Issn 305-9397. Ғылым және білім. 2022. №1-1 (66) issn 2305-9397
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| Materials and methods. The objects of research are grain crops and wild-growing cereals,
black bacteriosis (Xanthomonas campestris) and basal (Pseudomonas syringae) bacteriosis. In order to determine the area of distribution of bacterial diseases of grain and wild-growing cereals, samples of wild and agricultural cereals were collected in the north of Kazakhstan in Aktobe, Kostanay, Akmola, Pavlodar, and Karaganda regions. Genomic DNA was isolated using a lysing CTAB buffer (2%, 2M NaCl, 10 mM Na3EDTA, 100 mM HEPES, pH 5.3). DNA samples were dissolved in 1×TE buffer (1 mM EDTA, 10 mM Tris- HCl, pH 8.0) with RNase A (1 ng/ml) (Kalendar et al., 2021). DNA quality was checked using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and gel electrophoresis. All specimens were stored at -20° C to prevent DNA degradation. Genomic DNA extraction. Extraction of genomic DNA was carried out from leaves using a Qiagen DNeasy mini kit (QIAGEN), as well as a universal DNA extraction method with an acidic CTAB lysis buffer and RNase A, according to the PrimerDigital protocol ( http://primerdigital.com/dna.html ). Qualitative and quantitative DNA parameters were determined using gel electrophoresis and NanoDrop spectrophotometer ( http://www.thermoscientific.com/en/product/nanodrop-lite-spectrophotometer.html ). For all collected samples, screening was carried out sequentially using primers targeting 16S rRNA sequences for both genera, and then all the other primers targeting marker genomic regions - rpoD, dnaK, fyuA, ITS, recA, gyrB for Xanthomonas and ITS, rpoD, gyrB, gapA for Pseudomonas genera, respectively. DNA samples were dissolved in 1×TE buffer (1 mM EDTA, 10 mM Trizma, pH 7.5) with RNase A (1 ng/ml) and checked for quality using NanoDrop spectrophotometer (Thermo Fisher Scientific) and gel electrophoresis. The optimized composition of the PCR mixture is shown in Table 1. Forward and reverse primer sequences are listed in Table 2. Work has begun on the identification of tandem repeats in the genomes of pathogens of black (Xanthomonas campestris) and basal (Pseudomonas syringae) bacterioses, as well as the development of a PCR test system designed for the procedure for their analysis. |