Molecular characterization, cloning and sequencing of coat protein gene of a Pakistani potato leaf roll virus isolate and its phylogenetic analysis

Abstract

Total RNA was extracted from potato leaf roll virus (PLRV) positive potato plants and complementary DNA were synthesized. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. A 346 bp amplicon of PLRV- coat protein (CP) gene was amplified. Amplified CP-gene of PLRV was cloned in TA cloning vector, P^CR ® 2.1. The clone was confirmed subsequently through restriction digestion analysis. PCR amplification used cDNA clone as a template and nucleotide sequencing. Expected nucleotide sequence of amplified PLRV-CP gene show homology of 94 to 97% when compared to the sequences already reported in GenBank database. This explored novel PLRV-CP gene was submitted at NCBI GenBank for characterization of PLRV Pakistani isolate (Accession No. JN039286). Phylogenetic analysis was also carried out and tree was made by using MEGA 4.0.

Key words: Potato, potato leaf roll virus (PLRV), coat protein (CP) gene, reverse transcriptase polymerase chain reaction (RT-PCR), PLRV-CP Pakistani isolate.