Оригинальный русский текст: https://vavilovj-icg.ru/2023-year/27-4/
PLANT GENETICS AND BREEDING
High-molecular-weight glutenins play an important role in providing high baking qualities of bread wheat grain. However, breeding bread wheat for this trait is very laborious and, therefore, the genotyping of variety samples according to the allelic composition of high-molecular-weight glutenin genes is of great interest. The aim of the study was to determine the composition of high-molecular-weight glutenin subunits based on the identification of the allelic composition of the Glu-1 genes, as well as to identify the frequency of the Glu-1 alleles in bread wheat cultivars that are in breeding work under the conditions of the Pre-Ural steppe zone (PSZ). We analyzed 26 winter and 22 spring bread wheat varieties from the PSZ and 27 winter and 20 spring varieties from the VIR collection. Genotyping at the Glu-A1 locus showed that the Ax1 subunits are most common in winter varieties, while the predominance of the Ax2* subunits was typical of spring varieties and lines. In the Glu-B1 locus, the predominance of alleles associated with the production of the Bx7 and By9 subunits was revealed for both winter and spring varieties. In the case of the Glu-D1 gene, for all the wheat groups studied, the composition of the Dx5+Dy10 subunits was the most common: in 92.3 % of winter and 68.2 % of spring PSZ accessions and in 80 % of winter and 55 % of spring VIR accessions. The analysis of genotypes showed the presence of 13 different allelic combinations of the Glu-A1, Glu-B1, Glu-D1 genes in the PSZ varieties, and 19 combinations in the VIR varieties. The b b/al/с d allelic combination (Ax2* Вх7+Ву8/8*/9 Dx5+Dy10) turned out to be the most common for the PSZ spring varieties and lines, while for the PSZ winter accessions it was a с d (Ax1 Вх7+By9 Dx5+Dy10); the b с a and b с d genotypes (Ax2* Вх7+Ву9 Dx2+Dy12 and Ax2* Вх7+Ву9 Dx5+Dy10, respectively) occur with equal frequency among the VIR spring accessions; in the group of VIR winter varieties, the combination of the a b/ al d alleles (Ax1 Вх7+Ву8/8* Dx5+Dy10) prevails. The most preferred combination of alleles for baking qualities was found in the spring variety ‘Ekaterina’ and winter varieties ‘Tarasovskaya 97’, ‘Volzhskaya S3’, as well as in lines k-58164, L43510, L43709, L-67, L-83, which are recommended for further breeding programs to improve and preserve baking qualities in the conditions of the Pre-Ural steppe zone.
Juniperus deltoides is a relict species from the Tertiary Period. It is a typical representative of the Mediterranean group of the section Juniperus. It is included in the Red Books of the Republic of Crimea and the city of Sevastopol. Until recently, it was believed that a population of J. oxycedrus grew in Crimea. Currently, J. deltoides is described as a cryptic species, morphologically difficult to distinguish from J. oxycedrus. As a result, it became necessary to conduct a series of detailed studies to determine the morphological and phylogenetic features of the Crimean cryptic population in order to identify it as being one of the species of the cryptic pair. The studies were carried out in two stages: at the first stage, the morphological features of the vegetative and generative organs and their difference from J. oxycedrus were determined; the second stage included genetic research. The length of the needles of the Crimean population is 12.94 ± 0.19 mm, which corresponds to the Eastern Italian population of J. deltoides. At the same time, the width of the needles is 1.39 ± 0.02 mm, which is typical of the Portuguese population of J. oxycedrus. The dimensions of the cones are d1 (conditional height) = 7.54 ± 0.14 mm, and d2 (conditional width) = 9.11 ± 0.09 mm, which is more in line with J. deltoides. The shapes of the cones are very diverse. Some individuals have cones, the covering scales of which are visually indistinguishable, and their tops are completely fused. A similar phenomenon is characteristic of the Western Mediterranean populations of J. oxycedrus. Morphological analysis of the vegetative and generative organs of J. deltoides showed that when these two traits are combined, it is not possible to reliably distinguish between J. deltoides and J. oxycedrus individuals. Nuclear (ITS internal transcribed spacer) and chloroplast (petN-psbM, trnS-trnG) non-coding regions of the genome were used for genetic analysis. Studies have shown that the nuclear regions of genes have greater variability than chloroplast regions. The sequences obtained in this work formed a clade with J. deltoides samples 9430 and 9431 (BAYLU) growing in Turkey, which makes it possible to assign the samples studied to J. deltoides.
Grapes are one of the most common agricultural crops in the world. Currently, the analysis of genotypes directly at the DNA level is considered to be the most accurate method for studying the plant gene pool. The study of wild vines and ancient varieties in various regions of viticulture is an important direction of research in this field. The purpose of this work was to study the population of wild grapes growing on the territory of the Utrish Nature Reserve on the Black Sea coast of Krasnodar Region. The territory of the reserve is of interest as it is a site of ancient settlements, and the environmental conditions are suitable for the growth of wild grapes. During the survey of the territory, 24 samples of wild grapes were found, which were described according to the main morphological characteristics and analyzed by the molecular genetic method. The found vines were genotyped using 15 DNA markers, including nine commonly used for DNA fingerprinting (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) and VVIb23, which allows determining hermaphrodite and dioecious vines. Statistical processing of microsatellite loci polymorphism data was carried out using the GenAlEx 6.5 program. The genetic relationships of the studied vines were evaluated using the PAST 2.17c program. The samples were found to be morphologically and genetically polymorphic. The number of alleles identified in the sample varied from 5 to 18 and averaged 8 alleles per locus. Statistical processing of DNA analysis data made it possible to identify two genetically different populations among the wild discovered vines. An assessment of genetic similarity of the found vines with some local varieties of geographically close viticulture regions, rootstocks and representatives of Vitis sylvestris from other territories was made. One of the populations found in the Utrish Nature Reserve is close to a number of V. sylvestris genotypes, the DNA profiles of which are presented in the Vitis International Variety Catalogue.
Triticale (× Triticosecale Wittmack) is of great interest as an insurance crop that can ensure the stability of the gross harvest of feed and food grains at a lower cost. In Western Siberia, only winter triticale varieties are cultivated, however, spring triticales are important for cultivation in regions not suitable for winter crops. To create spring varie ties with high yields and good grain quality, it is necessary to study and enrich the gene pool, identify donors of economically valuable traits. One of the possible ways to solve this problem can be through the production of secondary hexaploid triticales with the involvement of the tetraploid wild-growing species of emmer wheat Triticum dicoccum (Schrank) Schuebl. The aim of this work was to create and study hybrids of emmer T. dicoccum (Schrank) Schuebl. with hexaploid triticale using genomic in situ hybridization for staining of meiotic chromosomes and analysis of plant productivity elements in F4–F8. DT4, DT5, DT6 plants and the prebreeding F6 forms obtained from them – DT 4/168, DT 5/176 and DT 6/186 – were selected according to the characteristics of the productivity and the nature of the grain in the F4 hybrid population. The offspring of hybrids DT4 and DT5 and prebreeding forms DT 4/168 and DT 5/176 had an increased grain nature (over 750 g/l), but low productivity. The hybrid DT6 and the breeding form DT 6/186 obtained from it had high grain productivity (785 ± 41 and 822 ± 74 g/m2, respectively), but, like the paternal form of triticale UK 30/33, had a reduced nature of the grain. In F8 DT 6/186 plants, 7 homologous pairs of rye chromosomes and from 27 to 30 wheat chromosomes were found in meiosis, which indicates the presence of a complete rye genome and two wheat ААВВ genomes. Rye chromosomes showed stable formation of bivalents in contrast to wheat chromosomes, which caused the presence of aneuploids in plant populations. Thus, hexaploid forms DT 4/168 and DT 5/176 with well-made smooth grain and high grain size were obtained, which can be used as a source of this trait for selection of food-grade triticale. DT 6/186 is a promising form for further breeding in order to obtain high-yielding forms of triticale.
ANIMAL GENETICS AND BREEDING
In animals, obesity caused by consumption of a sweet-fat diet (SFD) is the most adequate mouse model of human diet-induced obesity. Fibroblast growth factor 21 (FGF21) reduces body weight, beneficially affects taste preferences, and corrects glucose metabolism in obese mice. Sex is known to influence FGF21 effects in different models of diet-induced and hereditary obesity. In mice with SFD-induced obesity, the effects of FGF21 have been studied only in males. The aim of this study was to compare the effects of FGF21 on body weight, food preferences and glucose and lipid metabolism in C57Bl/6J male and female mice with SFD-induced obesity. Mice were fed with a diet consisting of standard chow, lard and cookies for 10 weeks, then they were injected with FGF21 (1 mg per 1 kg) or vehicle for 7 days. Body weight, weights of different types of food, blood parameters, glucose tolerance, gene and protein expression in the liver, gene expression in the white, brown adipose tissues, and the hypothalamus were assessed. FGF21 administration reduced body weight, did not alter total energy consumption, and activated orexigenic pathways of hypothalamus in mice of both sexes. However, sex dimorphism was found in the realization of the orexigenic FGF21 action at the transcriptional level in the hypothalamus. Metabolic effects of FGF21 were also sex-specific. Only in males, FGF21 exerted beneficial antidiabetic action: it reduced fatty acid and leptin plasma levels, improved glucose-tolerance, and upregulated hepatic expression of Ppargc1, Fasn, Accα, involved in lipid turnover, gene Insr and protein glucokinase, involved in insulin action. Only in obese females, FGF21 induced preference of standard diet to sweet food. Thus, in mouse model of obesity induced by consumption of a sweet-fat diet, the catabolic effect of FGF21 was not sex-specific and hormonal, transcriptional and behavioral effects of FGF21 were sex-specific. These data suggest elaboration of different approaches to use FGF21 analogs for correction of metabolic consequences of obesity in different sexes.
Alzheimer’s disease is the most common form of dementia, affecting millions of people worldwide. Despite intensive work by many researchers, the mechanisms underlying Alzheimer’s disease development have not yet been elucidated. Recently, more studies have been directed to the investigation of the processes leading to the formation of neurofibrillary tangles consisting of hyperphosphorylated microtubule-associated Tau proteins. Pathological aggregation of this protein leads to the development of neurodegeneration associated with impaired neurogenesis and apoptosis. In the present study, the effects of central administration of aggregating human Tau protein on the expression of the Bdnf, Ntrk2, Ngfr, Mapt, Bax and Bcl-2 genes in the brain of C57Bl/6J mice were explored. It was found that five days after administration of the protein into the fourth lateral ventricle, significant changes occurred in the expression of the genes involved in apoptosis and neurogenesis regulation, e. g., a notable decrease in the mRNA level of the gene encoding the most important neurotrophic factor BDNF (brain-derived neurotrophic factor) was observed in the frontal cortex which could play an important role in neurodegeneration caused by pathological Tau protein aggregation. Central administration of the Tau protein did not affect the expression of the Ntrk2, Ngfr, Mapt, Bax and Bcl-2 genes in the frontal cortex and hippocampus. Concurrently, a significant decrease in the expression of the Mapt gene encoding endogenous mouse Tau protein was found in the cerebellum. However, no changes in the level or phosphorylation of the endogenous Tau protein were observed. Thus, central administration of aggregating human Tau protein decreases the expression of the Bdnf gene in the frontal cortex and the Mapt gene encoding endogenous mouse Tau protein in the cerebellum of C57Bl/6J mice.
DNA repeat composition of low coverage (0.1–0.5) genomic libraries of four amphipods species endemic to Lake Baikal (East Siberia) and four endemic gastropod species of the fam. Baicaliidae have been compared to each other. In order to do so, a neighbor joining tree was inferred for each quartet of species (amphipods and mollusks) based on the ratio of repeat classes shared in each pair of species. The topology of this tree was compared to the phylogenies inferred for the same species from the concatenated protein-coding mitochondrial nucleotide sequences. In all species analyzed, the fraction of DNA repeats involved circa half of the genome. In relatively more ancient amphipods (most recent common ancestor, MRCA, existed approximately sixty millions years ago), the most abundant were species-specific repeats, while in much younger Baicaliidae (MRCA equal to ca. three millions years) most of the DNA repeats were shared among all four species. If the presence/absence of a repeat is regarded as a separate independent trait, and the ratio of shared to total numbers of repeats in a species pair is used as the measure of distance, the topology of the NJ tree is the same as the quartet phylogeny inferred for the mitogenomes protein coding nucleotide sequences. Meanwhile, in each group of species, a substantial number of repeats were detected pointing to the possibility of non-neutral evolution or a horizontal transfer between species occupying the same biotope. These repeats were shared by non-sister groups while being absent in the sister genomes. On the other hand, in such cases some traits of ecological significance were also shared.
To identify body systems subject to epigenetic transformation during in vitro fertilization (IVF), comparative morphological and functional studies were performed on sexually mature offspring of outbred CD1 mice, specific-pathogen-free (SPF), obtained by IVF (experiment) and natural conception (control). The studies included assessment of age-related changes in body weight and composition, energy intake and expenditure, and glucose homeostasis. To level the effects caused by the different number of newborns in the control and in the experiment, the size of the fed litters was halved in the control females. Males obtained using the IVF procedure were superior in body weight compared to control males in all age groups. As was shown by analysis of variance with experiment/control factors, gender, age (7, 10 and 20 weeks), the IVF procedure had a statistically significant and unidirectional effect on body composition. At the same time, IVF offspring outperformed control individuals in relative fat content, but were behind in terms of lean mass. The effect of the interaction of factors was not statistically significant. IVF offspring of both sexes had higher fat to lean mass ratios (FLR). Since adipose tissue contributes significantly less to total energy intake compared to muscle, the main component of lean mass, it is not surprising that at the same level of IVF locomotor activity offspring consumed less food than controls. When converted to one gram of body weight, this difference reached 19 %. One of the consequences of reduced utilization of IVF energy substrates by offspring is a decrease in their tolerance to glucose loading. The integral criterion for the effectiveness of restoring the initial glucose level is the area under the curve (AUC), the value of which was 2.5 (males) and 3.2 (females) times higher in IVF offspring compared to the corresponding control. Thus, the totality of our original and literature data shows an increase in the risk of metabolic disorders in IVF offspring, which is confirmed by epidemiological studies of a relatively young cohort of people born using assisted reproductive technologies.
The honey bee Apis mellifera is a rather difficult object for selection due to the peculiarities of its biology. Breeding activities in beekeeping are aimed at obtaining bee colonies with high rates of economically useful traits, such as productivity, resistance to low temperatures and diseases, hygienic behavior, oviposition of the queen, etc. With two apiaries specializing in the breeding of A. m. mellifera and A. m. carnica as examples, the application of genetic methods in the selection of honey bees is considered. The first stage of the work was subspecies identification based on the analysis of the polymorphism of the intergenic mtDNA locus tRNAleu-COII (or COI-COII) and microsatellite nuclear DNA loci Ap243, 4a110, A24, A8, A43, A113, A88, Ap049, A28. This analysis confirmed that the studied colonies correspond to the declared subspecies. In the apiary with A. m. mellifera, hybrid colonies have been identified. A method based on the analysis of polymorphisms of the tRNAleu-COII locus and microsatellite nuclear DNA loci has been developed to identify the dark forest bee A. m. mellifera and does not allow one to differentiate subspecies from C (A. m. carnica and A. m. ligustica) and O (A. m. caucasica) evolutionary lineages from each other. The second stage was the assessment of the allelic diversity of the csd gene. In the apiary containing colonies of A. m. mellifera (N = 15), 20 csd alleles were identified. In the apiary containing colonies of A. m. carnica (N = 44), 41 alleles were identified. Six alleles are shared by both apiaries. DNA diagnostics of bee diseases showed that the studied colonies are healthy. Based on the data obtained, a scheme was developed for obtaining primary material for honey bee breeding, which can subsequently be subjected to selection according to economically useful traits. In addition, the annual assessment of the allelic diversity of the csd gene will shed light on the frequency of formation of new allelic variants and other issues related to the evolution of this gene.
HUMAN GENETICS
The results of studies in Quaternary geology, archeology, paleoanthropology and human genetics demonstrate that the ancestors of Native Americans arrived in mid-latitude North America mainly along the Pacific Northwest Coast, but had previously inhabited the Arctic and during the last glacial maximum were in a refugium in Beringia, a land bridge connecting Eurasia and North America. The gene pool of Native Americans is represented by unique haplogroups of mitochondrial DNA and the Y chromosome, the evolutionary age of which ranges from 13 to 22 thousand years. The results of a paleogenomic analysis also show that during the last glacial maximum Beringia was populated by human groups that had arisen as a result of interaction between the most ancient Upper Paleolithic populations of Northern Eurasia and newcomer groups from East Asia. Approximately 20 thousand years ago the Beringian populations began to form, and the duration of their existence in relative isolation is estimated at about 5 thousand years. Thus, the adaptation of the Beringians to the Arctic conditions could have taken several millennia. The adaptation of Amerindian ancestors to high latitudes and cold climates is supported by genomic data showing that adaptive genetic variants in Native Americans are associated with various metabolic pathways: melanin production processes in the skin, hair and eyes, the functioning of the cardiovascular system, energy metabolism and immune response characteristics. Meanwhile, the analysis of the existing hypotheses about the selection of some genetic variants in the Beringian ancestors of the Amerindians in connection with adaptation to the Arctic conditions (for example, in the FADS, ACTN3, EDAR genes) shows the ambiguity of the testing results, which may be due to the loss of some traces of the “Beringian” adaptation in the gene pools of modern Native Americans. The most optimal strategy for further research seems to be the search for adaptive variants using the analysis of paleogenomic data from the territory of Beringia, but such genetic data are still very scarce.
Osteopetrosis (“marble bone”, ICD-10-78.2) includes a group of hereditary bone disorders distinguished by clinical variability and genetic heterogeneity. The name “osteopetrosis” comes from the Greek language: ‘osteo’ means ‘bone’ and ‘petrosis’ means ‘stone’, which characterizes the main feature of the disease: increased bone density caused by imbalances in bone formation and remodeling, leading to structural changes in bone tissue, predisposition to fractures, skeletal deformities. These defects, in turn, affect other important organs and tissues, especially bone marrow and the nervous system. The disease can be autosomal recessive, autosomal dominant, X-linked or sporadic. Autosomal dominant osteopetrosis has an incidence of 1 in 20,000 newborns and autosomal recessive one has 1 in 250,000. To date, 23 genes have been described, structural changes in which lead to the development of osteopetrosis. Clinical symptoms in osteopetrosis vary greatly in their presentation and severity. The mildest skeletal abnormalities are observed in adulthood and occur in the autosomal dominant form of osteopetrosis. Severe forms, being autosomal recessive and manifesting in early childhood, are characterized by fractures, mental retardation, skin lesions, immune system disorders, renal tubular acidosis. Clinical examination and review of radiographs, bone biopsy and genetic testing provide the bases for clinical diagnosis. The early and accurate detection and treatment of the disease are important to prevent hematologic abnormalities and disease progression to irreversible neurologic consequences. Most patients die within the first decade due to secondary infections, bone marrow suppression and/or bleeding. This article summarizes the current state of the art in this field, including clinical and genetic aspects, and the molecular pathogenesis of the osteopetrosis.
MEDICAL GENETICS
Asthma is a heterogeneous and often difficult to treat condition that results in a disproportionate cost to healthcare systems. Children with severe asthma are at increased risk for adverse outcomes including medication-related side effects, life-threatening exacerbations, and impaired quality of life. An important therapeutic focus is to achieve disease control, which is supposed to involve a personalized approach to treatment of asthma of any severity. Asthma is a multifactorial disease with a significant genetic determinant, however, the inheritance of asthma has not been fully elucidated. Polymorphic genes of inflammatory mediators, including cytokines, play an important role in developing various disease forms. In the current study, large-scale original data on the prevalence of cytokine gene genotypes (IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, IL31, IL33, IFNG, TNFA) among Russian children with asthma in Krasnoyarsk region have been obtained. Genotyping was carried out using real-time PCR. We identified markers predisposing to the development of different variants of the course of childhood asthma: the CT genotype and T allele of IL4 rs2243250 are associated with asthma (p < 0.05), especially in mild asthma and in controlled asthma. The TT genotype and allele T of IL13 rs1800925 are associated with severe and uncontrolled asthma (p < 0.05). The AA genotype of IL17A rs2275913, the TT genotype of IFNG rs2069705 and allelic A variants of TNFA rs1800629 are associated with mild asthma, and the TT genotype of IFNG rs2069705 is additionally associated with controlled asthma. The results obtained will supplement information on the prevalence of polymorphic variants of the cytokine genes in the Russian population and in asthma patients with different disease courses, which is likely to be used in order to shape a plan for Public Health Authority to prevent the development of severe uncontrolled asthma and to optimize personalized therapy.
Discovery of molecular mechanisms of primary osteoporosis development is fundamental to understand the pathogenesis of musculoskeletal diseases in general and for identifying key links in the genetic and epigenetic regulation of bone remodelling genes. The number of identified molecular genetic markers for osteoporosis is increasing but there is a need to describe their functional interactions. These interactions have been determined to be associated with the control of expression of a number of transcription factors and the differentiation of mesenchymal stem cells through the pathway of osteoblastogenesis or adipogenesis, and monocytic precursors through the pathway of osteoclastogenesis. The results of epigenetic studies have significantly increased the understanding of the role of post-translational modifications of histones, DNA methylation and RNA interference in the osteoporosis pathogenesis and in bone remodelling. However, the knowledge should be systematised and generalised according to the results of research on the role of epigenetic modifiers in the development of osteoporosis, and the influence of each epigenetic mechanism on the individual links of bone remodelling during ontogenesis of humans in general, including the elderly, should be described. Understanding which mechanisms and systems are involved in the development of this nosology is of interest for the development of targeted therapies, as the possibility of using microRNAs to regulate genes is now being considered. Systematisation of these data is important to investigate the differences in epigenetic marker arrays by race and ethnicity. The review article analyses references to relevant reviews and original articles, classifies information on current advances in the study of epigenetic mechanisms in osteoporosis and reviews the results of studies of epigenetic mechanisms on individual links of bone remodelling.
POPULATION GENETICS
Arid habitats have recently attracted increasing attention in terms of biodiversity research and the disco very of new bacterial species. These habitats are among the target ecosystems suitable for isolating new strains of actinobacteria that are likely to produce new metabolites. This paper presents the results on the isolation of actinobacteria from soils of the dry steppe zone of the Selenga Highlands, the characterization of their taxonomic diversity, as well as ecological and trophic properties. The bacterial counts on ISP 4 medium ranged from 6.6 × 105 to 7.1 × 106 CFU/g. The highest bacterial counts were observed in the subsurface and middle horizons of the studied soils. 28 strains of Gram-positive bacteria represented by thin-branched mycelium, coccoid and bacilliform forms were isolated. According to the results of 16S rRNA gene analysis, the isolated strains were representatives of Streptomyces, Arthrobacter, Glycomyces, Kocuria, Microbacterium, Micromonospora, Nocardioides, Pseudarthrobacter, and Rhodococcus (Actinomycetota). One isolate that showed low 16S rRNA gene sequence similarity with previously isolated and validly described species was a new species of the genus Glycomyces. It was shown that all tested strains are mesophilic, prefer neutral or slightly alkaline conditions, have growth limits in the temperature range of 5–45 °C and pH 6–9. The optimal NaCl concentration for growth of most strains was 0–1 %. The strains under study were capable of utilizing a wide range of mono- and disaccharides and polyatomic alcohols as a carbon source. The isolated strains were capable of using both organic (proteins and amino acids) and inorganic (ammonium salts and nitrates) compounds as nitrogen sources. The examinations of extracellular enzymes showed that all isolates were capable of producing catalase and amylase; 78.6 % of the total number of isolates produced protease and lipase; 53.6 %, cellulase; and 28.6 %, urease. The data obtained expand current knowledge about the diversity of microbial communities in soils of the Selenga Highlands and also confirm the potential of searching for new actinobacteria species in these soils.
CURRENT BIOTECHNOLOGICAL METHODS
The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pETTrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage.