Molecular detection, phylogenetic analysis and designing of siRNA against Potato Virus X

Shafique Ahmed, Idrees Ahmed Nasir, Tahir Yaqub, Muhammad Wasim, Bushra Tabassum, Faiza Masood, Anwar Khan, Shahid Javed Butt, Tayyab Husnain


Background: As potato (Solanum tuberosum L.) is one of the most liked food crops for human diet so increasing its production is an important goal for scientists to achieve. In this molecular study, we characterized the Coat Protein (CP) gene of Potato Virus X (PVX). CP gene is virulence mediator and integral part of viral structural assembly.
Methodology: We tissue cultured the PVX positive potato plants for viral RNA extraction. Total RNA was converted to cDNA for priming CP gene in PCR for amplification. To get the complete sequence of gene, we cloned CP gene into pTZ57R/T cloning vector. Upon double digestion of recombinant plasmid with EcoRI and HindIII restriction enzymes, 710 bp fragment was obtained which confirmed cloning. Recombinant plasmid was sequenced with M13 primers.
Results: Derived consensus sequence of 710 bp was found to be exact cds of CP gene showing 95% similarity with referenced genome. Phylogenetic analysis suggested Indian isolate of PVX as the nearest one. Multiple siRNA were designed against mentioned and optimized computationally to provide base for further studies.
Conclusion: Following facts may be established upon findings of this research; i) CP gene of Pakistani isolate of PVX has high homology with other PVX isolates found around the world, ii) in determining target for efficient siRNA mediated approach to silence PVX genome, this conserved nature can be proved very promising. Thus, to develop PVX-resistant potato crop in Pakistan through siRNA mediated strategy, CP gene could be the best target.

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