通常是从纯化的病毒制剂中提取的，其形式相对纯净且数量较大。这些策略依赖于从受感染植物中纯化病毒颗粒。然而，有许多顽固性病毒不能用现有的方法纯化，因此，无法获得标准的核酸模板来克隆它们。对于没有可用的抗血清的病毒，需要其他检测和诊断方法。对于其中的一些病毒，应用从草本或木本寄主植物中提取dsRNA技术可以检测到病毒复制核酸（RF-dsRNA）。本研究的主要目的是从病毒感染的植物组织中纯化的RF-dsRNA产生cDNA克隆。成功克隆了柑橘tristeza病毒（CTV）、草莓轻度黄边病毒（SMYEV）和甜菜伪黄边病毒，建立了检测这些病毒的RT-PCR方法。此外，还测定了CTV轻、重度分离株的全序列。在大肠杆菌中表达了SMYEV外壳蛋白基因，并纯化了该蛋白用于免疫家兔。免疫印迹法检测所得抗血清与感染草莓叶片反应。

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. These 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 purified from virus-infected plant tissues. The molecular clonings of citrus tristeza virus (CTV), strawberry mild yellow edge virus (SMYEV) and beet pseudo-yellows virus were succeeded and RT-PCR methods to detect these viruses were established. Furthermore, full sequences of mild and severe isolates of CTV were determined. SMYEV coat protein gene was expressed in E.coli and the protein was purified to immunize a rabbit. The resulting antiserum reacted with infected strawberry leaves in western blotting.