National Academy of Agricultural Sciences (NAAS)
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PRINT ISSN : 2319-7692 Online ISSN : 2319-7706 Issues : 12 per year Publisher : Excellent Publishers Email : editorijcmas@gmail.com / submit@ijcmas.com Editor-in-chief: Dr.M.Prakash Index Copernicus ICV 2018: 95.39 NAAS RATING 2020: 5.38 |
Tomato leaf curl virus (ToLCV) is a major Geminivirus which cause serious loss to tomato production in tropical and subtropical regions of the world. Hence, considered as major constraint to tomato cultivation. In the present study ToLCV coat protein gene was cloned and expressed using recombinant DNA technology approach. The ToLCV infected tomato leaf samples were collected from tomato fields near Krishi Vigyan Kendra, Dharwad. Further, the total DNA from ToLCV infected tomato leaf sample was isolated by following CTAB protocol. An 786 bp PCR product containing coat protein coding region of ToLCV was amplified using ToLCVCP (forward) and ToLCVCP (reverse) primers and the amplified product was cloned into the pTZ57R/T and further sub-cloned in to the pQE30. After transformation into JM109 and M15 cells the clones were confirmed through PCR and sequencing. Amplification with expected size of 786 bp and homology with other isolate showed integrity of the clone. Further, the coat protein was expressed by inducing with 1mM IPTG at 3hr after induction with. The expressed protein was analyzed through sodium-dodecyl sulphate-Poly acrylamide gel electrophoresis (SDS-PAGE). A band of 31 kDa on the gel confirmed that coat protein was really fused to the 6X His-tag. Further, 1.34 mg/100 ml of the coat protein was purified using His-tag purification kit (Genei).
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