Nucleotide sequence of coat protein gene for GPV isolate of barley yellow dwarf virus and construction of expression plasmid for plant

GPV is a Chinese serotype isolate of barley yellow dwarf virus (BYDV) that has no reaction with antiserum of MAV, PAV, SGV, RPV and RMV. The sequence of the coat protein (CP) of GPV isolate of BYDV was identified and its amino acid sequence was deduced. The coding region for the putative GPV CP is 603 bases nucleotides and encodes a Mr 22218 (22 ku) protein. The same as MAV, PAV and RPV, GPV contained a second ORF within the coat protein coding region. This protein of 17024 Mr (17 ku) is thought to correspond to the Virion protein genome linked (Vpg). Sequence comparisons of the CP coding region between the GPV isolate of BYDV and other isolates of BYDV have been done. The nucleotide and ammo acid sequence homology of GPV has a greater identity to the sequence of RPV than those of PAV and MAV. The GPV CP sequence shared 83.7% of nucleotide similarity and 77.5% of deduced amino add similarity, whereas that of the PAV and MAV shared 56.9%. 53.2% and 44.1%. 43.8% respectively. According to BYDV-GPV CP sequence, two primers were designed. The cDNA of CP was produced by RT-PCR. Full-length cDNA of CP was inserted into plasmid to construct expression plasmids named pPPI1. pPPI2 and pPPI5 based on different promoters. The recombinant plasmids were identified by using α-³²P-dATP labelled CP probe. α-³²P-ATP labelled GPV RNA probe and sequencing to confirm real GPV CP gene cDNA in plasmids.