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

Abstract


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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References


Karpova O, Zayakina O, Arkhipenko M, Sheval E, Kiselyova O, et al. Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’complexes. Journal of general virology, (2006); 87(9): 2731-2740.

Bostan H, Haliloglu K. Distribution of PLRV, PVS, PVX, and PVY (PVYN, PVYo and PVYc) in the seed potato tubers in Turkey. Pakistan Journal of Biological Sciences, (2004); 7(7): 1140-1143.

Hull R. Rapid diagnosis of plant virus infections by spot hybridization. Trends in biotechnology, (1984); 2(4): 88-91.

Rashid F, Khalid S, Ahmad I, Mughal S. Potato virus X (PVX) resistance in tomato cultivars. International Journal of Pest Management, (1989); 35(4): 357-358.

Lecoq H, Moury B, Desbiez C, Palloix A, Pitrat M. Durable virus resistance in plants through conventional approaches: a challenge. Virus research, (2004); 100(1): 31-39.

Vaistij FE, Jones L, Baulcombe DC. Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. The Plant Cell Online, (2002); 14(4): 857-867.

Hamilton A, Voinnet O, Chappell L, Baulcombe D. Two classes of short interfering RNA in RNA silencing. The EMBO Journal, (2002); 21(17): 4671-4679.

Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic acids research, (2004); 32(19): e149-e149.

Rozen S, Skaletsky H (1999) Primer3 on the WWW for general users and for biologist programmers. Bioinformatics methods and protocols: Springer. pp. 365-386.

Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution, (2007); 24(8): 1596-1599.

Yokota T, Sakamoto N, Enomoto N, Tanabe Y, Miyagishi M, et al. Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO reports, (2003); 4(6): 602-608.

Hasuwa H, Kaseda K, Einarsdottir T, Okabe M. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS letters, (2002); 532(1): 227-230.

Hadidi A, Yang X. Detection of pome fruit viroids by enzymatic cDNA amplification. Journal of virological Methods, (1990); 30(3): 261-269.

Nie X, Singh R. A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers. Journal of virological Methods, (2001); 91(1): 37-49.

Narayanasamy P (2011) Detection of Virus and Viroid Pathogens in Plants. Microbial Plant Pathogens-Detection and Disease Diagnosis:: Springer. pp. 7-220.


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