Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage

Antibodies raised against a Sunn bug (Eurygaster integriceps Put.) recombinant protease, rGHP3p2, can inhibit gluten‐hydrolyzing activity

Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten-hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat-bug gluten-hydrolyzing proteases

Identification 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

Zein patterns as effective markers of valuable agronomic traits in maize

The effective use of maize (Zea mays L.) in agriculture depends entirely on the knowledge of its gene pool, which, in turn, needs to undergo registration and certification as new data become available. Maize is one of the model objects for applying the marker methodology. This article summarizes the results of a long-term study of the maize gene pool at N. I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in two main areas: identification of the world genetic diversity of maize (including the most important wild relatives for breeding use), and search for reliable protein (zein) markers of traits valuable for breeding in order to use them in improvement, seed production, and seed control of this crop. Specific examples show the effectiveness of using zein markers in the development of new inbred lines and improvement of the existing ones. On the examples of such agronomic traits as early maturity, explosiveness, and waxiness of maize grain, the possibility of controlling the “inclusion” of these traits in the breeding material with the help of zein markers has been demonstrated. Evidence of the effectiveness of applying zein markers in the selection of parental pairs to obtain highly heterotic hybrids is presented. The nomenclature of zein adopted at VIR after its separation by polyacrylamide gel electrophoresis (PAGE) in an acidic buffer was compared with the nomenclature of zein after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) adopted among the foreign scientific community. The studies were performed on a large volume of accessions from the VIR collection as well as on breeding material provided by the country’s leading breeders

Weight method for determination of soluble β-glucans in barley grain

Background. Barley (Hordeum vulgare L.) is an important source of nutrients, such as starch, protein, and various dietary fibers. β-Glucans are soluble fibers found in high amounts in oat and barley grain, so they are becoming increasingly interesting due to their numerous functional and bioactive properties. The increased interest in β-glucans as a dietary supplement and a functional component of food calls for a convenient, inexpensive and affordable method for quantitative determination of these compounds.Materials and methods. An overview is given on the existing techniques for determining and isolating β-glucans in cereals: IR spectroscopy, enzymatic, colorimetric, and alkaline-enzymatic methods. Their advantages and disadvantages are shown. The disadvantages of the methods considered include high costs of reagents and equipment, duration of performance, and labor intensity.Results. This study promotes the weight method for isolation and quantitative determination of β-glucans in the grain of covered and naked barley. It is based on the modified alkaline method adapted to barley; we developed it earlier for oat grain. The amount of β-glucans in the grain of the studied barley accessions ranged from 4.12±0.23% to 5.34 ± 0.31% for naked cultivars, and from 3.57 ± 0.18% to 4.29 ± 0.32% for covered ones.Conclusion. Based on the conducted research, optimal conditions for the isolation and quantitative determination of β-glucans from barley grain were selected: centrifugation modes, temperature and extraction ratio, concentration of compounds, precipitation and drying procedures. The main advantage of the described method is its accessibility and practical applicability when conducting mass analysis, including studying a collection of cereal crops

Structural variability of sunflower gene for methionine-rich albumin SFA8

Background. The 2S albumins of sunflower and other oilseed plants possess a high nutritional quality, the defense activity against fungi diseases casual gents and also valuable functional properties. The major component of albumin fraction, the SFA8 protein consists of 103 amino acid residues among which methionine constitutes 15 Mole %. In the cultivated sunflower gene pool the SFA8 structural gene is represented by the two alleles the products of which have different isoelectric points and differ by the electrophoretic mobility, however molecular mechanisms of the polymorphism are still unknown. Results. The amplified sequences of the SFA8 gene from seven Helianthus annuus L. accessions and three accessions of wild Helianthus L. species from VIR collection were sequences. The intron of 258-303 bp length depending on the genotype was firstly found in the central part of the gene. The length of the first exon constitutes 99 bp, the second exon is of 210 bp length. The nucleotide and translated amino acid sequences are polymorphic among different genotypes. The line VIR 130 in which the two expressing SFA8 proteins, the normal polypeptide with isoelectric point (pI) approximately 6.0 (normal SFA8) and its allelic variant with pI 6.5 (variant SFA8) have been earlier revealed possesses two types of the SFA8 encoding sequence. In one sequence the substitution 108С—G is present that results in the substitution of the polar uncharged amino acid serine for the positively charged arginine and respectively in alteration of the protein charge and isoelectric point. The intron sequence is also polymorphic and characterized by the presence of indels of approximately 45 bp. The intron sequences of all accessions contain dinucleotides GT at the 5΄ end and AG at the 3΄ end which are characteristic for consensus sequences of splicing sites in the U2-type introns. The variants of the secondary structure of the SFA8 intron sequences of H. argophyllus Torr. & A. Gray and all the analyzed H. annuus genotypes are similar and differ from those of H. petiolaris Nutt. and H. giganteus L. Conclusions. The data on the SFA8 gene sequence polymorphism are important understanding the molecular mechanisms of genotypic differences in biochemical and functional properties of the protein, and he revealed differences in the intron secondary structure can be important for understanding expression patterns of the protein

The metabolomic approach to the comparative analysis of wild and cultivated species of oats (Avena L.)

Seed metabolomic profiles have been investigated in wild and cultivated forms (cultivars) of oat (Avena L.). Seed accessions from the VIR oat collection were used for the research. Metabolomic analysis employed gas liquid chromatography-mass spectrometry (GLC- MS) using an Agilent 6850 chromatographer (USA). The analysis covered the composition and content of organic and fatty acids, amino acids, polyatomic spirits and sugars. The content fluctuation range for the studied groups of compounds was found to be narrower (significantly in some cases) in cultivars than in the wild species. Along with a sharp increase in oleic acid content, cultivars demonstrated a decrease in that of linoleic acid. The general conclusions from the comparison of seed metabolomic profiles in wild species and cultivars are presented below. A number of wild species can be recommended as a potential source of biochemical quality traits for breeding purposes. A series of metabolites (compounds), the content of which changes during domestication or which differentiate wild oat species from cultivars has been identified was found. Along with such well-known healthy food chemical factors as oleic acid, glucose and fructose, etc., differences concerning monoacylglycerol compounds (MAG 16 : 0 and MAG-2 18 : 2, etc.) have been found. The latter have been proposed to be related to the formation of adaptive traits, in particular, resistance to diseases and pests, and to environmental abiotic stresses