Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression in Escherichia coli.
Total RNA from infected Physalis floridana was isolated to generate complementary DNA corresponding to the coat protein (GP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1-3) for fusion protein expression in E. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRVcub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.
López
L
L
Division of Plant Biotechnology, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Muller
R
R
Balmori
E
E
de la Riva
G
G
Ramírez
N
N
Doreste
V
V
López
M
M
Peréz
S
S
Oramas
P
P
Selman-Housein
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D003462:Cuba; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D017793:Luteovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000821:virology; D015678:Viral Structural Proteins / Q000235:genetics*
D018076:DNA, Complementary; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins