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

使用 RF-dsRNA 通过改良 RAPD 方法对顽固植物病毒基因进行分子克隆

• 批准号: 12660041
• 负责人: NATSUAKI Tomohide
• 金额: $ 2.05万
• 依托单位: Utsunomiya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660041/
• 关键词: recalcitrant plant viruses; replicative form dsRNA; PCR; simple detection method; molecular cloning; 難純化RNAウイルス; キュウリ黄化ウイルス; tomato infectious chlorosis virus; Cryptovirus; カンキツトリステザウイルス

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.

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相当大。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提取RF-dsRNA产生cDNA克隆。本研究建立了一种简便的单管RT-PCR检测方法。此外，还测定了柑橘tristeza病毒和黄瓜黄病毒轻、重度分离株的全核苷酸序列。番茄感染性褪绿病毒是日本新近发现的克里尼病毒属病毒。

项目成果

Suastika, G., Natsuaki, T., Terui, H., Kano, T., Ieki, H., Okuda, S.: "Nucleotide sequence of citrus tristeza virus seedling yellows isolate"Journal of General Plant Pathology. 67・1. 73-77 (2001)

Suastika，G.，Natsuaki，T.，Terui，H.，Kano，T.，Ieki，H.，Okuda，S.：“柑橘tristeza病毒幼苗黄化分离物的核苷酸序列”普通植物病理学杂志67・1。 .73-77 (2001)

Hartono, S., Natsuaki, T., et al.: "Yellowing disease of tomatoes caused by Tomato infectious chlorosis virus newly recognized in Japan"Journal of General Plant Pathology. 69. 61-64 (2003)

Hartono，S.，Natsuaki，T.，等人：“日本新发现的番茄传染性失绿病毒引起的番茄黄化病”普通植物病理学杂志。

Suastika,G., Natsuaki,T., Terui,H., Kano,T., Ieki,H. and Okuda,S.: "Nucleotide sequence of citrus tristeza virus seedlding yellows isolate"Journal of General Plant Pathology. 67-1. 73-77 (2001)

Suastika，G.，Natsuaki，T.，Terui，H.，Kano，T.，Ieki，H.

Hartono, S., Natsuaki, T., Genda, Y. and Okuda, S.: "Nucleotide sequence and genome organization of cucumber yellows vinos, a member of the genus Crinivirus"Journal of General Virology. 84-4. 1007-1012 (2003)

Hartono, S.、Natsuaki, T.、Genda, Y. 和 Okuda, S.：“克里尼病毒属成员黄瓜黄酒的核苷酸序列和基因组组织”普通病毒学杂志。

Suastika, G., Natsuaki, T., et al.: "Nucleotide sequence of citrus tristeza virus seedling yellows isolate"Journal of General Plant Pathology. 67. 73-77 (2001)

Suastika，G.，Natsuaki，T.，等人：“柑橘 tristeza 病毒幼苗黄化分离物的核苷酸序列”普通植物病理学杂志。