Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins.

Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one.

The Role of Carbonic Anhydrase αCA4 in Photosynthetic Reactions in Studied, Using the Cas9 and T-DNA Induced Mutations in Its Gene.

An homozygous mutant line of with a knocked out gene encoding thylakoid carbonic anhydrase αCA4 was created using a CRISPR/Cas9 genome editing system. The effects of the mutation were compared with those in two mutant lines obtained by the T-DNA insertion method. In αCA4 knockouts of all three lines, non-photochemical quenching of chlorophyll fluorescence was lower than in the wild type (WT) plants due to a decrease in its energy-dependent component.

Increasing the Efficiency of the Accumulation of Recombinant Proteins in Plant Cells: The Role of Transport Signal Peptides.

The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

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 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.

Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.

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 cells. To solve this problem, numerous methods and approaches have been designed and developed.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of .

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 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.

Cytological Techniques to Study Cytomixis in Plant Male Meiosis.

In this chapter we describe cytological techniques to study cytomixis, a process of nuclear migration between plant cells, in squashed plant male meiocytes of Nicotiana tabacum and Secale cereale. To perform immunostaining or fluorescence in situ hybridization (FISH) on meiotic cells involved in cytomixis common protocols are modified. During preparation of specimens for subsequent cytological analysis, it is necessary not only to make DNA and proteins accessible to DNA probes and antibodies, but also to preserve cell cytoplasm.

CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells.

Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cell lines carrying one to three gfp gene copies with a binary vector containing genes coding for Cas9 and guide RNAs targeting the gfp gene.

The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions.

The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response.

FlowerMorphology: fully automatic flower morphometry software.

The software FlowerMorphology is designed for automatic morphometry of actinomorphic flowers. The novel complex parameters of flowers calculated by FlowerMorphology allowed us to quantitatively characterize a polyploid series of tobacco. Morphological differences of plants representing closely related lineages or mutants are mostly quantitative.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response.

Cytoskeletal mechanisms in positioning of the second-division spindles and meiotic restitution in tobacco (Nicotiana tabacum L.) microsporogenesis.

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.

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues.

Cytomixis in the cereal (Gramineae) microsporogenesis.

The specific features in behavior of the nuclei and chromatin migrating through cytomictic channels as well as in formation of micronuclei in the cereal microsporogenesis have been studied. Immunofluorescence microscopy has allowed for demonstration that the tubulin cytoskeleton does not play a significant role in the intercellular migration of nuclei. Potential involvement of the actin cytoskeleton and SUN-KASH linker complexes in cytomixis is discussed.

Oral Immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10.

Two lines of transgenic carrot plants producing Mycobacterium tuberculosis proteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.

MatrixCatch--a novel tool for the recognition of composite regulatory elements in promoters.

Background: Accurate recognition of regulatory elements in promoters is an essential prerequisite for understanding the mechanisms of gene regulation at the level of transcription. Composite regulatory elements represent a particular type of such transcriptional regulatory elements consisting of pairs of individual DNA motifs. In contrast to the present approach, most available recognition techniques are based purely on statistical evaluation of the occurrence of single motifs.

New insights into cytomixis: specific cellular features and prevalence in higher plants.

The phenomenon of intercellular migration of nuclei in plant tissues (cytomixis) was discovered over a century ago, which has been followed by numerous attempts to clarify the essence of this process as well as to determine its causes and consequences. Most attention of researchers has been paid to cytomixis in microsporogenesis, since the transfer of part of genetic material between microsporocytes may influence the ploidy level of the produced pollen and, presumably, have an evolutionary significance. This review compiles the data on cytological pattern of cytomixis and proposes a scheme as to how cytomictic channels are formed and function in angiosperms.

Spontaneous spectinomycin resistance mutations detected after biolistic transformation of Daucus carota L.

Spectinomycin resistant mutant carrot (Daucus carota L.) callus lines detected in the experiments on biolistic transformation of plastome were analyzed. It has been found that this antibiotic resistance is determined by point nucleotide substitutions at two distinct sites of the chloroplast gene rrn16, coding for 16S rRNA, namely, G1012T, G1012C, and A1138G.

An ultrastructural study of cytomixis in tobacco pollen mother cells.

Intercellular chromatin migration (cytomixis) in the pollen mother cells of two tobacco (Nicotiana tabacum L.) lines was analyzed by electron microscopy during the first meiotic prophase. The maximal manifestation of cytomixis was observed in the pachytene.