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Obtaining of transgenic potato plants expressing human lactoferrin gene and analysis of their resistance to phytopathogens
SUMMARY. The transfer of human lactoferrin gene was carried out into genomes of a number of potato (Solanum tuberosum) varieties of Ukrainian selection with the use of Agrobacterium-mediated transformation. For this, the plasmid vector pBIN35LF was used carrying human lactoferrin gene hLf controlled by 35S promoter of Cauliflower mosaic virus (CaMV35S) and octopine synthase terminator, and a selective marker gene neomycinephosphotransferase II conferring the resistance to kanamycine. As a result of selection, 44 lines of Vernisage cultivar, 26 lines of cv. Levada, 25 lines of cv. Svitanok Kyivskyi and 16 lines of cv. Zarevo resistant to 100 mg/l of kanamycin were obtained. Intergration of the gene-of-interest into potato genome was confirmed with the use of PCR with the primers specific to lactoferrin. Identification of recombinant lactoferrin in the tissues of transgenic lines was carried out with the use of Western blotting with monoclonal antibodies to lactoferrin. The selected transgenic potato lines were tested on the resistance to bacterial and fungal phytopathogens. With the use of agar diffusion assay, the antibacterial effect of the juice of transgenic potato lines was established against phytopathogenic bacteria Ralstonia solanacearum (causing potato brown rot) and Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot). The resistance of transgenic potato plants to late blight was investigated by in vitro infection of plants with Phytophthora infestans isolate. As a result, the enhanced resistance to P. infestans of obtained transgenic potato lines was established compared to control. Thus, the obtained data show that transfer of hLf gene into potato genome enhance its resistance to bacterial and fungal pathogens.
Key words: human lactoferrin gene, genetic transformation, Solanum tuberosum, Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, Phytophthora infestans
E-mail: buziashvili.an gmail.com, yemets.alla gmail.com
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