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Characterization of a Pinus sylvestris thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Šņepste, Ilze, Šķipars, Vilnis, Krivmane, Baiba, Brūna, Lauma, Ruņģis, Dainis
Tree genetics & genomes 2018 v.14 no.4 pp. 58
DNA primers, Escherichia coli, Heterobasidion annosum, Pinus sylvestris, Pinus taeda, antimicrobial properties, bacteria, fungi, genes, methyl jasmonate, molecular cloning, open reading frames, pathogenesis-related proteins, pathogens, polymerase chain reaction, temperature
Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.