CONSTRUCTION OF A FULL-LENGTH CLONE OF THE STATICE (GONIOLIMON TATARICUM) ISOLATE OF THE TOMATO BUSHY STUNT TOMBUSVIRUS
T WETZEL and G KRCZAL
Staatliche Lehr- und Forschungsanstalt, Zentrum Grdne Gentechnik, Breitenweg 71, 67435 Neustadt/Weinstrabe, Germany
Background and objectives
A virus was isolated from commercially grown statice (Goniolimon tataricum) in southern Germany, in which flower crop damage is severe. The virus was identified as a tombusvirus closely related to tomato bushy stunt virus strain BS3, on the basis of host range, electron microscopy, symptomatology, physical properties and serology.
Materials and methods
Purified viral RNA was used as template for the different PCR-derived reactions. The AMV RTase (Pharmacia) and the Expand High Fidelity PCR system (Boehringer Mannheim) were used for long RT/PCR. The resulting PCR products were ligated directly into a dT-tailed plasmid (pBluescript, Stratagene).
Results and conclusions
Degenerate primers were designed by comparison of sequences from different tombusviruses. These primers were used in a ligation-anchored/polymerase chain reaction (LA/PCR)  to clone both 3' and 5' ends of the viral genome. The sequences obtained from these clones, corresponding to the 5' non-coding region and part of the 3' non-coding region of the viral genome, were used to design specific primers corresponding to the 3' and 5' ends of the viral genome. These primers were used in long-RT/PCR to amplify the full-length viral genome. The resulting PCR product was cloned and sequenced. Highest homologies (92% over the entire genome) were found with tomato bushy stunt virus (TBSV) . Assays are currently being done to cheek the infectivity of the full-length clone of the TBSV-Statice.
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