1999 Fiscal Year Final Research Report Summary
Molecular cloning of plant viruses genes using RF-dsRNA and production of antisera against viral proteins expressed in E.coli
| Project/Area Number |
09660042
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物保護
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| Research Institution | Utsunomiya University |
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Principal Investigator |
NATSUAKI Tomohide Utsunomiya Univ., Fac. Agri., Professor, 農学部, 教授 (10134264)
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| Project Period (FY) |
1997 – 1999
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| Keywords | plant viruses / dsRNA / molecular cloning / genetic analysis |
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| Research Abstract |
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.
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