Cauliflower Mosaic Virus Promoter Research Articles

Cassava torrado-like virus encodes a gene that facilitates the mechanical transmission to Nicotiana benthamiana of Cassava virus X

Cassava-torrado-like virus (CsTLV) is a bipartite single-stranded RNA virus belonging to the family Secoviridae. The virus has been reported in Brazil and Colombia, and is usually found in mixed infections, and in plants affected by Cassava Frogskin Disease (CFSD), an endemic cassava disease in the Americas. Genome analysis of CsTLV identifies a gene in RNA2 implicated in pathogen movement in other torradoviruses. This gene (RNA2-ORF1) and another one no related to virus movement (Maf/Ham1) were amplified by PCR and cloned into constructs under the 35S promoter of cauliflower mosaic virus (CaMV), which then were transfered to agrobacterium binary vectors. When agro-infiltrated in Nicotiana benthamiana plants, only RNA2-ORF1 had a positive effect on the mechanical inoculation of cassava virus X (CsVX), a potexvirus that has a low rate of mechanical infection in N. benthamiana. Efficiency of CsVX mechanical transmission was measured by the number of infected plants, presence of symptoms, and titers of CsVX as measured by ELISA, two weeks after infection. On average, CsVX could infect 2.3 times more plants when these were previously agro-infiltrated with A. tumefaciens binary vector encoding CsTLV RNA2-ORF1. We conclude that the novel secovirid CsTLV associated with leaf spot symptoms in cassava, encodes a gene that could enhance other viral infections in N. benthamiana. Further studies are required to elucidate this effect and its role in mixed infections, often observed in cassava plants affected by CFSD.

Stable and reproducible expression of bacterial ipt gene under the control of SAM-specific promoter (pKNOX1) with interference of developmental patterns in transgenic Peperomia pellucida plants.

Reproducible and stable transgene expression is an important goal in plant basic research and applications. Hence, we report the first stable expression of bacterial transgenes in medicinal plant, Peperomia pellucida (L.) Kunth. Two key elements relevant to the dynamic expression of the bacterial cytokinin biosynthesis gene, ipt (isopentenyltransferase) were examined. First, by designing a specific expression cassette driven by a tissue-specific promoter for the required levels of gene expression in the particular function of development, and second by using P. pellucida as a model plant due to its short developmental cycle that supported expedient tracking of transgene expression in the progeny. Transgenic frequencies of ipt gene obtained from different expression cassettes of pKNOX1 for tissue-specific promoter in shoot apical meristem were compared with the cauliflower mosaic virus (CaMV35S) promoter (p35S), a constitutive promoter investigated for T3 generation. It was clearly shown that transgenic plants with pKNOX1 showed percentage of survivals in T3 at about 2.2 folds more than those of p35S-transgenic. Transgenic P. pellucida under controllable expression of pKNOX1 showed increased leaf and seed size with a high percentage of fertile seed, whereas transgenic plants with p35S showed phenotypic features of bushy and small leaves, sterile pollen and lower reproductive fitness. Quantitative examination of ipt-positive gene expression in T3 generation of transformants with pKNOX1 were 100% (line k-14) and 50% (line k-20), while 33.3% was observed in transgenic line c-11 with p35S. Interestingly, the endogenous cytokinin biosynthesis gene (ipt3) was significantly upregulated (2-3 folds higher) in pKNOX1-transformants. The overall relative mRNA expression of bacterial ipt gene and overproducing of cytokinin contents (t-ZR and 2-iP) detected in p35S-transformants caused abnormality and low percentages of transgene reproducible Interestingly, pKNOX1-transgenic plants tended to maintain chlorophyll contents 4-5 folds and extending the developmental cycle to 12.4 weeks (wk), which was 2 folds more than wildtype (5.8 wk) and p35S-transformants (7.4 wk). The promotor effect on stable and reproducible transgene-expressions demonstrated prominent features of P. pellucida and also empowered further omics studies.

Genetic transformation of clone rootstock of stone fruits 146-2 using the green fluorescent protein reporter gene

For the form of dwarf winter-hardy clonal rootstocks of stone crops146-2 (Prunus pumilaL.xP.tomentosaThunb.), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated byAgrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by theA. tumefaciensstrain CBE21, with the binary vector pBINmGFP5ER containing thenptIIencoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reportergfpgene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration ofnptIIandgfpinto transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to producePlum pox virus(PPV) resistant plants.

Root-preferential expression of Newcastle virus glycoproteins driven by NtREL1 promoter in tobacco hairy roots and evaluation of oral delivery in mice.

Newcastle disease virus (NDV) is a lethal virus in avian species with a disastrous effect on the poultry industry. NDV is enveloped by a host-derived membrane with two glycosylated haemagglutinin-neuraminidase (HN) and Fusion (F) proteins. NDV infection usually leads to death within 2-6days, so the preexisting antibodies provide the most critical protection for this infection. The HN and F glycoproteins are considered the main targets of the immune system. In the present study, two constructs harboring the HN or F epitopes are sub-cloned separately under the control of a root-specific promoter NtREL1 or CaMV35S (35S Cauliflower Mosaic Virus promoter) as a constitutive promoter. The recombinant vectors were transformed into the Agrobacterium tumefaciens strain LBA4404 and then introduced to tobacco (Nicotiana tabacum L.) leaf disk explants. PCR with specific primers was performed to confirm the presence of the hn and f genes in the genome of the regenerated plants. Then, the positive lines were transformed via non-recombinant A. rhizogenes (strain ATCC15834) to develop hairy roots.HN and F were expressed at 0.37% and 0.33% of TSP using the CaMV35S promoter and at 0.75% and 0.54% of TSP using the NtREL1 promoter, respectively. Furthermore, the mice fed transgenic hairy roots showed a high level of antibody responses (IgG and IgA) against rHN and rF proteins.

Monitoring of plant raw materials and feed for animal in 2020 for the presence of GM-ingredients

The creation and the use of genetically modified products has become a tendency in the development of agricultural and food technologies. The area of agricultural land under genetically modified plants is constantly growing. Todays the process of using GMOs and the expediency of their creation is a debatable issue. The modification of the genome of traditional agricultural cultures gives them resistance to pesticides, pests, diseases, which cause to the significant an increase of harvest and improved quality and taste characteristics. However, the effects of GMOs on the environment and the body of animals and humans have not been fully studied, and therefore the thoughts of scientists are differ on the benefits and risks of genetic engineering. Recently, the scientific literature has data on the negative effects of GMOs on animals and humans, in particular, on the morphofunctional state of organs and systems of the body, reproductive function, immune status, biochemical parameters of blood and urine. Every year the number of new genetically modified plant lines is growing, so today, the need in research of plant raw materials and feed for animal on the presence of GMOs is very important and actual. The article presents the results of research on the detection of GM ingredients in plant raw materials and in products of its processing, feed for productive and unproductive animals, etc. In 2020, 1215 samples were investigated by polymerase chain reaction with detection in real-time (PCR-RT), and it was found out that only 0.3 % from total amount were positive. From the studied samples, the most positive samples were found in samples of rapeseed, soybeans and feed for productive animals. In 27 samples of rapeseed, the number of positive samples was 7.4 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium, and genes Pat and EPSPs. In 6 samples of soybean, the number of positive samples was 16.7 %, in them were detected the target sequences of the 35S promoter of cauliflower mosaic virus (CaMV) and the terminator NOS (T-NOS) T of the plasmid Agrobacterium. Also there was found GM ingredients in compound feeds for farm animals and poultry, in 6 samples the number of positive samples was 16.7 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium. Conducted studies indicate that transgenic plants are in circulation in the agricultural market, so it is necessary to constantly control animal feed, plant materials and seeds for the presence of GM sources.

Characterization of a strong constitutive promoter from paper mulberry vein banding virus.

Paper mulberry vein banding virus (PMVBV), a member of the genus Badnavirus in the family Caulimoviridae, infects paper mulberry (Broussonetia papyrifera), a dicotyledonous plant. Putative promoter regions in the PMVBV genome were tested using recombinant plant expression vectors, revealing that the promoter activity of three genome fragments was about 1.5-fold higher than that of the 35S promoter of cauliflower mosaic virus in Nicotiana benthamiana. In transformed transgenic Arabidopsis thaliana plants, these promoter constructs showed constitutive expression. Based on the activity and gene expression patterns of these three promoter constructs, a fragment of 384 bp (named PmVP) was deduced to contain the full-length promoter of the PMVBV genome. The results suggest that the PMVBV-derived promoter can be used for the constitutive expression of transgenes in dicotyledonous plants.

DISTRIBUTION OF TRANSGENIC PLANTS IN UKRAINE – CURRENT STATE

Scientific achievement in molecular biology and genetic engineering have made it possible to create new methods of selection k that are based on genome modification. Todays, more and more genetically modified plants are being intensively tested in the field, but their influence has not yet been fully studied on the environment, biodiversity, human health, and the animal body.
 The article analyzes the results of research on the presence of GM ingredients in plant raw materials, processed products, feed for productive and unproductive animals, etc. for the period 2019-2020. Studies on the presence of GMOs were performed by the method of polymerase chain reaction in real-time (PCR-RT), according to DSTU ISO 21569: 2008 (ISO 21569: 2005, IDT), DSTU ISO 21571: 2008 (ISO 21571: 2005, IDT).
 In 2019, 1134 samples were tested and the most positive were found in rapeseed and soybeans. In the study of rapeseed (31 samples) the number of positive samples was 6.5 %, in them were the target sequences of the terminator NOS (T-NOS) TІ plasmid Agrobacterium tumefaciens and Pat gene. In the study of soybeans (15 samples), the number of positive samples was 6.7 %, in them were the target sequences of the 35S promoter of cauliflower mosaic virus (CaMV) and the terminator NOS (T-NOS) TІ plasmid Agrobacterium tumefaciens.
 In 2020, 1215 samples were tested and was found positive samples in samples of rapeseed, soybeans and feed for productive animals. In the study of rapeseed (27 samples) the number of positive samples was 7.4 %, in them were detected the target sequences of the terminator NOS (T-NOS) TІ plasmid Agrobacterium tumefaciens, and genes Pat and EPSPs. In the study of soybeans (6 samples), the number of positive samples was 16.7 %, in them were the target sequences of the 35S promoter of cauliflower mosaic virus (CaMV) and the terminator NOS (T-NOS) T of the plasmid Agrobacterium. In the study of feed for poultry (6 samples) the number of positive was 16.7 %, in them were the target sequences of the terminator NOS (T-NOS) TІ plasmid Agrobacterium tumefaciens.

Induction of resistance to sugarcane mosaic virus by RNA interference targeting coat protein gene silencing in transgenic sugarcane.

Sugarcane mosaic virus (SCMV) is a serious disease of monocotyledonous plants, including sugarcane, causing deterioration in both growth and productivity. RNA interference (RNAi) inhibits gene expression through RNA-mediated sequence-specific interactions and is considered an effective approach to control viral infection in plants. In this study, the SCMVCp gene encoding the coat protein (CP) was inserted into the pGreen-0179 plasmid in both sense and antisense orientations. Cauliflower mosaic virus (CaMV) and Zea mays ubiquitin (Ubi) promoters were selected to drive the transcription of the intron-hairpin constructs, called HpSCMVCp-CaMV and HpSCMVCp-Ubi, respectively. Transgenic sugarcane expressing these constructs was generated through Agrobacterium-mediated transformation. This transformation method produced a high percentage of transgenic plants for both HpSCMVCp-CaMV and HpSCMVCp-Ubi, as confirmed by PCR analysis. Southern blotting revealed a single stable insertion of the DNA target in the genome of transgenic sugarcane lines. After artificial virus infection, lines that developed mosaic symptoms were classified as susceptible, whereas those that remained green without symptoms were classified as resistant at 42days post-inoculation. Immunoblotting revealed CP expression at 37kDa in susceptible and non-transgenic sugarcane, but not in resistant lines. RT-PCR analysis confirmed viral Cp and Nib gene expression in susceptible lines and their absence in resistant lines. Interestingly, upon comparison of effectivity, CaMV and Ubi promoter-driven gene expression resulted in 57.69% and 82.35% resistant sugarcane lines, respectively. Thus, we concluded that RNAi is effective for inducing resistance against SCMV and that the Ubi promoter is an effective promoter for producing transgenic sugarcane.

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

Comparison of gfp Gene Expression Levels after Agrobacterium-Mediated Transient Transformation of Nicotiana rustica L. by Constructs with Different Promoter Sequences

Promoters are key elements regulating gene expression levels, therefore their selection is an important step in genetic engineering research. The reporter gene gfp, which encodes green fluorescent protein (GFP), was transiently expressed in leaf tissues of Aztec tobacco Nicotiana rustica L. Compared to other species of the Nicotiana genus, Aztec tobacco has a large potential for expression of heterologous proteins, a large vegetative biomass, can be easily infiltrated, and is unpretentious in cultivation. Six genetic constructs were used with different promoter sequences: the 35S promoter of Cauliflower Mosaic Virus (35S CaMV), the double-enhanced 35S promoter (D35S CaMV), promoters of the RbcS1B and RbcS2B genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isolated from Arabidopsis thaliana (L.) Heynh., and promoters of the LHB1B1 and LHB1B2 genes from A. thaliana encoding chlorophyll a/b binding proteins. The gfp gene expression was detected visually, spectrofluorimetrically, and by protein content (Bradford assay) on the seventh day after infiltration. The highest level of expression was observed using the double-enhanced 35S promoter (D35S CaMV) and the lowest using the LHB1B1 gene promoter.

Developmental phenotypes of Arabidopsis plants expressing phosphovariants of AtMYB75

ABSTRACT The Arabidopsis transcription factor Myeloblastosis protein 75 (MYB75, AT1G56650) is a well-established transcriptional activator of genes required for anthocyanin and flavonoid production, and a repressor of lignin and other secondary cell wall biosynthesis genes. MYB75 is itself tightly regulated at the transcriptional, translational and post-translational levels, including protein phosphorylation by Arabidopsis MAP kinases Examination of the behavior of different phosphovariant versions of MYB75 in vitro and in vivo revealed that overexpression of the MYB75T131E phosphovariant had a particularly marked effect on global changes in gene expression suggesting that phosphorylated MYB75 could be involved in a broader range of functions than previously recognized. Here, we describe a range of distinct developmental phenotypes observed among Arabidopsis lines expressing various phosphovariant forms of MYB75. Expression of either MYB75T131E or MYB75T131A phosphovariants, from the endogenous MYB75 promoter, in Arabidopsis myb75− mutants (Nossen background), resulted in severely impaired germination rates, and developmental arrest at early seedling stages. Arabidopsis plants overexpressing MYB75T131E from a strong constitutive Cauliflower mosaic virus (CaMV35S) promoter displayed slower development, with delayed bolting, flowering and onset of senescence. Conversely, MYB75T131A -overexpressing lines flowered and set seed earlier than either Col-0 WT controls or other MYB75-overexpressors (MYB75WT and MYB75T131E ). Histochemical analysis of mature stems also revealed ectopic vessel development in plants overexpressing MYB75; this phenotype was particularly prominent in the MYB75T131E phosphovariant. These data suggest that MYB75 plays a significant role in plant development, and that this aspect of MYB75 function is influenced by its phosphorylation status.

Screening brinjal (Solanum melongena) accessions conserved in the National Genebank collected from states adjoining Bangladesh for adventitious presence of EE1 event

Systematic management of Plant Genetic Resources (PGRs) is the key to sustainable agriculture for food and nutritional security and in mitigating climate change. The National Genebank (NGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, acts as a repository of PGRs for future use. This study aimed at screening for the adventitious presence of transgenes in brinjal (Solanum melongena L.) accessions conserved in the NGB. The study targeted the collections made during 2007-2016 from areas adjacent to Bangladesh (Assam, Meghalaya, Mizoram, Tripura and West Bengal), where field trials of Bt brinjal event EE1 were conducted during 2005-2012 and commercial cultivation of this event was permitted in 2013.There could be an apprehension of both unintentional introgression and transboundary movement through borders. Adventitious presence of transgenes was checked in a set of 96 accessions of brinjal employing Polymerase Chain Reaction (PCR) and real-time PCR assays. As event EE1 carries cry1Ac gene for insect resistance with Cauliflower Mosaic Virus (CaMV) promoter (P-35S) and marker genes (nptII and aadA), so these genetic elements were targeted for qualitative GM testing. Based on the test results, transgenes were not detected in brinjal accessions conserved in NGB. Our study showed that brinjal and wild species collected from adjoining areas of Bangladesh, post field trials and release, do not contain the event EE1. The study presents an efficient and reliable method to ensure conservation of GM-free germplasm in the NGB.

Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum L.) with human lactoferrin gene and analysis of transgenic lines

Aim. Obtaining of tobacco lines (Nicotiana tabacum L.) stably transformed with human lactoferrin gene (hLf), analysis of transgenic lines. Methods. Agrobacterium-mediated transformation of tobacco was carried out with the use of pBin35LF plasmid containing human lactoferrin gene under control of 35S promoter of cauliflower mosaic virus (CaMV 35S) and selective npt II gene encoding neomicyne phosphotransferase II providing the resistance to kanamycin. The selection of transgenic lines was carried out for 3 months on MS medium supplemented with 100 mg/l of kanamycin. Integration of lactoferrin gene was confirmed with the use of PCR with primers specific to hLf gene. The investigation of karyotype of transgenic lines was carried out after the staining of the root cells with 1 % solution of acetoorseine. Results. After selection, 2 transgenic tobacco lines with confirmed integration of hLf gene were obtained. The transformation efficiency was 6.4 %. The chromosome number in transgenic and control lines was 2n=48. Conclusions. The obtained transgenic tobacco lines have the similar morphology and karyotype to control lines, so they could be considered to use as plant systems for the expression of recombinant human lactoferrin.
 Keywords: human lactoferrin gene hLf, Nicotiana tabacum L., Agrobacterium-mediated transformation, PCR, chromosomes, transgenic plants.

Obtaining Transgenic Potato Plants Expressing the Human Lactoferrin Gene and Analysis of Their Resistance to Phytopathogens

The human lactoferrin gene was transferred into genomes of several potato (Solanum tuberosum) cultivars of Ukrainian breeding using the Agrobacterium-mediated transformation. The plasmid vector pBIN35LF carrying the human lactoferrin gene hLf controlled by the 35S cauliflower mosaic virus promoter (CaMV35S) and the octopine synthase terminator, as well as the selective marker neomycin phosphotransferase II gene (nptII) conferring the resistance to kanamycin, was used. As a result of selection, 44 lines of Vernisage, 26 lines of cv. Levada, 25 lines of cv. Svitanok Kyivskyi, and 16 lines of cv. Zarevo cultivars resistant to 100 mg/L of kanamycin were obtained. PCR and Western blot analyses were carried out for transformed lines with primers specific to the hLf gene and a monoclonal antibody against lactoferrin to confirm the transgenic nature of selected tomato plants and hLf gene expression. The selected transgenic potato lines were tested for resistance to bacterial and fungal phytopathogens. The disk diffusion assay revealed that the juice of transgenic potato lines possesses an antibacterial effect against phytopathogenic bacteria Ralstonia solanacearum (causing potato brown rot) and Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot). The resistance of transgenic potato plants to late blight was investigated by in vitro infection of plants with the Phytophthora infestans isolate. As a result, it was found that the obtained transgenic potato lines have enhanced resistance to P. infestans as compared to the control. Thus, the obtained data show that the transfer of the hLf gene into the potato genome enhances potato’s resistance to bacterial and fungal pathogens.

Comparative analysis of Constitutive and fiber-specific promoters under the expression pattern of Expansin gene in transgenic Cotton.

Promoters are specified segments of DNA that lead to the initiation of transcription of a specific gene. The designing of a gene cassette for plant transformation is significantly dependent upon the specificity of a promoter. Constitutive Cauliflower mosaic virus promoter, CaMV35S, due to its developmental role, is the most commonly used promoter in plant transformation. While Gossypium hirsutum (Gh) being fiber-specific promoter (GhSCFP) specifically activates transcription in seed coat and fiber associated genes. The Expansin genes are renowned for their versatile roles in plant growth. The overexpression of Expansin genes has been reported to enhance fiber length and fineness. Thus, in this study, a local Cotton variety was transformed with Expansin (CpEXPA1) gene, in the form of two separate cassettes, each with a different promoter, named as 35SEXPA1 and FSEXPA1 expressed under CaMV35S and GhSCFP promoters respectively. Integration and Spatiotemporal relative expression of the transgene were studied in an advanced generation. GhSCFP bearing transgene expression was significantly higher in Cotton fiber than other plant parts. While transgene with CaMV35S promoter was found to be continually expressing in all tissues but the expression was lower in fiber than that expressed under GhSCFP. The temporal expression profile was quite interesting with a gradual increasing pattern of both constructs from 1DPA (days post anthesis) to 18DPA and decreased expression from 24 to 30 DPA. Besides the relative expression of promoters, fiber cellulose quantification and fluorescence intensity were also observed. The study significantly compared the two most commonly used promoters and it is deduced from the results that the GhSCFP promoter could be used more efficiently in fiber when compared with CaMV35S which being constitutive in nature preferred for expression in all parts of the plant.

Genetic transformation of Nicotiana tabacum with the yeast TPS1 and TPS2 genes involved in trehalose synthesis

Aim. The aim of the study was the creation of vector constructions with yeast genes of trehalose synthesis TPS1 and TPS2 and their using for Agrobacterium-mediated transformation of N. tabacum. Methods. Strain of Agrobacterium tumefaciens GV3101 carrying vector constructions — pGWВ2-TPS1 and pGWВ2-TPS2 with TPS1 and TPS2 target genes respectively under 35S promoter of cauliflower mosaic virus and selectable hpt gene of hygromycin phosphotransferase has been used for plants transformation. N. tabacum leaf explants were used for Agrobacterium-mediated transformation. The medium with the addition of hygromycin was applied to transgenic lines selection. Results. The created vector constructions pGWВ2-TPS1 and pGWВ2-TPS2 has been used in genetic N. tabacum transformation. Target genes TPS1 and TPS2 were integrated applied of Agrobacterium-mediated transformation and transgenic lines of N. tabacum were selected with addition at 25 mg/L hygromycin into the medium. Molecular analysis confirmed the transgenic nature of selected lines. Conclusions. Sensitivity of selected lines for sugars content into the medium was established for shoots and roots formation of tobacco plants. Conditions for increase of transformation frequency and rooting of transgenic lines of plant after integrated of target TPS1 and TPS2 genes were investigated. Keywords: trehalose, yeast genes TPS1, TPS2, genetic transformation, Nicotiana tabacum.

Inducible Gene Expression Systems in Plants

Gene expression is one of the major applications in plant molecular biology, which plays important role in theoretical and agricultural application researches. Due to the weak foundation and lack of tools, most of the early studies used constitutive promoters, such as the most commonly used 35S promoter of cauliflower mosaic virus, to overexpress. With the further researches, people have more understanding of the molecular biology of gene expression, and then derived better and more tools. In order to have spatiotemporal and quantitative fine-tune expression, several inducible gene expression systems have been developed. Classified according to different inducers, the commonly used expression systems are as such, the tetracycline induced, the steroid induced, the ethanol induced, the insecticide induced, and the copper induced. These systems have different working principles because of the different factors and components used; at the same time, there are also commonalities in each of them, they are usually composed of two elements, the activation element and the response element. In this paper, several plant inducible expression systems are reviewed, and their characteristics are analyzed, reviewed and prospected.

Studying the effect of tissue-specific expression of theK1gene encoding LysM-receptor-like kinase on the development of symbiosis in peas

To study the role of pea LysM receptor-like kinase K1 in the coordination of the infection process, starting in epidermis and nodule organogenesis in the root cortex of plants, during the development of rhizobium-legume symbiosis, the genetic constructs in whichK1gene was cloned under the control of tissue-specific promoter pLeEXT1 of tomatoLycopersicon esculentumextensin gene and the constitutive promoter of cauliflower mosaic virus (CaMV35S, cauliflower mosaic virus 35S) were obtained. During the transformation of the Nod-mutant line, thek1-1,with two types of constructs, the restoration of nodule formation was observed, which indicated the possible participation of K1 in the control not only early, but also later stages of symbiosis development in pea.

Genetic constructs creation using Golden Gate cloning method

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

A necessity of introduction of the system of registration of GMO is for Ukraine

The article presents the main methodological approaches and criteria for the necessity of development and introduction of the State Registration of GMOs sources in Ukraine. The list of analytical researches that are to be carried out at the registration of the GMO source, expert evaluation of GMO identification methods, reproduction of detection methods, identification and quantification of GMOs are given. Submitted and analyzed legislative documents regulating the procedure for registration and control of GMO circulation in the countries of the European Union and Ukraine. DNDKI of veterinary preparations and fodder supplements on the tasks of the State Committee of Veterinary Medicine of Ukraine during 2010-2018 a number of normative documents were developed regarding procedures related to the use and circulation of feed, feed additives, premixes and veterinary preparations that are composed, containing or made using genetically modified sources in Ukraine: the procedure for state registration, the procedure for carrying out the state veterinary and sanitary examination of GMOs for sources of forage feed additives and veterinary preparations ativ plan state monitoring GMO sources of feed, feed additives, premixes for livestock and poultry. The procedure determines the procedure for state registration of GMOs for sources of feed, feed additives and veterinary preparations containing genetically modified organisms or obtained from their use. It is envisaged that the state register of GMO sources is conducted in the form approved by the State Service of Ukraine for Food Safety and Consumer Protection. On the basis of the analysis, a draft list of feeds, feed materials, protein-vitamin concentrates and premixes that have to be monitored for GMO content has been developed. This project list includes soy, corn, rape and products of their processing, fodder, feed mixes, feed concentrates, etc. In the SSRCI of veterinary drugs and supplements, in 2018, the “Guidelines for identifying the GM soya line (Soybean MON40-3-2) GTS 40-3-2” were developed and approved. “Methodological recommendations are intended to identify the GM soya line (Soybean MON40-3 -2) GTS 40-3-2 (Genetically modified soybean that contains – genetically modified soybean containing (cp4 epsps gene inserted to confer tolerance to herbicide glyphosate) cp4 epsps gene inserted to provide resistance to glyphosate herbicide (glyphosate) in plant material, feed and food products by polymerase chain reaction in real time (PCR-RF )”. The qualitative determination of GMOs is based on the discovery of genetically modified (GM) targeted regulatory sequences of the 35S Cauliflower Mosaic Virus (CaMV) promoter or FMV promoter and/or the NOS (T-NOS) T1 terminator of Agrobacterium tumefaciens plasmids that are introduced into the genetic constructs of GM based on the use of the multiplex polymerase chain reaction method to obtain real-time research results – PCR RF.