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Information × Registration Number 0220U103778, 0120U103353 , R & D reports Title Structural and functional characteristics of Scots pine defensins as candidate genes for control of crop diseases. popup.stage_title Head Kovaleva Valentina A., Кандидат біологічних наук Registration Date 19-11-2020 Organization Ukrainian national forestry university popup.description2 The object of study: recombinant defensins of Scots pine, which are obtained by heterologous expression in different systems: their purification and biological activity The aim is to characterize the features of the structure and biological activity of Pinus sylvestris L. defensins to identify functionally significant structural motifs that are responsible for the antimicrobial properties of these peptides, for the purposeful construction of plants with given properties. Methods of research used in microbiology, molecular biology, gene engineering, bioinformatics and statistics. We developed the protocol for the expression of defensin 2 from Scots pine in the pET32 / BL21-CodonPlus (DE3) -RIL expression system and obtaining the target soluble peptide conjugated with thioredoxin (TRX). It was established that the highest yield of soluble TRX-PsDef2 is achieved by the induction of transformed cells with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and culturing them for 3 hours at 25 ° C. Chimeric TRX-PsDef2 protein was purified on HisPurNi-NTA resin and TRX-tag was subsequently removed by proteolytic cleavage with enterokinase. PsDef2 was isolated from the proteolytic mixture by ion-exchange chromatography on an SP-Sepharose Fast Flow column using a step-gradient from 0.05 to 1 M NaCl. We obtained one milligram of the target peptide with purity higher than 95% from one liter of bacterial culture. It was found that recombinant peptide PsDef2 has activity against phytopathogenic fungus Fusarium sporotrichiella and oomycetes Phythophtora gonapodyides at concentrations 5.0 μM. We developed a protocol for the expression of defensin 5.1 from Scots pine in the pET32 / Rozetta gami2 system and the preparation of the target peptide conjugated to thioredoxin (TRX) in soluble form. Product Description popup.authors popup.nrat_date 2020-11-19 Close