UA52574C2 - Molecule of separated two-chain dna including the dna sequence coding the enzyme glyphosate oxidoreductase (versions), method for obtaining genetically transformed plants, insulated peptide (versions), bacterial strain, culture of plant cell (versions) - Google Patents

Abstract

Genes encoding a glyphosate oxidoreductase enzyme are disclosed. The genes are useful in producing transformed bacteria and plants which degrade glyphosate herbicide as well as crop plants which are tolerant to glyphosate herbicide.

Description

This message is a continuation of our unfinished application number 07/543 236 dated June 1990.

Justification of the invention

Recent successes in genetic engineering have provided the necessary tools for the transformation of plants, causing foreign genes to be inserted into their genomes. Currently, it is possible to obtain plants with unique characteristics, which represent great value for agriculture. Undoubtedly, one of these useful features is the effective, in terms of low cost, control over the presence of weeds in the environment, which is caused by the tolerance of plants to herbicides. In the presence of such plants, it is not at all necessary to cultivate the soil for the purpose of fighting weeds, which significantly reduces the possibility of soil erosion.

One of the herbicides of this type, the study of which was dedicated! many studies, is available

M-phosphonomethyl-glycine, commonly known as glyphosate. Glyphosate has an inhibitory effect on the shikimic acid exchange pathway, leading to the synthesis of aromatic compounds, including amino acids! and vitamins. In particular, glyphosate suppresses the conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid into 5-enolpyruvyl-Z3-phosphoshikimic acid as a result of inhibition of the 5-enolpyruvyl-Z3-phosphoshikimic acid synthase enzyme! /"ERBR synthase or ERBR5B/.

It has been shown that plants that are tolerant to glyphosate can be resistant to glyphosate! by inserting into their genome factors that determine the ability of these plants to synthesize higher levels of EPOR synthesis, an enzyme that is preferentially tolerant to glyphosate (Pap et al., 1986). Introducing glyphosate degradation gene(s) into plants can be a way of ensuring tolerance to glyphosate in plants, as well as increasing the degree of tolerance in transgenic plants that already express glyphosate-tolerant ERBR synthase depending on the physiological effects of degradation products.

Glyphosate metabolism /degradation/ has been studied on various types of plants, and in most of these studies, a small degree of degradation was shown. In the examples where degradation is reported, the initial degradation product is casually represented by aminomethylphosphonate /AMPA/ /Sociriapa, 1985; Maizpai! with co-authors, 1987/. From these examples, it is not entirely clear whether glyphosate is metabolized by the plant or by microbes present on the surface of the leaves against which glyphosate is used. According to reports, AMPA is characterized by much less phytotoxicity than glyphosate for most types of plants /Egapo, 1985/, but not for all of them /Mayeg, 1983; Tapaka and others, 1938/. Degradation of glyphosate in soils is much more extensive and rapid /Tog5iep55op, 1985/. The main identified breakdown product is AMPA (Aperre! et al. 1977; Motyga a. Niop 1977), a phosphonate that can be metabolized by many microorganisms (Eugpik et al., 1963;

Mavia and others, 1965; SookK et al., 1978; Yuatsdniop et al., 1979a; 1979; 1979c; Maskey et al., 1987a/. White identified a number of pure cultures of bacteria that break down glyphosate in one of two ways / Mooge et al., 1983; TaiBroi et al., 1984; Zpiparadeg and Vgauteg, 1936; Vaipagog and

Nayaz, 1936; Kivpoge and uChasob et al., 1987; Muaskey et al., 1937a; Rirke et al., 1987a; Rirke et al., 1987B; NaiIakh5 with co-authors, 1988; Dasor et al., 1985 and 1989; Rizhke and Atipeip, 1988; Otsipp et al., 1988 and 1989; I egbz et al., 1990; Zspozmapek and Megigaye, 1990; UUeidnavze et al., 1990; Qi et al., 1991/. According to reports, the pathway involving "С-Р lyase", which destroys glyphosate to sarcosine and inorganic orthophosphate /Ри/, is characteristic of Rzepdotopavz species /Zpipabagdeg and Vgauteg 1986; Kizpoge and Dasobr, 1987/ and species of ApPphorasieg /Rirke et al., 1987/. Shown is pure culture! species Biamorasiept and Naiav, 1986/, species Rzendotopavh /dasob and co. In addition, a large number of isolates that convert glyphosate into

AMRA was identified from industrial activated sludge, considered as glyphosate waste /Naazkh et al., 1988/. However, the number and nature of the bacterial genes responsible for the above-mentioned degradation are still unknown, and besides, not one of them has been isolated.

Consequently, one of the aspects of the invention consists in providing new genes encoding a glyphosate-metabolizing enzyme, by means of which glyphosate is converted into aminomethylphosphonate and glyoxylate.

Another goal of the invention is to increase the activity of the glyphosate metabolizing enzyme in relation to glyphosate by replacing specific amino acid residues.

Another aspect of the invention consists in obtaining genetically modified plants in which the gene encoding the glyphosate-metabolizing enzyme is expressed, and which are characterized by increased tolerance to the glyphosate herbicide.

The second task of the invention is to show that the glyphosate metabolizing enzyme can target the plastid! using transit proteins of chloroplasts, and such enzymes, the targets of which are plastids, cause a high level of tolerance to glyphosate.

The next aspect of the invention is related to the development of a method of vibrating the tissue of a transformed plant using a glyphosate-metabolizing enzyme as a selective marker in the presence of inhibitory concentrations of glyphosate.

Zty and other goals, aspect! and the characteristics of the invention will become obvious to specialists from the following description and working examples.

SUMMARY OF THE INVENTION

The invention provides DNA structural constructs that encode the enzyme glyphosate oxidoreductase and that determine the ability of heterologous microorganisms (i.e., bacteria and plants) to degrade glyphosate, as well as the production of glyphosate-tolerant plants.

In addition to the above, according to one of the aspects of the invention, a method of obtaining genetically transformed plants, which are characterized by tolerance to the herbicide glyphosate, is provided, including the following steps!: (a) insertion of recombinant double-stranded molecules into the genome of a plant cell! DNA, the composition of which includes: () a promoter functioning in a plant cell to induce the synthesis of an RNA sequence, (I) the structure of a DNA sequence that determines the synthesis of an RNA sequence that encodes the enzyme glyphosate oxidoreductase, (I) 3 non-translated DNA sequences, by which, in plant cells, polyadenylated nucleotides are added to the 3' end of the RNA sequence; where the promoter is heterologous in relation to the coding sequence and is adapted to induce sufficient expression of the specified enzyme in plant tissue, including meristematic tissue, in order to increase resistance to glyphosate of a plant cell transformed with the specified gene; (b) obtaining a transformed plant cell; and (c) regeneration from a transformed plant cell of a genetically transformed plant, which is characterized by increased tolerance to the herbicide glyphosate.

In accordance with the second aspect, the invention provides for the production of recombinant double-stranded DNA molecules, sequentially including; /a/ a promoter, the functioning of which in plants causes the synthesis of an RNA sequence; and /v/ 3 is a non-translated site through which polyadenylated nucleotides are attached to the 3' end of the RNA sequence in plants.

In addition, in accordance with the invention, bacterial and transformed plant cells are obtained, which contain DNA, respectively, including the above-mentioned element! 7a/, /6/ and /c/.

In accordance with another aspect of the invention, differentiated plants were obtained, which consist of transformed plant cells described above and possess tolerance to the herbicide glyphosate.

According to the second aspect, the invention provided a method of selective control of weeds in the field with planting of agricultural crops in the form of seeds or plants, and the method includes the following stages: (a) planting of specified seeds of agricultural crops or plants, which are characterized by tolerance to glyphosate, due to instillation into the seed of crops or itself plant recombinant double-stranded molecule! DNA, and this molecule includes: (I) the promoter sequence that induces the synthesis of RNA sequences in plants, (II) the DNA structure of the sequence responsible for the synthesis of RNA encoding the enzyme glyphosate oxidoreductase, (PI) the 3" untranslated region that encodes a signal polyadenylation, providing in plants the addition of polyadenylated nucleotides to the 3' end of the RNA sequence, where the promoter is heterologous to the coding sequence and is intended to ensure sufficient expression of the specified enzyme in plant tissue, including meristematic tissue, in order to increase the tolerance to glyphosate of transformed plants, the cells of which contain the indicated gene, and (b) treatment of the indicated crops and weeds in the above-mentioned field with a sufficient amount of the herbicide glyphosate to control the indicated weeds without significant damage to the cultivated plants.

In the most preferred version, the double-stranded DNA molecule containing the gene for plant expression includes a DNA sequence structure encoding a fusion polypeptide containing an amino-terminal transit peptide of chloroplasts, which ensures the entry of the enzyme carboxyterminal glyphosate oxidoreductase into the chloroplast of the plant cell, carrying out the expression of the specified gene.

The next aspect of the invention concerns the use of the glyphosate oxidoreductase gene! as a selective marker for vibration and identification of transformed plant tissue.

Description of drawings.

Figure 1 shows the DNA sequence for the full promoter of the cone-nosed mosaic virus (EMU).

Figure 2 shows the DNA structure of the glyphosate oxidoreductase gene sequence! from bacterial isolate I VAA.

In Fig. 3, the results are reflected! structure comparisons! of the glyphosate oxidoreductase gene, subjected to manipulation, with a modified glyphosate oxidoreductase gene with a high degree of expression in plants. The manipulated glyphosate oxidoreductase gene is represented as the top DNA sequence. And only the changes obtained in the modified gene, readings in the lower sequences.

In Fig.4, the structure of the processed glyphosate oxidoreductase gene is compared with the synthetic glyphosate oxidoreductase gene, and is characterized by increased expression in plants.

The manipulated glyphosate oxidoreductase gene is depicted as the upper DNA sequence.

Figure 5 shows the structure of pMoOM 17032, and the pMOM 886 vector contains a modified glyphosate oxidoreductase gene, embedded as Tp-Samu355 - modified glyphosate oxidoreductase - MO5 3" cassette! in My! vector site. The pMOM 886 vector is described in the text.

Fig. b shows the nucleotide sequence for the transit peptide STR of the chloroplast, originating from the 55Sh1A gene from A. Inaiap.

Fig. 7 represents a gene-structural map of the plasmid PMOM 17066, while the vector PMOM 979 type contains a gene encoding a fusion peptide HTP/synthetic glyphosate oxidoreductase. Production, related to RMOM 979 type, represented by RMOM 17065 and RMOM 17073.

Figure 8 shows a gene/structural map of the plasmid PMOM 17138, an example of a vector of the PMOM 981 type, containing a gene co-iodizing the fusion poly peptide HTP/synthetic glyphosate oxidoreductase. In this example, the gene of HTP1-synthetic glyphosate oxidoreductase! cloned in RMOM 979 in quality

Crap! - Cotton wool fragment.

Figure 9 shows the nucleotide sequence of the XTP2 transit peptide of the chloroplast, obtained from the EP5P5 gene from A. IPpaiyapa.

Fig. 10 presents a structural map of plasmid pRMOM 17159.

Fig. 11 presents a structural map of the plasmid pMOM 17226.

Fig. 12 presents a structural map of the plasmid RMOM 17164.

Description of the invention

The expression of a plant gene, presented in the form of double-stranded DNA, includes matrix synthesis

RNA (mRNA) on one of the DNA chains using the enzyme RNA polymerase and subsequent processing of the mRNA of the primary transcript inside the nucleus. In this process, a 3' signaling site is included, which ensures the addition of poly-adenylate nucleotides to the 3' end of the RNA.

The transcription of DNA into mRNA is under the control of a DNA region called a "promoter". The promoter region contains a sequence of bases that provide signals for the interaction of RNA polymerase with DNA, which leads to the initiation of mRNA transcription when one of the DNA strands is used as a template, resulting in synthesis of the corresponding complementary RNA chain.

A number of promoters active in plant cells are described in the literature. This includes the promoter! nopaline synthase (MOB) and octopine synthase! (OS5) (which are found in tumor-inducing plasmids

Asgorasielitst Shshptegasiepv), promoters! caulimoviruses, such as the promoter! Cauliflower mosaic viruses (Samm ) 195 and 355, as well as the promoter of the pine cone mosaic virus (EMU) 355, an easily inducible small subunit promoter! ribulose bis-phosphate carboxylase! (55808І5СО, a very common plant polypeptide). Each of these promoters was used to obtain different types of DNA constructs expressed in plants; see, for example, communication PCT UMO 84/02913 (Aodeg» et al., Monsanto).

In the invention, it is possible to use a promoter that is already known or that is identified for the purpose of induction of DNA transcription in plant cells. Such a promoter! they can beat you! from a variety of sources, such as plants and plant virus DNA, including, but not limited to, the CaMmUu355 and EMUZ355 promoters, as well as promoters isolated from plant genes, such as the 55080OVIZSO gene or chlorophyll and/or binding protein genes . According to the above, it is preferable that the selected specific promoter is capable of inducing sufficient expression, causing the synthesis of effective amounts of glyphosate oxidoreductases, necessary to obtain a plant characterized by significant tolerance to glyphosate herbicides. The amount of glyphosate oxidoreductase required to achieve the desired degree of tolerance often varies depending on the type of plant.

It is preferable that the promoter used has a fairly high degree of expression in all meristematic tissues, in addition to other tissues, since it is currently known that glyphosate moves and accumulates precisely in this type of plant tissue. As an alternative), a combination of chimeric genes can be used, which will lead to the necessary total level of expression of the enzyme glyphosate oxidoreductase and to the production of a phenotype tolerant to glyphosate.

The mRNA synthesized on the DNA of the construct of the invention also contains a 5' untranslated leader sequence. This sequence can be obtained from a promoter selected for gene expression and specifically modified in such a way as to increase the amount of translation

mRNA. 5 untranslated regions can also be obtained from viral RNAs, from suitable zukaryotic genes or from synthetic gene sequences. The invention is not limited exclusively to the constructions described in the following examples, where the untranslated region originates both from the 5 untranslated sequence that follows the promoter sequence, and from part 5 of the untranslated fragment of the viral envelope protein gene. Most likely, as discussed above, the untranslated leader sequence may be derived from a foreign promoter or coding sequence.

The preferred promoter used in the invention is the promoter of the full transcript (355) from the kidney mosaic virus (EMU), which functions as a strong and permanent promoter of chimeric genes inserted into plants, in particular, dicots. In general, the resulting transgenic plants express the protein encoded by the inserted gene at a higher and more constant level in all tissues and cells than the same gene controlled by amplification

Samm355 promoter. According to fig. 1, the DNA sequence of the promoter is localized between nucleotides 6368 and 6930 (POS IO Me: 1) of the EMM genome. The 5' non-translatable leader sequence is preferably connected to the promoter and typically the leader sequence (POS IJOO Mo : 2), which can be seen from fig.1. The leader sequence can be a derivative of the EMM genome or foreign EMM genomic sources. 3 untranslatable section of the chimeric plant gene contains a polyadenylation signal that provides polyadenylation of nucleotides to the 3' end of the RNA. Examples of suitable 3' regions are: (1) 3 transcribed, untranslated fragment containing the polyadenylation signal of tumor-inducing genes (Ti) plasmids in Adgobasiegut, such as the nopaline synthase (MOB) gene, and (2) plant genes, such as reserve protein genes soybean and the small subunit of the ribulose-1,5-bisphosphate carboxylase gene (55808І5СО). As an example of a preferred 3" segment, a fragment from 55808I5СО of the pea gene (E9U), described in more detail in the examples below, can be given.

The DNA structures of the invention also contain the structure of the coding sequence in the form of double-stranded DNA, which encodes the enzyme glyphosate oxidoreductase, which is responsible for the conversion of glyphosate into aminomethylphosphonate and glyoxylate.

Brief information about the glyphosate oxidoreductase reaction.

The glyphosate oxidoreductase enzyme catalyzes the cleavage of the C-M bond of glyphosate, leading to the formation of aminomethyl phosphonate (AMPA) and glyoxylate as reaction products. In industrial conditions, oxygen is used as a reaction cosubstrate. In the same conditions, other carriers of electrons can be reaction stimulators, such as phenazine metho-sulfate and coenzyme 0. In the absence of oxygen, this is a factor! play the role of electron acceptors.

The enzymatic reaction can be studied in an oxygen absorption test using an oxygen electrode.

Glyphosate oxidoreductase from | VAA does not lead to the formation of hydrogen peroxide as a product of oxygen reduction. The enzyme is characterized by a stoichiometry of two moles of oxidized glyphosate per mole of consumed oxygen and forms two moles of both AMPA and glyoxylate as reaction products.

An alternative method for testing glyphosate oxidoreductases includes the reaction of samples with 2,4-dinitrophenylhydrazine and determination of the amount of glyoxylate-2,4-dinitrophenylhydrozone by NRI C analysis, which will be described in more detail in the following sections.

The third method for testing glyphosate oxidoreductase! consists of used 3771 - glyphosate as a substrate; radioactive AMPA, obtained with the help of an enzyme, is isolated from the substrate by HRI C on an ion-exchange column, which will be described below. The radioactivity associated with AMPA is an indicator of the degree of completion of the reaction with glyphosate oxidoreductase.

Glyphosate oxidoreductase from IVAA is a flavoprotein that uses EBAO as a cofactor. According to our assumption, one of the mechanisms of the reaction catalyzed by this enzyme is the reduction of ЕАУО in the active site of the enzyme under the influence of glyphosate. This leads to the formation of reduced RAJ and the Schiff base of aminomethylphosphonate with glyoxylate. The schiff base is hydrolyzed with water and hydrolyzed with the formation of components,

AMPA and glyoxylate. The reduced flavin is again oxidized by molecular oxygen. We assume that during the process of re-oxidation of reduced EAO, oxygenated flavin is formed as an intermediate compound. that intermediate flavin can catalyze oxygen saturation of glyphosate, which leads to the formation of AMPA and glyoxylate. This hypothesis is in accordance with the detected stoichiometry and our inability to detect hydrogen peroxide in the reaction mixture.

In addition to glyphosate, the enzyme glyphosate oxidoreductase from I VAA oxidizes iminoacetoacetic acid (IAA) to glycine and glyoxylate. The reaction rate with YAUK is much higher than with glyphosate.

Isolation of bacteria that effectively break down glyphosate to AMPA.

At present, there are bacteria capable of breaking down glyphosate. (NaiiIav) with co-authors, 1988; Maiyik et al., 1989). A number of these bacteria were subjected to screening for the rapid cleavage of glyphosate by the following procedure: 23 bacterial isolates were transferred from TSA (Tryptykase Soy Agar;

BB) cups in medium A, including medium with Dworkin-Foster salts, containing glucose, gluconate and citrate (0.195 each) as a carbon source, and also containing glyphosate in the amount

O.1mM as a source of phosphorus.

Dvorkin-Foster's minimal medium was prepared by mixing 1 ml of each of components A, V C and 10 ml of 0, thiamine NS /5 mg/, C - sources to final concentrations of 0.195 each and P - source /glyphosate in 1 liter (autoclaved water! or the second phosphonate or Pi / up to the required concentration:

A. 0-E Salts / Breeding X 1000; for 100 ml; autoclaving/

NzvVOz mg

Mm5oO» 7NgO mg 7p50x 7Н20 12.5mMg bi5O» 5NgO 8mg

Mamosz ZNgO 1.7 mg

V. Re5O»x 7NgO / Breeding X 1000; for 100 ml; autoclaving/ 0.1 g

S. Ma5oO»X 7NgO /Razvedenie X 1000; for 100 ml; autoclaving/ 20 g

O. (MNa)" 5054 / Dilution X 100; per 100 ml; autoclaving/ 20g

Yeast extract was added /UE; Difko/ to a final concentration of 0.01 or 0.001905.

Every ml of cultural media! also contained approximately 200,000 srt of glyphosate (Amersham;

SEA.745). The culture was incubated at 307 with constant shaking. Isolate I VAA showed significant growth within one day, while other test cultures! were characterized by weak growth until the third day. Determination of radioactivity (by scintillation counting) in the culture, cell sediment and culture supernatant (on day 4) showed a decrease in total 77 radioactivity, and the remaining radioactivity was distributed approximately 1:1 between the supernatant and the sediment, which confirms significant absorption and metabolism of glyphosate.

Table 1

Metabolism of glyphosate in I VAA culture. control 18631

On the 5th day, 75 μl of the culture supernatant from all test cultures were analyzed by HRIS as follows: a 5UMSONAORAKM A X 100 anion exchange column (R. Soregp) was used; the mobile phase included 65 mM KNeRox (pH 5.5 with Mahon ; depending on the requirements of the experiment, the concentration of the phosphate buffer varied from 50 to 75 mM in order to change the retention time of the material), was advanced isocratically, and the diluted material was subjected to continuous control using a radioactivity detector. The analysis showed that in one isolate (in particular, I! VAA) the peak glyphosate (retention time IVZ | - 7.0 minutes in this analysis) was completely absent, but a new radioactivity peak appeared with VZ, similar to the VZ of methylamine or M-acetylmethylamine (83 - 3.5 minutes).

The assessment of the collection of bacteria, part of which was strain I VAA, was carried out according to the degree of cleavage of glyphosate into AMPA (Nayaz et al., 1988); the detection of methylamine or M-acetylmethylamine suggests that AMPA or M-acetylAMPA are metabolized with the help of IVAA. "S-P lyase" activity, resulting in the release of phosphate, which is necessary to ensure growth in this experiment. Strain I VAA was studied in more detail.

Transformation of glyrosate into AMPA in microbial isolates.

For the sake of clarity and brevity of the presentation of the discovery, the description of the procedures for removing the genes encoding the enzyme! glyphosate oxidoreductase, is reduced to the description of the isolation of such a gene from a bacterial isolate (I VAA). For specialists, it will become clear that the same or similar tactics can be used to isolate such genes from other microbial isolates.

The pathway of exchange with the splitting of glyphosate in resting cells of the strain cultivated in the presence of glyphosate, GVAA, is characterized by the following features: cells from 100 ml cultures: IVAA, incubated in OE medium in the presence of glucose, gluconate, and citrate as sources of carbon, thiamine, yeast extract /0.0195 /in trace amounts /hsreda YuEZ5/; and also in the presence of glyphosate in a concentration of 0.2 mM as a source of phosphorus, collected at "KLETT" - 200, twice! 20 ml of OEZ5 medium were washed, and an equivalent of 20 ml of cells was resuspended in 100 μl of the same medium containing I372C| glyphosate /2.5 μl 52 microcuries/millimol/. The cell mixture was incubated at 30"C with constant stirring and a sample (20 μl) was collected after a certain interval. The sample was centrifuged and the precipitate and supernatant were subjected to VZ3LC analysis / the cell sediment was resuspended in 100 μl of acid - OEZ5 /-OEZ5, 0.65 N NOSI, subjected to boiling for 5 min., a short centrifugation, after which the obtained supernatant was studied; acidified glyphosate was studied as a control. In two hours, the amount of radioactivity in the glyphosate peak /B3 - 7.8 min./ of the supernatant decreased to about -3395 of the initial level; about 395 glyphosate was detected within the cells. The material that co-eluated with the methylamine standard was approximately -595 ot of the original label in the supernatant and about 1.595 in the cell pellet. A new peak of about 1.595 ot of the original radioactivity with a WZ of 7.7 minutes /B3Z of glyphosate - 8.9 minutes under the acidification conditions of this experiment/ protein was detected in the cell contents. A strong decrease in of radioactivity suggests that glyphosate is metabolized in this experiment at a significant rate. In the future, the experiment managed to elucidate a number of details related to the exchange pathway, by means of a comparative study of the metabolism of GHA. AMRA and IZYAS glyphosate / cm. more/, in resting cells collected at Cleet 165 and resuspended in the equivalent of 15 ml of cells per 100 μl

OEZ35 Wednesday. The study of the samples was carried out by VLC analysis, and the samples! consisted of whole cultures, acidified and processed as described above. During the first two hours of the experiment with glyphosate, 2595 radioactivity was detected in the methylamine/M-acetylmethalamine peak /ВЗ3 - 4 8 min./, 12.595 as AMPA /53 - b.4 min./, 3095 as the above-mentioned peak / B3 - 9.4 min./ and 30905 as glyphosate /B3 - 11.8 min./ In the AMPA experiment, 15956 of the radioactivity corresponds to M-acetylmethylamine/methylamine, 5995 to AMPA, and 1895 is found in the peak with VZ - 9.4 min.

The modified form of AMPA is defined as M-acetylAMPA. A similar stage of acetylation was the result of the products identified in B. soybean cultivated in the presence of aminomethylphosphonates as sources of phosphorus /Amiya Yeu1., 1987/ These data show that the path of splitting glyphosate in I VAA includes the following stages; glyphosate -» AMPA / -» methylamine / -» - acetylAMPA -» M-acetylmethylamine.

Cloning of Glyphosate oxidoreductase gene /genes/ in E. soybean.

After establishing the fact of conversion of glyphosate to AMPA in line I VAA, a direct approach was developed for cloning the gene(s) involved in the early process in E. soybean. Since there were no other methods, they were used! general cloning and genetic method for these purposes, described by Mapiai5 et al., 1982. The cloning method is as follows: introduction of a cosmid bank of line I VAA into ECs and selection for the gene/genes responsible for the transformation of glyphosate into AMPA, according to the required growth rate on the medium , including glyphosate as a source of phosphorus /P/. This selection is based on the use of AMPA, obtained with the help of an enzyme that metabolizes glyphosate as a source of phosphorus /P/, which is accompanied by the removal of Ri from AMPA by means of

E. Soyii "S-R Liaz". The majority of strains of E. soya are not capable of utilizing phosphonates as P sources in primary cultures, however, they adapt very quickly, regardless of VesaA, to the utilization of phosphonates /becoming Mry" //MaskKey! with co-authors, 1987/. E. soya "Mry" was isolated from E. soybean BA 200

Ley, Rgo, hesA, pzan, zurE, Zt/", YuyupA, as follows: a volume of fresh I-broth cultures of E. soybean ZA 200 were placed on MOR5 /Meiyadnagai et al., 1974/ complex agar /eg containing I-leucine and I-strait in the amount of 25 μg/ml and vitagin B (thiamine) in the amount of 1 μg/ml; agar - DIFKO "Purified"/ containing aminomethylphosphonate /"AMPA; 0.2 mM; Sigma/ as a source of phosphorus.

MORZB Wednesday includes: 10ml 10X MOR5 salts 2ml 0O.5mg/ml Theanine NOSE 1ml 2090 glucose!

X MOR5B salts include: per 100 ml d4Oml 1M MOV pH 7.4 4mMl 1M Tricin pH 7.4 1Tml 0.01M Re5O» 7H2gO

Bml 1.9M MNAaSI 1ml 0.276M K»50x 1T1ml 0.5mM Sasig 1Tml 0.528M Mass» 10ml 5M mass

IML 0.595 |! - Methionine 1 ml of microelements

Microelement! include:

Z x 1099M /yuNa/6Mp7Og4 4 x 10-7M NzVO»X

Z x 109M Sosi» 1.6 x 109M SibO4 8 x 109M Mpsig 1 x 108M 71504

After three days of incubation at 37°C, 6 individual colonies were sown! with a stroke from the indicated cup on MOP5 full agar containing either AMPA or methylphosphonate /Alfa/ as a source of phosphorus. One colony, designated as E.soyii 5A 200 Mry. white was selected from all colonies that showed equally uniform growth on both media with phosphonate.

Chromosomal DNA was prepared from the GVAA strain as follows: cell pellet from 100 ml of I-

Broth /Misheg, 1972/ culture in the late logarithmic phase and VAA was resuspended in 10 ml of solution 1 /Vigproit and Oou, 1979/, then DSN was added to a final concentration of 195, and the suspension was subjected to three times freezing - thawing, and each such cycle included immersion in dry ice for 15 minutes. and then in water at 707 for 10 minutes. The resulting lysate was extracted four times with equal volumes of phenol:chloroform/1:1; phenol, saturated TE/ /TE - 10mM Tris pH 8.0; 1.0 mm ZDTK/, and the phases were separated by centrifugation /150009; 1 min. The material treated with ethanol was precipitated from the supernatant by a short centrifugation /80009; 5 min./s with the subsequent addition of two volumes of ethanol. The precipitate was resuspended in 5 ml of TE and dialyzed for 16 h . at 4"C against 2 liters of TE. This procedure made it possible to obtain bml of DNA solution of 15 Ωkg/ml.

Partially restricted DNA was prepared as follows: three samples of DNA and VAA with a volume of 100 μg were treated for one hour at 377°C with restriction endonuclease Nipa I in doses of 4, 2, and 1 units of the enzyme per μg of DNA, respectively. Samples were collected, 0.25 mM was prepared solution! DNA with ZDTC and extracted with an equal volume of phenol:chloroform. After adding Ma Acetate and ethanol, the DNA was precipitated with two volumes of ethanol and the precipitate was obtained by centrifugation /120009; 1 min./.

The dried DNA precipitate was resuspended in 500 μl of TE and layered on a 10-4095 sucrose gradient /595 increments of 5.5 ml/ in 0.5M Mass, 50mM Tris pH 8.0, 5mM ZDTK. After centrifugation for hours at 2600006. in min. with the use of a 5M/28 rotor and a puncture of the test tubes of the assembly! fractions with a volume of 1 ml. 15 μl of sample from each third fraction was analyzed in 0.895 agarose gel, and DNA size was determined by comparison with linear lambda DNA and Nipa IP-abrasive lambda DNA standards.

Fractions containing 25 - 35kb fragments! The DNA of the congregations was beaten, they will be exposed! desalination on the columns of

AMIKONOM 10 /7000 rpm; 207C; 45 min. and concentration! by precipitation. This procedure gave 5 Ohmkg of IAA DNA of the required size.

Plasmid pHC79 DNA /Knopp and SoiYipe, 1980/ and treated with Nipa I -phosphotase vector were prepared according to the description /Mapiai5 et al., 1982/. The conditions for ligating whites are as follows:

DNA vector /treated with Nipa III and calf chain phosphatase/ - 1.6 μg.

The size of fractionated I VAA Nipa II fragments - 3.75 µg 1OH ligation buffer - 2.2 µl 250 µM Tris-HCI, pH 3.0; 100 mM Masi!d 100 mM dithiothreitol; 2MM Spermidine

TA DNA ligase /Boehringer - Mannheim/ - 1.0μl /400 units/μl/

NgO until 22, Omkl 18 hours. at 1670.

The ligated DNA /4μl/ was packed into lambda phage particles /5igaiadepe; Sidarask Со1а/,

using the manufacturer's method.

A culture of E. Soyii BA 200 Mry", which grew overnight in I-broth /with maltose 0.295/, was infected with 5 µl of packaged DNA. Transformants were selected on MOR5 complex agar containing ampicillin and glyphosate at a concentration of 0.2 mM as a source R.

Samples of certain volumes were also placed on MOR5 (Mei-nagai et al., 1974) complex agar with ampicillin containing Ri at a concentration of 1 mM for titration of packaged cosmids. On this medium, after 2 days of incubation at 377C, the cosmid transformant was isolated in a volume of approximately 105 per μg/ GVAA Nipa II DNA. On agar containing glyphosate, colonies appeared from 3 to 10 days with a final concentration of 71 per 200-300 cosmids. Plasmid DNA was prepared from 21 cosmid transformants formed in cups with glyphosate. These cosmids can be divided, at least, into two classes, based on the Nip type and restriction of plasmid DNA. In class 1, each of the cosmids indifferently cloned a 6.4 and 4.2 kb fragment of Nip II restriction, and in class II - a fragment! - about 23kb. Ten cosmids representing different cloneable fragments were subjected to retransformation in E. strain 5A 200 Mry", and utilization of glyphosate was checked by selection for growth on

MORB complex agar containing ampicillin and glyphosate. In addition, the final cell density achieved in a culture using glyphosate (0.2 mM in MOR medium) as a source of P was determined, and only small differences could be detected between individual transformants.

The transformants were also introduced into MOR complex broth with AMPA at a concentration of 0.1 mM as a source of phosphorus (to guarantee the presence of "C-P lyase" activity) and after 24 hours of incubation at 37"C, a hundredfold dilution was performed in MOR5 complex medium with glyphosate in concentration of 0.1 mm and

All cosmid-containing cells cleaved glyphosate and formed M-acetyl AMPA and M-acetylmethylamine, with small differences in the rate. In these tests, M-acetyl AMPA was detected in the culture supernatant. One of the Class I cosmids, identified as PMOM 7468, was selected for further study. The second glyphosate oxidoreductase gene was identified from the Class I cosmid clone.

Free from cells to lick! E. soybean 5A 200 Mry"/pRMOM 7468 were obtained from cells growing on

MORB5 in a complex medium with glyphosate at 1.0 mM (with the addition of I-phenylalanine, I-tyrosine and 1-tryptophan at a concentration of 100 μg/ml each and para-oxybenzoic acid, 2,3-dioxybenzoic acid and para-aminobenzoic acid at a concentration of 5 µg/ml, to minimize the effects of ERBZR synthase inhibition). The cell pellet (about 0.5 g of wet weight) was resuspended in a lysing buffer (40 mM MORB, pH 7.4; 4 mM Tricin, pH 7.4; 1095 glycerol; 1 mM DTT) and passed twice! via the French Press. Cellular remains were removed by centrifugation at 15,000 rpm. in min. within 10 minutes. The supernatant was analyzed after adding MosSi to 10 MM to destroy labeled glyphosate. The glyphosate substrate was used as glyphosate (final concentration - 17 μM). We detected a white product, mainly AMPA and a certain amount of M-acetyl AMPA; AMPA production is an indicator of the cloning of enzymatic activity from strain I VAA however, the presence of M-acetylAMPA could be due.

E. soyi zndogenic activity (Amiya et al., 1987). The specific activity for the formation of AMPA under these conditions was 13.3 pmol AMPA/min., mg of protein.

Characteristics of Glyphosate -» AMPA gene. The area of cloning responsible for this activity of the enzyme glyphosate oxidoreductase! white is then localized in the plasmid. Deletions of PMOM 7468 were isolated primarily within the cloned region by using restriction enzymes, which sometimes cut inside the insert, as follows: samples of plasmid DNA 0.5 - 2 μg were subjected to complete cleavage using restriction endonucleases Moi I, bas I, Va! II or

Vatn I, followed by extraction with phenol-chloroform, precipitation with ethanol, resuspension in

TE buffer and ligated for 2 - 4 hours at room temperature (or for 18 hours at 16"C) in a final volume of 50 μl of ligation buffer and T4 DNA ligase. The transformants were subjected to selection in E. soya 5A 200 Mry", after che zty deletions were investigated for the loss or inhibition of glyphosate-utilizing phenotypes.

This data is combined with restriction mapping of clones, if you use it! for the localization of the active region in the vicinity of the central part of the insert in Prom 468, which included two common NIP I fragments (6.4 and 4.2 kb). Next, a fragment of Nip and restrictions from this area were subcloned! in r Viye siri (Zigaiadepe) and their glyphosate phenotype was determined in E. soybeans. In MYO Mry" (Mry" derivative.) MISSION was selected according to the description for 5A 200 Mry"). Clones containing 6.4 kb. Nipa

And the fragment, in any orientation, was capable of utilizing glyphosate. After restriction mapping of that NIP I fragment, white isolated seryl clones with a deletion of two 6.4 kb. Nipa HER clones when using enzymes, which sometimes produce cutting in the insert, as well as in the region of the polylinker. Bly will also undergo subcloning of a number of restriction fragments internal to the Nip I fragment. 3.5 kb Pv I and 2.5 kb Vodi Ii fragments, in any orientation, had the ability to utilize glyphosate. This data, combined with data on deletions, was used to localize the active region directly near 1.8 kb Va! P-Khpo | fragment

In addition, the deletions obtained from the 6.4 kb Nip I fragment were characterized by the minimal size of the coding region, approximately 0.7 kb, with Eso V I and Bas I sites, possibly localized within the coding sequences.

The direction of transcription/expression of the locus responsible for the activity of the enzyme responsible for the conversion of glyphosate into AMPA was determined as follows: E. soybean UM 101 Mry" transformant rMOM 7469 Zh | i Zh 4 (Clones 2.5 kb Vo! I fragment WatH | site fig 118 ; opposite orientation) grew in MU - glucose-thiamine-ampicillin broth, in the presence and without

RI AC inductor IRTO, material collected in the late logarithmic phase (Klett 190 - 220); prepared as described above, the cells are free to lick! of four cultures, which were then examined for activity,

responsible for the transformation of glyphosate into AMPA in the presence of 17 μM glyphosate. The highest enzymatic activity was obtained for PMOM 7469 and | plys IRTa, where the Hpo I site is located distally to Rias, assuming that the genes expressed in Vo) II are directed to Hpo I.

Table II

Activity responsible for the transformation of glyphosate into AMPA in cell-free lysates of E. soybean transformants.

IRT Specific

Clone added activity pmol AMRA/min.mg rMoMmtiveni! | yes | 320 g of rmomtiveyanya| yes | "30

AMPA was the only detected product, thus showing that AMPA acetylating activity, which, as described earlier, was induced in T. soybean transformants incubated on glyphosate as a P source.

In a more late experiment, cell licks of RMOM 7469 and 1 and RMOM 7470 (Ba 1I-Hpo! 1.8kb in fig.

I8; formed from рРМОМ 7469 5 1 by deletion - 700 np KhpoI-Za!! fragment) white studies! on the activity responsible for the transformation of glyphosate into AMPA, in the presence of 2 mM glyphosate (sp.act. IZ" 1421 glyphosate - 3.7 millicuries/millimol; 0.2μcuries/reaction; culture! grew in the presence of IRTO in the medium), while white a much higher enzymatic activity was recorded, indicating more favorable conditions for the subject.

Table PI

Activity responsible for the transformation of glyphosate into AMPA in cell-free lysates of E. soybean transformants. nmoles AMPA/min.mg

Squirrels, let's code Va! It is a fragment of which we will determine the expression, using the T7 expression system (Tarog and

Visnagazop, 1985), after the cloning of that fragment in the Vatna site in the rVine vector, zepri (4) (RMOM 7471 яЯ1, 2; opposite orientations). The test and control plasmids were transformed into E. soybean KZ8 containing rari-2 (Tabrog and Vispagazop, 1985) and incubated at 30°C in I broth (2 ml) in the presence of ampicillin and kanamycin (100 and 5 Ωkg/ml, respectively) Klett shows about 50.

A certain volume was removed and the cells were collected by centrifugation, washed with MO salts (Miet, 1972) and resuspended in 1 ml of MU medium containing glucose (0.295), thiamine (20 μg/ml) and 18 amino acids at 0.0195 each (except cysteine and methionine). After incubation at 30"C for 90 minutes, the cultures were transferred to a water bath at 42"C and kept in those conditions for 15 minutes. Then Rifampicin (Sigma) was added to a concentration of 200 μg/ml and the culture was additionally incubated for 10 minutes. at 42"C, after which they were transferred to 30"C for 20 minutes. Try it! were kept with 10 microcuries of 55-methionine for 5 minutes. at 30°C, the cells were collected by centrifugation and suspended in 60 - 120 μl of kraging buffer (b0mM Tris-HCl 6.8/19560 DSN/195 2-mercaltostanol/1095 glycerol/0.0190 bromophenol blue). Determination of the volume of the samples was subjected to ! from electrophoresis in 12.595 DON-

PAAG and subsequent impregnation in the course of bomin. in 10 volumes of the mixture: acetic acid-methanol-water (10 : 30 : 60), the gel was soaked in EMHPaNTMIMSa tm (PVOROMT), according to the manufacturer's instructions, dried and exposed at -707C with X-ray film. Proteins labeled by using 355-methionine were determined only for Va II -» Chpo I directions, and the largest one was about 45 kd. In that case, when the Vai II - Hno I fragment was studied after cloning in the Wat NI - Hno I site of rVishye 5spiri (with the aim of forming RMOM 7472), the expression of this -45kb protein was still noted.

The effect of expression of the activity responsible for the transformation of glyphosate into AMKA on the tolerance to glyphosate of E. soybean was initially determined by monitoring the growth of recombinants in a medium containing inhibitory concentrations of glyphosate. The test compared the growth of E. soya UM 101 containing the control vector (ryC118; Miega and Mevvipd, 1987) or the ROS118 clone of a 2.5 kb fragment of Ba! (PrRMOM 7469 1, y4). A very clear correlation between the ability to utilize glyphosate and tolerance to glyphosate is shown. This tolerance phenotype (resistant to 15 mM glyphosate) was then used to perform rapid screening of the phenotypes of deletion clones, such as PMOM 7470 (VODI I -

Ho I 1.8 kb in pOS118; formed from RMOM 7469 Zh! by delenii -700np Ho And! - For! And fragment) and subsequent clones.

Nucleotide sequence of structures! glyphosate oxidoreductase gene!

The nucleotide sequence of Vodi Ii - Xpo I fragment (POS IO Mo 3) was determined using single-stranded DNA matrices! (obtained as a result of using phagemid clones and "helper"

M13 phage A 408) and the commercially available ZEOEMABE "M (International Biotechnology Ips) set. As a result of computer analysis of the sequence (POS IO Me 3) a single large open reading frame (ROS) was found in the direction from Ba Ii to Chpo I, which is presented in Fig. 2 and includes sections of some of the related restriction sites. The putative stop codon (SAA) is localized in position 2np 5 of the restriction site sa|. The data confirming that the ODA codon is a termination codon - 45 kd ROS, were obtained as follows: they were previously determined 3' limits, based on the phenotype capable of glyphosate utilization, which must be located between the bas and site 95np upstream of the 5sa site and the Ho site.

Zsa and the fragment were cloned in Vat NO - Zta and the site! rViye Zsgiri, and under conditions of protein expression in vivo, protein - 45kD continued to be synthesized. Wadi II - sa | The fragment was then re-cloned from that rMore than 5 clones as Khbva I - Nipa I in pOS118 Khbva I - Nipa II, as a result of which resistance to 15 mM glyphosate was confirmed for BE.soy UM 101 transformants. According to these data, the C-end -- 45kD protein is localized between bas I and sa | sites. The only stop codon, in any reading frame, between these sites is located directly upstream of the 5th site.

The two above-mentioned codons (AOS; located in positions 120 and 186) when used as EI will ensure the formation of proteins of 46140 and 44002kd, respectively, but will not precede the well-recognizable Schein-Dahl-grano sequence.

The starting point of the white squirrel is set more precisely next! image: sequence of recognition of Water! And the restriction site was introduced in the positions upstream of two potential start codons by site-directed mutagenesis of PMOM 7470, by substituting ASATST for the sequences АСАСТа ("Во 120") and аТАТАС ("Во 186"), 21 and U9np upstream of AOS 120 and AOS 186, respectively. Except where noted, oligonucleotide primers for mutagenesis included sequences characterized by flanking changes of 8-10 homologous bases on each side. Tolerance to glyphosate was determined for mutated clones. The introduction of the VDII site against the flow of ASHS did not have an effect on tolerance to glyphosate, while it was eliminated by mutagenesis, due to the introduction of the VII site against the flow of AOsSivv. Effect! these mutagenesis on - 45kD proteins were studied by subcloning the mutated sequences in the 77 zxpression vector, using a site in the RMOM 7470 (Krp I) polylinker, just upstream of the initial Ba II site, and downstream of the Nip II site. This complex fragment was cloned again in ri80T317 (PHARMACY) Krp I - Nipa III and tested in vivo as described above. -- the 45 kd protein continued to be expressed at comparable levels with each of the mutagenic sequences of Water II. If new 5891 II site! were used! in the quality of 5' ends (and along the course of Nipa

Ш site) for cloning in rVishye - gray Vat No - Nipa PP site, then - 45kd protein was still expressed when the new Vadi I site against the flow of ASHsSiig2o served as the 5 end, but not the one that was localized against the flow of ASHsSiivvb was 5' youth These data largely suggest that ASHStgo (or the codon itself is localized close to it) is the M-terminal end of the glyphosate oxidoreductase protein. Wow! And the site introduced against the flow of AOSiv does not lead to the formation of a prematurely terminated or highly unstable protein, and suggests that predictable changes in the coding sequence as a result of that mutagenesis (MaInv-Suviv -"

Agud1v-Aiato) affected the activity of the enzyme.

Further data to confirm the localization of the M end has been received! by separate introduction (through mutagenesis of RMOM 7470), the recognition sequence of Mso and the restriction site (SSATOS for STATatT; changes in the second codon from Serine to Alanine) or Maye! sequences (SATATO for

SSTATO) in AOSizgo and the expression of this ORS using efficient E. soybean expression vectors.

Below is a description of the Has I expression: Has I - Nipa Ii fragment beginning in the assumed

Atsa, cloned in RMOM 2123 (Mae I - Nipa II), replacing the E - ISE - | fusion fragment (U/opd et al., 1988) The obtained clone was injected into E. soya UM 101 and the cells were induced in the presence of nalidixic acid, according to the description, for 2 hours. (M/opds et al., 1988). The resulting protein differed in size from - 45 kd protein on SDS-PAGE, and the cell lysate from the induced culture had a specific activity of glyphosate oxidoreductase equal to 12.94 nmol AMPA/min.mg. When performing comparisons in a separate experiment, no differences were found for glyphosate oxidoreductase activity if the second codon was represented by Alanine instead of Serine.

The structural DNA sequence for the glyphosate oxidoreductase enzyme (POS IO Mo 4) begins at nucleotide 120 and ends at nucleotide 1415 of the Ba II - Khpo I fragment of Fig. 2, and the glyphosate oxidoreductase enzyme consists of 431 amino acids! (POS IU Mo 5).

Construction of vectors for the transformation of the plant gene glyphosate oxidoreductase!.

To optimize the construction of the structural glyphosate oxidoreductase gene, the internal sequences of recognition of restriction sites Eso VI and Mso | were removed by site-directed mutagenesis to replace the sequence of SAATTI with SAATTO and SSASOasn with SSATOO, respectively. The glyphosate oxidoreductase coding sequence, suitable for introduction into the vector transformation of plants and their expression, is formed as follows: Mso ("Mei-Aia") M-end is joined with Mso I and Eso VI by the deleted coding sequence, and the C-end is deleted to 5sa | site, using the internal 5rpi I and Eso VM site on a number of cloners! restrictions On those tributaries of Water II, the site was located directly upstream of the Mso I site, a

Eso VI and Nip AI assays were localized directly downstream of the stop codon.

The sequence of the manipulated glyphosate oxidoreductase gene (POC IO Mo 6) is shown in Fig.3. The manipulated glyphosate oxidoreductase gene is still capable of encoding the wild-type glyphosate oxidoreductase protein. Manipulations do not change the amino acid composition of the glyphosate oxidoreductase sequence. This is the structural sequence of glyphosate oxidoreductase (POS IO Me b), as a Ba! P/IMmso I - Eso Vi/Nip I fragment of 1321 np, easily cloned into the appropriate plant expression cassette. Ztot glyphosate oxidoreductase gene (POS

IO Me b) was cloned as a Va II - Eso VI fragment into the plant transformation and expression vector pRMOM 979 for the formation of RMOM 17073.

Modification and re-synthesis of the glyphosate oxidoreductase gene sequence.

The glyphosate oxidoreductase gene from IVAA contains sequences that can cause an adverse effect on the level of expression of this gene in plants. These sequences include potential sites of polyadenylation, which are often enriched

At 96 (34), which is higher than the values found in plant genes (5695 versus 50905), the concentration of bands of X and C residues, and codons, which are often not used in plant genes.

The high value (1-095 in the glyphosate oxidoreluctase gene has a number of potential consequences, including the following: higher use (C or C, compared to that shown for plant genes in the third is located in codons, and the possibility for the formation of strong, hairpin-type structures that can affect the expression and stability of RNA. A decrease in the content of C in the glyphosate oxidoreductase gene, breaks in bands (5 and C, the disappearance of potential polyadenylation sequences and optimization of codon usage, which is more often used in plant genes, can lead to a higher Expression of glyphosate oxidoreductase in plants.

In the first phase of the experiment, selected areas of the gene were modified by site-directed mutagenesis. These modifications are directed, first of all, but not exclusively, to the reduction of (1--C95 and to the destruction of some of the C44C clusters. The manipulated glyphosate oxidoreductase gene was first recloned into the phagemid vector PMOM 7258 as a Mso I - Nipa |! fragment for formation of RMOM 17014. A single DNA chip was prepared from the strain E. soya. protocols presented with this set.

For clarity, the reverse complement is presented: true primers. The position of the nucleotides in the sequence shown in Fig. 2 and 3, corresponding to the primers, indications! the first and second set of numbers, respectively.

IM table

Primer for modifying the coding sequence of the glyphosate oxidoreductase gene.

Primer 1 (149 - 210; 38 - 99)

SOSTOSASST SSAATSSTTS STOTATOSAS TOSTITSATo STTSAASSTe

STOSATTSAA Coll. (ZEO TO SHMO:27)

Primer 2 (623 - 687; 512 - 576)

OSASATSSTS TSTOSTbATO STTTOSSTSA TTTSSATSST vАСstТtТСsТСОС

ATOSTTTTTAS sliaso (BO TO MO:28)

Primer 3 (792 - 832; 681 - 721)

STSATSSOT? G?sSASToA MOOTOSTosT STSAAASOS t (BEF TO MO:29) )

Primer 4 (833 - 901; 722 - 790)

TASAASSAST AASSSTSTTS TOSSTOoTtTbA TOSASSSTsSTtT oTTOSASSTO

STOSASASTS TALATSAST (ZED by MO:30)

Primer 5(1031 - 1091; 920 - 980)

SZAAATOSOoT STtTOSstTOoTttTo STOSTASTOT TOSASsTTtTOST ssotstsAsAb stTostTostAA s (BVKO PIP) MO:31)

Primer 6 (1179 - 1246; 1068 - 1135)

TOSATOOSTI TTOSTOSTAS SATTOSTOAT TSISTTSSAb TOATTOSTSO

TOoSAASTOST ASASSSSsA (BO I MO:32)

Primer 7 (1247 - 1315; 1136 - 1204)

SSTAATSTAT OSTITIOSTS ASOSTSATST SOSTATSASA SSTOSTSSAA

TSASTOSAAEs Ttstsstosto (BO Io MO:a3)

The similarity of the obtained gene /VOC IO Me 7/ with the control gene subjected to manipulation, glyphosate oxidoreductase! was confirmed by the nature of its nucleotide sequence, as well as its ability to provide a level of tolerance to glyphosate comparable to the control gene. This modified gene /POS IO Mo 7/ is designated as the gene "modified glyphosate oxidoreductase!".

Sbya-Soy glyphosate oxidoreductase gene /LOS IUO Mo 6/ is reduced from, approximately, 5695, characteristic of the variant with manipulation, to approximately 5296 in the modified case /PLOS IUO Mo 7/,

A comparison of the gene obtained as a result of manipulations and the glyphosate oxidoreductase gene as a modified variant is shown in Fig. 3, and the first variant is presented at the top, and the changes obtained as a result of the modification are at the bottom. This modified, glyphosate oxidoreductase gene was cloned as a Vadi II - Eso VI fragment into a plant expression cassette, which includes the Ep - Ca MM355 promoter and the MO5 3" sequence. The cassette was then cloned as a My 1 fragment into the PMOM 886 vector to form PMOM 17032 /fig .5/.

The synthetic glyphosate oxidoreductase gene /VOC IO Mo 8/ is necessary to ensure, as far as possible, those unnecessary sequences that were discussed above. As a summary, the gene sequence was reconstructed with the aim of eliminating, as far as possible, the following sequences or individual sequence features /while avoiding the introduction of non-obligatory sites of restriction/: length (5 and C residues 5 or more; regions, enrichment Act / mainly/ which can perform the function of polyadenylation sites or the region of potential destabilization of RNA, as well as codons that are not so often found in plant genes. Comparison of glyphosate oxidoreductase genes obtained as a result of manipulations /PpOS IU Mo 6/ and synthetic /POS |IO Me 8 / is shown in Fig. 4, and the manipulated gene /POS IO Mo 6/ is presented at the top, and the differences caused by the synthetic gene /POS IO Me 8/ are at the bottom. 4-90 for the synthetic glyphosate oxidoreductase gene is - 5195 and potentially provides a rapid release a large amount of energy, and a "spire-shaped" structure! physical degree of reduction. This synthetic gene was cloned as Va! II - Eso VI fragment in rMOoM 979 with the purpose of formation of RMOM 17056 for introduction into plants.

Chloroplast expression directed by glyphosate oxidoreductase.

The target of glyphosate in plants, the enzyme 5-enolpyruvylshiki-mat-3-phosphate synthesis /ERBR5B/, is localized in the chloroplast. Despite the fact that glyphosate oxidoreductase activity, localized in the cytoplasm, reduces or prevents the entry of glyphosate into the chloroplast in transgenic plants, the direction of the glyphosate oxidoreductase enzyme directly into the chloroplast, according to the data, leads to a further decrease in the effects of glyphosate in relation to ERBR synthase. Many proteins localized in chloroplasts are expressed from nuclear genes as precursors and are sent to the chloroplast by means of the chloroplast transit peptide /khTP/, which is secreted during the required stage.

Examples of such chloroplast proteins are the small subunit /MSE/ Ribulezs-1,5-bisphosphate-carboxylase! /Vybizso/ 5-enol-pyruvyl-shikimate-3-phosphate synthase /"ЕРОРБ/", Ferredoxin, Ferredoxin oxidoreductase, light-harvesting complex protein 1 and protein 11, as well as Thioredoxin E. It was shown that these are not chloroplast proteins they can also be a target for chloroplasts by means of proteins fused to HTP, and that the sequence of HTP is quite sufficient for the protein to become a target for the chloroplast /ZePa-Siorra et al., 19877.

The glyphosate oxidoreductase protein becomes a target for the chloroplast as a result of the formation of a connection between the C-terminus of CTP and the M-terminus of glyphosate oxidoreductase. In the first example, a specific HTP was used, obtained from MSE IA gene from Agoridorvy (paiapa /Gitko et al., 1938/.

The HTP strain /designated as HTP1/ was formed by a combination of site-directed mutagenesis. HTP1 structure /LPOS IO Me 9/ /fig.6/ includes MSE 1A HTP/ amino acids! 1 - 55/, the first 23 amino acids! mature MSE 1A protein /amino acid 56 - 73/, a serine residue /amino acid 79/, a new segment that repeats amino acids 50 to 56 from MSE 1A HTP and the first two amino acids! from mature protein/amino acids! 30 - 37/, as well as alanine and methionine residues /"amino acids! 38 and 39/. The Mso | restriction site is localized at the 3 end /captures the Mei codon/ in order to ensure a more precise fusion with the 5' glyphosate oxidoreductase or other genes. at a later stage, the VaI site is introduced upstream of the M terminus of the MSE TA sequence to facilitate the insertion of the fusion into the plant transforming vector. Mso 1 site/ in ROEMZ g" 1 (c) vector with the purpose of forming PMOM 17034. This vector can be transcribed in vitro using 5Rb polymerase and subsequent RNA translation with the participation of 355-Methionine, which will ensure the production of material that can be introduced into chloroplast, isolated from the 1st axis, using the method described below by AePa-Siorra et al., 1936, 1937. However, it was found that this HTPI1-glyphosate-oxide oreduktase fusion is characterized by 995 efficiency of entry into the chloroplast! compared to the control, 255 markers of PreEgERBR5 /rMOM 6140; Aepa-Siorra et al., 1936/.

Next, the construction of XTP1-glyphosate oxidoreductase fusion with synthetic glyphosate oxidoreductase gene /VOC IO Me: 8/ was obtained, which was introduced as Water! I - Eso VI fragment in the plant vector PMOM 979 with the purpose of forming PMOM 17066 / fig.7/ After an intermediate cloning step to acquire a larger number of cloning sites, the HTP1-glyphosate oxidoreductase fusion was also subjected to cloning as a horse! - Vat No site in rMOM 981 for the formation of MOM 17133 /fig.8/.

In the second example, the HTP-glyphosate oxidoreductase fusion was formed between Agaridorvy

IShaiapa ERB5R5 /Kiee et al., 1937/ HTP and synthetic glyphosate oxidoreductase coding sequences. First, Agaridorvye HTPP was obtained by site-directed mutagenesis, so that! build a restriction site 5th year and a processing site in the HTP. As a result of this mutagenesis, Sish-IGUv in this localization is replaced by Suvz-Mei. The sequence of that HTP, designated as HTP2, /pOS O Me: 10/ is presented in Fig. 9, the site of the synthetic glyphosate oxidoreductase gene /POS

IO Mo: 8/ biil is replaced at the 5rn1 site, which overlaps the Mei codon. The second column was converted to a leucine codon at the same stage. This change had no obvious effect on the activity of in vivo glyphosate oxidoreductase! in E. soybean, the HTP2-synthetic glyphosate oxidoreductase fusion was cloned into rViss

For K5 (5) and the resulting matrix were transcribed in vitro using T7 polymerase, and the synthesized 355-methionine-labeled material, according to the data, was imported into the chloroplast with an efficiency similar to that of the XTP1-glyphosate oxidoreductase fusion. XTP2 - synthetic glyphosate-oxidoreductase fusion then cloned as Chra | - Vat NI fragment into the plant vector of expression for the formation of ROMOM 17164. The structural map of this plasmid is presented in Fig. 12.

Part of the plant vector PMOM 17164 /fig.12/ includes the following fragments. A chimeric kanamycin resistance gene obtained by genetic engineering for expression in plants,

ensures the selection of transformed tissue. The chimeric gene consists of the 355 promoter of the cauliflower mosaic virus! 0.35kb (P-355) / Wow! with co-authors, 1985/, 0.83kb neomycin phosphotransferase gene! type 11 /KAM/, and 0.26 kb of the 3'-non-translated region of the nopaline synthesis gene /MO5 3'/ /EgaIeu et al., 1983/; 0.45kb

Sia! in Oh! a fragment from the mouth of 15955 octopin Those plasmids that contain T-DNA left border region /VagKkeg et al., 1983/; 0.75k6 fragment containing the origin of replication from the AK2 plasmid /ogi-M/ /MaiKeg et al., 1981/; 3.0 kb 5ai I in the E5i I segment of rA 322, providing the origin of replication for the maintenance of BE.soy /ogi-322/ and the site for conjugational transfer into Adgobasiegpst Shptegasiepe cells 0.93kb fragment isolated from transposon Tp7, which encodes bacterial resistance to spectinomycin/streptomycin /5rs/5g/Riipd et al., 1935/, and is a selective factor for E. soy and

Adgobasiepyt steiasiep5 0.36kb Come on! I c All I fragment from the mouth of T37 plasmid, which contains nopaline-type T-DNA in the right border region /RgaIeu et al., 1935/. The expression cassette, which includes 0.bkb 355 promoter from pine cone mosaic virus /P-EMM/ /Bomaa et al., 1939/, several unique cloning sites, and 0.7kb 3 untranslated region of the pea gos 5-E9 gene /EU 37 / Sogpi27i and others, 1984; Mogeii et al., 1985/. The fragment of HTP2-synthetic glyphosate oxidoreductase fusion was cloned into the aforementioned expression cassette. In the following examples of descriptions! introduction of this plasmid! in Adhorasiegt and the subsequent transformation of the plant.

Specialists will understand that it is possible to obtain various chimerical constructions, where the function of specific HTP is used for the coupled import of the enzyme glyphosate oxidoreductase! in chloroplasty of plant cells. The entry of glyphosate oxidoreductase into the chloroplast can be tested by means of the following sample!

Chloroplast absorption test.

Intact chloroplast! isolate from lettuce / I ashsa zayima var. Iopoiyuia/ by centrifugation in

Perkol/. Ficol gradients according to the modification of Vapei et al., /1082/. The resulting precipitate of intact chloroplasts is suspended in 0.5 ml of sterile 330 mM sorbitol in 50 mM Nerev5-KOH, pH 7.7, tested for chlorophyll content /Agpop, 1949/ and the final concentration of chlorophyll in the solution is brought to 4 mg/ml by adding sorbitol/Nerevz. The yield of intact chloroplasts from one head of a fly is 3 - bmg of chlorophyll.

A common uptake test with a volume of 300 μl includes 5 mM ATP, 8.3 mM unlabeled methionine, 322 mM sorbitol, 58.3 mM Nere5-KOH /pH 8.0/, 500 μl translation products of reticulocyte lysate, and intact chloroplast from I.zaim /200 μg chlorophyll/ . The absorption mixture was slightly shaken at room temperature /in 10 x 75 mm glass test tubes/ directly in front of the optical lighting device at the maximum intensity of illumination (bulb 15O0w). Multiple samples of the absorption mixture /about 5bml/ were taken at different time intervals and fractionated in 100μl of a silicone-oil gradient /in 150μl polyethylene tubes/ by centrifugation at 11,000 x 9 for Zbmin. Under these conditions, the chloroplast is intact! a precipitate forms in the lower part of the silicone-oil layer, and the incubation medium /containing reticulocyte lysate/ floats to the surface.

After centrifugation, try! silicone-oil gradients are immediately frozen in dry ice. Next, the chloroplast sediment is resuspended in 50 - 100 μl of lysis buffer /LO0 mM Nere5-KOH pH 7.5, 1 mM PM5E, 1 mM benzamidine, 5 mM «-amino-p-caproic acid, and ZOmkg/ml aprotinin/ and subjected to centrifugation at 15,000 x dv for 20 minutes to obtain a precipitate of thylakoid membranes. The clear supernatant /stromal proteins/ from the centrifuge and the volume of the incubation medium of the reticulocyte lysate from each absorption test are mixed with an equal volume of 2X DSN-PAGE simple buffer for electrophoresis /cm. below/.

DSN-PAGE - the method was carried out in accordance with Iasttia /1970/ in Z - 1795 /w/v/ acrylamide gels on plates /bOmm X 1.5mm/ with 395 /"w/v/ acrylamide concentrating gels /5mm X 1.5mm /. The gel was fixed for 20-30 minutes in a mixture of 4095 methanol and 1095 acetic acid! Then the gel was soaked for 20-30 minutes in EM'NAMSE (by Ropib), after which it was dried in an apparatus for drying gels . The gel was viewed by autoreductography using an intensifying screen and overnight exposure to determine whether glyphosate oxidoreductase had entered the isolated chloroplast.

An alternative isolation protocol for the second structural genes of glyphosate oxidoreductase.

The identification and cloning of a number of second glyphosate oxidoreductase genes, including the second IVAA glyphosate oxidoreductase gene from the Class I cosmid, were carried out! rmoOmM 7477. The gene is localized, according to Southern hybridization, to the -23kb Nipa I fragment, which was discussed in the cloning section above, using the first glyphosate oxidoreductase gene as a probe. By means of Southern analysis, there are also revelations. of hybridization - 3.5 and - 2.5 kB, respectively. Wow! The II fragment from RMOM 7477 was subcloned in Vat NI site rMore gray vectors. Clone in E. soybean MISS! (PMOM 7482), in which the cloned fragment is oriented relative to the Ia5 promoter as in PMOM 7469 and I, was induced with IRTO and tested for glyphosate oxidoreductase activity. In this experiment, the specific activity was about 93nmol/min.mg. In the subsequent experiment, Class I and Class II spaceships! they were also isolated after infection with E. soy MIO! with the same preparation of packaged cosmids and selection directly for tolerance to glyphosate at Z - 5

MM glyphosate in MOU medium.

The glyphosate oxidoreductase gene was subcloned from the second microbial isolate, initially identified by its ability to utilize glyphosate as a source of phosphorus and, as it was later shown, containing the putative glyphosate oxidoreductase gene, judging by the results of hybridization with sample I! VAA glyphosate oxidoreductase gene. This gene was first cloned into cosmid T7 of the promoter using screening for tolerance to glyphosate in E. soybean NVIO/pORI-2 (Voueg and NkoPapa-bivzoikh, 1969; Tarog and Vispagazop, 1985) on MO medium containing glyphosate at a concentration of 3mMM. The presence of the glyphosate oxidoreductase gene was shown for the first time by a positive hybridization signal with the I VAA gene and by its localization on the 2.5 kb Vadi II fragment.

Ztot Va! Its fragment was cloned in Wat NI site in rVishye 5sgiri (RMOM 17183) and expressed from the Ias promoter as a result of addition of IRTO. In this experiment, glyphosate oxidoreductase with a specific activity of 5Nmol/min.mg was obtained, which confirms the isolation of the gene using the data method. Zty glyphosate oxidoreductase geniuses indirectly have the following features: genius! are located (shown by Southern hybridization using full-length glyphosate oxidoreductase gene probes) on - 2.5 kb Vo! I fragments, at - 3.5 Rei I fragments contain one Eso Vi site within the gene and do not include Nipa II site. The following diagram illustrates some common devil! from these genes, the ZR Esov titans

I- 2OX coding region

The high degree of similarity of glyphosate oxidoreductase genes also suggests the possibility of a second way of cloning new glyphosate oxidoreductase genes. The obvious preservation of regions flanking genes and the absence of specific restriction sites indicates the use of single-stranded oligonucleotide probes for flanking regions containing the site! restrictions for Wai II, Nipa I, Ra I, Wat NI, Maye I, or other suitable cloning sites, and PCR (Polymeraznaya

Chain reaction; see Episn, 1989, for more detailed information on PCR and its application) to amplify a fragment of the glyphosate oxidoreductase gene suitable for cloning. Fig. 2 shows the flanking sequences for 119 np upstream (POS IO Me: 11) of the glyphosate oxidoreductase gene! wild type (I VAA isolate) and for - 290 np (POS IYUO Me: 12) along the gene flow.

As a result of using the PCR approach, a number of glyphosate oxidoreductase genes were isolated from various sources. The presence of glyphosate oxidoreductase activity was confirmed by cloning the glyphosate oxidoreductase gene from chromosomal DNA obtained from Rzepdotopaz zr. strain I Vg (Chasob et al., 1988) and using primers homologous to the M- and C-termini of the IVAA glyphosate oxidoreductase gene, as well as the content of the following suitable restriction sites for cloning: 13) 3) apdf 5'-FbAASSAATOS AAOSTTSTSSA SbaASSOSSTA AOTAS-3 (520 Ii0

Y YUO:14), ---- s-g tyakh (POS IO Me: 14). Below are the tributes! cyclothermal parameters used in these PCRs:

Denaturation at 947 for min.

Annealing at 6070 for 2 minutes.

Polymerization at 7270 during Change. 30 cycles, without self-elongation, linked during incubation at 476.

The 1.3 kb fragment formed as a result of PCR was subjected to cleavage Has I! and Nipa I and then cloned in rMOM 2123 for the expression of the coded enzyme. The activity of glyphosate oxidoreductase was measured, as described above, and the Kt for glyphosate was similar to the corresponding value for enzymes from 1st BAA, presented above.

Source of glyphosate gene oxylreductase Kt (glyphosate: mm)

Rzepdotopaz 5 years strain

I Vg 25

Bacteria isolated during processes using glyphosate in waste treatment plants can also have the ability to convert glyphosate into AMPA. As two similar examples, we can name Reeepdotopaz strains I VAA and VI. This type of bacteria can also be used in cleaning plants.

The population of bacteria was isolated from a stationary column of cell immobilization, which was filled with particles of Mappmeier NA-635 diatomaceous earth, by placing it on Trypton Soy Agar (Difco) containing cycloheximide at a concentration of 10 µg/ml, and incubating at 28"C. Through the column for for three months, water with impurities from Mopsapio Sotrapu5 IShipd, M5, glyphosate production plant was passed through. The column contained 5Omg/ml of glyphosate and Mn as MNeaSi. The total organic carbon was Zb0bOmg/ml and BOD (Biological oxygen demand - a measure of "soft" availability carbon) was less than ZOmg/ml. Such a treatment column was described by Neikatre et al., (1990). One of the most preferred strains of this population, identified as

Adgobasiepit 5 years. strain 710 was found when growing in minimal broth, where the main source of carbon was glyphosate at a concentration of 10 mM (this broth was prepared for OE medium, but including glyphosate, which replaces glucose, gluconate, and citrate). Chromosomal DNA was prepared from that isolate and subjected to the same PCR procedure and with the same primers, which are described in more detail! for strain I Vg. After obtaining a fragment of the required size, it was cloned into the BE.soy expression vector.

The activity of glyphosate oxidoreductase was determined, as well as the Kt for glyphosate:

The source of the Kt gene (glyphosate: mM)

Adhorasiepit 5 years strain 28

The conversion of glyphosate to AMPA has been reported for many different soils (see Majik et al., 1989 for a review), and a number of methods are available for the extraction of total DNA from mixed samples taken from the environment, such as soil (Noirep et al., 1988; BieMap and APiav, 1988; Tzai and Oivop, 1991), which indicates the possibility of cloning glyphosate oxidoreductase genes in the absence of the first isolate, such as a decaying microorganism. Of course, the procedure described for cloning glyphosate oxidoreductase genes, based on ensuring the ability to utilize glyphosate or tolerance to glyphosate, for example E. soybean, gives a scheme by which you can clone other glyphosate oxidoreductase genes! or glyphosate-metabolizing geniuses, regardless of the homology specified for the descriptions of more glyphosate oxidoreductase genes. It is also possible to enrich glyphosate-degrading bacteria, for example, by re-using glyphosate in soil batches Opipp et al., 1988; Tairop et al., 1984). This enrichment stage can be used to increase the ease of extraction of glyphosate oxidoreductase genes from soil or other environmental sources.

Indications for the presence of the glyphosate oxidoreductase gene in soil bacteria and the procedure for isolating such genes are as follows: The population of suitable bacteria is enriched by selecting bacteria capable of growing on a liquid medium with the addition of glyphosate (at 100 mm) as a carbon source (This medium is prepared according to the description for Humo/iKkip-Bovieg environment, but with the release of a carbon source and with Ri as a source of R.) The material for infection was taken from the soil extract! (from a recently harvested soybean field in Jerseyville, Illinois), and the white population was selected by successful cultivation in the medium described above at 28°C (To prevent fungal growth, cycloheximide was added to the medium at a concentration of 100 µg/ml). Wednesday with

They were identified with agar! 5 types of colonies. Chromosomal DNA was prepared from 2 ml of cultures in I. broth of these isolates, and the presence of the glyphosate oxidoreductase gene was determined using PCR screening. Using the primers aSsSspAaAtTaAdsSsataassSaAAAlaSs (pos IOMe: 1553 and voAabAATaSSOapATaSTTSAASOoOaS (POS IO Me: 16), a DNA fragment of a given size with chromosomal DNA from one of the isolates (designated 53) was obtained. The PCR conditions were as follows: 1 minute at 947C; 2 minutes at 40"C; Change at 72"C; 35 cycles. The DNA fragment obtained in this way is used as a sample (after isotopic labeling) for the isolation of the 53 glyphosate oxidoreductase gene, a candidate from the cosmid bank, constructed as described for I VAA DNA, and this fragment facilitates the isolation of other glyphosate oxidoreductase genes.

We will use a primer homologous to internal sequences in | VAA glyphosate oxidoreductase gene. The use of PCR conditions allows a certain degree of inconsistency in the primers, and as a result it is assumed that the glyphosate oxidoreductase gene from 53 is not necessarily a closely related gene to the other glyphosate oxidoreductase genes that were successfully isolated! when using primers to the M- and C-ends of the I VAA gene.

A wide variety of methods are known for gene isolation. Some of these procedures are based on data on the function of genes, which allows you to plan phenotypic screening for the purpose of gene isolation. Other methods are based, at least on partial information about the DNA sequence, in connection with which it is possible to use samples and primers with partial or complete homology, or methods that are based! on the used antibody for detection of gene products. Each of the above options can be used for cloning glyphosate oxidoreductase genes!

Optimization of kinetic properties of glyphosate oxidoreductase.

Previous examples of herbicide resistance achieved by enzymatic inactivation of herbicides use an enzyme that can! bind and metabolize herbicides much more efficiently than glyphosate oxidoreductase metabolizes glyphosate.

The glyphosate oxidoreductase enzyme has a Kt for glyphosate of 20 - ZOmMM, and as a result, the rate of the glyphosate cleavage reaction can be increased with optimal efficiency in transgenic plants either by lowering the Kt or increasing the Mmax.

The technique of random mutagenesis combined with appropriate selection and screening is an excellent tool that can be successfully used to obtain large numbers of altered gene sequences and potential variants. The same approach! can be used for isolation and identification of glyphosate oxidoreductase variants with increased glyphosate cleavage efficiency. Acceptable methods of mutagenesis include: chemical mutagenesis of bacterial cultures containing the desired gene or purified DNA from that gene, as well as the PCR method used to obtain copies of the gene (or parts of it) under favorable conditions! for the incorrect inclusion of nucleotides (errors) in a new chain. An example of such a condition can be the amplification of the PCR reaction in the presence of MP".

Appropriate screening in vivo to obtain improved variants after mutagenesis can also be used to increase tolerance to glyphosate in E. soybean or increased growth in the presence of glyosate in Mri strains. For the purpose of screening, the glyphosate oxidoreductase gene is cloned into a vector containing a weak bacterial promoter and/or in a replicon with a small number of copies.

Phenotype, tolerance to glyphosate, of various glyphosate oxidoreductase constructs, as shown, vary in relation to glyphosate concentrations, and also correlate with the level of glyphosate oxidoreductase expression. For example, in the absence of induction, Rias-glyphosate oxidoreductase vector! express lower levels of glyphosate oxidoreductase than the RhesA-glyphosate oxidoreductase vector, and also show lower tolerance to glyphosate. the gene fragment after mutagenesis is cloned into a more suitable vector and the resulting library is screened. The variant is selected based on its ability to grow in the presence of certain levels of glyphosate, which inhibit the growth of the control strain containing the parent clone of glyphosate oxidoreductase. The activity of glyphosate oxidoreductase provides E. soya with the ability to convert glyphosate into AMPA, and, in suitable strains of E. soya, AMPA can serve as a source of phosphate due to the splitting of the C-P bond with the help of C-P lyase. Suitable strains of E. soy are recognized as V strain or Mry" derivative

K strains. The glyphosate oxidoreductase gene ensures minimal growth on glyphosate, as the only source of phosphorus in the E. soybean MIS strain! Mry" (- 248993). According to these data, the growth rate on glyphosate also correlates with the level of glyphosate oxidoreductase expression. The glyphosate oxidoreductase gene obtained as a result of mutagenesis is cloned into the appropriate vector and the library of variants is screened by different growth rates in cups or when cultivated in a medium containing glyphosate, as the only source of phosphorus.Clones that show more intense growth on cups compared to the control strain are again screened by growth curve analysis.

Option! glyphosate oxidoreductase, identified at each selection/screening, cloned into a vector with a significant level of expression and subjected to enzymatic analysis to determine Kt and

Mmax values for glyphosate. The best option! glyphosate oxidoreductase! undergo cleaning for full kinetic characteristics. Option! glyphosate oxidoreductases, which were characterized by lower values of Kt and szhnjmm or higher values of Mmax compared to the corresponding values of the wild-type enzyme, were analyzed by sequencing nucleic acids to determine the mutation (or mutations). The main purpose of the procedures! it results in the isolation of variants with a higher XauKt ratio for the cleavage of glyphosate catalyzed by glyphosate oxidoreductase.

An improved variant of this type is isolated. The mutagenesis procedure consisted of PCR using MP, and the matrix was a linear plasmid with a glyphosate oxidoreductase gene containing a synthetic glyphosate oxidoreductase gene (POSIU Me: 8). Oligonucleotide primers were homologous with regions of the vector flanking the glyphosate oxidoreductase gene.

PCR was performed under the following conditions: Cumin. at 94"С, 2 min. at 55"С and Zmin. at 72"C, with 35 cycles.

We used a ratio of ddDTPaDdGTPaTtTF to dATP equal to 5:1. Reactions included MpsSi2 at 125, 250, 375 or 500 μM. After the reaction, the amplified product was again cloned into a vector containing a weak E. soybean promoter. This vector is a derivative of pBA 327, including the AGA promoter and a suitable cloning site. One hundred colonies from this stage of cloning were then screened! in E. soybean 18993 with the purpose of selection for increased tolerance to glyphosate and utilization phenotypes in the medium consisting of MOR5 minimal medium with glyphosate and Ri or with glyphosate alone, respectively. Growth rates were determined by measuring Avzo over a 96 time period. During this screening, three clones characterized by the most intense growth rate were identified. You are a transformant! possessed a phenotype whose ability to utilize was 1.5-2.0 times higher. The glyphosate oxidoreductase gene! the protein was again cloned into a part of the expression vector, after which the verification of that phenotype was completed. All kinetic analysis was performed on intact E. soybean lysates. The putative protein variants of glyphosate oxidoreductase were subjected to additional expression after subcloning of the variant of the Mso I/Nip I glyphosate oxidoreductase gene in Rhes A-gene 101. expression vector. For additional expression in Rhes A gene 101 constructs, 248,993 cells containing the vector were induced at Klett - 110 - 120 in MU minimal medium with 50 μg/ml nalidixic acid and incubated! within 2.5 hours. at 37"C with vigorous shaking. The cells were then harvested by centrifugation at 4000 for 5 min. at 4"C and resuspended in 100mM Tris-HCII, pH 7.1, 1mM ZDTK, 35mM KSI, 2095 glycerol, and 1mM benzamidine in 3ml /g of cell sediment. The licks were prepared by breaking up the cells in a French press, both under pressure of 100 Orv (pounds per square inch). The insoluble residue was removed by centrifugation at 120,000 for 15 minutes. at 4"C, and the supernatant was subjected to desalting by passing it through a RO column (Sefadex 2-25, Pharmacy). The separated white fraction was used as a source of enzyme for kinetic analysis. The protein concentration was determined by using the Bio-Red binding dye protein sample. For determination of linear limits was carried out by testing at different times and with different concentrations. The enzyme test consisted of the following: lysate and glyphosate oxide reductase mixture (final concentration - 0.1 M MOR, 0.01 M Tricin, pH 7.4, 0.01 Mm

EAU, 10 Mm Masig) in 100 μl of the reaction mixture was pre-incubated at 30"C for 2 minutes before adding glyphosate (the analytical mother solution was prepared in water, the pH of which was adjusted to 7.0 using MaOnH). 10 minutes is considered the optimal time for testing the enzyme with using 10 μg of lysate. After 10 min of incubation at 307 with constant shaking, the

0.25 ml of dinitrophenylhydrazine (DNPH) (0.5 mg/ml in 0.5 M HCl) and then the reaction was carried out for an additional 5 minutes. at 30" with constant shaking. After that, a 1.5M Mahon solution (400 μl) was added to the test mixture, and again, the reaction took place for 5 minutes at 307 with shaking. The activity of the enzyme was determined based on the amount of the glyphosate product formed -

DNFH by measuring Avgo against the glyphosate standard. Enzyme testing was carried out twice, at least on two isolates of one colony of the suspected variant of glyphosate oxidoreductase. To determine Kt and Mmax, enzyme testing was performed using (0.2 - 2.0) x Kt limit of glyphosate concentrations. Kt and Mtakh were determined according to I ipemzheameg Vyk, Eadije-

Neue and hyperbolic kinetic curves, Mmax were established after determining the amount of immunoreactive glyphosate oxidoreductase protein in the lysates of the immunoblot analysis described below.

Immunoblot analysis was performed after SDS-PAGE and transfer of the beam from the gel to nitrocellulose at 500 mM in a Hoffer apparatus in 25 mM Tris/HCl, 192 mM glycine containing 0.195 SDS and 2595 methanol for 1-2 hours. After transfer, the nitrocellulose was incubated with 50 mM Tris-HCII, pH 7.5, 0.995 Mass, 0.0195 Tween 20, 0.0295 mM, containing 295 µg of serum albumin at room temperature with shaking for at least 30 minutes. After agglutination, the same buffer containing a 1:25,000 dilution of goat anti-glyphosate oxidoreductase anti-coagulant was added and placed on a filter with shaking at room temperature for 45 minutes. After incubation with primary glyphosate oxidoreductase antibodies, the filter was washed for 45 minutes. in a buffer containing no antibodies; then a buffer containing a 1:5000 dilution of rabbit anti-goat alkaline phosphatase-conjugated secondary antibodies (from Pierce) was added, and the filter was incubated for 45 minutes. at room temperature and shaking. After that, the filter was washed in a buffer without antibodies for 10 minutes. before adding MVT and VSIR (Rgoteda) for the purpose of staining. The amount of immunoreactive glyphosate oxidoreductase protein was also determined by blotting the lysate on nitrocellulose and subsequent processing of the filter as described above, except that 725I-Protein C was used to detect the protein. The amount of glyphosate oxidoreductase protein in the lysates was determined by counting the label and comparing the radioactivity with standard protein glyphosate oxidoreductase. One of the variants showed (var. 247) a 3- to 4-fold increase in the specific activity of glyphosate oxidoreductase! in the presence of 25 mM glyphosate, and the results of the immunoblot analysis showed that this fact was not associated with an increase in the level of glyphosate oxidoreductase protein. Subsequent testing showed that this option was characterized by size

Kt for glyphosate, which was 10 times lower than in the case of glyphosate oxidoreductase! wild type

In a similar way, the value of Kt was determined for YAUK. Ask this presentation! below

Kinetic analysis of variants of glyphosate oxidoreductase!: 1110 | Kt(mm) |Mt(U/m)| Utp/Kt wild you |2mo| in oe|o5| oz) le var247 | 26 | 07 | 06 | 0.7 | 23 | 1.0

The glyphosate oxidoreductase gene! from var.247 was sequenced (POC IO Moe:17) as a result of which five changes in nucleotides were detected. Because there are changes in connection! with codons, then they are indicated here: ST on 4SS (codon 43), absence of changes in amino acids; ACC on OS (codon 84), Ser on

Clay; AAS on AC (codon 153), Lyz on Arg; SAC on SOS (codon 334), Lyz on Arg; CSA to CSA (codon 362), without changes in amino acids. The amino acid sequence of the glyphosate oxidoreductase gene from 247 is presented as POC IO Mo: 18. The importance of these various changes in amino acids is initially estimated by re-cloning the changed sites in wild-type glyphosate oxidoreductase and determining the effect on the activity and kinetics of glyphosate oxidoreductase. This is carried out by recloning MsoI-Mpe! fragment (contains codon 84), MpeI-Ara! | fragment (contains codon 153) and Aragi-Nip II fragment (contains codon 334), separate into the wild-type gene.

Zty genius glyphosate oxidoreductase! they were compressed), after which the kinetic analysis was carried out. The presentation below shows that the change in the AragGi-Nip PI fragment (containing codon 334) is solely responsible for the change in the enzyme.

Kinetic analysis of domain changes

Clone Kt (mM) | Mt (U/mg) | Mp/Kt mm mam) 28.4 0.65 0.022 m.2A7(m1m2my3) 21 0.72 0.34 m1m2mZ 23.5 0.62 0.026 m1m2UZ 21 0.6 0.28 mg 2.0 0.75 0.375 m1zhmghUZ 2.6 0.55 0.21 interm2ghzhZ 28.0 0.75 0.027 m1m2zhZ 26.7 0.55 0.021 "mi - ZEN 84; m2 - 1 5 153; mZ3 - NI5 334 u1-01 84; m2-AnNa153 х3-АНОЗЗ34

These results are confirmed and supplemented as a result of the repeated change of His to Arg in codon 334 and the introduction of other specific changes into the remainder by site-directed mutagenesis. The following list uses primers!:

Arg- SaTTSTSTAS ASTSSTOSTOO aTAAIatTTOaS (POS IO Mo: 19); Liz - SETTOTSTAS ASTAASOaStTo atTAAattas (POS IO Mo: 20); Glu - SET STSTAS ASTSAALDOaStTo STAASsTTas (POS IO Mo: 21); Ala - SETTTSTSTAS ASTaSTAsto atTAAstTaSs (POS 10 Me 22). (These sequences are recognized as anti-sense words in relation to the sequences used at the present time.) The presence of these changes is confirmed by the sequencing of glyphosate oxidoreductase genes after mutagenesis. A kinetic analysis of the expressed glyphosate oxidoreductase enzymes was completed, the data of which show that a number of substitutions are possible in this position and that, in turn, leads to the synthesis of the enzyme with changes in kinetic properties.

Kinetic analysis of variants of glyphosate oxidoreductase

Wild type 27.0 2.8 0.8 0.5 1.03 | 8 c. 247 2.6 0.7 0.6 | 0.7 | 23 | 1.0

Arg334 | 2.6 0.5 0.6 | 0.6 | 231 1,2

Liz 334 | 9.9 1.3 0.7 | 0.8 | .07 | .62

Chapter 334 | 19.6 3.5 0.6 | 0.7 | .03 | .20

Ala 334 | 26.7 3.5 0.2 10.2 1.007 |1.057

Additional mutagenesis was carried out for substitutions! His 334 residue on the second amino acid residue. To perform this procedure! the following primers were used! and a new codon:

Tr STSTASASTTaaasStTSaTtAAaSTTTSTTSSAas (pos I Me: 23)

Пе СТСТАСАСТАТСОЯСТСІаТААІаСТТСТТТОСАаС (POS I Me: 24)

HER en STSTASASTSOT)OISTSatA5AaTaststssSAas (POS IYUO Me: 25) sish STSTASASTaAA0asStTSaTataAAaTast st ССАаs (pos IO Me: 26) (These sequences are antisense words in relation to the sequences used at the present time; these primers also attach a "silent" Nip I, which facilitates identification in the population of offspring obtained as a result of mutagenesis.)

The SI 334 variant retains significant glyphosate oxidoreductase activity, while the TAR 334, PE 334 and I BI 334 variants show significantly lower activity.

From the options of the first generation, an option! with a higher .Ksa/Kt ratio are preferably subjected to the second cycle of mutagenesis, which is accompanied by subsequent screening and analysis.

An alternative approach may consist in obtaining the second generation of variants of glyphosate oxidoreductase by combining point mutations identified among the variants of the first generation.

Transformation of plants

Using this invention, it is possible to ensure tolerance to glyphosate in the following types of plants, however, it should be noted that this list is not exclusive: soybean, cotton, grain, flax, sunflower, potato, tobacco, tomato, wheat, rice, alfalfa, lettuce , apple tree, poplar, pine, sugar beet, rapeseed, canola.

The double-stranded DNA molecule of the invention ("chimeric gene") can be integrated into the plant genome by using a suitable method. Suitable vectors for the transformation of plants are the vector obtained from the plasmid Adgobasiegt Shshpteiasiep5, as well as those found, for example, Ne!tega-

Evi gePa (1983), Vemap (1984), Kiviee (1985), and submission to ERO publication 120516 (ZspiPregoopy et al.). In addition to plant transformation vectors derived from Ti or root-inducing plasmids (Bi)

Adhorasiegisht, they can use an alternative method for inserting the DNA constructs of the invention into plant cells. These methods include, for example, the use of liposomes, electroporation, chemicals that increase the absorption of free DNA, delivery of free DNA by means of a micro-projecting bombardment device, or transformation using a virus or a virus. рРМОМ 979 vector of plant transformation/expression is derived from РМОМ 886 (described below) by replacing the neomycin phosphotransferase gene! type II (KAM) in pRMOM 886 by a 0.89 kb fragment containing the bacterial gene for gentamicin-3-M-acetyltransferase type I (AAS(3)-PI) (Nauiga et al., 1988).

The chimeric P-355/АА(3)-Ш/МО5 Z'gene encodes resistance to gentamicin, which ensures the selection of transformed plant cells. pRMOM 979 also contains a 0.95-kb expression cassette consisting of an amplified Samm 355 promoter (Kau et al., 1987), several unique restriction sites and a MO5 3" end (P-Ep-Samm 355/MoO5 3). The rest of PMOM 979 DNA segment! are recognized exactly the same as in RMOM 886.

Plasmid rMOM 886 is constructed from the following DNA segments. The first one is represented by a 0.93 kb Ama IV in connection with Eso VAM fragment, isolated from transposon Tp 7, encoding bacterial resistance to spectinomycin/streptomycin (Zrs/5i1), thus being a determinant for selection in E. soya and Adgobasieyts istegasiepe. This segment combines with a 1.61kb DNA fragment encoding chimeric kanamycin resistance, which ensures the selection of transformed plant cells. The chimeric gene (P-355/KAM/MO5 3) contains the 355 promoter of cauliflower mosaic virus (Samu), the neomycin phosphotransferase gene! type II (KAM) and the 3'-untranslated region of the nopaline synthase gene (MOB 3) (Egaiieu et al., 1983). The next segment is represented by 0.75 kb of M, containing the origin of replication from the AK2 plasmid. It is connected with 3.1 kb of bai I in connections with Pmy segment from pBA 322 (oii 322), which provides maintenance of the origin of replication in E. soya, as well as a site for conjugational transfer into cells Adhorasiyepyit istegasiepe. The next segment is 0.36 bkb Rmuy | in compounds with All I from rt/137, which carries nopaline-type T-DNA of the right edge (RgaIeu et al., 1985). pRMOM 981 plasmid contains the following DNA segments: a 0.93 kb fragment isolated from the Tp7 transposon and encoding bacterial resistance to spectinomycin/streptomycin (5rs/5i0); determinant for selection in E. soya and Adhorasiegt (Stegasiepv (Bipu et al., 1985); a chimeric gene for resistance to kanamycin, obtained by genetic engineering for expression in plants, which provides selection of transformed tissue, including 0.355b promoter 355 of the cauliflower mosaic virus (P-355) (Egaieu et al., 1985), C.83 kb neomycin phosphotransferase type I gene (KAM) and 0.26 kb 3'-untranslated region of the nopaline synthase gene (MOB 3) (Egaieu et al. ., 1983); 0.75kb origin of replication from VK2 plasmid! (ogiMm) (iaiKeg et al., 1981); 3.1kb Zai I in connections with Pmy | segment from rBA 322, which provides the origin of replication for maintenance in BE. soybean (ogpi-322) rot site for conjugational transfer into cells Adgobasiepyit Shtei?asiep5, as well as 0.36 kb of Rmy I in compounds with All fragment from plasmid rt/137, including the right border region of nopaline-type T-DNA (Rgaieu et al. ., 1985). The expression cassette consists of a 0.6 kb 355 promoter from the pine cone mosaic virus (P-

EMU) (Somaa et al., 1989) and the 0.7 kb 3'-untranslated region of the pea gene press 5-E9 (E9 3) (SOVI et al., 1984; Motgeii et al., 1985). 0.6kb 55r | the fragment containing the EMM 355 promoter (Fig. 1) was engineered to accommodate suitable cloning sites downstream of the transcription start site.

The plant vector is used in the AVI Adgorasiepyt strain. AVI strain is A208 Adhorasiyit

A strain carrying the armless Ti plasmid rtiC58 (rMROONK) (Kopsl and 5enei, 1986). That plasmid does not possess the T-DNA genes of phytohormones, and therefore the strain is not capable of causing root cancer. The combination of the plant vector with AVI is achieved by means of systems! triparental conjugation using

PeIreg plasmid pAK2O13 (I Sha et al., 1980). This is the case if plant tissue is incubated with the conjugate

If the vector is a plant, then the vector is transferred to the cells of the plant as a result of the functions coded by the armless rtiS8 oplasmid. The vector opens the right marginal region of the T-DNA, and the complete sequence of the plant vector can be integrated into the chromosome of the host plant. rtis5v ti plasmid is not transferred into plant cells, but remains in Adgobasiepyit.

Regeneration of plants

When the necessary activity of glyphosate oxidoreductase is achieved in transformed cells or protoplasts, the cells (or protoplast) regenerate into a whole plant. The method used for joint regeneration is considered not so exclusive in each specific case, and suitable prescriptions are available for plants from I editiposaye (alfalfa, cultivated soybeans, clover),

Otrveiiegaye (carrots, celery, parsnips), Stpisigegaye (cabbage, radish, rapeseed, etc.), Sysigtrigaseaye (melons and cucumbers), Sgatipsaye (wheat, rice, corn, etc.), Boiapaseaye (potatoes, tobacco, tomato, pepper) and various flower crops. See, for example, Attigayu, 1984; Questionnaire, 1989; Egott, 1990; Mivia, 1990.

Below are examples that introduce the use of the invention in more detail, but their interpretation is not necessarily limited to the scope of the present invention.

Specialists will understand that the presentation of the methods and the description of the genius can be modified or reduced accordingly, without affecting the very essence of the invention.

Try it!

Expression, Activity and Phenotype Glybosal oxidoreductase! in transformed plants.

In the following examples of descriptions! transformation, expression and activity of glyphosate oxidoreductase, as well as tolerance to glyphosate phenotype, obtained in plants with the help of oxidoreductase genes introduced into Misoyapa iabasit su. "Zatveip" and/or Vgazzisa of the sg UUeiag pair using vector rMOM17073, rMOM17032, rMOMO17065, rmoMO17066, rmoMO17138, rMOMO17164. According to the initial data obtained for tobacco, the expression of the glyphosate oxidoreductase gene, obtained as a result of manipulations (POS IO Me 6), under the control of the Ep-Samu355 promoter (see the data for RMOM17073 in table. MIII and IX, as an example) is characterized by low levels . Transcription of the gene was confirmed in 3 - 4 plants by Nosern and 51 analysis, however, the glyphosate oxidoreductase protein was not detected! (detection limit in this sample was about 0.0195 level of expression). Analysis of plants after spraying 0.4 lb/acre (approximately 0.448 kg/ha) of glyphosate also indicated very low levels of tolerance. The modification of the gene sequence (described in this message) led to increased expression in tobacco plants, as a result of the use of the EMMU promoter and the fusion of HTP with the glyphosate oxidoreductase gene. In this case, most of the data were obtained on transgenic plants transformed with the help of vectors containing all the instructions for the optimization of the glyphosate oxidoreductase construction. One of the series of experiments with the modified glyphosate oxidoreductase vector PMOM17073 is described in example 1. Example 2 presents the result of the glyphosate oxidoreductase studies obtained as a result manipulations, synthetic glyphosate oxidoreductase, as well as HTPP 1-synthetic glyphosate oxidoreductase. Transformation and expression of glyphosate oxidoreductase in canola! descriptions in example 3.

Example 1

The protocol of transformation of tobacco leaf discs represents the tissue of normal leaves at the age of about 1 month. After 15-20 minutes of surface sterilization with 1095 Clorox in combination with a surfactant, leaves three times! washed in sterile water. Using a sterile pocket punch, leaves were punched and placed upside down in M5104 medium (M5 salts 4.3g/l, sucrose

Zog/l, vitamin B5 500X 2ml/l, MAA 0.1mg/l and VA 1.Omg/l) with the purpose of preincubation during one day.

Then the disks were infected with a night culture of besplechnoi Adgobasieegpts AVI, containing the necessary vector, and the culture was diluted 1/5 (that is, about 0.6OD - optical density).

Infection was carried out by placing discs in centrifuge tubes with culture. After 30-60 seconds, the liquid was aspirated, and the discs were soaked with sterile paper filters. Then the disks were placed upside down in cups with nutrient medium M5104 on the filter! for co-cultivation.

After 2 - From the day of co-cultivation, the disks were transferred, again upside down, into cups for selection with M5104 medium. After 2 - From a week, the formed callus or separate white shoots separated from the leaf disks. The shoots were cut off from the callus when they became large enough and could be distinguished from the stems. The selected shoots were placed on a hormone-free medium favorable for root growth (M5O: M5 salts 4.Zg/l, sucrose Zbg/l and B5 vitamin 500X 2ml/l). After 1-2 weeks, roots formed. Any testing of leaf callus was preferably performed on root shoots that maintained sterility. The root shoots were placed in the soil and kept in conditions of increased humidity (that is, in plastic containers or bags). The roots took root more and more as a result of maintaining humidity in the environment.

A total of 45 kanamycin-resistant RMOM17073 tobacco strains were investigated (Table M).

Table M

Expression of the modified glyphosate oxidoreductase gene in tobacco plants. (KI transgenic variant RMOM17073) Western use

I glyphosate in new analysis

Plants with calluses (0.5MM - glyphosate) plants 45 (9) 11 34 24 21 same average values 0.5 - 2ng/50mcg of protein - values « 0.5ng/50mcg of protein

Leaves forming callus again in the medium with plant tissue culture showed a low level of tolerance to glyphosate (defined as w/- phenotype), at least in 11 of the investigated lines. At least 24 lines showed a detectable level of glyphosate oxidoreductase expression! in the range of 0.5 to 2 ng per 50 μg of extracted protein. Tolerance to glyphosate, which was found in the tested leaves with newly formed callus, and a higher level of glyphosate oxidoreductase expression, testify in favor of the fact that changes in the coding sequences of glyphosate oxidoreductase, due to the construction of a modified glyphosate oxidoreductase gene (POS IO Me:7), had a noticeable effect on the ability of this gene to be expressed in plants. The same effect can be achieved as a result of the expression of the glyphosate oxidoreductase gene, obtained with the help of manipulations (POS IU Me:b) with the use of strong plant promoters, by using the best sequences of the 3' polyadenylation signal, optimizing the sequences near the initiation codon for ribosome loading and initiation of translation, or by combinations of these or other regulatory or expressing sequences or factors. YAN! progeny of a number of the specified lines, including those that showed the highest level of glyphosate oxidoreductase expression (Zh518854 and 18848), were subjected to glyphosate spraying in the amount of 0.4 and 1.0 pounds per acre (0.448 and 1.12 kg/ha, respectively) , after which vegetative symptoms were assessed over a 4-week period (Table U1).

MI table

Spraying of tobacco for РМОМ17073 В1 plants is given

volume

The line of preparation C days) 14 days (28 days. kg/ha 112 1 1 2

Via shi: WE PENG SHE SHE SHI 112 2 Z

Vol shi YN I SHNSH SHE 112 2 2

Will s: MINE SHI SHE SHO 112 2 5 112 1 2 4 112 2 1 2 112 1 2 Z 112 1 1 (9) x Vegetative signs: o - death xx effect not detected

After the initial Iad phases, these lines showed vegetative tolerance to glyphosate at both spraying regimes (which increased over time), and this was especially true of plants characterized by the highest levels of glyphosate oxidoreductase expression. Activity of the enzyme glyphosate oxidoreductase! tench was determined in two of RMOM17073 (Zh5 18858 and 18881). Leaf tissue (1 g) was frozen in liquid nitrogen and stored at -8070 until preparation of the extract. For the purpose of extraction, the tissue of the leaves was crushed in a mortar and ground with a pestle with liquid nitrogen. One ml of extracting buffer (100 mM Tris CI, pH 7.4, 1 M ZDTC, 2095 glycerol, 35 mM KSI, 1 mM benzamidine NCI, 5 mM ascorbate Ma, 5 mM dithiothreitol, and 1 mg/ml bovine serum albumin, 4"С) was added to the powdered leaf tissue. , after which the sample was triturated for 1 min. The resulting mixture was centrifuged for 5 min (high speed, Errepaogy) and the supernatant was treated with a saturated solution of ammonium sulfate to obtain 7090 of the final concentration (2.33 mL of saturated solution/ml of extract). The precipitated protein was collected as indicated above, by centrifugation, and the sediment was resuspended in 04 ml of extraction buffer. After centrifugation, the sample was subjected to desalting in order to remove particles, passing it through Sephadex (550, contained in an iml syringe and balanced extraction buffer, according to the method of RepeivzKu (1979). Plant desalting The extracts were stored on ice, and the protein concentrations were determined by the method of Vgadiog (1976). Ifosate oxidoreductase was added in duplicate samples for 10 minutes. at 30"C in a mixture of 01 MORB/0.01 tricine buffer, pH 7.4, containing 10 mM MoCoO", 0.01 mM flavin adenine dinucleotide (EVO, Sigma), and 1 mM coenzyme O (Sigma). Plant extract! (75 ml ) were pre-incubated in the above-mentioned mixture for 2 minutes, after which reactions were initiated by adding iminoacetoacetic acid (IYAUK, 20 μl) as a substrate to a final concentration of 50 mM (total sample volume 0.2 ml). The reaction mixtures were cooled and subjected to derivatization, as described below.

Control reactions were carried out in the absence of JAUK and plant extract. Determination of glyoxylate was carried out by using 2,4-dinitrophenyl-hcdrazine (2,4-DNPH) derivatization and phase-reversed high-performance liquid chromatography (HPLC), using a modification of the method of Oshgezpi et al., (1982). The reaction mixture with glyphosate oxidoreductase (0.2 ml) was cooled with 0.25 ml of DNFH reagent (0.5 mg/ml DNFH (Alagisni in 0.5 M NOI) and subjected to derivatization for 5 minutes at 2570. After that, the sample was extracted with ethyl acetate (2 x 0.3ml) and the combined ethyl acetate extract was extracted with 1095 MgCO3 (0.3ml). The Mg2bOz phase was then washed once with ethyl acetate (0.2ml) and the Ma2CO3 phase was introduced (100μl) into a Westup ObBigazrpeg C18 IR VZJHU column (5μ, 4 ,6mm x 25cm), using a YK5 211 binary VLC system with a U/aiet5 990 photodiode mm ШМ/I5 VLC detector, with the help of an UmMaiet5 MIR VLC autoinjector. The isocratic mobile phase is represented by a mixture: methanol-water-acetic acid (60:38.5 :1.5) with 5 mM tetrabutylammonium phosphate (Pierce). The DNFG - glyoxylate peak (retention time - 6.7 min) was detected at 365 nm. A comparison of this peak with the glyoxylate standard (Sigma, 20 μM in 0.2 ml), subjected to derivatization was carried out exactly in the same way.

Table MI!

Glyphosate oxidoreductase activity in transgenic tobacco plants. nmol/min mg

Plug O (not detected) 18881 0.039 18858 0.018

Example 2

A series of transformed white tobacco lines was obtained as a result of the use of "isogenic" glyphosate oxidoreductase vectors rmoOM17073 (glyphosate oxidoreductase obtained by manipulation) (POS IUOMe:b), rMOM17065 (synthetic glyphosate oxidoreductase) (POS IOMe:8), and rmMoOM17066 (HTPI-synthetic glyphosate oxidoreductase). Through Western analysis of a number of silent plants (see Table 7), it was shown that plants with manipulated glyphosate oxidoreductase express up to --

O, Bng glyphosate oxidoreductase is 50 omkg of plant protein, with synthetic glyphosate oxidoreductase - up to - 0.5 - 2 ng per 50 mcg and with HTPI-synthetic glyphosate oxidoreductase - from - 2 to 2 ng per 5 omkg of protein.

MI table

Zxpression glyphosate oxidoreductase! in tobacco plants.

Western rMOM17073 21270 (9) (manipulated) 21281 (9) 21286 1 21929 1 (HTPI-synthetic) 21830 (9)

21845 Z 21872 Z 21889 1 21891 (9) rmMoOM17065 21199 (0) (synthetic) 21208 2 21212 2 21217 (0) 21218 2 21792 1 21795 (0) 21811 2

Western scale of evaluation for 5 omkg of protein: 0 - no glyphosate oxidoreductase detected 1- « 5ng 2 - ng - 2ng 3-5 2ng

A number of primary No transformants expressing manipulated or synthetic glyphosate oxidoreductase or HTPiI-synthetic glyphosate oxidoreductase were treated with glyphosate at a concentration of 0.4 lb/acre (0.448 kg/ha) and then evaluated as described above.

Table IX

Glyphosphate treatment data: РМОМ17066 (ХTP1-glyphosphate oxidoreductase).

Tobacco (plants)

Line, Volume, Vegetative control, Western treatments, sign and assessment lb/sheet kg/ha (days after processing)

A C (9) (9) is not detected in C 1 (9) glyphosate (9; C 1 1 | oxidoreductase 22933 1 C 1 (9) (рРМОМ17073) 22741 2 2 1 9 (рРМОМ17065) 22810 З 31416 (РрМОМ17066) 22825 1 2 1 1 (pMOM17066) 22822 Z 10|170|170 (PrMOM17066) 22844 Z 10|170|170 (PrMOM17066) 22854 Z 9 |10|10 (PrMOM17066) 22860 Z 8 |10|10 (PrMOM17066) 1281 1982 1231 (PrMOM17066) 22881 2 2 (9) (9) (PrMOM17066) 22886 Z 9 |10|10 (PrMOM17066) 22887 Z 9 |10 | 10 rmMOM17066

The scale of Western evaluation (per 5 omkg of protein) 0 - - no glyphosate oxidoreductase was detected! 1 - « 0.5pd NG 2-0.5: 219

From -» 2p9

Vegetative sign o - death 5 effect was not detected

The line with synthetic glyphosate oxidoreductase shows a response similar to the response observed in Ni plants with oxidoreductase, which were characterized by some direct effects of glyphosate, which disappeared over time as a result of metabolizing the herbicide glyphosate oxidoreductase to produce AMPA and glyoxylate. Since the target of glyphosate (ERB5R synthase) is localized in the chloroplast, the activity of glyphosate oxidoreductase should lead to a decrease in the level of glyphosate in this organelle as a result of the removal of the herbicide before it enters the chloroplast.

Plants with HTP1-synthetic glyphosate oxidoreductase showed an increased tolerance to glyphosate, which was manifested in the fact that glyphosate did not have any effect on plants at this treatment concentration. In general, treated tolerant plants were characterized by normal development, flowering and fertility. хХТПП1-synthetic glyphosate oxidoreductase plants showed a significantly higher level of glyphosate oxidoreductase expression compared to other glyphosate oxidoreductase constructs. This increased level of glyphosate oxidoreductase may be due to an increase in the translation of the structure represented by the fusion, or the release of phosphate oxidoreductase inside the chloroplast, which, in turn, leads to a longer half-life of the protein. A higher level of glyphosate oxidoreductase and/or its localization in the chloroplast may cause higher levels of tolerance to glyphosate due to the rapid destruction of glyphosate in the chloroplast. The presence of glyphosate oxidoreductase within the chloroplast has been established. Five leaves from each of the four plants (422844, 22854, 22886, 22887), which, according to Western analysis, were positive for glyphosate oxidoreductase, were subjected to homogenization in a U/agpd mixer in 0.9 HA buffer (Vapiei et al., 1982) for 3 x 3 sec. at high speed. The homogenate was filtered through 4 layers of Migasoi and centrifuged at 6,000 rpm in a (5-3 rotor. The precipitate was resuspended in 4 ml of the CA-buffer mixture and layered on a 40/8095 Regsoi step gradient, after which it was centrifuged at 9,500 rpm for 10 min.. Intact chloroplast (lower strip) was washed once (SA - buffer Vapei et al., 1982) and centrifuged at a speed of up to 600 rpm without braking. Then they were subjected to resuspension in 300 μl of 50 mM mM Neiez pH 7.7, 330mM Sorbitol and subsequent lead on ice with the help of ultrasound (small sample, 30950-3 freezing in micropipettes x1Osec). After sedimentation of the remains, the supernatant was passed through a column with Sephadex (50 in 50mM Neiez, pH 7.5.

The concentration of dissolved protein was 2.4 mg/ml. Enzyme testing was carried out as indicated above, using both 50 mM JIAUK and 50 mM glyphosate as substrates (Z0 min. probe), but without the addition of 1 mM cofactor 0.

Table IX

Glyphosate oxidoreductase activity! in isolated chloroplasts of transgenic tobacco.

Substrate Specific activity (nmol/min.mg) acid

Example C

A number of transformed flax cannoli! obtained using vectors PMOM17138 (XTP1-synthetic glyphosate oxidoreductase) and PMOM171648 (XTP2-synthetic glyphosate oxidoreductase) as follows.

Plant material

Seedlings of Vgazvia were steamed and transplanted into 2-inch (-5 cm) pots containing Meigo Mich 350. Plants were grown in a growth chamber at 2470, 16/8 hour photoperiod, light intensity 4000Et" sec" (NIO lamps). Wednesday was enriched by using Reiegz 20-10-20 Sipiga! Rygroze Zresiai. After 2 1/2 weeks, they were transplanted into 6-inch (- 15 cm) vessels and left to grow in a chamber at 15/1070 day/night temperature, with a 16/8 hour photoperiod, light intensity of 800 andEt? sec! (NIU lamps). The environment was enriched with the use of Reiegz 15-30-15 Nirnoz Zresia|.

Transmission/Selection/Regeneration

Four terminal internodes were removed from the plants immediately before entering the shoot or during this process, but necessarily before flowering, and their surface was sterilized in 7095 vol/vol ethanol for min. , 295 wt/vol of sodium hypochlorite for 20 min., after which they were washed three times with sterile deionized water. Stems with leaves can be frozen in moist plastic bags for up to 72 hours. before sterilization. From 6 to 7 segments of the stem, they were cut into Bb5mm disks with the help of Neiso Medeiarie Biiseg 200, which ensures the orientation of the basal end. -Adhorasiepyts were grown overnight on a rotator at 24"7C in 2 ml of Lurie broth containing 500 mg/l kanamycin, 24 mg/l chloramphenicol and 100 mg/l spectinomycin. A 1 : 10 dilution was made in M5 (Myhavnpide and 5Kosod) medium, which, approximately , amounted to 9 x 10 8 cells per ml. Growth under the same conditions as in the case of plant culture. After three weeks, leaf samples with newly formed calluses were assessed for tolerance to herbicides (calluses or tissue of green leaves) or sensitivity to them (bleaching) .

Transplantation

At the time of cutting, the stems with shoots were placed in NooiYopeFf and then in a 2-inch (- 5 cm) vessel containing Meito-Mix 350 and placed under humid conditions. Plants were placed in a growth chamber at 242C, 16.8-hour photoperiod, 400 and 0Et? sec" (NIO lamps) for about 3 weeks with the aim of rooting.

The seed collected from the No plant is the No seed that gives rise to the No plant. To assess the tolerance to glyphosate, the plants and their offspring are tested. Since it is believed that the No plant is hemizygous at each position of the insertion, self-pollination leads to maximum genotypic segregation in No. Each insertion acts as a dominant allele, so in the absence of linkage and the assumption that only one hemizygous insertion is required for the manifestation of tolerance, one insertion will segregate as Z : 1, two insertions as 15 : 1, three insertions as 63: 1. Therefore, to detect at least one resistant phenotype, it is necessary to grow several No plants accordingly.

The seeds of No plants are collected, crushed and dried before sowing for the test of spraying with glyphosate. Various methods of growing plants were used to conduct research on the assessment of processing. Testing was carried out both in greenhouses and in growth chambers.

Two systems were used! sowing; their 10 cm pots or sowing trays containing 32 or 36 cells.

The soil used in this case was either Meigo 350 with the addition of three types of slow-release fertilizer, or the Meigo 350 installation. In the greenhouses, irrigation was carried out from above, and subirrigation was used in the growth chambers. Fertilizer was applied as much as required with water for irrigation. The temperature regime met the requirements established for canola!

A 16-time photoperiod was used. At the beginning of flowering, the plants were transferred to - 15 cm pots for the formation of seeds.

The processing batch consisted of several sets of Vi, descendants, and they were all processed at the same time. Some batches included plants other than Vi, the evaluation of which also had to be carried out. In addition, each batch included both treated and untreated, non-transgenic genotypes representing the genotype! a separate party, which is presumably due! battle of transformations". In addition, the batch includes one or more non-segregating transformed genotypes, previously identified as having a certain resistance.

From two to six plants from each separate Ko progeny are not processed and serve as controls for comparison and measurement of tolerance to glyphosate, as well as for studying any changes caused not by glyphosate, but by other factors. When the rest of the plants reach the stage of 2-4 leaves, which is usually observed 10-20 days after sowing, treatment with glyphosate is carried out in concentrations varying from 0.28 to 1.12 kg/ha, depending on the object of research. A technology of low doses, characterized by small volumes of herbicide, was developed. In laboratory studies, spraying doses are adjusted in such a way as to obtain concentrations equivalent to those/m in field conditions.

A scale from 0 to 10 is used to evaluate the vegetative resistance of treated plants.

The scale also applies to untreated plants from the same No plant. 0 - means the death of the plant, while 10 is an indicator of the absence of any visible differences from the untreated plant. Numbers between 0 and 10 reflect a gradual decrease in the degree of damage to plants when compared with generally untreated individuals. The evaluation of plants is carried out on 7, 14 and 28 days after processing (DPO), or during the period of arrow growth, and the line is characterized by the average evaluation of processed plants within the family of plants No.

Six integers are used to qualitatively describe reproductive disorders caused by glyphosate; 0: absence of flower bud development 2: flower buds are present, but they stop developing before opening; 4: flower! open, but without anthers, or the anthers are unable to extend beyond the petals 6: sterile anthers 8: partial sterility of anthers 10: full fertility of flowers

Evaluation of plants using this scale is carried out at the beginning or soon after the beginning of flowering, depending on the speed of development of structures! a flower

Table X

Evaluation of glyphosate treatment of canola plants containing PMOM17138

Name

Vegetative! Reproductive 17138-22 79 9 10 17138-30 79 9 10 17138-145 79 10 10 17138-158 79 8 10 17138-164 80 8 10

Untransformed 77 Z (0) peaceful!| 79 1 (8)

Tables! Х and ХІ represent estimates of vegetative and reproductive characteristics for canola plants transformed using, respectively, ЯМОМ17138 (treatments at a concentration of 0.5 bkg/ha) and РМОМ17 164 (treatments at a concentration of 0.84 kg/ha). Here is the result! show that canola plants acquire tolerance to glyphosate as a result of the expression of the glyphosate oxidoreductase gene in plants!

Table XI

Evaluation of glyphosate treatment of canola plants,

containing RMOM17164

Vegetative! Reproductive 17164-6 82 7 10 17164-9 83 8 10 17164-20 82 7 10 17164-25 83 8 10 17164-35 84 7 10 17164-45 83 9 10 17164-61 83 7 7 1 8 64-10 10 17164-85 84 7 10 17164-97 84 B 10 17164-98 83 9 10 17164-105 83 7 10 17164-110 83 9 10 17164-115 83 7 10 17164-129 83 83 8 -140 83 8 10 17164-164 83 7 10 17164-166 83 8 10 17164-175 83 8 10 17164-186 83 Z 10 17164-202 83 8 10 17164-218 84 b 10 17164-212 1912 19164-294 84 7 10 17164-225 83 7 10 17164-227 84 7 10 17164-230 83 83 8 10 17164-243 83 7 10 17164-247 84 7 10 17164-287 84 7 10 17164-289 83 83 8 10 17164-337 83 8 10

Example 4

The glyphosate oxidoreductase gene! also introduced into the plants of ordinary soybeans, where it was expressed, as a result of which these plants acquired tolerance to glyphosate. The XTP2-synthetic glyphosate oxyloreductase fusion gene /described above/ was introduced into soybean plants under the conditions of the EMM promoter and with MOZ 3" sequences in the vector ROMOM 17159, the map of which is shown in Fig. 10. In addition to the sequences of the glyphosate oxyloreductase gene, this vector includes the following elements: origin of replication, MRT11 bacterial selective marker gene /kanamycin/ and beta-glucuronidase gene /305; UeMyegzop et al., 1986/ under the control of EZ55 promoter and with E9 3 sequences. The latter gene provides an evaluation marker to facilitate the identification of transformed plant material.

The transformation of ordinary soybean plants is carried out with the use of RMOM 17159 by means of the microprojection injection method using the technology of targeted bombardment of particles, described by Spiiziop et al., 1988/. Seeds, a collection of No plants, are designated as Vi seeds, which give rise to No plants. To evaluate the tolerance of plants to glyphosate, the offspring of those plants are evaluated. Since the NKo plant is considered hemizygous in the tile of the insert, self-pollination leads to maximum genotypic segregation in No. Since each insertion functions as a dominant allele, in the absence of linkage and the assumption that only one hemizygous insertion is necessary for the expression of tolerance, one insertion will segregate as Z : 1, two insertions as 15 : 1, three insertions as 63 : 1, etc. .d. Therefore, to detect at least one resistant phenotype, it is necessary to grow several Vi plants accordingly.

Seeds of common soybean plants are collected and dried before sowing for the glyphosate treatment test. Seeds are planted in 4-inch /- 5 cm/ square pots containing Meigo 350. For testing, 20 seedlings per plant is considered sufficient. Plants are kept and developed in conditions typical for greenhouses. A photoperiod of 12.5 - 14 hours and a temperature of 30"C during the day and 24"C at night are generally maintained. If necessary, Wednesday is enriched

Reieg» Reiie And this is a fertilizer, soluble in water.

The processing batch includes several sets of Vi offspring that are sprayed at the same time. Some batches may also include excellent plants for evaluation purposes. In each lot, there are also treated and untreated non-transgenic genotypes, representing the genotypes of a separate lot, which are presumably transformed. In addition,

the batch contains one or more non-segregating, transformed genotypes, pre-identified as possessing certain resistance.

One or two plants from each individual Ko progeny are not processed and serve as a control for comparison and characterization of tolerance to glyphosate, as well as for the study of any variability caused not by glyphosate, but by other factors. When the rest of the plants reach the first three-leaf stage, which is usually observed 2-3 weeks after planting, treatment with glyphosate at a concentration of 128 ounces per acre /8.895 kg/ha/ VoipairF is carried out. During laboratory studies, treatment doses are calibrated in such a way that! to obtain concentrations equivalent to those in field conditions.

Uses! the vegetative rating is from 0 to 10. The rating also applies to unprocessed offspring of the same plant. 0 - means the death of the plant, while 10 - the absence of obvious differences from the untreated plant. Numbers between 0 and 10 reflect a gradual decrease in the degree of damage to plants when compared with untreated individuals. Plants are evaluated 7, 14, and 28 days after treatment /DPO/.

Table ХИЙ

Evaluation of glyphosate treatment of common soybean plants containing PMOM17159

Estimate 8.895 kg/ha 17159- 14 9 17159- 14 9 17159- 14 (5) 17159- 14 4 17159- 14 4 17159- 14 10 17159- 14 9 17159- 15 4

Raw 14 (9)

Example 5

The glyphosate oxidoreductase gene was also injected into the cells of wheat grains! Viask Mehisap Zmeei /VM5 / with the expression of the protein determined in the callus.

Plasmid pRMOM 19632 was used to introduce the glyphosate oxidoreductase gene! in grain cells. The basis of this plasmid was formed by inserting the 355 RNA promoter /Ez355/ 0.6bkb of the cauliflower mosaic virus /Samm/, containing the duplication of the -90 and -300 regions /Kau et al., 1987/, 0.58kb of the fragment containing the first intron of the alcohol gene dehydrogenase /Saiv et al., 1987/, 3 terminal sequences from the fossil synthase gene /МО5/ Egapyu et al., 1933/ in rОС119 /Mapivek - Regop et al., 1985/. RMOM 19632 was formed by embedding 1.7kb Va! I/ EsoBi fragment from rMOM 17064, contains Agaridorziz 950 HTPP in connection with the sequence coding for synthetic glyphosate oxidoreductase /POS IUO Me:8/.

Plasmid pRMOM 19632 was introduced into BM5 grain cells by the method of joint bombardment with EC9, a plasmid containing a sulfonylurea-resistant form of the acetolactate synthase gene! corn! 2.5 μg of each plasmid was layered on tungsten particles and injected into VM5 cells in the logarithmic phase, using the ROB-1000 particle bombardment method, described in its essence in

Kiivip et al., 1939. Transformant! were selected on M5 medium containing 20 parts per billion of chlorosulfuron. After the primary selection for chlorsulphoron, I swear! white research! by means of

Western blot glyphosate oxidoreductase!.

VM5 callus (3 g of wet weight) was dried on a paper filter (Whatman Zh 1) under vacuum, weighed again and the extraction buffer (500 μl/g of dry weight) was added; 100 mm Tris, iM ZDTK, 1095 glycerin/. The tissue was homogenized with the help of a M/peap suspension stirrer for 10 seconds. at 2.8 unit power. After centrifugation /Zmin, Errepiop/, the supernatant was removed and the amount of protein was calculated /Vio Best protein test/. Break through! applied (5Omkg/well) to the SDS PAAG (uiis, Z - 1790) together with the glyphosate oxidoreductase standard! (1Ong), subjected to electrophoresis, and then transferred to nitrocellulose, similarly to the previously described metol (Ryddeye, 1987). The nitrocellulose blot was examined using goat anti-glyphosate oxy-doreductase immunoglobulin (IgD) and treated with 1-125 Belkom and The blot with the radioactive label was examined by autoradiography.

The result! were calculated using the method of densitometry using LKB UltrasScan Hi laser densitometer, and presented in the form of tables XII.

Table ХИЙ

Evaluation of glyphosate treatment of common soybean plants containing RMOM17159 (95 extractable protein)

EC9 (without COX) (0)

T13-17 0.016

T13-16 0.0065

T13-15 0.016

Tt13-14 0.003

Tt13-14 0.003

Tt13-12 0.0079

T13-7 0.01

T13-5 0.004

T13-18 0.026 T13-8 0.019 T13-9 0.01 T13-4 0.027

Table XII shows that glyphosate oxidoreductase can be expressed and, therefore, it can be found in monocots, such as wheat.

Example 6

The glyphosate oxidoreductase gene can be used as a selective marker for plant transformation directly on a medium containing glyphosate. The possibility of selection and identification of transformed plant material depends, in most cases, on the use of a dominant selective marker gene to ensure the possibility of preferential and continuous growth of transformed tissues in the presence of normally inhibitory substances. Gen, responsible for resistance to antibiotics and tolerance to herbicides! almost exclusively as a dominant, selective marker gene in the presence of the appropriate antibiotic or herbicide. Most often, obviously, the selection scheme at 11/kanamycin is used.

It was shown that glyphosate oxidoreductase is also useful, which can be an excellent selective marker in the selection scheme for obtaining and identifying transformed plants.

In this scheme, Ptop17226 is used as a plant transformation vector (Fig. 11). This plasmid resembles many other plasmid descriptions and, basically, includes the previously described bacterial replication system, which ensures the replication of this plasmid! in E. soy, and its introduction and replication in Adgobasiept; bacterial selective marker gene (Zre/50), i

Chtin glyphosate oxidoreductase synthetic gene in the EMM promoter-E9 3 cassette, localized between the T-DNA of the right edge and the left edge. This plasmid also has separate sites for a number of restriction enzymes, localized inside the borders or outside the cassettes! zxpressions This makes it easy to add other genes or genetic elements to the vector that is introduced into the plant.

The protocol for direct selection of transformed tobacco plants on glyphosate is presented. For precultivation, the plants were prepared according to the standard method described in Example 1: sterilization of the surface of leaves from tobacco plants at the age of 1 month (15 min. in 1095 chlorox, the same surface-active substance; ZXaNgO smyiv)); the plants were cut into squares! 0.5 x 0.5 cm, with the removal of the edges of the leaves, the middle veins, the apex and the ends of the petiole to obtain a uniform fabric; to transplant! placed in one layer, upside down on M5 104 cups and 2 ml of 4СО0О5К medium for surface moistening; the precultivation period was 1-2 days. Plant! were infected using a night culture of Adhorasiegpts, containing a plasmid for plant transformation, and the titer was brought up to 1.2 x 109 bacteria/ml of 4C005K medium. The explant was placed in a centrifuge tube, where Adhorasiyst suspension is also added, and the mixture of bacteria and explants is subjected to maximum mixing using "Mopeh" for 25 seconds. for a full guarantee of penetration of bacteria.

The bacteria were pooled, and the explants were placed between layers of dry sterile filter paper to remove excess bacteria. After soaking, the explants were placed upside down in M5 104 cups with 2 ml of 4C005K medium - a filter disk. Socultivation continued 2 - Since the day. Plant! Carbenicillin 1000 mg/l and cefotaxime 10 mg/l were transferred to M5 104 I on the third day (delay phase). Then the explants were transferred to M5104 with glyphosate 0.05 mM and carbenicillin 100 Ωg/l and cefotaxime 10 Ωg/l for the selection phase. For 4-6 weeks, the shoots were separated from the callus and placed in M5O with Carbenicillin 5b0Omg/l root medium. Roots were formed for 3-5 days, and at that time, pieces of leaves can be taken from the root cups to confirm tolerance to glyphosate, as well as the transformation of the material.

The presence of glyphosate oxidoreductase protein in these transformed tissues was confirmed by immunoblot analysis of leaf discs. Further introductions! data from one experiment with RMOM17226: shoots, formed on glyphosate and 100 transplants, were infected with Adgobasiyit AVI/rMoOMm17226; 15 of them turned out to be positive in relation to repeated callus formation on glyphosate, and 19 of them were positive for glyphosate oxidoreductase protein, detected by immunoblot analysis. According to these data, the degree of transformation was 15 - 19 per 100 explants, which indicates high efficiency and time saving thanks to the transformation procedure for the plant. A similar degree of transformation was obtained when using the РМОМ17226 derivative (РМОп17241) containing the glyphosate oxidoreductase gene! v.247 (POS IYUO Me:17). As it was shown, the glyphosate oxidoreductase gene provides direct selection of transformants in other types of plants, including

Adgobrasiegiit, potato, sugar beet.

From all of the above, it is clear that the invention allows you to realize all the goals and tasks that were mentioned earlier, which indicates the advantages and value of this work!

It is clear that it is not necessary to use all combinations and characteristics, but to apply them regardless of other properties of the invention, however, with all changes, it is necessary to study the set of requirements for the use of the invention.

Taking into account the requirements for the invention of possibilities, however, different options! its application, and it is necessary to remember that the presentation here is premature, and further schemes and drawings should be considered only as illustrative, and not limiting material at all.

ATITASSASS ATTSSASSATT SOSTTSLATS VASAASSSTAS SASSSATATS ASTTTATTSA vo

AATTOSTATS SOSALALSSA ASLASOLAST SSSATOSTSA AASOTTTTSTA LOSAAOKATT 120

STSASTSSAA ASOSTOAASA ASOTSASOST ASASASTSTO SALASSATTA SSSLAAMOST 180

ASASSASATS AATOAASAAT STISAATSAA ASTALASTAS TOTTSSASSA SATOSATSAT 240

SOTSASTAAS TTTSASLAAK ASASATSSAS SSAASASTTA AASTIASTOS OSATSTITOA 300

HASTAATSTT OTSAAASATOS ASSASSTOOS TTSTOOOOSAS SASASAALLA ASOAATOSTO 360

SASAATTSTT ASOSOSASST ASSALAASSA TSTTTOSSTI TATTOSALAA SATALASSAYA 420

ATTOSTSTAS TASKASTOOOO SAASKALATA ASSTOSAAAAA SLOSTOTOST SASASSOSS 480

TSASTAATOS STATSASSAA SOSASTOASO ASSASALLAS AATTITOSST STATATAAOHA 540

ASSSATTTSA TTSSSATTTO labo 564 (2) Information about POS IYUO Me:2: (i) Sequence characteristics: (A) Length: 27 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type of molecules !: DNA (genomic) (hi) Sequence description: POS IYUO Me:2:

ATSATSASAAT ASTAAASSAAT AtTtTISTe 27 (2) Information about POS IU Me:3: (i) Sequence characteristics: (A) Length: 1689 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Molecular type : DNA (genomic) (hi) Sequence description: POS IUO Me:3:

MNSATOSASOT STOATOSALA TSSTOSTTAS SOSASSALOS TALOSSASOS SSAATITTAT 50

SASSTASSOS SALASSSTOS STASOSASSO ASASASToTS СОоСТОСОСоа ОАОСАСТСТА 120

TOTSTOASAAA SSASALAALA STASOSATOO StToSABSSSa AATOSTOSOS STAToSASAoo 180

SSSTSATOST TSASSOSSOOS SSATTSAAAAO TSASSTTSAT TSASSSSSAAS SSToSsTObsSo 240

AAOSTOSATS OTTTOSOAAT SSOSSATOST TSAASSOSTS ATSSSToOTS SSTATOTSSSA 300

TOSSSOOAAA STTSASSSKOS STSSSOAAST SOSTOSTTOA SSOSATOSOS SSTTOTSKMAT 360

SSSSTTSASS TATTTOSLAS SATSATOSST SOTTOATTOO STTTSTOTTA bSSOSAKSAS 420

SALASAAOOT SAASSASSAS OSOALASSAS TSSOSALATST SATSAKOTOS ASssToSSTe 480

TSATSAASTO ATTSSSOSAS SASOSTSATO SSLOSSATSI SATSSOSSAT SALOSTSATS 540

TIASSSTATA TOSTOSASAAA SSASASTTOYA SSAAASSASSO SSSASSTTOS SAASTOOSOSOS 600 сТСТСААСоО TOTTSOSASO САSATOSTСА ССОСССАТОС OTTOSSOOAT TTSOАTeSoA bo

ASTTSTSOSA TOSOTITAS AASSOSATTS TTATASKLASA SAASOSTSAS ASSATTAATS 720

СсОСААССОСТ ОСТСАСОСТО ТТСТТТСОос СТИТТАТООС СААСОСТООС СААТТСОТАТ 780

СТОоСОСОТОТ SATOSOSTTI SASASTOAMO STASSSSOST TALASOSATT ASLASSASSOH 840

МСООСОоТТСТ оОССССТТОАТ ОСАОСОСТТО ТСОСАССОСО СОСАСАСТСО АААТСАСТТЕ 900

STAATTSOST ASSOSATSAS ATSOSOSTOO ATASSOLASO TOSATATSAT ATOSTSATOYA 950

ССААТССОСА AОССОСТОСА СОСАТССССА СОАСССАТОС СТСАССАЛАА ТТСАТСССОА 1020

SASSTATOSA AATOsbOSTT SosoTobsoo OTASOOTTOA oTTSosSTobo STSASAOSSO 1080

STSSTAASTO SALASOTOSO SATOTOSTST ATASOSASOS TSSAALASTT STtTeSLOSSS 1140

TSOSOSSTOS SASTISTOAA SLASSATATT SSALATOSAT bo00Ttss00 СSOASSATOS 1200

SOSATTSOST SSSSOTSATT SSSSSSSOAA SSSOSASASO OSASSTAATS TATOSTTTOV 1260

СССАТССТСА ТСТОСОСАТО АСАСОССОССОССОССОСТСОСТОСТОСТСОСТОСТСОСТОСТСОСТСОСТСОСТСОССОСТСОССОССОСТОССОССОСТСОССОССОСТСОССОССОССОСТСОССОСТОССОСТОССОСТОССОСТСОССОСТСОССОССОСТСОССОСТСОССОСОССОССОСОССОССОССР

TSOSASOSOA AKASASSTSA ATSOASATTT SOSOSTTOSS ASSALASSOS TTTOOTATTO 1380

OSALATSSLA OSYAVSOSOT SSOOSAASTI AASTASTTAS OSOSTOOOTOA STASSSSOSA 1446

САСССОСТОТ СЛАСАТСААТ СТОСАССТОО СЯТСКОСТС ССАСАСОССА ААТСОСССАА 1500

АТАСААСАСА TATTААССАС ТСАСОССССС ААСССТTTОb СТСАСТАСАЕ ТСАбСосСоСС 1550

SSASSOSSTO OATTSATTSA TOTITSOSOT SAOSTTSSSSA TSAAAASSASA ASoSSASTSYA 1620

SAOSAATSTO ASSATSTSOT SSATAASSYA SSSASTSTSo TISTOSOSAA Tttostoves 1680 stASsTosai 1689 (2) Information about POS IYU Me:4: (i) Sequence characteristics: (A) Length: 1293 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type of molecules: DNA (genomic) (their) Feature (A) Purpose/key: (B) Localization: 1293 (xi) Sequence description: POS IU Me:4:

ATO TST SAS AAS SAS ALLA ALLA STA SOS ATS ST Oba SS OSA ATO ote 16

Mes bek Sich lep Niv Tsuye Muye Uai Siu 116 Aia Sbiu A1ia biu i1e Chai 3 5 10 15

SSS STA TS ASOo osSs ST ATS STT sSAb SOS sb boA To MAA ote AS 96 biu mmai Sat Tg liz ce Meb i4i bib Ago Ago biu RBe uz chai Tit 29 25 30

TT AT SAS sob AMS SST SST oo SAL OST bsSA TO TT bo LAT ss 1414 tsech 116 lLer Rgo yap Rgo Rgo b1u biz biu A1iv bek Rije siu Avp liv

OSA TOS TS AAS boss TsA TOS ST ste OST ATO TosS AT sob so AS 192

F1u Suv Ri. Azo b1iu beg bek Uzi chai Rgo Neb beg Nei Rko biu lap bo

To ASo Abs sto sob AAb To sto STT САС СО ATO bos sot st SA 210 иФы ТАК bet Chai Rgo fur coat Tgr vch Ge Avr Rgo Me b1iu Aho Suv bip 55 70 15 80

TSS OST TSA OST ATT TOS AAS SAT SAT osSS tob TTo AT sos tM se 288 beg b1iu Bek Aia I16 Zeg Aza Ni Niv Aia Tkr ii 116 Agu Re ivo 85 90 95

TTA OSS ObA AS SSA AAS ALO STO AL bab sb oso ALLA OSA sto SOS 316 taz Aa siu Ago Rgo KAap Bua Chai buye biz s1p lia in A1a im Ag9

100 105 110

AAT Sto ATO AAS TOS AS oto ssStT Sto ATS AAO TSA To bob cm vAb 384

Avp ieFa 116 Suye beg Tr Uai Rgo vy 1316 bue beg yFi Aza 019 bi 115 120 125 bST OAT OSS AOS SAT STO ATS SOS SAT SALA OST SAT STO ASS OTA TAT 432

Aia der Aia beg Mia yech T11e Ag Niv bi biu Niv ce Ts hUai Tug 130 135 140

SoT OSA SAA OSA bAS TS OSS AAo SAS SOS OSA OST Tbo sla sto so 480

As biu biu Aza Avr RiF Aia tsuv Avr Agu s1u F1u Tgr 019 vy Ag 145 150 155 160

SOT ste AAS Ost TT SOS ASO SAB ATS Sto les SOS SAT oso to sob 528

KE Sech delo biu Uaii As TVg Fi 116 Fo bek Aza Avr Aia im Ago 165 170 175

SAT TT SAT SSO AAS TO tob SAT boss TT ACC AAS Ob AT STI ATA 576 dar RF Ar Rgo Aep I4m beg Ni Aia Re TP Buv F1u 116 Ge i16 180 185 190

SAA SLE KAS OST SAS ASO ATT AAT sota sla Oso ste ste AS ste Ma 524 bis sich Avp biu Ni Tvg I19 Aap Rgo bip s1u em Uai TI Shvy i4o 195 209 205

TTT C00 SOT TT ATO oso AS ObT o0S SAA TT STA TST sso sst ote 512

Rpe Ag Ako RpF 116 Aia lap biu siu b1h RF Khai bak A1ia lgo Uvy 210 215 229

ATS SOS TT sb AST OAA OST Or FS STI ALLA bos AT ASA ASe AS 720 112 biu Ripa bi Tlg biz O1u Ag9 Aza Teo uz biu 116 TI Tvph Tk 225 1230 235 240

KAS boss otTt hundred SS ST bAT OSA oso STI ste osa SS boss OSA SAS 1768

Azp b1u Mai yay lia Uai Ayur Aia Ali Mai Mai Aza Aia S1u dia Niv

245 250 255

TO AAA TSA STT OST AAT tob sb sob SAT sAS Te so Sto SAT ss 816 besg tsuye ber Pec Aza dep Beg iz biu Ler ler 116 Rko ez Avr Tg 260 265 270

SAA SST SOA TAT SAT ATO STO ATO bo AAT SSO OA oSs ost ssA SOS 864

Sish Agd biu Tut Niv 116 Uai 116 Aia lyap Rko bi dia Aza Rgo Akd 215 280 285

ATT SS ACC AS SAT Oso TSA bbA AAA TTS ATS Oso ASA SST ATO BAH 312

IiF Rgo Tig Trg Avr Liva bek biu tsu» RF 119 Aza Tru Reo Mei Jan 290 295 300

Kto bob stt SOS hundred sso bot AS st svo TT ost ro ste AsSA oss 960

Me Siu im Ag Mai Aiv biu Tr o Mai 019 Re Aia 91u Ii Tok Khia 305 310 315 320 bost OST LAS TO AAA SOT oso SAT STO STS TAT ASO SAS OST SsA MAH 1008

A1a Rgo Map Tgr Kuye Ag Aza Ni Hai yeech Tug Trg Niya A1a Ak tsuye 325 330 335

STI STT SsA bos ste oso SST oso AST TST SALA SAA SA TAT TOS AH 1056 yeei Gaz ?ho Aza Gech liwa Rgo A1ia Zeg Zek biz sich Agu Tug begh Tsuv 340 345 350 tob ATO obo TT sb SSO ABS ATS SSo SAT tob hundred SOS hundred At boss 1104

Tgr Nes biu Rre Ag Rko Beh 119 Rko Ar beh Immyi Rkgo Mai 11 biu 355 360 365

SO SA SS About ASA SSS SAS STA ATS TAT OST TTS boss SAT SOT SAT 1152

As9 Kia Tvzh Ak Tak Rgo Aar Uai 11e Tut Aiz Re biu Niv biu Niv 370 375 zvo

Sto obsS ATO ASA obo ss sSo ATO ss osa ASo ste ote TSA sao ste 1200 yFc biu Keb Tg biu Aia Rgo Meb Tik Aia TIE Ge Uai bek biz yFu 385 390 395 400 stTs OSA bos baAA AL ASS TSA ATO sSSS ATI To sSos tt OSA SsA ks 1248 yeech Aia biu 015 tsue Tvg Zeg 116 lLyur 11 Zeg Rko Rv. Aia Rgo ley 405 110 415

SOS tTtTtT sot ATT ObS AAA TS AL SAA AS OST sb osSA AT TAH 1293

Ag Rpe siu 116 biu buye bes buye Sip Tizh biu Rgo AiiF Zag 420 425 430 (2) Information about POS IU Me:5: (i) Sequence characteristic: (A) Length: 430 amino acids (B) Type: amino acid (C) Structure: double chain (C) Topology: linear (y) Type of molecules: protein (hi) Description of the sequence: POS IYUO Me:5:

Meai beg chip lap yiya Shshchuyu but baii Siu 119 Aza 91u Aza Z1u 116 Uai 1 s! 19 15 biu chi Suvye TV Aza Pshsh Kes zaz iv lgu Agu b1u Vp Husband Chuschi TV 49 15 30

Tshi 116 zr Rebs Anya Rgs Rgo y1u b1ich 01xK li Zag Ra 41u yel dia ab 45 biu Suv Ra Ayap sS1u Beg bek Mai chai Rgo Mat beg Maye Rgo biu db ho 55 a yiv TgobBek Uai Rto Sub Tk sao TFo Air ogo Mah biu who Su Ffii

Be 79 t i)

God siu tak Liya 116 Zag lai Kish Nizh Aza bur aa 114 At RNe Ii 85 for 95 yeech left siu Ag Rgo lap tsuye Uai tsuye biz bil liwa Bug liz Se Aga 100 105 116 dep ich Ii Puye beg Tik Uai Rgo Tem I1e uyu b5eg Tem liv 01 biz 115 1295 125 liva ler Aia bek Yaiv i 116 AkF Ni biz biu ie Mei Tv Mai Tug 130 135 140

Ag9 b1u b1ich lia Avr Re Aiv tsu Avr At o1u b1i1u Ter bij ce Ag 145 150 155 169

Kg food Ap O)u Mai Age Tvpgobip 11 Her bek Aia Ar left is Ag 165 179 175

Mayor of the Russian Federation Avr Rgo Avp ate Zeg Ni A1ia Rye Te pub biu 116 yeech I18 180 185 190 b1z biz Avp biu Ni TE I16 Ava Rgo bip biu imo Uai TV Ilki Geo 195 290 205

Rpe Ag Ag RpF 116 Aza dep biu biu biz Re waiii Zeg Aza Ago Chai 210 215 220

Ii b1u Re bi Tvh biyu Siu Ag9 Aia iz buye Siu 116 Tpg Twk Ta 225 230 235 240 lap biu Hai Bo Aia Uaii Lor Aza Aia Uai Ugi di Aia biu lia Niv 245 250 255 beg Buv beg Gets A1iv Ayap beg Pes biu Avr Ler Ii6 Rto yeech der TE 260 265 270 .

bi» Ak b1iu Tuk Niye 11 Ua) 119 liv two Rgo 01h Aia Aia Rgo AgF 275 280 285 116 Rgo Tag Tig Avr Aia bZeg biu tsu Re 11 Aia Tag Rtgo Meb 01 2990 295 300

Keye s1u iz Ag uai Aia siu Taj Mai bi RBF Hia S01u wo Tak ia 305 310 315 320

Aia Rgo lap Ter tsu Ag Aia Niya Mai Mech Tug TB Niye A1a AgF Buv 325 330 335 yeech Mi Rko Aii Her Aia Rgo A1iaa bek Beh biz biz Ago Tuk beg Tsuye 310 345 350

Tgr Mee biu Rle AgF Rgo bek Ii Rgo Aer bas Ge Rgo Chai 116 siu 355 360 355

Ag Aza Tlt Aka Tvg Rgo ALur uai 119 Tukh Aia RBe biu niyu biu Niv 370 375 380 ivy biu mes Tlh 01u Aia Rgo Mebye TE Aza Tpg IGats UMai god sich ate 385 390 395 400 isi Aia biu Fiz bue TVg ber 118 Avr I1yu Rkgo Re Aia Reo dep 405 1410 415

Med RpF biu 116 Siu Shtsuav bek tsuz siv Tk biu Rgo Aia bek 420 425 «30 (2) Information about POS IYU Me:6: (i) Characteristics of the sequence: (A) Length: 1296 np (B) Type: nucleotide (C) Structure: double chain (C) Topology: linear (y) Type of molecules: DNA (genomic) (hi) Sequence description: POS IYUO Me:6:

ATOOSTSASA ASSASALALA ASTASOSATS SSTOSASOSO SAATSSTOSO SOTATOSASO because

OSoSToATOS TTSAbOSOSO SOSATTSALA STSASSTTSA TTSKSSSSAA SeSsToSToOS 120

SALSSTSSAT SOTTTOOSAK TOSOSOATOS TTSAASOSSST SATSOOTSOT SSSTATSTOS 180

KTOSSOOSAL ASTTSASSAS SoToSOsSALO TOSSTOSTTO ASSSSATOSO oSSSTToTSA 240

АСССОСТСА ОСТАТТИСС ААССАТСАТО СОСТОСТСА TTSOSTTTST ОТАСССОСА 300

ASASSALASA ASSTSAASSA OSASOSSALA OSASTOSOSA ATSTSATSAA OTSSASOSTO zvo

SSTSTSATSA ASTSATTOOS OSLOSASOST SATOSOXSS ATSTOATOSO SSATOAAOST 420

SATSTOASOYA TATATSOTOS ASALOSASS TTSOSSALOS ASSOSOsKSo TTOOSKASTO 480

ССОССТСТСА АСОСТОТТSO САСОСАСАТС СТСАСССОСОС ATOSOTTOSO СSATТTOSAT 540

СССААСТТСТ СОСАТОССТ TASSААСОбС АТТСТТАЭ ЛАСАСАЛСОЙ TSАСАССАТИ 600

MATSSOSAAS SOSTOSTOAS SSTSTIOTTI SOOSOTTTTTA TSOSSALSSOo TOSSSAATTT 660

STATSTOSOS STSTSATSOS STTTTSASAST OAASOTASOYA SOSTTALASO SATTASKASS 720

ASSAASOoSO TTSTbOSOST TSATOSASSo STTISTSOSAb SOSOSSSASA STOSAAATSA 780

STTOSTAATT SOSTAOSOSA TSASATSOSO STSSTASSO AASOTOSATA TSATATOSTS 840

АТСоССААТО СОСААССООС ТОСАСОСАТ СССАСОАССО ATOSOTSASO КААААТТСТЕ 900

СССАСАСТА TOSАЛАТОСО oSTTSbSoTo bSSOSTASSO TTOASTTSOS TOSOSTSASA 960 bSSOSTOSTA ASTOSALASO TOSOSATOTO STSTATASOS ASOSTOOAAAA ASTISTTSSA 1020

ССССТСОСОСС СТОСОАСТСТСТОАЛОААССА TATTSSALAT ССАТоСсоОТТ СссессосАСС 1080

ХТоССОбАТТ СОСТССССОТ SATTOоССОБ ОСАССОбСА САССССАССТ ХАТSTATOST 1140

ТТСбССССОо ОТСАТСТСОО SATСАССОС ОСОССОАТОА ССОСААСОСТ СОТСТСАсАй 1200

СТеСТСОСАй ССОАЛАААСАС СТСААТССАС ATTTСОСССТ TSOSАССААА СоОСТИТоСТ 1260

ATTSOSALAT SSALSSALAS OSOTSSSOSA ASTTAH 1296 (2) Information about POS IO Meo:7: (i) Sequence characteristics: (A) Length: 1296 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type molecule!: DNA (recombinant) (hi) Sequence description: POS IU Me:7:

ATOSSTOASA ASSASADLALA ASTASOSATS OSTOSASSTO SAATSOTTOS TOTATOSAST bo

OSTTTOKTOS TTSAASSTSSO TOBATTSALA STSASSSTISA TTSASSSSSAA SoSTeSsToOsS 120

SAASSTOSAT SOTTTSSSAA TOSSOSATOS TISAASSOST SATSSOTOST SOSTATOTOS 180

ATOSSOOASAA ASTTISSOAS SoTOoSesAAo T9OoSsTeSTTo veSesTooo oSSTTTOtsA 240

ATSSOSTTSSA SSTATTTOS ALSSATSATO SOSTOSTTOA TTSOSTTTST STTASSSOsa 300

ASASSAMASA AOSTSALOSA OSASOSSOAAAA OSASTSSOSA ATSTSATSAAA SToSASOSTO zbo

SSTSTOATSA ASTSATTOOS SOASSASOST SATOSSASSASS ATSTOATOSO SSATSAAOST 420

SATSTOASSO TATATSSTSO ASAASSAOAS TTSoSSAAOO AosSoSsosKos TTsOsAASTO p 489

СООСОТСТСА АСОСТОТТОС ССОСАСАТС ССТСТСТОСТО ATOSTTTOSo TSATTTOSAT 540

SSTAASTTOT SOSATOSTIT TASSAASOSS ATISTITATAS LASASADOFa TSASASATT 600

AATSSOSADO SOSTOSTOAS SSTSTISTIT SSOSSTTTTTA TSOSSAASOo TOOSOAATTT 560

STATSTOSOS OTOTSATOSO TTTTSASAST SALOSTOOTO STSTSALASO SATTASAASe 720

ASTAASOSTO TTSTOSSTOT TOATOSAOST OTToTToSAO STOSTOSASA STSTAAATSA 780

STTOSTAATT SOSTASOSSA TSASATSSSO STOSATASSO AASSSTOSATA TSATATSSTS 840

ATSOSSAATS SOSALOSSOS TSSASOSATT SOSASSASOS ATOSOTSASO AALATTSATS 900

СССАСАСТА ТОСАААТОСЯ ТСТТООТОТТ ОСТОСТАСТО ТИСАСТИТОС TOOTSTSASA 960

OSTOOSTOSTA ASTOSALASO TOSOSATOTO SISTATASOS ASOSTOBAKA ASTTTSTTOSA 1020 оСССТСОСОСССТОСБАСТИС TSAASAААССА TATTOSALAT ООАТОоSaTTtТТ ТОСТОстТАС 1080

ATTOSTSATT STSTTOSAST SATTOSTOOT SSAASTSOTA SASSSSASST AATSTATOST 1140

TTTOOTSASO STSATSTOSO TATSASASST OSTSSAATOA STOSAASTST SOTSTOAM 12009

STOSTOOSAS OOOAAALASAS STOAATSSAS ATTTSSSSST TOOSASSAAAA SSOSTTTOST 1260

АТТООССАААТ ССАЛССАЛАС СОСТССОССЯ АСТТАХ 1296 (2) Information about POS IU Me:8: (i) Characteristics of the sequence: (A) Length: 1296 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type molecule!: DNA (synthetic) (hi) Sequence description: POS IUO Me:8:

ATSOSTSASA ASSASAASAAA OSTTOSTATS OSTOSASSTO SAATOOTTOO TOTTTOSAST bo

SSTTTsAToS TTSALOSTSO TOSATTSALO STTASSTTSA TTOATOSALA СсСсСАСССССТ 120

SMASOTOSTT STITSOSTAA SoSToSTTOS TTSAASOSTI SSTSOsTTotT TOSAAtoToS 180

KTOSSASSAA ASTTOASTAF SOTTSSAAALAO TOOSTISTTYA ASSSAATOoso TOSATToToS 240

ATSSSTTTSA OSTASTTTSS AASSATSATO SSTTOSTTTSA TTOSOTITSTT OSTToSTobA 300

ASASSALASA ASOTOALSSA SSALOSTALO SSASTOSSTA ASSTSATSAAA STOSASToTO 360

SSTITSATSA ASTOSTTOOS TSAbSASOST SATOSTASSS ASSTIATOSO TSASOAAASOT 420

САССТАССО TСАСССТob АСАЛОСАСАС TСОССМАСО АСОСТОСАСО ТССССААСТ 480

540

ССТААСТТОТ СТСАСОССТИ TASSKAАСОСА АТССТТАТОС ААСАСААСОС TSАСАССАТе 600

KASSSASAAS OTSTOSToAS TSTSTIOTTT OSTOSTTTSA TSoSTAASOo TosasASTTe 660 oToTsSTostTo SToTTATOSO ATTOSASAST SAASOTSSTo STSTSAABOYA TATSASSASS 17120

ASSAASOSTo TTSTTOSTOT TOATOSASSST STTOTTOSAS STOOTOSASA STOSAASTST 780

STISSTAAST SOSTTOSTOA TSASATSSSA TTOSATASSO AASSTSSSATA SSSATSSTo 640

ATOSSSAASS SASAAOSTOS TOSASSSTATIT SSAAASTASSO ATOSTISTOO AAASTTSATE 900

OSTASTOSTA TOSASATOSO TSTTSOTOTT OSTTSSAASOS TTSASTTSOS TOSTSTSAST 950

OSTOSTOSTA ASTOSAMOSO TOSTSASSTT STSTASASTO ASOSTSOTAA OTToSTTosA 1020 oSTSTSOSTS STOSSASTITS TSAASAASOT TASTSSALOT OOATOSOTTtT СостТосААвС 1080

ATSSSASSATT SSSTISSAST SATIOOTSOT OSTASSSSTA SToSASASOT TATsSTASsOoSsT 1140

TTSsotTSASO STSASSSTSO4 TATSASTOST OSTOSAAATOA SSOSAASOST osTTtSToMOa 1200

STOSSToOSAS STSASMLASAS STSTATSSAS ATSTSTOSAT TSOSASSAAAA ssottttsost 1260

ATTOSOTAAST SSAASSAAAS TOOTSSTOSA TOSTAA 1236 (2) Information about POS IU Me:9: (i) Sequence characteristics: (A) Length: 279 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type molecule!: DNA (recombinant) (hi) Sequence description: POS IUO Me:9:2.

ASATSTSSAS AATOSSTTOS TSTATOSTST STTOSOSTAS TATsOTTOosSS tTetosvoste vo

ASOSSASTAT СсТСоСТОСТ ТТСАЛСОСАС ТТААСТоСТо СоСсТосСстТто ССАСоСсАССС 120

SSAAASSSTAA SALSSASSATT ASTTOSATSA SAAOSALOSO SSOAASASTT AASTOSATOS 180

АСоТСТобсС ТСССАТТОСА АЛОЛАСААСТ ТТСАСАССТST STСТАССТ ОСТОАССТТА 240

СССАТСТСОСО ТОСТСОСОТС ААСТОСТОС АосссТОв 2739 (2) Information about POS IYUO Me:10: (y) Characteristics of the sequence: (А) Length: З1внп (В) Type: nucleotide (C) Structure: double-stranded (С) Topology: linear (y) Type molecule!: DNA (recombinant) (hi) Sequence description: POS IUO Me:10:

ASVISTATOS ATALOSTTaA TOTAATIOVA SSAKSATOVA AATTTISKT SOSSATTSI 5

SOKtTIOStTtv AATICHALOTI TSISSSSATOI SOSAAOTIVO SASAATSTOES ATO 120

ASadsSSStS TOTTATFTSS AvTSTSTOSA AATOSASTSA ASaSALATST SOSTTATSCHI i tTTISTtTstToKA SAsosAssSka SAtoSASSAO SITATSSOvAT TRSOTSOTO0 TOROSSTTOA 316

АСААСАСТОО САТСАСОТТА ATTSOSTSTO AOSTTSOTOS TSTTAASOTO AToTSsTISTO zo0 tTttTosSASSoS STOSATOS 318 (2) Information about POS IYUO Me:11: (i) Characteristics of the sequence: (A) Length: 119 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (y) Type of molecules: DNA (genomic) (hi) Sequence description: POS IUO Me:11:

NSATOSASST STOATSOALA TOSTOSTTAS SOSASSANOS TAASOSASOS SSAATTITAT 60

SASSTASSOS SALASSOTOO STASOSAOSO ASASASTOTO О09СТСССОСОССАСТОСТ 119 (2) Information about POS IYUO Me:12: (i) Sequence characteristics: (A) Length: 277 np (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) ) Type of molecules: DNA (genomic) (hi) Sequence description: POS IO Me:12:

STASTTASOS OSTOSTSAOT ASLOSOSMA SSOSSTOTSA ASATSAATST OSASSTOZSA 60

АТСССТСОО АСССПССАЛА ТОССОСАЛАТ АСАЛСАСАТА ТТААСОМОТС ВСОССССбАА 120

SOSTITSBOT SASTASASTS ASOSooSSSS ASSSoSTosSA TTSATTSATO TTTSSSOTSA 180

OSTTSSOATS ALASSASALO SSSASTSOSA FSAATSTOAS SATSTOSTOYA ATALSSASbS 240

САСТСТССТИ СТССОСАААТИ ТОСТОСОССТ АСТОбАЙ 277 (2) Information about POS IYUO Me:13: (i) Characteristics of the sequence: (A) Length: ZZnp (B) Type: nucleotide (C) Structure: double-stranded (C) Topology: linear (i) Type molecule!: DNA (synthetic) (xi) Description of the sequence: POS IO Me:13: ,adadsAstat saAstosasSa valasSATSAT ATO (2) Information about POS IUO Me:14: (i) Characteristics of the sequence: (A) Length: 35np (B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) Type of molecules: DNA (synthetic) (xi) Sequence description: POS IYUO Me:14:

СААСаААТОСС АДОИСТСТСА СОАССЯСАИТА ДаТАС (2) Information about POS IYUO Me:15: (i) Characteristics of the sequence: (A) Length: 24 np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules !: DNA (synthetic) (xi) Description of the sequence: POS IO Me:15: aСсСсОаАСАТад ssataassaA AAsa (2) Information about POS IХО Me:16: (І) Characteristics of the sequence: (А) Length: 24 np (В) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules: DNA (synthetic) (xi) Sequence description: POS IO Me:16: vadpAATaSSOa aATaSTTSAA SOS (2) Information about POS IYUO Me:17: ( i) Characteristics of the sequence: (A) Length: 1296 np

(B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) Type of molecules: DNA (recombinant) (their) Features: (A) name/key: (B) Localization: 1... 1296 (xi) Sequence description: POS IO Me:17:

ATS OST SAS KAS SAS AAS ALS OTT 00 ATS OST Both OST OSA ATO TT che

Mes Aia 91 Aep Niv pu bue Mai 91u 116 Aiv 01u A1a b1u 116 Uai 1 5 10 15

OST OT TOS AST OST To ATO STI SALA SOT SOT ObA TS AAo ot As 96 biu Chai Suye Trt Aia Pez Meb iz bip AKku Agu 91u Re puya Uai Ti

ТТ АТ AT СС АС СС СсА ost сАА Ost ss TstT Ts ot KAS OST 144

Tem I1e ler Rko Aep Rho Rgo biu bi b1u Aia beg Rie b1u lyap Aia ot TosS TT AAS ost TosS TosS ST TT SsSA ATO TS ATO SSA OSA Le 132 s1u Sub Ra Ap s1u 5eg Beg Mai Mai Rgo Meik beg Mesye Rkgo z1iu Ava 60

TT AT AOS ST SsSA AAb Ts STT STT SAS SSA ATO OT SsSA TtTo TOos 249 tsez Te bek Uaii Rko tsue Tt9» yech tsech Azr Rgo Meb biu go Tse 5eg 65 70 75 80

ATS sot TT boss TAS TT SSA ASS ATS ATO SstT 99 TT ATtT osot To 288 116 Ak Re b1u Tuk Re Rgo Tik 119 Mei Rko Tgr Ge I19 Ak9 ve 25 90 95

TTo STt ost SA ASA SSA KAS Aa oto Abo SAS SA ost Ako osA ste 336 cez ce lia biu Ak ko Aep tsuye Chai was biz b1p Aza buye Hi 14h 100 205 110

SsoT AAS ST ATS AL TS AST OTO OST TTo ATO AAb o Ttos Tta ost bab 384

Aha lap yeeez Her Pub back TP Maii Rgo shop 116 bue Beh yeech Aiv c19 115 120 125

SA OST SAT OST AS SAS STI ATS SOT SAS SAA OsoT SAS STI des st 432 si Aia dar Aia beg Ni Pecs I16 Agu Niye biz biu Ni Tsez Tph Uai 130 135 10

TAS SOT SsA OAA OSA bAS To OSS Or SAS OST SbA sot TO0 SAA STI "80

Tu Ag Chiu beach Aia dar RpF Ai1ia Ag Agr Ak biu biu Ter 01 svi 145 150 155 160

SOT sot Sto AS OST TT OST AST SAA ATS STO AbS OST SAT OSA TI 528

Ag Ag Be Ava biu Mai Ag Ts o bip 116 Tez Zag Aza Har left ate 165 170 175

SOT AT TTS SAT SST AAS TO TST SAS OSS TT ASS ALLO OSA ATO ST 576

Ago Mer Raf dar Rgo Dap tsi bZeh Ni liv Rpe Tph uz 91u 116 yigy 180 185 1939

ATS SAA SA MAS SOT SAS ASS ATS AS SSA SA bot Sto sto ASTT ste 624 112 bich 0165 Depp biu Niye Tak 116 leva Rgo bib biu ach Mai TiK gu 195 200 295

TTO TT SS OsStT TTo ATS OST AAS sSI bSA SA TS oto St ost sot 572 yez Re Ag Ag Re 119 liya Aalobiu biu biv RBe Uai Zeg Aza Ago 210 215 229

STT ATS OSA TTS SAS AST SAA bot SOT OST Sto AAb bot ATS As ss 720

Uai 116 biu Rpe 01 Tk bib biu Ak9 lia Tez Buz b1u 11yu Tg Tx 215 230 235 240

АСС ААС OST ST ST OST TT ОАО ОСА OST st ot bsSA ost ost bSA 768

Tpg dep b1u chai izch lia Uaii Aer lia Hia Ua! Uai Aia lia siu Li 215 250 255

SAS TOS AAi TST STI OST AAS TOS STT OST SAT SAS KTS SSA TO bAT 816

Niv bZeg Guz 5eg iz liy lap b5eg Gem siu Ler ler 114 Rgo tsvch lvr 250 265 270

ACC САА сот ббА TАС САС ATO STO ATS SS AS СсСА SA OST ost ss v64

Tit biz Ak9 biu Tut Ni I16 Uai 119 Kha1ia Avp Rho Fich Aza A1a Rgo 375 280 285

SOT ATT SSA AST ASS bAT OST TST OSA AAO TS ATS OST AST SST ATO 312

Aha i1yu Rgo Tk Tr Avr Hlia Zek Fi1u Puye Re I1F Aza Trth Rgo mes 2590 295 300

SAS ATO SOT ST SOI ST? OST Oba ASS ST blo te ost bot ste m 960 bi mes bsiu ceyu Ag Uai Aia biu TBzh Uai 0195 RBe Aza siu Se TV 305 310 315 3120

OST ost ost LAS tob AAo sot ost SAS ST ste TAS ASstT sas ost sot 1008 liv Aiva Rgo lap Tgr Bug Ah A1iza Niya chai tse" Tug TLR Ag9 Liv Ago 325 330 335

Klo To STI SSS ost ST OST OST osSS AT TST ol SAA sot TAS tes 1056 ruye Fo cem Rgo Aia tech Aia Rgo Liva bZeg Beh 0615 015 Ag9 Tug bog 345 350

MAO TO0 ATO o9T TT SOT SSA AS ATS SSO SAT TOS STT SSA STO ATT 1194

Buv Ttr MNeb 91u RF Ago RtFo 5Zeg 1316 Rko Avr et Tez Rgo Uai 116 355 360 355 bsST SOT OST ASS SOT AST SSS SAS OTT ATS TAS ost To obt ss sot 1152 biu Ag9 Mia Tpg Ag So Rgo Avr Mai 11 Tut Aza Rie biu No s1u 370 375 380 I

SAS STO OST ATO AST ST OST SSA ATO AS osa ACC ste st tTst SA 1200

Niv ate biu Mek Tk b1u Aza Rgo Mebye Tik Lia TE Pec Uai bZeg biz 385 390 3958 400

СсТС Sto OSA OST SAS AK ASS TST ATS OAS ATS TST SSA TT OSA SsA 1248 tse ie Aia biu biz buye Tvh Zeh Y18 Avr 119 Bek Rgo Rple Aza Rgo 405 410 415

MAS SStT tTtTS ost AT SOT LAS TOS AAO SAA AST sot OST OSA TS TA 1296

Aap Ag RP. siu 11ie siu uz Zeg Buv Sip TP biu Rgo Aza 5eg 420 425 430 (2) Information about POS IYUO Me:18: (i) Sequence characteristics: (A) Length: 431 amino acids (B) Type: amino acid (C) Topology: linear (y) Type of molecules: protein (hi) Sequence description: POS IO Me:18:

Mep. vii is Khvy Yaiya ruz buye Uvi F1u 114 A1ia yiu Liya aiu 1 Uzi 1 in 19 15 siu chai Suyu Tyi Az Bach Ka bach 012 Asu As 21 va buzh Uai Tg 29 25 za

T.Ch.

Fiu Suya Ri dep bi1u Khaikh Zag chai Uvi Rso Mas bei Nes vgo 21u dep o i5 bo shyai Tit bak Uuai ha buyaye Thr Tbn By dar Rgo Mah Fiu fgo ach bag vh 7 7 vo 114 Aga Rimn biu Tut RF ho Tvt 114 Keb Ro Tr Gea 116 Agu Re

VE for 5 rez That action biu Akd Ugo Avloguv wai uyu bim biz liz uyu Aza Gats i1so 195 112 aku lap rach iiya iush bah Tk Udi Kto i4m 14 pui sas bez kii b1ch 115 129 125 biz Via der diva Zag Kii ii 114 AK No bi» b1u No food Tvh Uai iso 135 o tuh Kg4 siu 29 Left Az Rie left and: AJr Ag z1iu 91u Tgr bij mu 15 150 155 160

Agu AgF Pa dyp biu Mai Ak Tlg bil Y16 Hashek ate Aar A1 a 165 179 175 zt Azhr o Ryzh dar Rgo dai there be Kivsh Aa Ryzh Tk uz O1u T1zh bai i8o 135 139 116 Sich bib la biu biz Tvpe tiv lal RKO bil o biu im wai TK a 195 200 20 izu Red dko AKU ?pv 11zh A1ia lyap b1u b1u z1h RBe Ua! Beh dia Aga 210 215 229 teas 11« siu Ra biz Te sich biu Ah liv food was b1iu ii Tl TE 115 230 235 240

Tag Anya Siu Mii food Aia Uai Ler liya Aia wai chai Liya liv F1iu ice

259 255

Nie beg tsue Zak ts Liv Avp beg tsech b1u Avr lar 119 Rgo Te lvr 260 265 110

Tvh biz Agu Fiu Tug Niya I1e Uai Ii Liva Aep Rgo beach Liz Aia rgo 275 280 285

Ag9 119 Rgo Tlh Tk Aer Liya bek siu Tsuye RpF I16 Aia Tig Rgo Nes 290 295 300 biz Nes siu yeech Agu Mai A1iz biu TE Uai beach Rpe Aiia siu tech tah 305 310 315 320

Kiz Aia Rgo lap Tgr bue Ak9 Aia Ni Uai iea Tuk tv mg Kia gu 325 330 335 tsuve yeech cem Rgo A1ia iz Aza Rgo A1ia beg 5eg biz bi Ak9 Tuk Zeg 340 345 350 tsuye Tgr Mebs Fiu RF Asu Rko 5eg I1 Rgo ALer Zeht Her Rgo Uai Ii 355 zbo 365 b1u Ag liv TpE leFdf TI Rgo Avr Ua! 116 Tug left Rije biu niv biu 370 375 380

Nie Iv Fbiu Meb TP Fiu liz Rho Mebs Tk Aiia Tk ih Uzi Zek biz 385 390 395 400 iiech rech lia biu 01 Shuz Tit Zach 13e Avr 116 5eg Rgo RBF Aza Rgo 4105 410 415

Mep Agu Rpe b1iu Ii 91u tsu bek buye ip Tpg Fiu Rko Hi 5Zeg 420 425 450 (2) Information about POS IYUO Me:19: (i) Characteristics of the sequence: (A) Length: 29 np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (y) Type of molecules: DNA (synthetic) (hi) Sequence description: POS IO Me:19: sattstTstTAS ASTSOatTOaSsTto atAlattas (2) Information about POS IYUO Me:20: (y) Characteristics sequence: (A) Length: 29 np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules: DNA (synthetic) (xi) Sequence description: POS IO Me:20:

SattstTotTAS ASTAADOasto atTAAstTTas (2) Information about POS IO Me:21:

(i) Characteristics of the sequence: (A) Length: 29 np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules: DNA (synthetic) (xi) Description of the sequence: POS IO Me :21:

SattstTotTAS ASTSADAsSsto atAlattas (2) Information about POS IYUO Me:22: (y) Characteristics of the sequence: (A) Length: 29 np (B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (y) Type of molecules! : DNA (synthetic) (xi) Sequence description: POS IO Me:22:

СсаттстТстТАС АСТОСТОСТОо аТААСстТас (2) Information about POS IYUO Me:23: (y) Characteristics of the sequence: (A) Length: З2np (В) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (y) Type of molecules! : DNA (synthetic) (xi) Sequence description: POS IO Me:23:

СТСТАСАСТТ сСОаStToаТtАА аСТТСТТоСА СС (2) Information about POS IYUO Me:24: (i) Characteristics of the sequence: (A) Length: 32np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules !: DNA (synthetic) (hi) Sequence description: POS IO Me:24:

STOTASASTA TSISTSOaTAAA SSTTSTTSSA SS (2) Information about POS IO Me:25: (i) Sequence characteristics: (A) Length: З2np (В) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules !: DNA (synthetic) (xi) Sequence description: POS IO Me:25:

СТСТАСАСТО ТаИССТСОаТАА ИСТТСТТОСА СС (2) Information about POS IYUO Me: 26: (i) Characteristics of the sequence: (A) Length: З2np (В) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules !: DNA (synthetic) (xi) Sequence description: POS IO Me:26:

СТСАТСАСТО АДаСТСатАА ССТТСТТОСА СС (2) Information about POS IO Me:27: (i) Characteristics of the sequence: (A) Length: b2np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules !: DNA (synthetic) (xi) Sequence description: POS IO Me:27: seasSstapdasStT aaAdATSaTTa atTaTATasSAS TaASTTTaATOa STTSAASO,TO atasAtTttTSAA (2) Information about POS IUO Me:28: (i) Characteristics of the sequence: (A) Length: b5np ( B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) Type of molecules: DNA (synthetic) (xi) Sequence description: POS IO Me:28:

аСАСАТОСТО ТСТасСстТОАТОа СТТТаСатаА ТТТСОАТОСТ ААСТТастТО АТаСТТТТАС СААСа (2) Information about POS IYUO Me:29: (i) Characteristics of the sequence: (A) Length: 41 np (B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) ) Type of molecules!: DNA (synthetic) (hi) Sequence description: POS IO Me:29:

STSATSOSTT TTOASASTOA AOSTSOTOST STSAAAASasA TT (2) Information about POS IU Me:30: (i) Characteristics of the sequence: (A) Length: b9np (B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) Type molecule!: DNA (synthetic) (hi) Sequence description: POS IUO Me:30:

TASAASSAST AASOSSYATOatto tasstattald taslastat attasaasta ataSASASTO TAAATSAST (2) Information about POS IYUO Me:31: (i) Sequence characteristics: (A) Length: binp (B) Type: nucleotide (C) Structure: single-stranded (C) Topology: linear (i) ) Type of molecules: DNA (synthetic) (xi) Description of the sequence: POS IO Me:31: cOAAAATasat stttatatta staatASTat tadattta7ast vaatotoASAa STaSTtoStTAA (2) Information about POS IUO Me:32: (i) Characteristics of the sequence: (A) Length: banp (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Molecule type!: DNA (synthetic) (xi) Sequence description: POS IO Me:32: tapATOSOSTTT TTsatostAa SATTOStadT TtetToTtoSAa Taltta!tTSa TaSAASTOSST ASASSSOA (2) Information about POS IYUO Me:33: (i) Characteristics of the sequence: (A) Length: b9np (B) Type: nucleotide (C) Structure: single-chain (C) Topology: linear (i) Type of molecules: DNA ( synthetic) (hi) Description of the sequence: POS IO Me:33:

SatAATSTAT SSTTTTaaato ASOatSATST SOSTATaASA CMtaSTOoSAA TOASTOSAAS TSTSSIaTSTO

TSAASAASOTASOVOSSATATSASTTTATTSADATToo

TATSVSSLADASSAAAVAAVSAASTOSTST SSTSADAOO

PTOTAAVLAAVAATTSTSAOTSSAAASSSTSAASAAS

BTSABOBTASAPASTSTOSADASSATASSSAVADOST

ASASOABATSAATOVAAVAATSTTSAATSVAASTAAAST

ASTOTTSSAOSASATOSATSATOSTSAVTADO TT To

AAAAAASASATSSASSTLAVAST TRAT TABTOOOSAT

SETTTOADAVTAATE TO SAASATOVAVSAOSTOSTI

STOOOASSAVASADAAALOSAATOSTOSAOAATTOT

VOBSVSASSTASSLAVAVSATSTTTOSSTTTATTOSAA

BABATAAAASSASATTSSTSTAYTAASAASTOVOSAASAA

AVTAASOTOOLAAAALAVSTVTTSSTOASAAVSSSASTSAYE

TAATOSOTATSASOAASOSASTSASOASSASAADAODA

TIPESSSTSTATATAABAVOVSAT TT SATTOSSTTTO

AABOATSATSASASATASTAAASSAATATTTTSTO 6954 years

Fig. 1

AND NSATOBASOTSTOATSVLAATOSTTSOT GASSBSAVSAAV TAASOSASOSSVAATT TAT

AND SASSASSOSSSADASOVTOOSTAOSA OSbASAVASTTOVOSTOSSSVOBAPSATSSTA

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IC TOTSTODOAAASSASAAAAAAOTAVOSA TOVSTA SSSOAKT SOT OOBSYATATOSASOS 5ENNKKMbIvshaivvbiIbUstTtA

Ib1 SOSToATEEGTSAOOVOSOSObATTSAAAOTSASSST TOATTVASSSTDASSSTOSTOVSO i KivbA8RBEKUTI1RNRRBE 241 AAOBTOSATOT t TOObAAATOSSObATOSTSAASSSSTSATSSTSSSTOSSTATOTOSA

SA 5 EBNAbSENOY:imMUvYHK

ZO TOOSOPOADASTEBASVAVSOTOSSVAASTVOSTEST TIAOSSBATOVOOSSV TT OTSAD

RO NI TT UR.KMAi VRYBRIII

ZI TeSoOTTeAOSTATTTOSAASSATSATOSSSTOVT TOKAR STO TABSSVVDA

ЕЕ5 ЦЕТ ИНРУУИИТЕЕ Ав 421 БАССАДАСААВТ TVA САССАВСВАААВСАСТЕСССЛА ESTSATSAAVTSАССССТОЕ

RnyKkUKkKEIVNAKASENIIIKU TM "81 STOTOATSAASTSATTVOSOVBAVOLOSTOATVSVAVSSSATSTVATOSOSSATOAAEST o

GIKKAEEEAZAYINKNIIENESN

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BE boss gSstTeAAASieT OT TEBSASOSAOATSTSAOSOSSSOATOSTTOSVBOV ETTSOATS

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BI SoDASTTateSATOSYAT TASSADOVOSATTSTTTATASAASAOVASOVTSASASOA AND

NEZNARTKEGETEENNI AND IK 721

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901

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About TSVOSAATO SAS OST A SAT OSS BASOSATOOST SABBAADATTSATOE

A NREVAREIRTIVA; KA A

TORI SBASASSTATOSALATOOSSTT OSS TAYEST TATO TSASA

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Claims (17)

1. A molecule of isolated double-stranded DNA, including the DNA sequence encoding the enzyme glyphosate oxidoreductase, and the linear amino acid sequence of the specified enzyme is selected from the group including 5EO IU MO:5 Mee bek 515 Avp Niv Shtsuv Puv Mai biu i1e Aia Ssiu Aza siu Iie wai 1 5 10 15 si1iu mmmai Suv Tph A13 Pem Mee Pe bip Aga Ak biu Vpe- Buv Ma1i1 TNg zo Pec T1ie Avr o Rgo Azp Rgo Rko biu biu biu biu A1i1a Beh Rne biu Avp AI1a sSiu Suv RpOe Avp piu Bek bek Uai Uai Rgo Mees beh Mee Rko biu Ahp in . 55 bo Hem Tak o Bek Uai Rho Puv Thr this cem Avr orto Mes Siu Rko Tem Bek 65 to 75 in I1e AkYA4 Rpe Beh Tukhk Rpe Rko Tpk i116 Mes Rgo Tktr Pes I1e Ak RPEe 85 o 95 Tem iey Aza siu AkhaYA Rko Avp oBuv My pub pi bip Aiia ytsuv Aia Gay 100 105 110 Akch Avpoiei yT1e pbuv bek Tik Mai Rgo Petc Ii Muv Beh Petc Aia Z1y 115 120 125 biz dia Avr A1a bek Niv Tez i1e Ahd Niv dich y)1u niv cemsh Tk uv 130 i35 140 tuk Akach biu siy Aza AvroRBe Aza tsuv Avr o Aka s1iu siu Ter bich Pech 145 150 155 1650 Aga Aga iem Avp o spiu uai Ako Tk obip i1ie Pes beh Aia Avr Aza Pec 165 178 175 AhYA ABrpe Avr Rgo Avp o Gei bet Niv A1a Rpe Tik tsuv biu T1e Pem 180 185 190 i1e siy bij dvp o siu Niya Tvh I1e Ap o rko bip b1iu Pe Uzi Tk oBbem 155 29 205 iiey Rre Ah AhkYa Rie I1e dia Avp s1u siu siy Re uv Zeg Aza AhF 210 215 22 wai I1e biu Vpe bij TVh 915 51u Ah A1ia Bey bu Z1u i1e Tk TBE 225 230 235 240 Twk Avp b1iu in Pets Aza uai Avr visa Aza Mai Uai dia dia s1u Aza 245 259 255 Niz Beh Puv Beh Pets Aia ADvp bek 1u Avr Avr I18 RH ho Geli Avr 260 265 a7o Tvh sii Ah biu Tut Niv i11e Uai i1e Aia Avp Rko bii A1ia dia Rho 275 2vo 285 Vuchomie Rho TP Tpk o Avbr Aza Beh s1iu Buv Rne I1ie Aia t rko Mei 79 295 zp biz Mes sbiu tsets Ahku Uai Aza Siu tТgoUai b3iy Re Aza p1u Hem TVE 305 310 315 329 ia dia Rtgo Avp Tour was Aka Aza Niv Ma1 rech tukh Tph Niv Aia AhF 325 zzo , 335 Buv Gay Ped Rkgo Aia ep Aza Rgo dia bek Beh bi sich Dku Tukh zek zao z45 350 ace Tgr Mei Siu rBe Aku rRho Bek i11e rRho Avr Bek cem rRho Chai I1e 355 ZBO 365 s1u AhYA Asia tt Aku Tk o rko Avroua1i I1ee Tukhg A1a Rbe 01u Niv siu 370 375 380 niv tsets biu mes Tik b1u A1a Rrko Mes Tih Aia TPg Te Uai Bek b1c z85 390 395 400 Bez Pec Aza biu Sich tsuv Ts bek I1e Avr I1e Zek Rgo Rne A1a. Rho 405 410 415 Avp Akta Re p01u i11e b1u Puv bek Puv bip Tk s1u Rko Aia bBei a20 425 t" 430 and the residue in the prescribed 334, denoted by X, is selected from the group containing histidine, arginine, lysine, glutamine, alanine, tryptophan , leucine, isoleucine and glutamic acid and BEO IU MO:18 Mebe A1a Sich lyap Niv tsTsuv yub Mai Siu 112 Aia b1u lia biu t1ie Uvi 1 5 19 15 s1iu Mai Sue TBg liy Iach Kei rech bip Agu Aku F1u Re bue Chai TE 2o 25 zo yeech Ii6 Aar Rkgo dep Rko Rgo O1u beach biu Aia beg Rpye 01u Azp lLia siu Su Rpe dep b1u Beh beg Mai Mai ?Rko Mb Beh Nei Ro b1u Mep 50 ivch TI Zach Chai Rgo buye Tsr Gechm Tez Ayar Rto Keb b1u Rho without back 65 70 75 in 118 Aku Re biu Tug Riye Rgo TBk Ii MNeb Rgo Ttr yiv Ii114 Agu Re 85 90 . 75 iz tsech left 01u Ak Rgo Avp tsuya Maiii tsu im Sip Aia Bush Aia iz 100 105 119 Mk9 da om 119 rub bak Tpg Mai Rgo cem I16 Puyeya Zag vy A)x b1z 115 120 125 biz" Aiv Avr Aia bag Niv yeech 11 AsFo niv 015 bi1u Niv Gats Tizhg Ugi 130 135 140 Tuk Agu siu Fiz Aza Avr Rie Liv Ago Avpr Ahu b1u biu TER Sich ieh 145 150 155 160 Ako Ag baz dep siu Uvi Agu Tvg o bip iii Pec Zah Aiv Aar Aza te 155 it 175 Ak Aar Riye Avr Rko lap Ta beg Niya Aza Rle TVg was 01u 118 yiv 180 185 130 11 b1z biz lap biu No Tpk Ii Avp Rko bip biu Tvz Mai Tpg im 195 200 405 yeech Rpe Agu Asd Rpe 116 Aia Avp biu s1iu beach Re Uai beg Aia Ag9 210 7515 220 chai I1a Siu Be b1z TK biz Siu AgF A1ia ce» Su b01u 116 TVE TE 225 230 235 240 TYE lap s1u uai ces Aii Uai Avr Aiy Aiia Uki Ua) li Aza s1u hiv Ni 245 459 255 bZeh Shuye bak Tech liva lLep Zak Ge Oiu Lur Avr 116 Rgo Gach dar 256 265 270 Tv 01z Ag s1iu Tuye Niv 11 Ua1i TI Aia Aep Rgo 01 Liv Liv Rto 275 280 285 Agu 116 rgo Tpg Tve Avr Div bag biu Shuya RF I1yu liv Tkh Rgo MNees 290 295 300 biz Mebe 01 iu Mei Ag Ma) Aia sSiu tTkh Uvi biz re Aiv biu Sech Tvg 305 3160 315 320 Kiv div Rgo Aep Tkr tsuya Ag Aza Nizh Maiii Pes Tukh TI As Aia Ag 325 330 335 tsu bez Se Rgo Aiv y4u A)v Rko Aza Zeg bek biz biz Ah Tuk Zak 340 345 350 uv Tgr Keb biu RBa Ago Rgo bek 11 Rgo Mer Bek Imy Rgo Mai 11 355 360 365 biu hg9 Kia Tig Ago Tk Rgo Avr Mi 11 Tuk Aiia Rie biu Niv siu 370 375 zvo Niya tse biu Mas Tik siu liv Rgo KMeb Tpg dia Torg yiai Uai behg biv 385 390 395 400 yiv ilyech Aia biu biz buya Tag bas Y1a Har 119 god Rgo Re Aiv ro 405 410 415 Avp Ak Re biu Ii Siu Bush bZeg Tu biv Tlk siu Rgo Aza back 2. Молекула вьіделенной двухцепочечной ДНК, включающая ДНК последовательность, кодирующую фермент оглифосат ооксидоредуктазу, причем указанную ДНК последовательность вьібирают из группьі, включающей 5БЕО ІЮ МОЗ МСАТОСАСОТ СТаАТССААА ТОСТООТТАС СОСАОСААСС ТААОССАСасС СОААТІТТАТ І) САССТАССОС СААдСОосСТОС СТАСССАССОо АСАСАСТОТО СОСТОСОСОО САССАТОСТА 120 ТОТСТСАСАА ССАСАААААА ПТАСССАТСО СТОСАСССОС ААТОСТОООС СТАТОСАСаС 180 СОСТОАТОСТ ТСдасСоСссоС ОСАТТСАААС ТСАССТЕСАТ ТаАССССААС ссТОосТевСо 240 АдастТОсСАТО СТТТОСОААТ СССОСАТОСТ ТСААСаОСсТо АТоСатостТо ССТАТОТССА 300 ТЕССОССААА СТТОАССАСС СТасосААСТ ОССТОСТТСА ССССАТОСОО СССТТСТСАА з6о ТССОоСТТСАС СТАТТТТССА АССАТСАТОС сССстТОоаттТсАТ ТСОоСТТТСТО ВЕ ТАСССОСАд 4720 САССАААСАА ООТСААОСАС САССССАААС САСТОСОСАА ТСТСАТСААсС ТоСАСсссТОаС зво СТОТОАТСАА СТСАТТООСО САСаАСОСТО АТОССАСССА ТСТОАТССООС САТОАДАсоСсТО 540 АТСТОАСОСТ АТАТОСТОСА СААССАСАСстТ ТСОССАдДОСА ССасСобдоСТ ТОаСААСТОоС боб ОСССТСТСАА ССОТТТФОС ТТТССАТС бе ССААСТТСТС ССАТСССТТТ АССААССОСА ТТСТТАТАСА АСАСААСОСТ САСАССАТТА 720 дтососАлос остостТодДос стоттотттТо ОсОоСаттТтТтТАТ сСОосСОАвАсоотТ соСсОовАттоео тво ТАТСТОСОСО ТОаТСАТОСООС ТТТОАбАСТО ААСОТАСОСС ОСТТААДоСС АТТАСААССА 40 СОААСОоссстТ тостТаССсСОоТТ бАТОоСАОасСос тТтТаТСОсСАоС ССасосАСАС ТООАААТСАС зоо ТТОСТААТТС ОСТАСОССАТ САСАТСССОС ТОСАТАСССА АССТОСАТАТ САТАТССТСА 960 ТСОССААТОС ССААсССасСтТ ССАСОСАТТС СБАСаАСССА ТасСатТСсАсОА АААТТСАТОО 1020 СОАСАССТАТ пСАААтТосос сттТооСотос сооотТАСсотТ ТОАстТТСОосСтТ сООСстТСАсСАС 1080 ССОСТОСТАА СТОСАЛАССТ ССОСАТОТОС ТСТАТАСОСА СОСТОСАААА СТТСТТССАС 1140 СССТСОоСоСС ТОССАОТТСТ СААСААСОАТ АТТССАААТО бАТОоСостТТС СОбСССАССА 1200 ТСССОобАТТСо ССТОСССОТО АТТОСССОСС СААСССССАС АССССАСОТА АТСТАТОСТТ 1250 ТСОСССАТОПС ТСАТСТОООС АТОСАСАОСОосС СОССОАТСАС СОСАдСсаСстТо стТетТоАсдос 1320 ТОСТСоСАСО ССАЛААСАСС ТСААТСОАСА ТТТСОСОСТТ СОСАССАААС СОСТТТОСТА 1380 ТТаССАААТС СААССАААСО ССТОСООСАА СТТААСТАСТ ТАСОСОСТСО ТОАСТАСАоС 1440 ССАСАССССО ТСТСААСАТО ААТСТОСАСС ТСОСААТСАС стТСОСАСАСа СОАААТООСО 1509 САААТАСААС АСАТАТТААС САСТСАСОСС СССААСССТТ ТОООТСАСТА САСТСАбОоСОо 1560 СССССАССОС СТОСАТТСАТ ТСАТОТТТСС ОСТСАССТТС ССАТСАААСС АСААССССАо 1620 ТСОСАССААТ СТСАССАТСТ ССТСОАТААС САСОССАСТо ТОСТТСТССО СААтТТтТОоСстТО 1680 вссстТАСТОС А 1692, АТО ТСТ САС ААС САС ААА дАдД СТА СОС АТО ОСТ ОСА SSS OSA ATS ste av Mes bek sbip Azp Niv P puk Puv Ma1 siu I1e A1i1a 51u A1a s1u I1e Uai 1 5 10 - 15 BOS STA TOS dSo ssSa STO and STT SAS SbS SOS OobA TTO AAA hundred dos 96 S1u Chai Suv WEEK A1ia Hz Mes pes bip Akchach Akgu bSiu Re uv mma1i tih zo TTO ATT SAS SS AS OSST SStT POS SA SOT OSA TSO TTT SOS AAtT SSS 134 is 11th Avr VBgo AvBp o Rko Rko siu sitz biu Aia bek Riv Siu Ap Aia 240 45 OSA TOS TT AAS boss TSA TO STO Sto SST ATO TOSS ATO SSO OSAD dAS 192 siu Suv Rrpe Avp biu bek bek ma1i Uai Rko Mee bek Mes RRRgo S1u Avp BO 55 vo TTOo ASOo AS OsTo SSO AAs tob Sto SstTtT BAS SSO to opo SSS To TsA 240 Petc Tbg bek Uai1 Rko puv Ttr Bay Petc Avr Rko Mee siu Rko Petc beg 65 70 75 . in ATO sob TTS AOS TAT o TTT SsSA ASS ATS ATO SOS otTOo TTO ATT SOS TTT y 288 I12e Ak RBe bBekht Tuk Rpe Rko TPI I12e Mee Rgo Tgr Gets I11e At Rrge 85 out of 25 STO TTA OSS OSA ASA SSA AASOAAS STO AAS SAS SAb SSO AAA SSA STO 336 eem Pe Aza Siu Acad Rrko Avp o Puv Ua1 Puv 211 pip dia Puv A13 Bbech 100 105 110 SOS OAAT ST ATS AAo tes So sto SST STO ATS AAb TSA TTOo sb САС zva Ag Ayep Pec I1e Ppuv bek Tit Uuai Rko Pep I1e Puv Beh Bec A1a Jan 115 120 125 SAS OSST SAT SSO AS SAT STO ATS SOS SAT baAA bST SAT STO ASS atA az32 si A1a Avr A1a bek niv te I1v Akud Ni biz b1iu Ni PesheTnNIt Uai 130 135 150 TAT SOT Both SAD OSA SAS OTTO OSS AL SAS SOS OSA ST TOO SAA STO 4avo Tukhk Ak p1u biz Ata Azr Re Aza tsuv Avr AgYA Z1u yiu Tur bi Gay 145 150 155 160 so sot sto AAS ost tt SOS ASo SA ATS STS AOS ss bAT oso TTo IV Aka AhYa Pem Avposiu Ma1! Ak Tk sip I1e Bez bek Aza Avr ATa Gets 165 170 175 SeS SAT TTOo SAT SSO AAS OTTO TSO SAT SSS TTtT ASS AAS o OoS ATT STT 576 Agch ABr Rpe Avr Rgo Avp obets bet Niz Aza Rpae Tph Bush S1Uu I1v Pec . 180 185 1980 ATA ZAA bAb AAS o ObT SAS AS ATT AAtT SS SAA sob STO sto AS sto 624 I1e bij bish ABp o b1u niv tt t11e Avp o Rgo biu pei wai Tk Bay 195 20a 205 tTTs tTtTt sob sstT TISH ATO Sa Ado sot pOS bAA tTtS TA tot o bso sot 672 yeech Rre AkCh Ako Rpe I1e Asia ABp biu s1u bi Rbe Uua1i bek A1a dha 210 215 220 STO ATS OS TTT SAS AST SbAA sobtT do oso STT AAA bbsS ATT ASA des 720 uai Iie b1u Rre bij TiK bi 41u Ag Aa be Ppuv 41u I1e Tpk tk 225 230 235 240 sa las sosS stt sto OSS TT AT OSA osa tt stos OSA pSS boss OSA 768 TP Avp osiu Ua1 Pec A1ia uaii Advr A1i1a A1a Uai Uuai A1a A1ia siu lia 24 250 255 САС TSb AAAA TSA STI OST AAT TSO STA BOS OAT SAS ATO SSO STO SAT vie Niv Bek Ptsuv Bek Ietsp Aza ABp bet Pei b1iu Avr ABr I118e Rko this Avr 260 265 270 АСС САА SST OSA TAT SAT ATO STO ATO SO LAT SSO OAA so OST SsA ve4 Tykh sich Aku bi1iu Tuk Niv I11e Uai I116 Aia ABp Rgo bis A1ia dia Rgo 275 25o 285 SOS ATT SSO ASIA ASO SAT SSO TSA OSA AAA OTTO ATS SSO ASA SST ATS 912 Ag Iie Rkgo Tbu Tpk AvBvr o Aia Beh SsS1u Puz RRBe Iie A1ia Tph rko Mes 290 235 Zoo SAA Ata bob stt sSososo ssso ost AS STT SAS TTS OST sob STO As, z6o bi Mes p1u Pec Act Mai dia biu tik wai bii Re Aza s1u cei tk 305 z10 z1i5: 320 SSS OST SST AAS OTO AAA SstT oso SAT STO STO TAT AS SAS ost sb, 1008 A1ia dia Rko Ap o Tgtr tsuv Ahu Aza Niv wai ei Tkh Niv A1a AkaCh 325 330 335 AVA OSTI STT SSA bos ste sss sstT oso AST TST ODA SAA SSA TAT tes 1056 pub pem Mei Rgo Aza Pec A1i1a Rgo Aza bek bek s1c bid Agud Tuk beh 340 za5 35O AvAA Tai ATO sb TS sob SSO AOS ATS SSO SAT tob sto ss vBta AtTt 1104 puv Tkr Mes b1u Rpe Ak Rgo Beh Ii Rgo Azr Zek Bay Rko Chai Iie 355 zvo ze as sa OSA ASS SOS ASA SSS SAS STA ATS TAT SST TT boss SAT sot 1152 51u AkchCh A1a Tk Ak Teh Rgo Avr o Mai I1e Tug A1i1a Rre SsS1u Niv s1iu 370 375 zvo SAT STO bas ATO ASA sos o sSa sob ATO DSS OSA dob Sto Oto tTSA ss 1200 Niv pecs siu Mee TI biu Aza rgo Mee Trt Aia Tit Pecs wai b5eth bij 385 zao 395 400 STO Sto OSA spsS pAA LA o ASS TSA ATS SAS ATT TOO SSS TT S OSA SSA 1248 yeech prep A1i1a s1iu Si Puv tBE Bek IZe Avr I1e Bek Rko Rre dia Rho " 405 a1t10 415 AAS SOS RTT OST ATT OSS AAA ToS AAS SAA ASsS bat SSO OSA AOst 1293 Avp AkYA RBe s1u I1e p01u Ppuv bek Buv bSip Tk S1iu Rko Aza Vek. 420. 425 430 TAd 1296 АТСОСТСАСА АССАСАДАДА АСТАСОСАТС ССТОСАСССО СААТоСТОССо СОТАТоСАСо ео ССССТОАТОС ТТСАСоСОС ССОСАТТСАЛА СТСАССТТОА ТТСАССССАА СоСТоСсТОоСе 120 СААССТОСАТ ССТТТСССАА ТСОСОСАТОС ТІСААСоОСТ САТоСоТостТ СеСТАТоТеС 180 АТССССОСОАА АСТТСАССАС ССоТоСССААС тТооСстТосСТТС АССССАТОСО ССССТІСТСА 240 АТССССТІСА ССТАТІТТОСС ААССАТСАТО СССТОСТТОА ТТСОСТТТСТ СТТАСССОСА зоо ВСАССАААСА АССТСААЛССА ССАСОССАДА ССАСТССОСА АТСТСАТСАА СсСТоСАСосТо 360 ССТСТОАТСА АСТСАТІССС СОАССАСОСТ САТОССАССС АТСТСАТССО ССАТОАЛОСТ 420 САТСТСАССО ТАТАТОССТОС АСААССАСАС ТТССССААСо АССоССОАСо ТТОССААсСтТо 430 СсССССТСТСА АСОСТоТТСо САСССАСАТС СТСАССоССО АТОССтТТОСо СОАТТТССАтТ о) СССААСТТСТ СОСАТОСОТТ ТАССААСОСС АТТСТТАТАС ААОАСААСОС ТСАСАССАТТ 5О0 ААТОСОСААС СоСТоСТСАС ССТСТІСТТТ СООССОСТТТТА ТССССААСбо ТООССААТТТ 6вво СТАТСТОСОС СТОТСАТССоО СТТТСАСАСТ СААбСТАСоСо СОСТТАААСО САТТАСААСС 720 АСОААСоССс ТІТСТбОоССоТ ТОАТоСАССо СТТСТСОСАС ССОССОСАСА СТССАААТСА 780 СТТОСТААТТ СОСТАССССА ТОАСАТСССС СТОСАТАССС ААСОТОСАТА ТСАТАТОСТС в4о АТССССААТС СОСААСССоС ТССАСОСАТТ СССАССАССО АТОССТСАСС ВДАВАТТСАТС 900 СССАСАССТА ТССАААТосСо ССТІСоССТо СССОСТАСоо ТТСАСТТСоС ТеСОСТСАСА 950 СССОСТССТА АСТОСАААСС ТОСОСАТОТо СТСТАТАСОС АСОСТСОААЛА АСТТСТТОСА 1020 СССсСтТооСсс СТОССАСТТС ТСААСААСОА ТАТТССАААТ сСАТОоОбоТТ СосбоСсссвос 1080 АТССССОСАТТ ССОСТСССССТ САТТСССССС ССААСССОСА САССССАСОТ ААТСТАТОСТ 1140 ТТСбоССАСО СТСАТСТСОС САТСАСАССо ССбСССАТСА ССОСААСОСТ СОТСТСАСАС 1200 СТОСТСОСАС СССАДАВСАС СТСААТССАС АТІТСССССТ ТСОСАССААА ССССТТТОСТ 1260 АТТОССАААТ ССААССАААС ССОТССОБСА АСТТАА 1296 , ЗЕО І МО:7 АТСОСТСАСА АССАСААААА АСТАСССАТС ОСТОСАССТО СААТССТТСС ТСТАТОСАСТ 6о ОСТТТСАТСС ТТСААССТОО ТОСАТТСАЛА СТСАССТТСА ТІСАССССАА СоСТОСТОСС 120 СААССТССАТ ССТІТСССАА ТОССОСАТОС ТТСААССОСТ САТСССТОСТ СССТАТОоТОоС 180 АТОССОССАА АСТТСАССАС ССТОССОААС ТоССТоСТТС АССССАТоСо СОСОТІСТСА 240 АТССОСТТСА ССТАТТТТСС ААССАТСАТО СССТОСТТСА ТЕСОСТТТСТ СТТАСССОСА 300 АСАССАААСА АССТОААСОА ССАСССОААА ССАСТССОСА ATSTSATSAA SToSASSoTo zeo SSTSTOATSA АСТСАТТІСОС ССАССАСССТ САТОоССАССС АТСТОАТССС ССАТСАдОоСТ 420 САТСТСАСОС ТАТАТССТОС АСААССАСАС ТТСОССААСО АССоСосАССС ТТСССААсСТо а4во СССССТСТСА АСССТСТТСС САСССАСАТС СТСТСТОСТО АТОСТТІТОСС ТСАТТІССАТ 540 ССТААСТІСТ СОСАТОСТТІ ТАССААСССС АТІСТІТАТАС ААСАСААСОС ТСАСАССАТТ 60оо ААТССОСАвО СОСТОСТОАС ССТІСТТОТІТ СООССОТІТТА ТОСССААСоо ТОБССААТТТ 560 СТАТСТОСОС СТОТФАТСОо ТІТТСАСАСТ СААССТОСТО СТСТСАААбО САТТАСААСС 720 АСТААСОСТО ТІСТООСТОТ ТОАТОСАССТ СТТОТТОСАС СТОСТОСАСА СТСТАААТСА 780 СТТССТААТТ СОСТАСОССА TOASATSSSO STSSATASSS AASSTOSATA TSATATSSTo vo АТССССААТС СССААСССОС ТССАСОСАТТ СССАССАССО АТОССТСАСС АЛААТТСАТС зо0 СССАСАССТА ТОСАААТОСо ТСТТОСТСТТ ОСТОСТАСТО ТТОАсСТТТОС ТОСТСТСАСА 9Зво ОСТОСТССТА АСТОСАААСС ТОСССАТОТО СТСТАТАСОС АСОСТОСАЛА АСТІСТТОСА 1020 ссСссСтТСосСос СТОССАСТТС ТСААСААССА ТАТТССААДАТ ОСАТОСОТТІ ТСОТОСТАбС 1080 АТТССТОАТТ СІСТІССАСТ САТТОСТССТ СОСААСТОСТА САССССАСОТ ААТСТАТОСТ 1150 ТТТОСТСАСо ОТСАТСТСОС ТАТСАСАССТ ОСТССААТСА СТОСААСТСТ СОТСТСАСАС 1200 СТССТОбСвО ССОААААСАС СТСААТОСАС АТТТСОСССТ ТСОСАССАЛА ССОСТТТООТ 1250 АТТОССАААТ ССААССАААС СОСТоССОСА АСТТАА . 1296, ЗЕО ІЮ МО:8 АТОССТСАСА АССАСААСАА ОСТТОСТАТС ССТОСАССТо СААТОСТТоС ТСоТТТоСАСТ во ССТТТСАТОС ТІСАВССТСО ТОСАТТСАЛС СТТАССТТОА ТІСАТССАдА СоСАССАССТ 120 СААССТОСТТ СТТТСССТАА СоСТОсСТТоС ТТСААСОСТТ ССТОСОТТОТ ТССААТСТСС 180 АТСССАОСАА АСТТСАСТАС ССТТССАААС ТСОСТІСТТО АСОСВАТОССо ТОСАТТСТоС 240 ВАТОСОоТІТСА ССТАСТІТСС ВАССАТСАТо ССТтТСОотТТоА тТтТосаТтІТСсТтТ ССТТоСтТовА 300 АСАССАААСА АСОТОСААССА ССААОСТААС ССАСТОССТА АССТСАТСАА СТССАСТОоТо 350 ССТтІТСАТСА АСТОСТТОСС ТСАССАСССТ САТОСТАССС АССТТАТССС ТСАССААоСсТ 420 САССТТАСОС ТОТАСССТОС АСААССАСАС ТТСОССААСС АСОСТССАСС ТТСССААСТТ аво ССТОСТСТСА АСОСТОТТСС ТАСТСАААТС СТСАССОСТо АТОСАТТОССо ТСАТТТССАТ 540 ССТААСТТСТ СВСАСОССТТ ТАССААСОСА АТОСТТАТСС АВСАСААСОС ТСАСАССАТС воо ААССсСАСАдДо сТСстТссТсАС ТоТСсТтТотТтТІ СОТСОТТТСА ТСССТААСОС ТОСАСАСТТС 6вео сТстТСТосСтТС СТСТТАТОСО АТТССАСАСТ СААССТОосСТо СТІСТСАДССо ТАТСАССАСС 1720 АССАвСоСТо ТТСТТОСТСТ ТОАТоСАССТ СТІСТТОСАС СТОСТОСАСА СТОСААСТСТ 780 СТТОСТААСТ СОСТТССТОА TSASATSSSA TISS АТАССС ААССТОСАТА ССАСАТОСТОо в40 АТССССААСС САСААССТОС ТОСАССТАТІ ССААСТАССО АТОСТТСТОС АААСТТСАТС Зоо "ССТАСТОСТА ТОСАСАТосСс ТСТТоОоТОТтТ оСТОоСААССС ТТОАСТТосс ТооТСТСАСТ 960 ОСТОСТОСТА АСТОСААССО ТОСТСАССТТ СТСТАСАСТС АСОСІССТАА СТТОСТТОСА 1020 ССТСТСССтТС СТОССАСТТС ТСААСААССТ ТАСТССААСТ ССАТоОСТТТ СССТоСАвОоС 1080 АТСССАСАТТ СССТТССАСТ САТТОоСТССТ ССТАССССТА СТОСАСАССТ ТАТСТАСОоСТ 1140 ТТСОСТСАСС СТСАССТССС ТАТСАСТОСТ ОСТССААТСА ССССААСсСТ ССТТТСТоАо 1200 СТОСТСОСАС СТОАСААСАС СТСТАТССАС ATSTSTOSAT TOSSASSAAAA SoSTTTOoST 1260 ATTOSTAAST SSAASSAAAS TOSTOSToSA TOSTAA I 1296 , and 5EO IJU MO:17 ATO OST SAS AAS SAS AAS AVS STT SST ATS OST bSA ST OSA ATS SsTT 48 Mei A1a Sic Avap Niv Kpuv Puv Mai Siu I1e Aiwa biu 1 5 10 15 ost STI ToS ASI OST TT ATO STT SAA SST SOT OSA TT Ado stt ves 96 s1u MUMai Suv Tig Aza Gesh Mee Pech bip Agud Akgu siu Rpe Tssuv Uai Tpg za TT ATT SAT SSA AASOSSA SSA OST sAA sot ssS o tTstT TT bot AAssS o sstT 144 cem Iie Avr Rgo Avp Rgo Rgo siu 51: b1u A1a beg Rie biu Avp dia OST TOS TES AAS ost ToS ToS STI STI SSA ATO TOS ATS SSA SSA As 192 S1iu Suv Re Avp 2iu Bek bek Uai UMai Rgo Mebs beg Mezh Rko siu dyap 5o 55 in TtTo AST AS STT SSA AAS OTOo STI STT SAS SSA ATO st SsSA tto tes 240 Ietsp Tig Bek Mai Rgo tsuv Tgr Pecsem Avr Rgo Meb Siu Rko vy 5eg 65 7o 15 vo ATS Sst TtTo bos TAS TTT SSA ASS ATS ATO SST TSO TtTb ATtT o sst Tts 288 I1e Akd Re siu Tug RBe Rgo TNg I1e Mee Rkgo TER Pecs I16 Agu Re 85 90 95 TTo STT OST SSA ASA SSA AAS AAS STO AAS SAC SAA OST AAb o ssSA SsTS 336 tsech Pecs Aia Siu Ak Rgo Avp o Puv Mai TsPuv 010 Sbip A1ia Tsuv Aza Gets 100 105 110 sot AAS Hund ATS AAb o TSS o ASstT o hundred SST TTo ATS AAb tes TTo ost svo 384 Agu two this I1ie Tsuv bek TBHg Ua1i Rgo Pyemsh I1e Puv Behg Pep A1ia si 115 120 125 SAS OST SAT OST AOS SAS STT ATS SOT SATS SATS SATS SATS OST es hundred 4z2 sich dia Azr Aia bBeg Niya Pech I1ee Agud Niv Sish biu Niv ces Tng Uai 130 135 140 TAS SOT SSA SAA SSA SAS o TTS SS ASo SAS SST SSOA OST tob SAA ST 480 Tut Agd biu 51 A1ia Adzr Rpe A1ia Agu Avr A gu s1u biu Ter beach Bay 145 150 155 160 sSsT SOT ST AAS ot oTI SOT AST SAA ATS STO AS OST bAT OSA TIS 528 vk Ag pech Avp o biu Uai Agu Tk Sip I1e6e Hey Bek A1ia Avr A1iA Gey 165 170 175 SST SAT TTS SAT SST AAS OTTO TST SAS SSS TTT ASS AAb boby ATS SsTtT 576 AkCh Avzr Re Avr Rko Avp Petc bei Niv Aia Rpe Tik tsu biu 11e yeech 180 185 190 ATS OAA SAS AAS OST SAS ASS ATS AAS SSA SAA ost stos sto St sIS 624 I16e biz bib dvo biu Niv Tk I1ie Avp Rko sip siu Gen Uai Tpg Gech 195 200 205 tTtTo TtTI SStT SStT TT ATS OST AAS Sst oBbA SAS TTo sto Ttst ost sst 672 Iz Rie Agu Ag Re I1e Aia Avp Siu biu Sish Rpe Uai bah А1а Аг9д 210 215 220 СТТ АТС ОСА ТС САС АСТ САА сот СОТ ST ST AAb OST АТС АСС ss 720 Uai Ii siu rRpe bi Trg bich siu Agu A1ia Pesh Puya biu T16 TVg Tvg 225 230 235 240 ASS AAS o bstT stTI STt OST stTt SAT SA OSI TT STI OSA SST OST OSA 768 Tg dvp biu Ua! Petz Aia Mai Avr Aiia Aia UMai UMai Aia A1ia siu A1a 245 250 o avv SAS OTOSS AAS TST STI OST AAS o TOS STT OST SAT SAS ATS SSA TTb SAT 816 Niv Beg PCuv beg Pesh Aza Avp bek tseu ADvr Avr T1e Rgo tech Avr 260 265 270 ASS OSAA SST bSA TAS SAS ATS STO ATS SSS AAS SSA SAA OST SST SsSA vva TpkK biz Ag biu Tug Niv i1e Ua1i Iie A1ia Avp Rgo Si A1ia Aza Rgo 275 280 285 SST ATT SSA AST ASS SAT sST TST o soA AAS TT ATS ost AST SsstT AT 912 Akch I1e Rko TI Tig Avr A1ia back biu tsPuv RBe I16e dia Tik Rko Maye 290 295 300 SAS ATO OST sSttT SoT STI bST OSA ASS STT SAS TTS ost OT Sto ASstT 60 bits Meb biu tsets Agu Mai dia biu Trg oUai biv Re Aza b1u Ge TE zo5 310 315 320 SST OST SStT AAS tob dDAS sSstT ost SAS o stTtT STS TAS AsStT sosSs sstT sot 1008 A1a dia Rgo Avp Ter Duv Ag A1a Niz Mai Ten Tug Tii Agu Aia Aga 325 zzo 335 Av OTIS StTtT sSsSA OST ST OST OST OSS AST TST SAA SAA SOT TAS ToS 1056 Buv Pez Pez Rgo Aza Pez dia Rko Aza beg Bek Sbits bitsh Aku Tuk bZeg 340 345 350 AAS TOS ATO OST TTS SStT SSA ASS ATS SSO AT TOS STT SSA STO ATT 1104 tsu Tkgr Meb biu Re Aku Rro be g I1ie Rkgo Avr 5etg Betsh Rko Uai I1e 355 zvo z65 bot Sst SsST AS SOT AST SSA SAS OStTT ATS TAS OST TtS bot SAS OST 1152 siu Akd Aza tTBg Asu TBg o Rgo Avr o Ua! Ii Tug Aia RBe SsSiu Niv siu 370 a75 zvo SAS STS SST ATS AST ssStT OST SSA ATS ASSOSSA ASS sto stt o tTst SAS 1200 Niv tep biu Mei Tik Siu A1ia Rgo Mei tng Aiia trE Goz wai bet pizza zv5 - zz0 395 : 400 STO Stos OSA bot SAS AAb ASS TST ATS ATS ATS ATS TST SSA TT OSA SsSA 1248 yech tsech dia biu bi Tsuz TlyuE Beh I1e Avr I1e Bek Rgo Rie A1a Rgo 405 410 415 AS Sst tTTS OST ATT ST AAS TSS AAS SAA AST bST SST OSA TOS TAA 1296 Azp Aku Re sSiu I16ee biu Muv bet Puz bip Tpg biu Rgo A1a Bak 420 425 azo I 3. A molecule of isolated double-stranded DNA, including in its sequence a) a promoter whose action in plants produces RNA sequences, b) a structural DNA sequence represented by a molecule according to item I, which is capable of producing RNA sequences encoding the enzyme glyphosate oxidoreductase, c) 3 the non-translated region, the effect of which in plants is the addition of polyadenylated nucleotides to the 3" end of the RNA sequence, and the promoter is heterologous to the structural DNA sequence and is capable of driving sufficient expression of the specified enzyme in plant tissue, including meristematic tissue, with increased plant cell tolerance , transformed by gene instructions, to glyphosate. 4. The isolated double-stranded DNA molecule according to claim 3, characterized by the fact that the indicated structural DNA sequence encodes a fusion polypeptide, which includes the amino-terminal chloroplast transit peptide and the enzyme glyphosate oxidoreductase. 5. The isolated double-stranded DNA molecule according to claim 4, characterized by the fact that the promoter is a viral promoter of plant DNA. 6. The isolated double-stranded DNA molecule according to claim 5, characterized by the fact that the promoter is selected from the group including Sama 355 and EMU 355 promoters. 7. The method of obtaining genetically transformed plants tolerant to the herbicide glyphosate, characterized by the fact that the method includes the following stages: a) insertion of a recombinant double-stranded DNA molecule into the genome of a plant cell, consisting of: 1) a promoter, which acts in plants to produce RNA sequence, i) the structural DNA sequence represented by the molecule according to claim 1, which is responsible for the production of the RNA sequence encoding the enzyme glyphosate oxidoreductase, 11) 3 untranslated region, which acts in plants to attach polyadenylated nucleotides to the 3 end of the RNA sequence, and the promoter is heterologous with respect to structural DNA sequence and is capable of carrying sufficient expression of the indicated enzyme in plant tissue, including meristematic tissue, with increased tolerance of the plant cell transformed with the indicated gene to glyphosate, b) obtaining a transformed plant cell, and c) regeneration from a transformed plant cell of a genetically transformed plant with increased tolerance to the herbicide glyphosate. 8. The method according to claim 7, characterized by the fact that the specified structural DNA sequence encodes a fusion polypeptide, including the amino-terminal chloroplast transit peptide and the enzyme glyphosate oxidoreductase. 9. The method according to claim 8, characterized by the fact that the promoter is a viral promoter of plant DNA. 10. The method according to claim 9, characterized by the fact that the promoter is selected from the group including Sama 355 and EMU 355 promoters. 11. An isolated peptide including an amino acid sequence encoded by a DNA sequence selected from the group consisting of 5EO IU MO:3, 5EO IU MO:4, 5BO IU MO:6, 5EO I MO:7, 5BO I MO: and 5EO I MO:17, description of the sequence of some of the claims in claim 2. 12. The isolated peptide according to item 1, characterized by the fact that the peptide contains an amino acid sequence that is encoded by the DNA sequence 5EO Y MO:18, the described sequence of which is given in claim 1. 13. The isolated peptide according to item 1, characterized by the fact that the peptide contains an amino acid sequence encoded by the DNA sequence 5EO Y MO:5, the described sequence of which is given in claim 1. 14. Bacterial strain including a double-stranded DNA molecule encoding the enzyme glyphosate oxidoreductase, and the amino acid sequence of the specified enzyme is encoded by a nucleotide sequence selected from the group including 5EO IU MO:3, 5EO IU MO:4, 56EO IU MO:6, 5EO IU MO :7, 5BEO IJU MO:8 and 5EO JU MO:17, description of the sequence of their occurrences! in claim 2. 15. Plant cell culture transformed with a molecule of isolated double-stranded DNA according to claim 1, encoding an amino acid sequence according to claim 13. 16. The isolated double-stranded DNA molecule according to claim 2, characterized by the fact that it includes the DNA sequence 5EO IU MO 4. 17. The isolated double-stranded DNA molecule according to claim 2, characterized by the fact that it includes the DNA sequence 5EO IU MO 17.