DEVELOPMENT OF SYSTEMS FOR GENETIC DETECTION OF CORONATIN AND SYRINGOPEPTIN PHYTOTOXINS IN GENOMES OF PSEUDOMONAS SYRINGAE BACTERIOPHAGES

Abstract

The article presents results of studies on development of systems for genetic detection of coronatin and syringopeptinphytotoxins in genomes of Pseudomonas syringae bacteriophages. A team of authors designed detection systems for fragments of determinants of pathogenicity factors (phytotoxins) – coronatin and syringopeptin with application of electronic resources of NCBI: BLAST nucleotide and PRIMER BLAST. Nucleotide sequences were determined, homologues characteristic of other phytopathogens were studied. The system of oligonucleotides (primers and probe) for detecting a fragment of Pseudomonas syringaephytotoxincoronatin genes (cma A gene) includes primers: forward - CAATTGCATCTCGTCGGCTG, reverse - ATTACAAGCGGCTAACGCCT, probe - BHQ1-ACCAACACGACGGCTTTCAGCC-Fam and syringopeptin (syp C gene): forward primer - CTTGCAGCTT , reverse - TTGCATCGGTTCGTCCAGTC, probe - BHQ1-TGCGCACTGCACTGGTCTGG-Fam. A number of experiments were carried out to improve the amplification protocol: DNA denaturation at a temperature of 95 0C for 300 seconds once; primer annealing - at a temperature of 95 0C for 10 seconds, at a temperature of 60 0C for 30 seconds - 5 cycles; elongation - at a temperature of 95 0C for 5 seconds, at a temperature of 60 0C for 10 seconds fluorescence - 40 cycles. According to the amplification data, these determinants of coronatin and syringopeptin were not identified in the genomes of bacteriophages Ps.s.7 UlGAU and Ps.s.27 UlGAU, which are part of biological product. When studying the genome of the bacterial strain Pseudomonas syringae № 3, used as a mother culture in production of bacteriophage biomass, coronatin and syringopeptin determinants were identified.