1993 Fiscal Year Final Research Report Summary
Molecular cloning of plant virus gene from RF-dsRNA and the production of antisera against proteins encoded by the gene.
| Project/Area Number |
04660042
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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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.Agric., Associate Professor, 農学部, 助教授 (10134264)
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| Project Period (FY) |
1992 – 1993
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| Keywords | molecular cloning / dsRNA / citrus tristeza virus / plant viruses / fusion protein / serology / gene |
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| Research Abstract |
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.
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