In silico Analysis and Expression of Osmotin-EAAAK-LTP Fused Protein
Publish place: Journal of Genetic Resources، Vol: 6، Issue: 1
Publish Year: 1399
نوع سند: مقاله ژورنالی
زبان: English
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شناسه ملی سند علمی:
JR_SGR-6-1_005
تاریخ نمایه سازی: 1 مرداد 1399
Abstract:
Antifungal agents are causing different problems in the agriculture industry. Plants are using various defense mechanisms for resistance against fungal pathogens. Some examples of these mechanisms are making physical barriers, producing chemical components and pathogenesis-related proteins such as lipid transfer protein (LTP) and Osmotin which can inhibit the growth of fungi at micro-molar concentrations. In this study, Osmotin and LTP genes were fused by the EAAAK linker to produce a single-fused gene construct. An in silico approach was used to predict and analyze Osmotin-EAAAK-LTP fused protein. Secondary and tertiary structure and mRNA formation of fused protein were predicted using bioinformatics tools. The designed construct was chemically synthesized and cloned in the pUC57 cloning vector. To express the fused protein gene was subcloned in expression vector pET-21b (+) with a hexahistidine tag. This gene was used for prokaryotic expression in E. coliBL21 (DE3) host. Different expression conditions were examined for expressing of fused protein. The fused protein was expressed with 1 mM IPTG after 3 hours of incubation at 28°C. The expression of 36.5 kDa protein was confirmed by western blotting. The study of antifungal activity of expressed fused protein was achieved by radial diffusion assay. This protein was able to exhibit antifungal activity towards experimented plant pathogenic fungi under in vitro conditions.
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Authors
Minoo Partovi Nasr
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, IRAN
Mostafa Motalebi
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, IRAN
Mohammad Reza Zamani
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, IRAN
Esmat Jourabchi
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, IRAN
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