Abstract
Potato (Solanum tuberosum) is one of the largely consumed staple food crop of the world, including Pakistan. In Pakistan, potato shares a large percentage in country’s agriculture GDP. In Pakistan majority of good quality virus free seed potato is imported by multinational and local seed companies. This costs a huge burden on country’s economy. Potato Virus Y (PVY) belongs to Potyviridae family, severely damaging leaves of the Solanaceae family including potato. Plant lectins are the potential proteins that specifically bound and cross linked with carbohydrates and target glycans present on the cell surface of viruses through a pathway termed as lectin activation. The present in-vitro study shows the effect of lectin extracted from the seeds of Chenopodium murale against the downregulation of the CP (coat protein) gene of PVY. Stable interaction was found between PVY coat protein and CML in-silico. Full length CP gene from PVY virus was amplified and cloned in mammalian expression vector. CML protein was purified to sub fractions through anion exchange chromatography on SP-sepharose gel. The non-cytotoxic effect and safe limit of CML extract was indicated by CC50 value. The hemagglutination activity of CML against the rabbit erythrocytes was revealed to be 35 µg/ml. The real-time PCR revealed the anti-PVY activity of CML extract. CML extract in concentration of 30, 60 and 90 µg/ml was found effective against PVY CP gene in comparison with control. Keywords: Anion Chromatography; Coat Protein gene; Haemaglutination assay; HepG2 cancer cells; Lectins extract; Potato Virus Y (PVY); SDS-PAGE http://dx.doi.org/10.19045/bspab.2019.80059
Highlights
CML extract in concentration of 30, 60 and 90 μg/ml was found effective against Potato Virus Y (PVY) CP gene in comparison with control
Resistance shown by this gene is conferred during early stages of viral infection while in case of RTM1 lectin, the resistance is observed in advanced stages of infection which implements that resistance mediated by lectin plays a vital role in the diversity of plant-virus relation [6]
Current study is aimed to outline the effect of mannose-binding lectin, isolated from seeds of Chenopodium murale, against the potato virus Y CP gene
Summary
Two-fold serial dilutions were prepared of CML in PBS (phosphate buffer saline) and an equal volume of 2% erythrocyte suspension was added to it. Control was prepared by adding PBS to erythrocyte suspension. Total RNA isolation from PVY infected potato plants PVY infected potato plants were surveyed in various regions of Punjab for sample collection. From the collected viral infected plant samples, leaves were used for the isolation of total RNA by suing TRIzol reagent method with some minor changes [16]. Construction of expression plasmid For the construction of a plasmid, CP gene fragment conserved in the PVYo strain was taken for primer designing using the primer software (http://frodo.wi.mit.edu/primer3/). After gel purification of the amplified fragment, it was later inserted into the mammalian expression vector pcDNA 3.1 (+) to attain pcDNA-CP clone. Cell culture Cell culture lab of CEMB, Lahore, Pakistan, provided HepG2 cancer cells, which were grown using DMEM (Dulbecco’s modified Eagle medium) including penicillin (100U/ml), streptomycin (100ug/ml) and Fetal bovine serum (10%), in 5% CO2 atm. at 37 ̊C
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