Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Abstract

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.

Sequence analysis of Potato leafroll virus isolates reveals genetic stability, major evolutionary events and differential selection pressure between overlapping reading frame products

“…Another isolate from Poland (PLRV-P) is also more closely related to the three former isolates than to the latter (Palucha et al ., 1994 ). Similarly, sequencing of ORF3 of Cuban and Brazilian isolates suggests that these isolates are more closely related to the Northern temperate country isolates than to the Australian isolate (López et al ., 1994 ; Caram de Souza-Dias et al ., 1999 ). Finally, comparison of ORF3 and ORF5 sequences between Scottish isolates has shown that these isolates are as divergent from each other as they are from overseas isolates, except for the one from Australia (Jolly & Mayo, 1994 ).…”

Incidence and Molecular Analysis ofPotato leafroll virusin Iran

“…Enzyme‐linked immunosorbent assay (ELISA) is a common technique for detecting PLRV in infected plants (Casper, 1977; Clarke, 1981). Reverse transcription‐polymerase chain reaction (RT‐PCR) using PLRV‐specific primers and sequencing have been also developed for detection and classification of the viral isolates (López et al., 1994; Djilani Khouadja et al., 2003; Mukherjee et al., 2003). We have used double‐antibody sandwich‐enzyme‐linked immunosorbent assay (DAS‐ELISA) to assess the incidence of PLRV infection in potatoes in four Iranian Provinces.…”