MOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE OF THE COAT PROTEIN GENE OFA CUBAN ISOLATE OF POTATO LEAFROLL VIRUS AND ITS EXPRESSION IN ESCHERICHIA-COLI

Total RNA from infected Physalis floridana was isolated to generate co mplementary DNA corresponding to the coat protein (CP) 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 expr ession vectors pEX(1-3) for fusion protein expression in E. coli. The product was detected by antibodies specific for the PLRV CP. The codin g sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduc ed 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 PLRV(cub) CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applica ble to other plant viruses of low abundance or of cumbersome isolation , since this method is less time consuming than the traditional method s of cloning coat protein genes of plant Viruses with known sequences.