[Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put].

cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes:

Metabolomic prof iles of Ribes nigrum L. and Lonicera caerulea L. from the collection of the N.I. Vavilov Institute in the setting of Northwest Russia.

Recently, the trend of using fruit and berry crops as ingredients for functional and dietary nutrition, the development and implementation of f lavors, pigments, new medicines and dietary supplements has been actualized. Because the direction of use depends on the biochemical properties of fruits, which are determined not only by species and varietal characteristics, but also by reproduction conditions, the study of the biochemical composition of fruits grown in various regions of the world continues to be relevant. In this regard, the collection of N.I. Vavilov Institute (VIR), which has a wide diversity of fruit and berry crops, is of great interest for study. Ribes nigrum fruits have a balanced set of sugars, organic acids, essential oils, microelements, a high content of vitamins, anthocyanins, pectins. Lonicera caerulea fruits are characterized by high values of phenolic substances: biof lavonoids, hydroxycinnamic acids, f lavonols, polyphenols, anthocyanins, as well as vitamins, carotenoids, iridoid glycosides and other natural antioxidants. The investigation of L. caerulea and R. nigrum fruit's accessions from the VIR collection using gas-liquid chromatography with mass spectrometry allows us to obtain new information about the biochemical characteristics of fruits, to identify L. caerulea and R. nigrum varieties with optimal economically valuable characteristics, to determine the specif icity of L. caerulea and R. nigrum metabolomic spectra in the setting of Northwest Russia. As a result of the analysis, typical compounds of the metabolomic prof ile of each culture were identif ied. Organic acids, phenol-containing compounds and polyols prevailed in L. caerulea, while mono- and oligosaccharides, in R. nigrum. The qualitative composition of the black currant varieties 'Malen'kii Printz', 'Dobriyi Dzhinn', 'Tisel', 'Orlovskii Val's', and blue honeysuckle 'S 322-4', 'Malvina', 'Leningradsky Velikan' was optimal for food consumption; the varieties of blue honeysuckle 'Bazhovskaya' and black currant 'Aleander' had a good representation of biologically active compounds, which makes samples attractive as raw materials for the production of biologically active additives, including with the use of microorganisms' cultures.

Identif ication of duplicate accessions in the sweet maize collection by means of zein electrophoresis.

Of all the subspecies of Zea mays L. cultivated in the world, sweet maize is the most important for the global economy. The leading seed-growing companies and research institutions around the world are engaged in breeding this crop. To meet the increasing demands of the industry to grain quality, it is important to select appropriate local varieties and lines for hybridization. Local (usually heterogeneous) varieties are a valuable source material for creating self-pollinated lines that contribute to a significant broadening of the genetic base of parental forms used in breeding. The advantages of sweet maize varieties and the interest of the food industry in them make it possible to consider accessions from the maize collection of the N.I. Vavilov Institute (VIR) as a potentially valuable source material for breeding. The present research concentrated on 19 local sweet maize varieties with different grain colors from the VIR collection, that is, 9 varieties with the blue color of ripe grain, 4 with white (colorless) grain, 3 with yellow, and 3 with red. The research included an analysis of zein electrophoretic patterns (protein markers); a study of their biotype composition and the nature of genetic polymorphism, as well as the creation of a protein pattern database for each accession. For a series of accessions with the same varietal name, but different catalog numbers, the degree of their identity was determined from their biotype composition in order to exclude duplication. Zein electrophoresis was carried out in vertical plates of 10 % polyacrylamide gel according to the standard ISTA technique developed with the participation of the Biochemistry and Molecular Biology Department of VIR. Zein patterns were used for the first time to electrophoretically study sweet maize varieties with different grain colors. Unique zein patterns were established for all the accessions studied, which makes possible their identification by specific marker components. The results of this work characterize zein electrophoresis as a useful tool for the identification and registration of duplicate accessions in the VIR collection of sweet maize varieties.

Metabolomic approach to search for fungal resistant forms of Aegilops tauschii Coss. from the VIR collection.

Broadening of the genetic diversity of donors of resistance to biotic environmental factors is a challenging problem concerning Triticum L., which can be solved by using wild relatives of wheat, in particular, Aegilops tauschii Coss., in breeding programs. This species, believed to be the donor of D genome of common wheat (T. aestivum L.), is a source of some traits important for breeding. This greatly facilitates the possibility of crossing Ae. tauschii with common wheat. Aegilops L. species are donors of effective genes for resistance to fungal diseases in wheat. For instance, genes that determine resistance to rust agents in common wheat were successfully introgressed from Ae. tauschii into the genome of T. aestivum L. The aim of our study was to identify differences in metabolomic profiles of Ae. tauschii forms (genotypes), resistant or susceptible to such fungal pathogens as Puccinia triticina f. sp. tritici and Erysiphe graminis f. sp. tritici. These indicators may be used as biochemical markers of resistance. A comparative analysis of groups of Ae. tauschii accessions showed that metabolomic profiles of the forms with or without resistance to fungal pathogens differed significantly in the contents of nonproteinogenic amino acids, polyols, phytosterols, acylglycerols, mono- and oligosaccharides, glycosides, phenolic compounds (hydroquinone, kempferol), etc. This fact was consistent with the previously obtained data on the relationship between Fusarium resistance in oats (Avena sativa L.) and certain components of the metabolomic profile, such as acylglycerols, nonproteinogenic amino acids, galactinol, etc. Thus, our studies once again confirmed the possibility and effectiveness of the use of metabolomic analysis for screening the genetic diversity of accessions in the VIR collection, of Ae. tauschii in particular, in order to identify forms with a set of compounds in their metabolomic profile, which characterize them as resistant. Ae. tauschii accessions with a high content of pipecolic acids, acylglycerols, galactinol, stigmasterol, glycerol, azelaic and pyrogallic acids, campesterol, hydroquinone, etc., can be used for creating wheat and triticale cultivars with high resistance to fungal pathogens causing powdery mildew, brown rust, and yellow rust.

[Genetic differentiation of hexaploid wheat inferred from analysis of microsatellite loci].

Landraces of wheat can serve as important potential sources for extending the genetic basis of selection cultivars. Analysis of microsatellites and typing of polymorphism in a representative sample of 347 genotypes, including landraces and selection cultivars, was performed using a set of 38 selected oligonucleotide primer pairs. Classification of genotypes with respect to the level of their similarity was performed using cluster analysis. The data obtained pointed to genetic differentiation of hexaploid wheat. The groups of cultivars, the formation of which was thought to be associated with the main old areas of wheat cultivation in Europe and Asia, were identified. The basis of each of the groups was formed by landraces of common wheat. The differences between the groups identified were associated with multiple changes in the wheat genome and were expressed as quantitative differences in the allele frequencies of microsatellite loci. The results of the study are of interest in terms of understanding the structure of wheat genetic diversity and revealing the pathways of evolution of this culture.

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds.

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin (Ki=100 pM). The crystal structure of this peptide in complex with bovine beta-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60-70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.

[Study of a collection of Triticum spelta L. wheat species using gliadin polymorphism]. / Issledovanie kollektsii vida pshenitsy Triticum spelta L. po polimorfizmu gliadinov.

Using gliadins, endosperm storage proteins of kernels, as markers, the genetic diversity of 170 samples from the Triticum spelta L. collection of the Vavilov Institute of Plant Industry was studied. High intraspecific polymorphism of the gliadin electrophoretic patterns was revealed. On the basis of similarity of the gliadin electrophoretic patterns, groups of samples were isolated, and the genetic structurization of the collection was performed.

[Seed proteins as markers in resolving the problems of genetic plant resources, selection and seed production]. / Belki semian kak markery v reshenii problem geneticheskikh resursov rastenii, selektsii i semenovodstva.

The article discusses the main directions and prospects of usage of protein and DNA markers in decision applied and theoretical problems of plant genetic resources (gene banks), plant breeding, seed production and seed testing. The examples of usage of protein and DNA markers for solving important problems of applied botany, genetics and plant breeding were considered.

[Identification of albumin 0.19 in wheat and other cereal proteins]. / Identifikatsiia al'bumina 0,19 v belkakh zerna pshenitsy i drugikh zlakov.

It was found that albumin 0.19, being an inhibitor of alpha-amylases from human and insect saliva, is not specific for T. aestivum and is also revealed immunochemically by means of antiserum in the proteins of T. durum. Using rocket immunoelectrophoresis, it was shown that the albumin component specific for T. aestivum is not identical to albumin 0.19. The levels of albumin 0.19 in the proteins of wheat, aegilops. secale, agropyron and barley grains were studied.