Abstract:
Abstract
An experiment was conducted in Molecular Biology and Plant Virology
Laboratory under the Department of Plant Pathology, Sher-e-Bangla Agricultural
University, Dhaka-1207, Bangladesh. Total RNA was extracted from Potato leaf
roll virus (PLRV) positive leaves and complementary DNA (cDNA) were
synthesized from total RNA. Reverse transcriptase polymerase chain reaction
(RT-PCR) based detection conditions were optimized by using coat protein (CP)
gene specific primers. In PCR amplification cDNA and in nucleotide sequencing
PCR product was used as a template. A 346 bp amplicon of PLRVCP gene was
amplified and amplified gene region was sequenced. The expected nucleotide
sequence of amplified PLRV-CP gene showed 95 to 98% homology when
compared with the isolates sequences reported in Gene Bank database. This
explored novel PLRV-CP gene was characterized as a PLRV Bangladeshi isolate
(Accession number, Bankit 2274496, MN605963). PLRV-CP gene protein
modeling was carried out using Expert Protein Analysis System (ExPaSy),
DNASTAR's protein tools server used for 3D protein modeling. Phylogenetic
analysis was also carried out, the tree was made by using MEGA 4.0 software and
maximum parsimony method was selected to construct phylogenetic tree. The
RT-PCR based molecular technique optimized in this study, would be a useful for
early detection, epidemiological studies of PLRV as well as in seed tubers
certification program and the novel hyper variable sequenced region of PLRV -CP
gene will be useful in pathogen derived resistance breeding program against the
PLRV local strain.
Keywords: potato, PLRV-CP Gene, PCR-Based Cloning, PLRV-Bangladeshi isolate.
