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International Journal of Current Microbiology and Applied Sciences (IJCMAS)
IJCMAS is now DOI (CrossRef) registered Research Journal. The DOIs are assigned to all published IJCMAS Articles.
Index Copernicus ICI Journals Master List 2019 - IJCMAS--ICV 2019: 96.39 For more details click here
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National Academy of Agricultural Sciences (NAAS)
NAAS Score: *5.38 (2020)
[Effective from January 1, 2020]
For more details click here

ICV 2019: 96.39
Index Copernicus ICI Journals Master List 2019 - IJCMAS--ICV 2019: 96.39
For more details click here

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Original Research Articles

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

Int.J.Curr.Microbiol.App.Sci.2018.7(4): 141-151
DOI: https://doi.org/10.20546/ijcmas.2018.704.016


Cloning and Expression of Tomato Leaf Curl Virus Coat Protein Gene in E. coli
S.H. Honnesh, M.S. Patil, Narayan Moger, V.P. Savalgi, L.S. Rashmi and Ratna Bergi
Department of Biotechnology, College of Agriculture, Dharwad University of Agricultural Sciences, Dharawad-580005, Karnataka, India
*Corresponding author
Abstract:

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).


Keywords: Geminivirus, Recombinant coat protein, TA cloning, His-tag purification
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How to cite this article:

Honnesh, S.H., M.S. Patil, Narayan Moger, V.P. Savalgi, L.S. Rashmi and Ratna Bergi. 2018. Cloning and Expression of Tomato Leaf Curl Virus Coat Protein Gene in E. coli.Int.J.Curr.Microbiol.App.Sci. 7(4): 141-151. doi: https://doi.org/10.20546/ijcmas.2018.704.016