Lakhneko O.R. Development of biotechnological approaches for wheat evaluation in terms of quality and adaptability. Thesis for the degree of Doctor of Philosophy in the field of knowledge 16 Chemical and bioengineering in the specialty 162 Biotechnology and bioengineering. - National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, 2021. The development of biotechnological approaches for wheat assessment in terms of grain protein quality and adaptability to water scarcity is a promising task of modern biotechnology in the current realities of climate change and the growing issues of reducing allergenicity/toxicity of wheat. The purpose of the work is to develop biotechnological approaches for wheat assesment in terms of grain storage protein quality and polymorphism of genes of drought adaptability. The object of research is biotechnological approaches for wheat assessment in terms of grain storage protein quality and plants drought adaptability.
The subject of research is the rational parameters of biotechnological approaches of wheat assessment in terms of grain storage protein quality and drought adaptability.
It was established that under the conditions of soil drought the varieties of winter wheat Astarta, Odeska 267 and Podolianka were tolerant. It was found that grain productivity under the action of soil drought depends on the regulation of the aperture of the stomata on the abaxial side of the leaf.
It was found that the nucleotide sequences of homologous TaWRKY2 genes from six contrasting wheat varieties are characterized by different levels of polymorphism: 7 SNP for TaWRKY2-A1, 12 SNP and 1 insertion for TaWRKY2-B1, 1 deletion for TaWRKY2-D1 of which 11 polypeptides; and 3 insertions, 3 deletions, and 20 SNPs for the TaWRKY2-D1 promoter region, of which 3 SNPs potentially affect regulatory elements of expression. The functional dependence of the change in the level of expression of TaWRKY2 genes on the water regime of plant cultivation was revealed. Two drought-resistant varieties Poliska 90 and Natalka showed a rapid and strong increase in the expression of TaWRKY2 genes during the simulation of drought conditions in the flag leaf. Among drought-resistant varieties, only the Odeska 267 variety showed increased expression of TaWRKY2 genes and only the variety Podolianka showed an increase in relative expression in the ear among drought-resistant varieties.
36 SNPs, 3 insertions and 1 deletion in the sequences of wheat stomatal biogenesis genes EPF1, EPF2 and MUTE from three subgenomes of six contrasting varieties were characterized. However, only 15 polymorphisms cause changes in the polypeptide chain. It has been studied that the genes EPF1, EPF2 and MUTE are expressed at a high level only in the young leaves.
The biotechnological approach for wheat evaluation based on a modified PCR method using 9 reproducible primer systems for TaWRKY2-A1 (PA1), TaWRKY2-B1 (PB1, PB2 and PB3) and TaWRKY2-D1 (PD1, PD2, PD5, PD4) and 7 systems to EPF1-A1 (E1A1), EPF1-B1 (E1B1 and E1B2), EPF1-D1 (E1D1 and E1D2), EPF2-A1 (E2A1) and MUTE-D1 (MD1). For each of the systems, rational chemical conditions, temperature and PCR modes for enzymatic replication were selected in order to obtain products in the form of amplified DNA. It is shown for the samples of varieties, wild wheat and hybrids that the developed technology can be effectively used for genotyping of varieties, for phylogenetic studies of wheat, characteristics of germplasm donors for crossings, discrimination of genetic material derived from wheat subgenome D. The biotechnological approach for estimatimation the proteomic composition of wheat grain based on single-stage protein extraction and complementary 2DE-LC/MS and GF-LC/MS was developed, which in the complex produced 20.4% more protein identifications than the use of 2DE-LC/MS. Rationalized enzymatic cleavage of differentially expressed proteins by three enzymes in the 2DE-LC/MS approach increased the number of identifications from 57.6% to 74.2%. The majority of differentially expressed grain proteins belong to the group of metabolic proteins – 27, 44%; then gliadins - 20 proteins, 33%; and the least glutenins – 14 proteins, 23%. In contrast, among allergenic/toxic proteins, glutenins with 14 proteins showed the greatest diversity, 48%; gliadins included 13 proteins, 45%; and the non-gluten group consisted of only two proteins, 7%. The highest coefficient of accumulation of allergenic/toxic proteins in Ukrainka (48.7% of detected proteins) was revealed, which is explained by genetic heterogeneity. Sotnytsia was characterized by 4 of 39 allergenic/toxic proteins (10.3%). The procedural schemes for wheat assessment of grain protein quality and drought adaptability have been developed. Biotechnological approaches for wheat assesment in terms of quality and adaptability involve obtaining DNA fragments and protein identifications.
