Construction of a chimeric viral gene expressing plum pox virus coat protein

The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA ( cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA [synthesized by in vitro transcription of a double-stranded (ds) cDNA c...

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Veröffentlicht in:Gene 1992-10, Vol.120 (2), p.167-173
Hauptverfasser: Ravelonandro, Michel, Monsion, Marie, Teycheney, Pierre Yves, Delbos, René, Dunez, Jean
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Sprache:eng
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Zusammenfassung:The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA ( cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA [synthesized by in vitro transcription of a double-stranded (ds) cDNA clone], using an ad hoc synthetic oligodeoxynucleotide as primer. The resulting cDNA was rendered ds and cloned into the plasmid, pBluescribe M13 +. Transcription of this chimeric construction produced RNA molecules of 1250 nucleotides in length, which were used as messengers in the in vitro protein-synthesizing systems. The major product of this transcript consists of a 36-kDa polypeptide and was identified as the PPV coat protein (CP) by molecular weight estimation and by immunoprecipitation with a polyclonal antiserum to PPV. Transfer of this cDNA via Agrobacterium tumefaciens into plants was successfully performed. Transgenic Nicotiana plants producing the PPV CP were subsequently obtained.
ISSN:0378-1119
1879-0038
DOI:10.1016/0378-1119(92)90090-C