Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.
Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic “safe harbors”) using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of “euchromatization” of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.
Behavior of nucleolus during the nuclear migration between plant cells (cytomixis) is studied for the first time in the tobacco male meiosis. As is shown, the nucleolus is located in a nonrandom manner in the migrating nuclei. In the majority of cases, the nucleolus resides on the nuclear pole strictly opposite to the cytomictic channel. Owing to this localization, the nucleolus extremely rare enters the recipient cell, so that the nucleolar material is in most cases undetectable in the micronuclei formed after cytomixis. When a whole nucleus migrates from a donor cell to recipient, the nucleolus can leave the nucleus and remain in the donor cells either alone or with a small amount of chromatin. The causes underlying a nonrandom location of the nucleolus in cytomictic cells are discussed. It is assumed that the nucleolar material contacts the cytoplasmic cytoskeleton, which prevents migration of the nucleolus into another cell within the nucleus. The potential use of cytomixis as a model for studying the nuclear motion is discussed.
The structure of cytoskeleton in the tobacco male meiocytes involved in the migration of nuclei between cells (cytomixis) is studied. The tubulin and actin components of cytoskeleton are examined using specific antibodies and phalloidin. The presence of microtubules and actin filaments inside cytomictic channels directly when the nuclei migrate through these channels is demonstrated for the first time. The actin and tubulin cytoskeleton is shown to retain its reticular structure in early prophase I before the beginning of cytomixis, during nuclear migration, and after completion of this process and emergence of micronuclei in the cytoplasm of recipient cells. It is demonstrated that if migration takes place in late prophase I, the nucleus leaves the perinuclear tubulin cage, encompassing it at this stage. After migration, the cytomictic micronuclei in recipient cell reside beyond the perinuclear tubulin cage in both meiotic prophase I and II. A microtubule-organizing activity of the migrated chromatin in meta-telophase I and II is demonstrated for the first time as well as the ability of this chromatin to form independent minispindles and radial microtubule arrays and contact the spindle of recipient cell. The role of cytomixis in production of aneuploid and unreduced pollen is discussed.
Microsporogenesis patterns of the polyploid (2n = 4x = 96) and diploid (2n = 2x = 48) Nicotiana tabacum L. (cv. Havana Petit line SR1) plants have been analyzed and compared. Four types of abnormal positions of the second-division spindles-tripolar, parallel, proximal, and fused-have been observed. Of these abnormalities, only tripolar (2.4%) and parallel (1.4%) spindles are observable in diploid plants. As for polyploids, the increased ploidy is accompanied by an increase in the incidence of tripolar (22.8%) and parallel (8.1%) spindle orientations and emergence of two remaining abnormalities (proximal and fused spindles, 3.3%). As has been shown, the spindle position abnormalities in diploid plants have no effect on the meiotic products, whereas both dyads and triads are detectable among the tetrads in polyploid plants. Analysis of cytoskeletal remodeling has allowed for the insight into the role of interzonal radial microtubule system in spindle positioning during the second division. The reason underlying the change in spindle positioning is disturbed polymerization-depolymerization processes and interdigitation of microtubule plus ends within the interzonal cytoskeleton system in late telophase I-interkinesis and prophase II. As has been demonstrated, fused second-division spindles are formed as a result of fused cytoskeletal structures in prophase-prometaphase II in the case when the nuclei are drawn abnormally close to one another.
Extreme environmental factors decrease seed setting and form unseeded or thinning panicles. Since meiosis underlies formation of gametes, it largely determines further processes associated with pollination, fertilization and development of a full-fledged seed. In this regard, studies meant to identify the peculiarities of male meiosis for plants cultivated in the severe natural and climatic conditions of Central Yakutia are relevant and of particular interest. The goal of this study was to identify the characteristics of male meiosis in oat (Avena sativa L.) varieties adapted to local conditions. We should emphasize that no studies of meiosis in common oats under the conditions of Central Yakutia have been performed earlier. We used four varieties of common oats (Pokrovsky, Vilensky, Pokrovsky 9, and Khibiny 2) as study material. All the varieties under analysis were hexaploids (2n = 6x = 42). The studies were conducted in 2020 at the Pokrovsky Department of Yakut Research Institute of Agriculture, in Khangalassky ulus of the Republic of Sakha (Yakutia) located in the middle taiga zone. The weather and climatic conditions during the growing season were characterized by an insufficient provision of soil with productive moisture at the early phases of grain and forage crop growth and development. To analyze meiosis, we used oat spikelets sampled when they were emerging into the tube. The spikelets were fixed on June 25-27, 2020 in duplicate using Carnoy’s solution. We analyzed microsporogenesis and pollen fertility on the temporary squashed preparations of meiocytes using the generally accepted method with 4% acetocarmine staining. The results were statistically processed using Statistica 7.0 (StatSoft. Inc., USA). The samples were compared through one-way ANOVA. The general cytological picture of meiosis in the oat varieties under study mostly corresponded to the conservative scenario typical of monocotyledonous plants. However, all the oat varieties showed some deviations from the general meiosis pattern. One of such deviations was cytomixis - chromatin migration as part of nuclei between microsporocytes through one or several cytomictic channels (Fig. 1, 2). As a rule, the phenomenon of cytomixis in microsporocytes was observed at middle prophase 1 at the pachytene stage (Fig. 1). Nuclei migration was also observed at late prophase 1, for example, at the diplotene stage. Chromatin movement between two adjacent microsporocytes clearly correlated with the formation of micronuclei and cytoplasts. Quite often, this process was also accompanied by nuclei pycnosis (Fig. 2). We should note that rarely we observed massive destructive cytomixis in microsporocytes and tapetum cells. When studying common oat cultivars, we also found microsporocytes with separately lying bivalents that were not included in the metaphase plate (Fig. 2), as well as chromosomal bridges (Fig. 2) at the anaphase-telophase 1 stage. The proportion of microsporocytes with meiotic deviations in all the four cultivars was small (Fig. 3) and varied from 2.99 to 3.23% (Table 1). We found no consequences of the revealed deviations at the tetrad stage. Probably, either most deviations were compensated for by intracellular mechanisms or defective microsporocytes were excluded from the microsporogenesis and eliminated. Besides, these deviations of meiosis did not significantly decrease pollen fertility (Table 1). The results indicate that the observed deviations of meiosis do not significantly contribute to seed formation in such an important grain fodder crop as common oats in the climatic conditions of Central Yakutia. The article contains 3 Figures, 2 Tables, 27References. The Authors declare no conflict of interest.
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