RU2799014C1 - Pro-smamp-d1 promoter from stellaria media l. plant for plant biotechnology - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a DNA fragment for genetic modification of a plant cell in order to express a recombinant gene in a plant. Also a genetic construct containing the specified DNA fragment is disclosed. A method for expressing a recombinant gene in a plant cell is disclosed.

EFFECT: invention makes it possible to efficiently express a recombinant gene in a plant.

3 cl, 10 dwg, 1 tbl, 6 ex

Description

The field of technology to which the invention belongs

The invention relates to the field of biology, molecular biology, biotechnology and plant genetic engineering. The invention can be used to create genetic constructs for the purpose of increased expression of recombinant genes and production of recombinant proteins. The invention can be used to produce recombinant proteins in plant cells by transient expression, to create transgenic plants producing recombinant proteins, to select transgenic cells, calluses, shoots and seedlings of plants on a nutrient medium with a selective agent. The present invention can be applied in basic plant biology research using forward and reverse genetics methods.

State of the art

A gene promoter is a fragment of genomic DNA that initiates the transcription of a gene. Promoters are essential for coordinated gene expression in plant cells under changing environmental conditions. In plants, full-length promoters of protein-coding genes consist of distal, proximal, and "core" promoter regions. The "core" region contains, as a rule, the sequence of the known cis-element TATA-box, localized at about - 40-20 bp. upstream (or to the left) of the transcription start site (TSS) where the proteins of the main transcription machinery (RNA Pol II, TATA-binding proteins and TBP-associated factors) start transcription (Hernandez-Garcia and Finer 2014). The change in gene expression is achieved due to the physical interaction between the main transcription mechanism and regulatory proteins (transcription factors) that bind the proximal promoter region adjacent to the "core". The distal promoter region, which is farthest from the TSS, can affect the efficiency of gene expression due to the three-dimensional mobility of DNA and its spatial proximity to the "core" region.

The functional architecture of a full-length promoter is determined by multiple regulatory elements (enhancers, silencers, insulators, and cis-elements) - specific nucleotide sequences for binding by regulatory proteins, distributed along the entire nucleotide sequence of the promoter. Interactions between various regulatory elements and their binding regulatory proteins play a major role in determining the efficiency and specificity of the eukaryotic promoter.

Isolated nucleotide sequences of promoters and their individual structural regions in the expression cassettes of genetic constructs are essential for regulating the expression of recombinant genes in transgenic plants. The term "recombinant gene" is usually understood to mean any artificially created gene, consisting of components of different genes. A recombinant gene is created by combining individual components of different genes into new combinations using genetic engineering techniques. Any gene within the expression cassette of a genetic construct for plant transformation may be considered recombinant.

Isolated promoters can retain their main functions as part of genetic constructs integrated into the genome of transgenic plants; however, qualitative and quantitative variations in the expression of recombinant genes under their control are not uncommon and should be studied for each promoter proposed for plant biotechnology (Hernandez-Garcia and Finer 2014).

Promoters used in plant biotechnology can be classified as constitutive (active in most tissues at different stages of plant development), spatiotemporal (active in certain tissues and/or at certain stages of plant development) and inducible (activated when exposed to chemical or physical agents) . Synthetic promoters consisting of regulatory elements of various origins can be classified as a separate class (Hernandez-Garcia and Finer 2014).

Strong constitutive promoters are most in demand in plant biotechnology. Promoters of this class, along with the effective expression of target genes (recombinant genes intended to change the plant phenotype), are able to control the expression of selective genes (recombinant genes intended to select transformed plant cells) at a level sufficient for the selection of transgenic cells, calli, shoots and seedlings of plants on nutrient environment with a selective agent.

Previously, in plant biotechnology, almost all transgenic plants contained two recombinant genes, one of which, under the control of a constitutive promoter, was necessary for the selective selection of transformed cells, and the other target gene, under the control of any type of promoter, was intended to change the plant phenotype. Currently, the possibilities of multigene transformation make it possible to import entire metabolic pathways into plants, including the expression of several target genes, and the development of transgenic crops that simultaneously produce a range of compounds (Zhu et al. 2007; Peremarti et al. 2010). Each additional recombinant gene needs its own promoter, which requires the use of different promoters with a similar level and expression profile in one genetic construct, or one promoter several times. In the first case, there is a shortage of available promoters with the necessary parameters, and in the second case, the introduction of repeated sequences into one or different loci of one transgenic plant may have a negative effect on the expression and inheritance of the recombinant genes included in them due to the effect of homologous silencing (Mette et al. 1999; Lessard et al. 2002; Potenza et al. 2004; Mourrain et al. 2007). It is now highly recommended to drive different recombinant genes introduced into transgenic plants using different promoters to avoid the effect of homologous gene silencing due to homology of repetitive promoters (Peremarti et al. 2010; Dutt et al. 2014; Nuccio 2017).

So far, the most popular strong constitutive promoter for plant genetic engineering is the CaMV35S promoter, derived from the cauliflower mosaic virus 35S RNA promoter region CaMV (Odell et al. 1985). The CaMV35S promoter provides a high level of expression in most tissues and organs of plants and is widely used in the development of transgenic plants, especially dicotyledonous species; therefore, it is often used as a comparative control (prototype) in studies of other promoters. Other strong constitutive promoters are known from plant pathogen genomes. For example, the promoter from the genome of the dahlia mosaic virus (Kuluev et al. 2010), the promoter from the genome of the dahlia mosaic virus (Acharya et al. 2014), the nos promoter of the nopaline synthase gene of Agrobacterium tumefaciens (Verdaguer et al. 1996), etc. However, promoters isolated from plant pathogens can disrupt the development of transgenic plants (AH and Kim 2019), they are less favorable from an ecological point of view, and their effectiveness can be suppressed if genetically modified plants are infected with viruses (Al-Kaff et al. 2000). Plant pathogen promoters in expression cassettes can be replaced with plant-derived promoters to produce cis-gene or intra-gene plants free of unwanted foreign (non-plant) sequences (Holme et al. 2013). Therefore, in the last two decades, the search for and testing of new effective promoters of plant genes and the creation of synthetic promoters have been actively carried out (AH and Kim 2019).

There are very few known strong constitutive promoters of plant origin that are simultaneously effective for the expression of target and selective genes. One of the few is the AtTCTP promoter from Arabidopsis (Arabidopsis thaliana), whose constitutive activity is sufficient both for high-level expression of the gus reporter gene in various organs of transgenic Arabidopsis plants and for controlling the selective phosphinothricin acetyltransferase gene when selecting transgenic calli and plants on a nutrient medium with the herbicide phosphinothricin. bent grass (Agvyrostis stoloniferd) (Han et al. 2015). The Actinl promoter from rice (Oryza sativa) proved to be a strong constitutive promoter in the selection of transgenic calli and maize (Zea mays) plants on a medium with the antibiotic paromomycin (Prakash et al. 2008). Relatively recently, a strong UBI10 promoter from Brachypodium distachyon was proposed for the genetic engineering of monocot crops, which is up to 35 times more efficient than CaMV35S (Coussens et al. 2012). For the genetic engineering of dicots, a whole spectrum of strong promoters of various plant genes was cloned, but their superiority over CaMV35S was not as significant as in monocots. The promoters of the UBQ1 and UBQ6 genes from A. thaliana are active in all tobacco tissues at the level of the CaMV35S viral promoter (Callis et al. 1990). The strong MtHP promoter from Medicago truncatula in plants of Arabidopsis, creeping clover (Trifolium repens), and alfalfa (Medicago sativa) was about 1.5 times more efficient than CaMV35S (Xiao et al. 2005). Compared to CaMV35S, the promoter of the ACC synthase gene VR-ACS1 from cowpea radiata (Vigna radiata) was up to six times more efficient in transgenic tobacco and Arabidopsis plants, but its clear superiority was the result of translational rather than transcriptional activation (Cazzonelli et al. 2005).

Previously, we cloned the promoters of the pro-SmAMP1 (MF461278) and pro-SmAMP2 (KX196447) antimicrobial peptide genes from the S. media plant, which are comparable or superior in efficiency to the CaMV35S viral promoter (Komakhin et al. 2016; Vysotskii et al. 2016; Madzharova et al. 2018). Promoter efficiency was initially assessed by the expression of the gus reporter gene by measuring the activity of its GUS protein product. It was found that these promoters, when transiently expressed in Nicotiana benthamiana plants, were up to 3.4 times more efficient than the CaMV35S viral promoter. In homozygous lines of transgenic tobacco plants (Nicotiana tabacum), the pro-SmAMP1 and pro-SmAMP2 promoters were two times stronger than the CaMV35S viral promoter. The pro-SmAMP2 promoter is as efficient as controlling the neomycin phosphotransferase II (nptII) gene in the selection of transgenic tobacco and Arabidopsis plants on media with the antibiotic kanamycin at recommended concentrations as well as the duplicated viral 2x CaMV35S promoter. But at the same time, it turned out that the nucleotide sequences of the pro-SmAMP1 and pro-SmAMP2 promoters are 94% identical, which prevents their use to control the target and selective genes in the same genetic construct due to the possible effect of homologous silencing. Therefore, it is currently necessary to search for new plant promoters with a similar pattern of activity in plant cells and a genetic basis (nucleotide sequence) that differs from the pro-SmAMP1 and pro-SmAMP2 promoters.

Thus, despite the existing achievements in the field of molecular biology and biotechnology, the need for high, constitutive, stable, and accurate expression of recombinant genes in transgenic plants necessitates the search for new gene promoters for plants with different genetic bases. The use of such promoters for the expression of recombinant genes will make it possible to create stable transgenic plants that efficiently express many target genes and produce valuable secondary metabolites or complex proteins consisting of several polypeptide chains.

Disclosure of the essence of the invention

The object of the invention is the nucleotide sequence of the promoter region of the SmAMP-D1 defensin gene from the genome of the plant Stellaria media. The pro-SmAMP-D1 promoter isolated from the genome for the first time is represented by two polymorphic versions and their 5'-deletion variants of various lengths. The first version of the pro-SmAMP-D1 promoter, designated as pro-SmAMP-D1-1, is represented by 5' deletion variants -303 bp. (SEC ID NO 1) and -118 b.p. (SEC ID NO 2) relative to +1 b.p. TSS of the SmAMP-D1 defensin gene. The second version of the pro-SmAMP-D1 promoter is designated as pro-SmAMP-D1-2 and is represented by 5'-deletion variants -304 bp. (SEC ID NO 3) and -118 b.p. (SEC ID NO 4) relative to +1 b.p. TSS of the SmAMP-D1 defensin gene. The versions of the pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoters are 93% identical and differ in the substitutions of individual nucleotides in their sequences.

Isolated nucleotide sequences of the pro-SmAMP-D1 plant promoter are designed to create genetic constructs to express recombinant genes at high and constitutive levels in genetically modified plant cells and genetically modified plants.

Genetic constructs carrying a recombinant gene (target or selective) under the control of the pro-SmAMP-D1 promoter, also containing a transcription terminator, are intended for genetic transformation of plant cells, including those using Agrobacterium strains.

Agrobacterium strains containing genetic constructs with target or selective genes under the control of the pro-SmAMP-D1 promoter as part of a binary vector for plant transformation are intended for agrobacterial infiltration of plant tissues or for co-cultivation with plant explants.

Infiltration of plant tissues using strains of agrobacteria containing the target gene under the control of the pro-SmAMP-D1 promoter as part of a binary vector for plant transformation ensures the accumulation of the target protein at a level that is comparable to or exceeds the level of accumulation of the target protein when using the strong viral CaMV35S promoter.

Agrobacterium strains containing genetic constructs with the target gene under the control of the pro-SmAMP-D1 promoter as part of a binary vector for plant transformation, after co-cultivation with plant explants, make it possible to create transgenic plants that produce the target protein at a level that is comparable to or exceeds the level of accumulation of the target protein. protein using a strong viral promoter CaMV35S.

Agrobacterium strains containing genetic constructs with a selective gene under the control of the pro-SmAMP-D1 promoter as part of a binary vector for plant transformation, after co-cultivation with plant explants, make it possible to create and select transgenic cells, calli, shoots and seedlings on a nutrient medium with a selective agent plants without the use of promoters from plant pathogens.

The main technical result of the invention is to expand the arsenal of new effective promoters of plant origin for plant biotechnology, including for the expression of recombinant genes at a high and constitutive level without the use of promoters from plant pathogens.

Brief description of the drawings

Fig. Fig. 1. Nucleotide sequence of the promoter region of the SmAMP-D1 gene. (a) -303 bp pro-SmAMP-D1-1 promoter version. relative to +1 b.p. TSS together with 5'-UTR of the SmAMP-D1 gene; (b) Promoter version of pro-SmAMP-D1-2, -304 bp in length. relative to +1 b.p. TSS together with the 5'-UTR of the SmAMP-D1 gene. The 5'-UTR is underlined, the main cis-acting elements in the promoter sequence are boxed and labeled. The translation start site of the atg protein is highlighted in red. Vertical red arrows and green numbers mark the starting points of the nucleotide sequences of the 5' deletion variants of the promoter.

Fig. 2. Genetic constructs based on the plant expression vector pCAMBIA1381Z, in which the intron-containing gus reporter gene is controlled by the -303 and -118 bp 5' deletion variants. versions of the pro-SmAMP-D1-1 promoter and 5'-deletion variants -304 and -118 bp. versions of the pro-SmAMP-D1-2 promoter. The pMOG35SintGUS plant expression vector containing the gus intron gene under the control of the CaMV35S viral promoter was used as a comparative control. The coding region of the gus gene is shown in black; Int - modified castor catalase intron within the translated region of the gus gene; PIV2 is a modified intron of the potato ST-LS1 gene within the translated region of the gus gene. Promoters are depicted as arrows with corresponding labels.

Fig. 3. Genetic constructs based on the plant expression vector pCAMBIA2300, in which the selective nptII gene is under the control of the -303 and -118 bp 5'-deletion variants. versions of the pro-SmAMP-D1-1 promoter and 5'-deletion variants -304 and -118 bp. versions of the pro-SmAMP-D1-2 promoter. The pCAMBIA2300 vector with the nptII gene under the control of the duplicated 2x CaMV35S promoter was used as a comparative control. The coding region of the nptII gene is shown in blue. Promoters are depicted as arrows with corresponding labels.

Fig. Fig. 4. Activity levels of the GUS reporter protein in the leaves of N. benthamiana plants with transient expression of the gus gene under the control of different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter and the CaMV35S promoter (control). Horizontal lines mark deletion variants based on pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoter versions. The numbers below the abscissa show the sizes of the 5'-deletion variants in bp. relative to +1 b.p. TSS. Error bars correspond to standard errors. * - values significantly different from control at p≤0.05.

Fig. Fig. 5. Histochemical localization of GUS reporter protein activity in 2-week-old _T2 generation transgenic A. thaliana plants expressing the gus gene under the control of different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter and the CaMV35S promoter (control). Horizontal lines mark 5'-deletion variants based on pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoter versions. The numbers show the sizes of the 5'-deletion variants in bp. relative to +1 b.p. TSS. The localization of GUS activity (blue tissue color) in leaves, petioles, stems, roots, and cotyledons of transgenic plants was shown in comparison with similar organs of intact Arabidopsis plants of the Columbia-0 ecotype.

Fig. Fig. 6. Activity levels of the GUS reporter protein in the leaves of 3-week-old _T2 generation transgenic A. thaliana plants expressing the gus gene under the control of different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter and the CaMV35S promoter (control). Horizontal lines mark 5'-deletion variants based on pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoter versions. The numbers below the abscissa show the sizes of the 5-deletion variants in bp. relative to +1 b.p. TSS. n is the number of independent lines of transgenic plants. Error bars correspond to standard errors. * - values significantly different from control at p≤0.05.

Fig. Fig. 7. Morphogenesis of shoots from somatic cells of tobacco leaf explants on a nutrient medium with the antibiotic kanamycin at a high concentration of 350 mg/l. To control the expression of the selective nptII gene, different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter and the 2x CaMV35S promoter (control) were used. The numbers show the sizes of the 5'-deletion variants of the promoter in bp. relative to +1 b.p. TSS.

Fig. Fig. 8. Segregation of T ₁ generation tobacco seedlings on a selective nutrient medium with the antibiotic kanamycin. Green plants are resistant to kanamycin. Plants sensitive to kanamycin are white. Different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter and the 2x CaMV35S promoter (control) were used to control the expression of the selective nptII gene. The numbers show the sizes of the 5'-deletion variants of the promoter in bp. relative to +1 b.p. TSS.

Fig. Fig. 9. PCR analysis of the genomic DNA of _T1 generation tobacco plants for the presence of a DNA segment consisting of the nucleotide sequences of the nptII gene and 5'-deletion variants of the pro-SmAMP-D1-1 promoter version, the pro-SmAMP-D1-2 promoter version and 2x CaMV35S promoter. M - DNA marker. "K+" - positive control. "K-" - negative control. Numbers show individual plant samples.

Fig. 10. Histochemical localization of GUS reporter protein activity in transgenic potato plants expressing the gus gene under the control of a -304 bp 5'-deletion variant. the pro-SmAMP-D1-2 promoter; and the CaMV35S promoter. The localization of GUS activity in leaves, petioles, stems, flowers and tubers of transgenic plants was shown in comparison with similar organs of intact plants of the variety Vector (K). The word "no" indicates the absence of flowers in the plant.

Implementation of the invention

The above disclosure describes the invention in general terms. A fuller understanding can be gained by presenting relevant examples. The examples presented here are for illustrative purposes only and are in no way intended to limit the scope of the present invention within the scope of these examples. Modification of the form or substitution of equivalents is allowed on the basis of its expediency in the relevant conditions. Although specific terms will be used here, they are used for descriptive purposes only.

Example 1. Cloning of the SmAMP-D1 defensin gene promoter from the genome of the Stellaria media plant

The stellate plant genome has not yet been sequenced; therefore, it is not possible to directly amplify the nucleotide sequence of the SmAMP-D1 defensin gene promoter from intact S. media plant DNA. However, the nucleotide sequence of the SmAMP-D1 gene, including the 5'-untranslated (5'-UTR) and protein-coding regions, was previously cloned (Slavokhotova et al. 2011). Therefore, in this work, to clone the promoter together with the 5'-UTR of the SmAMP-D1 gene, we used a method that was previously developed to detect T-DNA integration loci in the genome of Arabidopsis plants (Pogorelko and Fursova 2008). To do this, the genomic DNA of stellate plants was isolated and hydrolyzed using the HinfI restriction enzyme, for which a restriction site was found in the adjacent coding region of the SmAMP-D1 gene. The DNA was then ligated according to the procedure and used as a template for sequential amplification with two pairs of primers oriented oppositely. For this, primers complementary to the 5'-UTR and the adjacent coding region of the SmAMP-D1 gene were developed: "outer" smDa1 (5'-aaatccagtgcattattttctgtgtcga-3') and smDa2 (5'-aggaaagtaacatggatggatacccaacaca-3') and "inner" smDb3 (5' -aacttacatattcctcttaggctaat-3') and smDb4 (5'-tcttttttgtttagtttaagtcgatca-3'). As a result of successive rounds of amplification, first with "external" and then with "internal" primers, a single amplicon of about 750 bp was obtained. During its sequencing, it was found that between the sequences for annealing the "internal" primers, there are known nucleotide sequences of the SmAMP-D1 gene, between which the putative promoter region is located. In order to test the existence of this region in the intact genome of starfish, as well as to clone the promoter, two new primers were developed. The first primer smD4.3(E) (5'-gaattcaactataacattttgtattctttt-3') is complementary to the 5' end of the putative promoter nucleotide sequence and contains the EcoRI restriction site. The second, smD11(N) (5'-ccatggtttctttttgtttagtttaagtc-3'), is complementary to 5-UTR up to the atg initiation codon of the SmAMP-D1 gene and contains the NcoI restriction site. (N) received an amplicon of about 350 bp. When sequencing it as part of the T-vector, it was found that it consists of the known 5-UTR of the SmAMP-D1 gene, 42 bp in size. and the adjacent proximal and core promoter regions of the SmAMP-D1 gene, about 300 bp in size. By comparing ten clones, it was established that the genome of the stellate average contains at least two 93% identical versions of the SmAMP-D1 gene promoter, designated as pro-SmAMP-D1-1 (-303 bp in size relative to +1 bp . TSS) and pro-SmAMP-D1-2 (-304 bp relative to +1 bp TSS), differing in individual nucleotide polymorphisms (Fig. 1).

Using the Blast program, no other nucleotide sequences identical to those of the new pro-SmAMP-D1 promoter were found in GenBank (https://www.ncbi.nlm.nih.gov/).

In silico analysis of the nucleotide sequences of both versions of the pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoters was performed using the New place A Database of Plant Cis-acting Regulatory DNA Elements program (https://www.dna. affrc.go.jp/PLACE/?action=newplace). Both versions of the promoter were found to contain the conserved cis-element TATA-box at the expected -34 bp position. to the left relative to +1 b.p. TSS. In the region of the minimal promoter at position -107 b.p. relative to +1 b.p. TSS found a conserved cis element of the CAAT-box (FIG. 1). In addition, other potential cis-elements in the pro-SmAMP-D1 promoter were identified that can provide tissue-specific or inducible expression of the SmAMP-D1 gene or act as enhancers or suppressors of its expression. They included cis-elements responsive to light (GATABOX, GT1CONSENSUS) and phytohormones (PYRIMIDINEBOXHVEPB1), required for tissue- and organ-specific expression (CACTFTPPCA1, WRKY710S, RAV1AAT), for response to abiotic stress (CCAATBOX1, CBFHV and MYCCONSENSUSAT), for metabolism carbohydrates (DOFCOREZM, WBOXHVISO1), to respond to the attack of pathogens (BIHD10S, SEBFCONSSTPR10A), etc.

Example 2. Creation of 5'-deletion variants of the pro-SmAMP-D1 promoter and genetic constructs for agrobacterial transformation of plants

Based on the analysis of the nucleotide sequences of the pro-SmAMP-D1-1 and pro-SmAMP-D1-2 versions, primers were selected to create 5'-deletion variants in order to subsequently assess the promoter efficiency in controlling the expression of recombinant genes. Created one new "forward" primer SmD12(E) (5'-gaattcgcatgtcacaatt-3') containing an EcoRI restriction site, limiting the region of both versions of the promoter to -118 bp. relative to +1 b.p. TSS (Fig. 1). The previously created primer smD11(N) was used as the reverse.

As templates for the amplification of new 5'-deletion variants of both versions of pro-SmAMP-D1-1 and pro-SmAMP-D1-2 -118 bp in size. used plasmid DNA with their longer 5'-deletion variants.

Using the new primers, two new short 5'-deletion variants of the promoter versions, designated as pro-SmAMP-D1-1-118 and pro-SmAMP-D1-2-118, as well as their previously created longer 5'-deletion variants pro-SmAMP-D1-1-303 and pro-SmAMP-D1-2-304. Scheme of the resulting genetic constructs, designated pC1381Z-pro-SmAMP-D1-1-303, pC1381Z-pro-SmAMP-D1-1-118, pC1381Z-pro-SmAMP-D1-2-304, and pC1381Z-pro-SmAMP, respectively -D1-2-118 is shown in FIG. 2.

The created genetic constructs are intended for expression in plants of the target gene, which is under the control of the pro-SmAMP-D1 promoter. The gus intron-containing reporter gene (gusA or uidA) was used as the target gene. The pMOG35SintGUS plant expression vector containing the gus gene under the control of the known strong and constitutive CaMV35S viral promoter was used as a comparative control. The genetic constructs listed above were used to create agrobacterial strains based on GV3101 Agrobacterium tumefaciens cells.

When creating transgenic plants, the plant (binary) vector pCAMBIA2301 (Cambia, Australia) was used to optimize the selection of transformed cells and seedlings on a nutrient medium with a relatively cheap antibiotic kanamycin. For this, fragments from the pC1381Z-pro-SmAMP-D1-1-303, pC1381Z-pro-SmAMP-D1-1-118, pC1381Z-pro-SmAMP-D1-2- 304 and pC1381Z-pro-SmAMP-D1-2-118, containing the nucleotide sequences of the corresponding 5'-deletion variants of the promoter and the gus gene, and pre-cut at the Eco91I and EcoRI sites. As a result, pC2301-pro-SmAMP-D1-1-303, pC2301-pro-SmAMP-D1-1-118, pC2301-pro-SmAMP-D1-2-304 and pC2301-pro-SmAMP-D1-2 genetic constructs were created. -118.

To assess the possibility of using the pro-SmAMP-D1 promoter to control the expression of the selective neomycin phosphotransferase II (nptII) gene, which confers resistance to the antibiotic kanamycin in plant cells, genetic constructs based on the pCAMBIA2300 binary vector (Cambia, Australia) were created. For this, the nucleotide sequences of the -303 and -118 bp 5'-deletion variants were cloned at the EcoRI and NcoI restriction sites into the pCAMBIA2300 vector. versions of the pro-SmAMP-D1-1 promoter and 5-deletion variants -304 and -118 bp. versions of the pro-SmAMP-D1-2 promoter. Created genetic constructs designated as pC2300-pro-SmAMP-D1-1-303, pC2300-pro-SmAMP-D1-1-118, pC2300-pro-SmAMP-D1-2-304 and pC2300-pro-SmAMP-D1- 2-118 contain the nptII gene under the control of various 5' deletion variants of the pro-SmAMP-D1 promoter (FIG. 3). An intact pCAMBIA2300 vector with the nptII gene under the control of a duplicated 2x CaMV35S viral promoter was used as a comparative control.

Genetic constructs based on plasmids pCAMBIA2300 and pCAMBIA2301 were used to obtain agrobacterial strains using A. tumefaciens AGLO cells.

Example 3. Transient expression of the reporten gus gene under the control of different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter in agroinfiltrated Nicotiana benthamiana plants

Leaves of N. benthamiana plants were infiltrated with five variants of agrobacteria of strain GV3101 with plasmid genetic constructs pC1381Z-pro-SmAMP-D1-1-303, pC1381Z-pro-SmAMP-D1-1-118, pC1381Z-pro-SmAMP-D1-2- 304 and pC1381Z-pro-SmAMP-D1-2-118 and pMOG35SintGUS as a comparative control. Initially, agrobacterial strains were cultivated on LB agar medium with antibiotics kanamycin (100 mg/L), rifampicin (100 mg/L), and gentamicin (25 mg/L). Then, to perform agroinfiltration of plant leaves, the strains were grown in a liquid LB nutrient medium at the same concentrations of the above antibiotics during the day at a temperature of 28°C and stirring (160 rpm). To suppress RNA interference in plant cells, the A. tumifaciens GV2260/C58C1 pLH7000 p19 strain was used, which was grown in liquid LB nutrient medium with antibiotics rifampicin (100 mg/l) and streptomycin (50 mg/l) during the day at a temperature of 28°C. C and stirring (160 rpm).

Agrobacterium cell suspensions were diluted to an optical density of 0.6 at a wavelength of 600 nm. Then the strains GV3101 pMOG35SintGUS, GV3101 pC1381Z-pro-SmAMP-D1-1-303, GV3101 pC1381Z-pro-SmAMP-D1-1-118, GV3101 pC1381Z-pro-SmAMP-D1-2-304 and GV3101 pC1381Z-pro- SmAMP -D1-2-118 was mixed in a ratio of 1:1 with a suspension of agrobacteria GV2260/C58C1 pLH7000 p19. The prepared mixture of agrobacteria was injected into the leaves of N. benthamiana plants with a syringe without a needle from the inside. After infiltration, the plants were kept at 21° C. and 16/8 hours of artificial light. On the 7th day, cuttings weighing about 10 mg were taken from the inoculation site and frozen at -70°C.

Then, protein extracts were isolated from each cut, in which the activity of the recombinant β-glucuronidase enzyme was measured using the substrate 4-methylumbelliferyl-β-D-glucuronide Trihydrate (MUG, PhytoTechnology Laboratories) and the concentration of water-soluble proteins by the Bradford method; Based on the results obtained, the activity of the GUS reporter protein was calculated in pmol 4MU/mg⋅min or pmol/mg min. For each genetic construct, 60 to 100 cuttings were analyzed, obtained in three repetitions of the experiment. The mean level of GUS activity using each deletion variant of the pro-SmAMP-D1 promoter and the CaMV35S viral promoter is shown in FIG. 4.

As follows from FIG. 4, the novel pro-SmAMP-D1 promoter is functional for transient expression of the gus reporter gene and production of its GUS protein product in heterologous plant cells. The average level of GUS activity in N. benthamiana leaves agroinfiltrated with pCambia1381Z genetic constructs carrying various 5'-deletion variants of the pro-SmAMP-D1-1 and pro-SmAMP-D1-2 promoters varied from 29914 to 98338 pmol/min⋅mg, which is both lower and higher than the activity stimulated by the CaMV35S promoter (53161 pmol/min⋅mg). In particular, the long 5'-deletion variant -304 bp. version of the pro-SmAMP-D1-2 promoter was 1.9 times significantly stronger than the CaMV35S viral promoter. Short 5'-deletion variant -118 b.p. versions of pro-SmAMP-D1-2 and a long 5'-deletion version -303 b.p. the pro-SmAMP-D1-1 versions were comparable in efficiency to the CaMV35S promoter. Short 5'-deletion variant -118 b.p. version of pro-SmAMP-D1-1 was 1.7 times significantly weaker than CaMV35S. In general, short 5'-deletion variants of pro-SmAMP-D1 are expected to be less efficient than their longer 5'-deletion variants.

Example 4 Transgenic Arabidopsis thaliana Plants Expressing the gus Reporter Gene under the Control of Different Versions and 5' Deletion Variants of the pro-SmAMP-D1 Promoter

Using constructs pC2301-pro-SmAMP-D1-1-303, pC2301-pro-SmAMP-D1-1-118, pC2301-pro-SmAMP-D1-2-304 and pC2301-pro-SmAMP-D1-2-118 performed the genetic transformation of Arabidopsis (A. thaliana) plants of the Colambia-0 ecotype by immersing inflorescences in a suspension of agrobacteria. When using all genetic constructs on a selective nutrient medium with the antibiotic kanamycin at a concentration of 50 mg/l, resistant T ₁ generation plants producing the GUS reporter protein were selected. As a result of self-pollination of transgenic plants, seeds of the T ₂ generation were obtained. With the help of their selection on a nutrient medium with the antibiotic kanamycin, plant populations with monogenic T-DNA inheritance were selected, from which individual homozygous plants were selected (according to the level of GUS activity and the absence of segregation on the selective agent of T ₃ generation seedlings), which were used for the study (Fig. 5).

All versions and 5' deletion variants of the pro-SmAMP-D1 promoter were found to be able to drive gus gene expression in different tissues and organs of transgenic A. thaliana plants (Fig. 5). GUS reporter activity visualized using the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt (X-Gluc, PhytoTechnology Laboratories) was present in roots, stems, cotyledons, and leaves, although staining intensity tissues visually differed depending on the used version of the genetic construct.

To clearly differentiate the levels of gus expression among versions and 5' deletion variants of the pro-SmAMP-D1 promoter, GUS activity was quantified in the leaves of 3 week old transgenic plants (FIG. 6). The average level of GUS activity in the leaves of transgenic A. thaliana plants varied from 5054 to 20770 pmol/min⋅mg, which is both lower and higher than the activity stimulated by the CaMV35S promoter (about 8050 pmol/min⋅mg). In particular, the long 5'-deletion variant -304 bp. the pro-SmAMP-D1-2 version is 2.1 times significantly stronger than the CaMV35S viral promoter. Short 5'-deletion variant -118 b.p. pro-SmAMP-D1-2 version, long 5'-deletion variant -303 b.p. and a short 5' deletion variant -118 bp. the pro-SmAMP-D1-1 versions are comparable in efficiency to the CaMV35S promoter. The pattern of the distribution of GUS activity levels depending on the promoter version used and the 5' deletion variant in transgenic Arabidopsis plants (Fig. 6) generally corresponded to the distribution of activities during transient expression of the gus gene in N. benthamiana plants (Fig. 4).

Example 5. Use of the selective nptll gene under the control of different versions and 5-deletion variants of the pro-SmAMP-D1 promoter for the selection of transgenic cells and tobacco plants

The method of agrobacterial transformation of leaf explants (disks) of tobacco plants (Nicotiana tabacum) was used to evaluate the efficiency of selection of transgenic cells, calluses and shoots on a nutrient medium with the antibiotic kanamycin. To do this, agrobacterial transformation of tobacco plants was performed using genetic constructs pC2300-pro-SmAMP-D1-1-303, pC2300-pro-SmAMP-D1-1-118, pC2300-pro-SmAMP-D1-2-304, pC2300-pro -SmAMP-D1-2-118 and pC2300 by the "lawn" method we used earlier (Komakhin et al. 2010; Madzharova et al. 2018). It was found that all versions and 5'-deletion variants of the pro-SmAMP-D1 promoter are sufficient for the constitutive level of expression of the selective nptII gene, since they allow the selection of transformed cells, calli and shoots of tobacco on a nutrient medium with kanamycin (Fig. 7).

In particular, on a nutrient medium with kanamycin at the recommended concentration of 100 mg/L (Draper et al. 1991), after three months of selection, both versions and all 5'-deletion variants of the pro-SmAMP-D1 promoter and the 2x CaMV35S promoter allowed the formation of kanamycin-resistant calli and shoots of all tobacco leaf explants. Both versions and all 5'-deletion variants of the pro-SmAMP-D1 promoter allowed the formation of kanamycin-resistant calli and shoots in 35-55% of tobacco leaf explants on a nutrient medium with kanamycin at an excess concentration of 350 mg/L for four months of selection (Table 1). 1, column "B/A × 100%").

Under the same conditions, using the 2x CaMV35S promoter, morphogenesis was observed in 95–100% of explants (Table 1, column “B/A × 100%”).

The regeneration of tobacco shoots resistant to the antibiotic kanamycin was evaluated during three months of cultivation on a selective nutrient medium with a recommended concentration of 100 mg/l and an excess concentration of 350 mg/l. Formed shoots were separated from the callus and selected on a nutrient medium with kanamycin at a concentration of 100 mg/l for 1.5 months.

Rooting shoots at the rate of 1.5-1.8 shoots per explant taken initially for transformation were obtained using both versions and all 5'-deletion variants of the pro-SmAMP-D1 promoter and the 2x CaMV35S promoter on a nutrient medium with kanamycin at the recommended concentration of 100 mg/l. Under these conditions of shoot selection, no significant differences were found between the variants of genetic constructs.

Rooting shoots with an efficiency of 0.3-0.6 shoots per explant were obtained using both versions and all 5'-deletion variants of the pro-SmAMP-D1 promoter on a nutrient medium with kanamycin at an excess concentration of 350 mg/l (Table 1, column "D / A, pieces"). When using the 2x CaMV35S promoter, the efficiency of shoot formation was 1.2-1.3 shoots per explant. The higher efficiency of the 2x CaMV35S promoter under conditions of excess kanamycin was expected, since in the intact pC2300 plasmid it contains two copies of the CaMV35S promoter, which obviously determine its higher efficiency for nptII expression (Fig. 3). These results are intended to further demonstrate the ability of the pro-SmAMP-D1 promoter to work efficiently under conditions of a multiple excess of the selective agent. Previously, in tobacco plants, only the pro-SmAMP2 promoter from the chickweed was comparable in efficiency to the 2x CaMV35S promoter for the production of shoots on a nutrient medium with kanamycin at a concentration of 350 mg/L (Madzharova et al. 2018). Under the same conditions, the pro-SmAMP1 promoter from chickweed was three times less effective.

Using the PCR method and previously created primers virE ₂ F and virE ₂ R (Komakhin et al. 2010) to the VirE2 gene sequence of agrobacteria, it was established that tobacco plants rooted on a nutrient medium with kanamycin did not have agrobacterial contamination. To check the presence of the nptII gene under the control of different promoters in rooted shoots, we used the PCR method and initially created primers to the sequences of long and short 5'-deletion variants of the pro-SmAMP-D1 promoter, respectively, smD4.3(E) or SmD12(E). The previously developed 35Splus primer (Madzharova et al. 2018) was used to analyze plants created using the 2x CaMV35S promoter. The originally designed olgminus primer, which is complementary to the site in the nptII gene sequence, was used as a “reverse” in all analyzes (Madzharova et al. 2018). Using DNA samples from ten rooted plants of each genetic construct variant, it was determined that they all contained nucleotide sequences corresponding to a hybrid region consisting of the sequence of the corresponding 5' deletion variant of the promoter and the nptII gene. In particular, for long 5'-deletion variants of the pro-SmAMP-D1 promoter and the nptII gene fragment, amplicons of the expected size of about 400 bp were obtained, for short 5'-deletion variants of the pro-SmAMP-D1 promoter and the nptII gene fragment about 300 b.p. When using DNA samples from transgenic plants with a duplicated 2x CaMV35S promoter, two amplicons of about 500 and 800 bp, respectively, were obtained for each of the copies of CaMV35S and the nptII gene fragment. Thus, primary tobacco transformants were created, which were designated as T ₀ 2300-pro-SmAMP-D1-1-303, T ₀ 2300-pro-SmAMP-D1-1-118, T ₀ 2300-pro-SmAMP-D1-2 -304, T ₀ 2300-pro-SmAMP-D1-2-118 and T ₀ 2300.

In the next generation of T ₁ , tobacco seedlings carrying nptll under the control of both versions and all 5'-deletion variants of the pro-SmAMP-D1 promoter segregated depending on resistance to kanamycin into resistant and susceptible plants, in some populations in a ratio of 3:1 ( resistant:susceptible), suggesting monogenic inheritance of the transgene (Fig. 8). All kanamycin-resistant tobacco plants (from 8 to 14 for each construct) were free from Agrobacterium contamination and, according to the PCR results, all contained hybrid nucleotide sequences between the promoter variants and the nptII gene of the expected size (Fig. 9).

Thus, the new pro-SmAMP-D1 promoter is constitutive and certainly sufficient for the selection of transformed cells, calluses, shoots, and seedlings on a medium with the concentration of the antibiotic kanamycin recommended for the creation of transgenic tobacco plants from 50 to 100 mg/L (Draper et al. 1991 ). The pro-SmAMP-D1-2 promoter is not inferior in efficiency to the constitutive viral CaMV35S promoter when selected on a nutrient medium with kanamycin at the recommended concentration of 100 mg/l. From an ecological point of view and from the position of modern science in the field of plant cell biology, the pro-SmAMP-D1-2 plant promoter is preferable for genetic modification of plants than the CaMV35S viral promoter.

Example 6 Transgenic potato plants expressing the gus reporter gene under the control of the pro-SmAMP-D1 promoter

To test the effectiveness of the pro-SmAMP-D1 promoter for the genetic modification of agricultural plants, we performed agrobacterial transformation of potato plants (Solanum tuberosum) of the variety Vector (originator of the variety is the Federal State Budgetary Scientific Institution "All-Russian Research Institute of Potato Farming named after A.G. Lorch") . Genetic transformation was performed in accordance with the method published by us earlier (Vetchinkina et al. 2016). As a result of the research, nine independent potato transformants were created using each genetic construct pC2301-pro-SmAMP-D1-2-304 and pMOG35SintGUS, which were resistant to the antibiotic kanamycin at a concentration of 75 mg/l and produced the GUS reporter protein.

It was found that the 5'-deletion variant -304 b.p. version of the pro-SmAMP-D1-2 promoter, which is the most promising for plant biotechnology, is able to control the expression of the gus gene in various tissues and organs of transgenic potato plants (Fig. 10). GUS reporter activity, visualized by tissue staining using X-Gluc substrate, was present in stems, petioles, leaves, tubers, and flowers of transgenic plants at a high level using both pro-SmAMP-D1-2 and CaMV35S promoters. Noticeable differences between promoter patterns were observed only when staining leaves. In particular, the pro-SmAMP-D1-2 promoter promoted a more intense staining of leaf conductive tissues, while the CaMV35S promoter promoted a more uniform staining of the leaf parenchyma.

Thus, the new pro-SmAMP-D1-2 promoter can be used for potato plant biotechnology and is not inferior to the strong viral CaMV35S promoter in terms of the efficiency of recombinant gene expression in various plant organs. From an ecological point of view and from the position of modern science in the field of plant cell biology, the pro-SmAMP-D1-2 plant promoter is preferable for genetic modification of plants than the CaMV35S viral promoter.

Conclusion

The pro-SmAMP-D1 promoter can be recommended for biotechnology as a regulatory element that effectively controls the expression of recombinant genes in the cells of genetically modified plants. The pro-SmAMP-D1 promoter can be recommended for controlling target or selective genes within expression cassettes of genetic constructs. The use of different versions and 5'-deletion variants of the pro-SmAMP-D1 promoter can be used to optimize the expression of recombinant genes at different levels, including those exceeding the expression level when using the strong CaMV35S viral promoter. The advantage of the pro-SmAMP-D1 promoter is that it is of plant origin and is represented by a nucleotide sequence that differs from the nucleotide sequences of other known plant promoters, and therefore can be used as part of the same genetic construct with other plant gene promoters.

Bibliography

Acharya S, Ranjan R, Pattanaik S et al. (2014) Efficient chimeric plant promoters derived from plant infecting viral promoter sequences. Planta 239:381-396. https://doi.org/10.1007/s00425-013-1973-2

Ali S, Kim W-C (2019) A Fruitful Decade Using Synthetic Promoters in the Improvement of Transgenic Plants. Frontiers in Plant Science https://doi.org/10.3389/fpls.2019.01433

Al-Kaff NS, Kreike MM, Covey SN et al. (2000) Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene. nature biotechnol. 18:995-999. https://doi.org/10.1038/79501

Cazzonelli CI, McCallum EJ, Lee R, Botella JR. (2005) Characterization of a strong, constitutive mung bean (Vigna radiata L.) promoter with a complex mode of regulation in planta. Transgenic Res. 14(6):941-67. doi:10.1007/s11248-005-2539-2

Coussens G, Aesaert S, Verelst W, Demeulenaere M et al. Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize, J. Exp.Bot., 2012, vol. 63, pp. 4263-4273.

Dutt M, Dhekney S, Soriano L, Kandel R, Grosser JW (2014) Temporal and spatial control of gene expression in horticultural crops. Hortic Res. 1:14047. https://doi.org/10.1038/hortres.2014.47

Callis J, Raasch JA, Vierstra RD (1990) Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. J Biol Chem. 265:12486-12493.

Han Y, Kim Y, Hwang O et al. (2015) Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation. Plant Cell Rep 34:265-275. https://doi.org/10.1007/s00299-014-1705-5

Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Science 217:109-119. https://doi.org/10.1016/j.plantsci.2013.12.007

Holme IB, Wendt T, Holm PB (2013) Intragenesis and cisgenesis as alternatives to transgenic crop development. J Plant. Biotechnol. 11:395-407. https://doi.org/10.1111/pbi.12055

Komakhin RA, Komakhina VV, Milyukova NA et al. (2010) Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination. Russ. J. Genet. 46:1440-1448. https://doi.org/10.1134/S1022795410120069

Komakhin RA, Vysotskii DA, Shukurov RR et al. (2016) Novel strong promoter of antimicrobial peptides gene pro-SmAMP2 from chickweed (Stellaria media). BMC Biotechnol. 16(1):43. doi: 10.1186/s12896-016-0273-x.

KuluevBR, Knyazev AV, Chemeris, AV and Vakhitov VA. Morphological features of transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the dahlia mosaic virus promoter. Russ. J. Dev. Biol., 2013, vol. 44, pp. 86-89.

Lessard PA et al. (2002) Manipulating gene expression for the metabolic engineering of plants. metabolic engineering. 4(1):67-79.

Madzharova NV, Kazakova KA, Strelnikova SR et al. (2018) Promoters pro-SmAMPl and pro-SmAMP2 from Wild Plant Stellaria media for the Biotechnology of Dicotyledons. Russ J Plant Physiol 65:750-761. https://doi.org/10.1134/S1021443718040040

Mette MF et al. (1999) Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans // The EMBO journal. 18(1):241-248.

Mourrain P et al. (2007) A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production // Planta. 225(2):365-379.

Nuccio ML (2017) A Brief history of promoter development for use in transgenic maize applications. Methods Mol Biol. 1676:61-93. https://doi.org/10.1007/978-1-4939-7315-6_4.

Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313(6005):810-812.

Prakash NS, Prasad V, Chidambram TP et al. Effect of promoter driving selectable marker on corn transformation, Transgenic Res., 2008, vol. 17, pp. 695-704.

Peremarti A et al. (2010) Promoter diversity in multigene transformation // Plant molecular biology. 73(4-5):363-378.

Pogorelko GV and Fursova OV (2008) A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants. Journal of Genetics. 87(2):133-140.

Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: Promoters used in plant transformation. In Vitro CellDevBiol-Plant. 40(1):1-22.

Slavokhotova AA, Odintsova TI, Rogozhin EA et al. (2011) Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds. Biochimie 93 (2011) 450e456 doi:10.1016/j.biochi.2010.10.019.

Verdaguer B, de Kochko A, Beachy RN, and Fauquet C. Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter. Plant Mol. Biol, 1996, vol. 31, pp. 1129-1139.

Vetchinkina EM, Komakhina VV, Vysotskii DA et al. (2016) Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens. Russ. J. Genet. 52(9): 1055-68.

Vysotskii DA, Strelnikova SR, Efremova LN et al. (2016) Structural and functional analysis of new plant promoter pro-SmAMPl from Stellaria media. Russ J Plant Physiol 63:663-672. https://doi.org/10.1134/S1021443716050174.

Xiao K, Zhang C, Harrison M, Wang Z-Y (2005) Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants. Mol Breeding. 15(2):221-31. https://doi.org/10.1007/sll032-004-5679-9.

Zhu C et al. (2007) Transgenic strategies for the nutritional enhancement of plants. Trends in plant science. 12(12):548-555.

Draper D, Scott P, Armitage F et al. Plant genetic engineering. Laboratory guide. Educational edition. Moscow: Mir. 1991. 408 p.

--->

SEQUENCE LIST

<210> SEC ID NO 1

<211> Length: 303

<212> Type: DNA

<213> Organism: Stellaria media

<400> Sequence: 1-1-303

aactataaca ttttgtattc ttttttttttg ctaccctatt tataaatttc ttgatcctcc 60

cctggtttga ttatcacccg atgttgctaa attttaattc ttaaaaaata atagtaaaac 120

gatagttttt tccaaaaaac gaaaatcgac gtccctgcta tccccttcct cttgttgtcg 180

cccgtgcatg tcacaattat tttatttgct cttatcatga caacacttgc gtcgccttac 240

atgaatattc ttcgaagagg cctataaaac accccattgt cactctaaaa tgatcaaaat 300

ctt 303

<210> SEC ID NO 2

<211> Length: 118

<212> Type: DNA

<213> Organism: Stellaria media

<400> Sequence: 1-1-118

gcatgtcaca attattttat ttgctcttat catgacaaca cttgcgtcgc cttacatgaa 60

118

<210> SEC ID NO 3

<211> Length: 304

<212> Type: DNA

<213> Organism: Stellaria media

<400> Sequence: 1-2-304

aactataaca ttttgtattc ttttttttgc taccctattt atgaatttct ggatcctcac 60

ctgatttgat taccacccga tgtcgctaca ttttaattct taaaatataa tattaaaatg 120

180

gcccgtgcat gtcacaattc tttcatttgc tcttatcatg acaacacttg tgtcgcctta 240

catgactatt cttcgaagag gcctataaaa taccccattg tcactctaaa atgatcaaaa 300

tctt 304

<210> SEC ID NO 4

<211> Length: 118

<212> Type: DNA

<213> Organism: Stellaria media

<400> Sequence: 1-2-118

gcatgtcaca attctttcat ttgctcttat catgacaaca cttgtgtcgc cttacatgac 60

tattcttcga agaggcctat aaaatacccc attgtcactc taaaatgatc aaaatctt 118

<---

Claims (3)

1. DNA fragment for genetic modification of a plant cell to express a recombinant gene in a plant, having a nucleotide sequence selected from the group consisting of SEC ID NO 1, SEC ID NO 2, SEC ID NO 3 and SEC ID NO 4. 2. A genetic construct containing a DNA fragment according to claim 1 and a recombinant DNA sequence, including a gene for a recombinant protein and a transcription terminator, for the expression of a recombinant gene in a plant. 3. The method of expression of a recombinant gene in a plant cell, including the use of a genetic construct according to claim 2, consisting of the steps: creating a genetic construct according to claim 2, containing the nucleotide sequence according to claim 1; introduction of genetic constructs according to claim 2 into a plant cell; selection of the transformed cell on a nutrient medium and regeneration of the transformed plant; expression of the recombinant gene in the plant.

Images