UA118470C2 - Hppd variants and methods of use - Google Patents

Abstract

In the present invention, HPPD enzymes and plants containing them showing a full tolerance against several classes of HPPD-inhibitors arc described. A set of HPPD enzymes have been designed which have either no or only a significantly reduced affinity to HPPD inhibitors and, at the same time, the rate of dissociation of the HPPD inhibitors of the enzyme is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become folly reversible inhibitors, ha particular, isolated polynucleotides encoding HPPD inhibitor tolerance polypeptides are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.

Description

The field of technology

The invention relates to the field of plant molecular biology, in particular, to new GFPD polypeptides that provide increased resistance to GFPD inhibitor herbicides.

State of the art 4-hydroxyphenylpyruvate dioxygenase (HFIPD) is an enzyme that catalyzes the reaction of converting para-hydroxyphenylpyruvate (henceforth abbreviated as HFP), a product of tyrosine degradation, into homogenizate (henceforth abbreviated as GH), a precursor of tocopherol and plastoquinone in plants (Stoisi M .R. et al. (1997), Neuroscience, 53, 20, 6993-7010, Ege et al. (2004), Research Journal 134:1388-1400). Tocopherol plays the role of a membrane-associated antioxidant.

Plastoquinone, firstly, acts as an electron carrier between RBZII and the cytochrome Bb/ complex, and secondly, it is a redox cofactor of phytoene desaturase, which participates in the biosynthesis of carotenoids.

To date, more than 1,000 nucleic acid sequences in the MSVI database have been annotated as encoding a putative protein with a HFPD domain. However, most of these proteins have not been shown to have GFAP enzymatic activity in an in vitro or in vitro assay, nor that similar GFAP proteins can confer resistance to GFAP inhibitor herbicides when expressed in a plant. Some HFPD proteins and their primary sequences have been described previously in the art, in particular HFPD proteins of bacteria such as Rheendotopah (Kieivspi et al., Eig. U. Viospet., 205, 459-466, 1992, MO096/38567), Kogaia (МО2011/076889) with Zupesposossiv5 (М/О2011/076877) and Kpodosossiv (ММО2011/076892), protists such as Vierpagista (МО2011/076882), euryarchaeotes such as Rishorpyi5 (УМО02011/076885), plants such as

Agaridorziz (M/O96/38567, SEMVAMKF ARO47834), carrots (MO 96/38567, SEMVAMKF 87257),

Auepa zaina (UO2002/046387, UMO2011/068567), wheat (UMO2002/046387), Vgaspiagia riaturpuiia /(M/02002/046387), Sepsigy5 espipaiv (M/O2002/046387), I oijsht hydidit (M O2002/046387), EReviisa agipaipasea (M/O2002/046387), bZeiapa iabei (MO 2002/046387),

Eieivzipe ipaiisa (M/02002/046387), sorghum (M/O02002/046387, UO2012/021785), corn (M/O2012/021785), Sossisoides (ZEMVAMKF SOITKR), Soriiv |aropisa (MO2006/132279),

Spathudotopazh heippagaiia (E5 2275365; IMO2011/145015), or mammals such as mouse or pig.

Inhibition of GFIPD leads to the uncoupling of photosynthesis, the insufficiency of auxiliary light-harvesting pigments and, most importantly, the destruction of chlorophyll by UV radiation and

With active forms of oxygen (discoloration) due to the insufficiency of photoprotection, normally provided by carotenoids (Moggi5 et al. (1995), Riapi Seiyi 7: 2139-2149). Discoloration of photosynthetically active tissues leads to growth inhibition and plant death.

Some HFPD inhibitor molecules, which inhibit the conversion of HFP into homogenate and which bind specifically to the enzyme, have proven to be effective as herbicides.

Currently, most commercially available HFPD inhibitors belong to one of the following chemical families: 1) triketones, e.g., sulcotrione (ie, 2-(2-chloro-4-(methylsulfonyl)benzoyl|-1,3- cyclohexanedione, mesotrione ( i.e. 2-(4-(methylsulfonyl)-2-nitrobenzoyl|-1,3-cyclohexanedione|; tembotrione Igobto 2-(2-chloro-4-(methylsulfonyl)-3-((2,2,2,-trifluoroethoxy) methyl|benzoyl|-1,3-cyclohexanedione; tefuryltrione; 2-(2-chloro-4--"methylsulfonyl)-3-|(tetrahydro-2-furanyl)methoxy|methylbenzoyl|-1,3-cyclohexanedione|; bicyclopyrone Igobto 4-hydroxy-3-(2-(2- methoxyethoxy)methyl|-6-(trifluoromethyl)-3-pyridinylcarbonyl|bicyclo|3.2.Poct-3-en-2-one; benzobicyclone (gobto 3-(2- chloro-4-mesylbenzoyl)-2-phenylthiobicyclo|3.2.zoct-2-en-4-ones; 2) diketonitrile, e.g. 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane -1,3-dione and 2-cyano-1-(4--("methylsulfonyl)-2-trifluoromethylphenyl|-3-(1-methylcyclopropyl)propane-1,3-dione; 3) isoxazoles, eg isoxaflutol | hobto (5-cyclopropyl-4-isoxa zolyl)(2-(methylsulfonyl)-4-(trifluoromethyl)phenyl|Imethanones. In plants, isoxaflutol is quickly transformed into DKN, a diketonitrile compound that exhibits the properties of a HFPD inhibitor; 4) pyrazolinates, e.g., topramesone i.e. I3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl)(5-hydroxy-1-methyl-1H-pyrazole-4- yl)methanone and pyrasulfotol |hobto (5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluoromethylphenyl)methanone; pyrazofen |hobto 2-

14-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxyacetophenone; 5) M (1,2,5-oxadiazol-3-yl)benzamides (М/О2011/035874) and M-(1,3,4-oxadiazol-2-yl)benzamides (МО2012/126932), e.g. 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (hereinafter also referred to as "Col. 17"); b) M-(tetrazol-5-yl)- or M-(triazol-3-ylarylcarboxamides (M/O2012/028579), e.g., 2-chloro-

3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (hereinafter also called "Spol. 273; 4-(difluoromethyl)-2-methoxy-3 -c"methylsulfonyl)-M-(1-methyl-1H-bo tetrazol-5-yl)benzamide (hereinafter also called "Col. 3"); 2-chloro-3-(methylsulfanyl)-M-( 1-

methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)benzamide (hereinafter also called "Spol. 4"); 2-(Methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)benzamide (hereinafter also called "Spol. 5"); 7) Pyridazinone derivatives, as described in UMO2013/050421 and M/O2013/083774; 8) Substituted 1,2,5-oxadiazoles, as described in UMO2013/072300 and MMO2013/072402; and 9) Oxoprazine derivatives as described in UMO2013/054495.

These GFAP inhibitors can be used against grass and/or broadleaf weeds in fields with metabolically resistant crops such as maize (7ea tauh), rice (Ogu/a Zaiiima) and wheat (Tgyisit aevziimit) in which they quickly decompose (Zespii? et al. (1993), REEVZ IeNegv5, 318, 162-166; Miispei! et al. (2001), Rezi Mapadetepi Zsiepse, Moi 57, 120-128; Sagsia et al. (2000), Viospet ., 39, 7501-7507; Raiei et al. (2001), Rezi Mapadetepi

Zsiepse, Moi 57, 133-142). In order to expand the range of application of these inhibitor herbicides

GFPD, some efforts have been made to provide plants, especially plants with no or insufficient metabolic resistance, with a level of resistance sufficient in field agronomic conditions.

In addition to an attempt to bypass the production of homogentisate mediated by HFPD (05 6,812,010), overexpression of a sensitive enzyme was also performed in order to make a sufficient amount of the target enzyme in the plant in relation to the herbicide (M/096/38567). Overexpression of GFPD resulted in predominant pre-emergence resistance to the diketonitrile derivative (DKN) isoxaflutol (IFT), but the level of resistance was insufficient to induce post-emergence resistance (Maigipde et al. (2005), Rebzi Mapadetepi bsiepse 61: 269-276).

A third strategy was to mutate HFPD in such a way as to produce a target enzyme that, while retaining the ability to catalyze the conversion of HFPD to homogenizate, is less sensitive to HFPD inhibitors than the native HFPD before the mutation.

This strategy was successfully applied in the production of plants resistant to 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and to 2-cyano-1-|4-(methylsulfonyl )-2-trifluoromethylphenyl|-3-(1-methylcyclopropyl)upropane-1,3-dione (ЕР496630), two GFPD inhibitor herbicides belonging to the diketonitrile family (M/099/24585).

Rho215I ey, SiuZzZbOoim, SiuZ36b Pe, and more specifically SiuZ36bTgr (position of mutating amino acids

Zo labeled relative to GFIPD Reepidotopach Pyogezsep5) were identified as mutations responsible for increased resistance to treatment with these diketonitrile herbicides.

Not so long ago, the introduction of the GFPD gene Rzeidotopazh Pyogezsep5 into the plastid genome of tobacco and soybean showed greater efficiency compared to nuclear transformation, providing resistance to post-emergence treatment with isoxaflutol (Oshoigtapgei et al. (2007), Riapi Vioyesppoi U).5(1):118- 33).

In MO2004/024928, the inventors set themselves the goal of enhancing the biosynthesis of prenylquinones (e.g., the synthesis of plastoquinones, tocopherols) in plant cells by increasing the influx of the GFP precursor into plant cells. This was achieved by joining the synthesis of the mentioned precursor to the "shikimate" pathway through the overexpression of the enzyme prephenate dehydrogenase (PDH). They also noted that the transformation of plants with the gene encoding the PDI enzyme and the gene encoding the HFPD enzyme makes it possible to increase the resistance of the mentioned plants to HFPD inhibitors.

In M/O2009/144079, the sequences of nucleic acids encoding hydroxyphenylpyruvate dioxygenase (HFPD) with specific mutations at position 336 of the HFPD protein were disclosed

Rzeiidotopach Pyogezsep5 and their use for obtaining plants resistant to GFPD-inhibiting herbicides.

In MMO2002/046387, several domains of GFPD proteins of plant origin were identified that may be relevant in conferring resistance to various herbicide inhibitors

GFPD, but neither ip riapy nor biochemical data are shown to support the influence of the described domains' functions.

In M/O2008/150473, a combination of two different resistance mechanisms was shown - the modified Amep gene takes a mutant enzyme encoding GFPD and the corn monooxygenase SUR450 (pvia gene) - in order to obtain improved resistance to GFPD inhibitor herbicides, but the data were not disclosed. demonstrating the synergistic effects of the combination of both proteins.

Further, in О052011/0173718, a method of creating plants resistant to HFPD inhibitors is described by overexpression of not only a gene encoding a resistant HFPD, for example, from Amep zaiima, but also a combination with several genes of plants encoding the Goth protein (homogentizazolanesyltransferase). However, the level of resistance to some selected herbicides - GFPD inhibitors was extremely limited. 60 In UMO2011/094199 and O52011/0185444, the resistance of several hundreds of wild-type soybean lines to the HFPD inhibitor isoxaflutol was evaluated. Very few lines showed an acceptable level of resistance to herbicides. A putative LCP (quantitative trait locus) responsible for resistance was identified. In this region of the genome, the gene encoding the ABC transporter was recognized as the main feature responsible for the observed improved resistance to HFPD inhibitors. However, transgenic plants expressing the identified genes did not show any improvement in resistance to the tested GFPD inhibitor herbicides.

In UMO2010/085705, several mutant GFPDs of Amepa zaziima were described. Some variants were shown to show improved resistance to the triketone mesotrione, but very few mutants were expressed in tobacco plants. In addition, none of the tobacco plants expressing these mutants showed increased resistance to mesotrione or isoxaflutol compared to tobacco plants expressing the wild-type GFPD Amep gene.

OB 2012/0042413 describes polypeptides exhibiting HFPD activity but also showing some insensitivity to at least one HFPD inhibitor, and also suggests a specific set of mutations at various positions of HFPD enzymes, and ultimately discloses biochemical data and resistance levels of plants containing some such mutated GFPD In EP 2453012, several mutant GFIPDs are described; however, increased resistance of the described ip riapia mutants to several GFPD inhibitor herbicides was not shown.

Described at this time and partially commercialized GFPD inhibitors act as slow binding or slow, strong binding inhibitors (see Mogtizop (1982) Tgtepaz Viospet. si. 7, 102-105).

These inhibitors bind slowly (i.e., with a low association rate, Kop) but not covalently to the GFIPD enzyme (i.e., they give a time-dependent binding) and are released very slowly (i.e., with an extremely low dissociation rate, Koi) due to extremely strong interaction with the enzyme.

These inhibitors bind so strongly that stoichiometric titrations with the enzyme become possible.

It is becoming increasingly clear that slow binding or slow, strong binding inhibitors are not only exceptionally strong inhibitors of GFAP, but also have features that make them attractive agrochemicals for weed control. The low rate of dissociation enhances the effectiveness of the inhibitor to such an extent that, in the ideal case, only one molecule is sufficient

From the inhibitor to the active site of the enzyme to completely inhibit its activity and maintain the same degree of inhibition for a long period of time even in the absence of free inhibitor molecules in the plant cell. This results in a small dose of inhibitors required to control unwanted weeds in crop growing areas.

Properties of slow binding or slow, strong binding inhibitors have an advantage in the case when the goal is to achieve inhibition of HFPD and herbicidal activity.

However, these same properties are a major drawback when it is necessary to design GFPD enzymes resistant to these inhibitors. Mutations in the GFAP enzyme that only reduce the affinity of the inhibitor for the enzyme (ribo) cannot completely overcome GFAP inhibition, because inhibitor binding and inhibition of the GFAP enzyme can still occur and, accordingly, the achieved level of inhibition will be maintained for a long time even under time of absence of free inhibitor in the plant cell.

Due to the above-described kinetic properties of all currently described and partially commercialized GFPD inhibitor herbicides, to date no plants have been created that are fully resistant to GFPD inhibitor herbicides, despite numerous efforts to create them.

Brief description of the invention

The present invention describes HFPD enzymes and plants that contain them, which demonstrate complete resistance to several classes of HFPD inhibitors. It turned out that for the creation of such enzymes

GFAP with maximum or complete resistance to GFAP inhibitors, it is more important to increase the dissociation rate (Koi) of slow-binding or slow, strong-binding inhibitors than to decrease their affinity for the enzyme (ribo) if resistance to the inhibitor is to be achieved. Ideally, it is necessary to simultaneously achieve a decrease in the affinity of the inhibitor to the GFPD enzyme (ri5BO) and increase the rate of dissociation of the inhibitor from the GFPD enzyme (CoOy) in the mutant enzyme in order to obtain a high level of resistance to the inhibitor.

In the proposed invention, this goal was achieved by developing a set of enzymes

HFPDs that have no or significantly reduced affinity for HFPD inhibitors, and at the same time the rate of dissociation of HFPD inhibitors from the enzyme is increased to such an extent that HFPD inhibitors no longer act as slow-binding or slow, strong-binding inhibitors, but as fully reversible inhibitors.

The proposed invention provides compositions and methods for obtaining HFPD enzymes, because having the above-mentioned properties (ie, having no or significantly reduced affinity for HFPD inhibitors, an increased rate of dissociation of HFPD inhibitors from the enzyme; HFPD inhibitors no longer act as slow-binding or slowly, tightly binding inhibitors, but as fully reversible inhibitors). The compositions include HFPD and isolated, recombinant or chimeric nucleic acid molecules encoding such polypeptides, as well as vectors and host cells containing these nucleic acid molecules. The compositions also include antibodies to these polypeptides. Nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide sequences can be synthetic sequences designed for expression in an organism, including, but not limited to, a microorganism or a plant.

The compositions include nucleic acid molecules encoding herbicide-resistant polypeptides, including nucleic acid molecules encoding the Rzendotopav HFPD protein

Pyogeseps5 containing a proline at a position corresponding to position 335 5EO IU MO-1 and a phenylalanine or tyrosine at a position corresponding to position 336 5EO IU MO and optionally one or more amino acid substitutions at positions corresponding to position 188, 189, 200, 215, 226, 339 and 340 5EO IU MO:1, including the GFPD protein set forth in any of the ZEO IU MO:7-33, as well as fragments thereof.

The compositions also include transformed plants, plant cells, tissues and seeds resistant to GFPD inhibitor herbicides by introducing a nucleic acid sequence of the invention into the genome of the plants, plant cells, tissues and seeds. The introduction of the sequence allows applying GFIPD inhibitor herbicides to plants for the selective destruction of weeds sensitive to the GFIPD inhibitor or other non-transformed plants, but not the transformed organism. The sequences can also be used as a selection marker for plant cells grown in the presence of one or more herbicide inhibitors

GFPD

In addition, the methods of identifying the HFPD enzyme with the activity of resistance to HFPD inhibitors are described.

Compositions and methods according to the invention are useful in creating organisms with increased resistance to GFPD inhibitor herbicides. These organisms and compositions containing the organisms are desirable for agricultural purposes. Plants or seeds containing a nucleic acid sequence encoding a GFPD according to the invention can be grown in the field and harvested to obtain a plant product. The compositions of the invention are also useful in detecting the presence of GFIPD inhibitor-resistant proteins or nucleic acids in products or organisms.

Brief description of the drawings

Figure 1 shows an alignment of the amino acid sequences of the GFPD of microbial and plant species, including Rzeidotopach Pyogeseps (RI, 5EO IU MO:1), Amepa zaina (5EBO IU MO:38), the GFPD variant of Amepa zayima (ZEO IU MO:39), 7ea tauz5 (5EO IYUO MO:40), bBigeriotuse5 amegtiyiiv (ZEO IYU MO:44), Agaridorziv Inaiyapa (5EO IYU MO:41), Nogaeit muciIdage (ZEO IYU MO:42), Baisiv sagoia (ZEO IYU MO:43), MusozrpaeegeiIa dgatipisoia (ZEO IU MO:45) and Saussisoide5 ittiiy5 (ZEO IU

MO:46).

Figure 2A shows an example of the time-dependent inhibition of the time-dependent mutant GFPD enzyme in the presence of 1 μM GFPD inhibitor. Figure 28 shows an example of reversible inhibition of the reversible mutant enzyme HFPD in the presence of 10 µM inhibitor.

Detailed description of the invention

The present invention will be described more fully below with reference to the accompanying drawings in which some, but not all, embodiments of the invention are shown. In fact, the present invention may be embodied in many different forms and should not be construed as limited to the following embodiments; rather, these options are provided to satisfy applicable legal requirements for this disclosure. The same number designations denote the same elements everywhere.

Many modifications and other embodiments of the invention may occur to one skilled in the art to which this invention relates, having the benefit of the information provided herein in the foregoing descriptions and associated drawings. Therefore, it should be understood that the inventions are not limited to the specific embodiments described and that modifications and other embodiments are intended to be encompassed within the scope of the appended claims. Although specific terms are used herein, they are used in a generalized and descriptive sense and not for the purpose of limitation.

Review

Several attempts have been made to provide plants with an agronomically acceptable level of 60 resistance to a wide range of GFOD inhibitor herbicides, including bypassing GFPD-mediated production of homogentisate (05 6,812,010), overexpression of a sensitive enzyme in order to produce sufficient amounts of the target enzyme for the herbicide in the plant (M/O96/ 38567), and mutation of GFPD in such a way as to obtain a target enzyme that, while retaining the ability to catalyze the conversion of GFP to homogenizate, is less sensitive to GFPD inhibitors than native GFPD before the mutation.

Despite these advances in the development of plants showing resistance to several GFAP inhibitor herbicides, there is still a need to develop and/or improve plant resistance to newer or several different GFAP inhibitors, particularly GFAP inhibitors belonging to the triketone class (e.g., sulcotrione , mesotrione, tembotrione, benzobicyclone and bicyclopyrone), pyrazolinates (e.g., topramesone and pyrasulfitol), M-(1,2,5-oxadiazol-Z3-yl)benzamides (M/O2011/035874), M-(tetrazole-4 -yl)- or M-(triazol-3-ylluarylcarboxamides (M/O2012/028579), pyridazinone derivatives (M/02013/050421 and UUO2013/083774); substituted 1,2,5-oxadiazoles (M/O2013/072300 and УМО2013/072402); and oxoprazine derivatives (ММО2013/054495).

Thus, the present invention provides improved compositions and methods for regulating resistance to GFPD inhibitor herbicides. Herbicides-inhibitors of GFIPD, such as the class of M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-yllauarylcarboxamides, such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5- yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide; M-(1,3,4-oxadiazole-2 -yl/benzamides, such as 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-methylsulfonyl)-4-(trifluoromethyl)benzamide (Col. 1); M -(tetrazol-b5-yl- or M-(triazol-3-ylarylcarboxamides, such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl) benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro- 3--(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3-(methylsulfinyl)-M- (1-methyl-1 H-tetrazol-5-yl)-4--trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (МИО2013/050421 and ММО2013/083774); substituted 1,2,5-oxadiazoles (МИО2013/ 072300 and UMO2013/072402); and oxoprazine derivatives (M/O02013/054495); triketones, such as tembotrione , sulcotrione and mesotrione; the isoxazole class, such as isoxaflutol; or the pyrazolinate class, such as pyrasulfotol and topramesone, have outstanding herbicidal activity against a wide range of economically important monocotyledonous and dicotyledonous annual weeds.

Z Active substances also effectively act on perennial harmful plants that grow shoots from rhizomes, woody mass or other perennial organs and are difficult to deal with.

Within the framework of this invention, "herbicide" should be understood as a herbicidally active substance by itself or such a substance that is combined with an additive that changes its effectiveness, such as, for example, an agent that increases activity (synergistic agent) or an activity-limiting agent (safe ). The herbicide may also contain solid or liquid adjuvants or carriers commonly used in formulation (e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, emulsifiers, growth promoters, etc. ), as well as one or more pesticides (eg, insecticides, viricides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides, etc.)

Methods include transforming organisms with nucleotide sequences encoding a GFAP inhibitor resistance gene according to the invention or otherwise introducing genes that introduce resistance to a GFAP inhibitor into organisms that do not contain them (eg, by mating, cell fusion, or crossing organisms, containing the introduced gene of the HFPD inhibitor according to the invention with organisms that did not contain it, and obtaining offspring containing such a gene).

Nucleotide sequences according to the invention are useful for creating plants that show increased resistance to HFPD inhibitor herbicides, especially increased resistance to HFPD inhibitor herbicides of the M-(1,2,5-oxadiazol-3-yl)benzamide class; M-(tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-methylsulfonyl)-M-(1-methyl-1H-tetrazol-5 -yl/benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole-

B-yl) benzamide; M-(1,3,4-oxadiazol-2-yl)benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-methylsulfonyl)-4 -(trifluoromethyl)benzamide (Spol. 1); M-(tetrazol-5-yl)- or

M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 2), 4--difluoromethyl)-2-methoxy-3-(methylsulfonyl)-

M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro-3--"methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4 --trifluoromethyl)benzamide (Sol. 4), 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethylubinzamide (Sol. 5); pyridazinone derivatives (M/O2013/050421 and UO2013/083774); substituted 1,2,5-oxadiazoles (M/O02013/072300 and

UMO2013/072402); and oxoprazine derivatives (ММО2013/054495); triketones, such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, such as isoxaflutol; or a class of pyrazolinates such as pyrasulphotol and topramesone. The GFPD inhibitor resistance gene according to the invention can also show resistance to "coumarone-derivative herbicides" (described in UMO2009/090401,

UMOo2009/090402, UMO2008/071918, UMO2008/009908). In this regard, any of the GFAP inhibitor resistance genes according to the invention can also be expressed in a plant that also expresses a chimeric homogenizatolanesyltransferase (HST) gene or a mutant HST gene as described in

УМО2011/145015, УМО2013/064987, УМО2013/064964 or ММО2010/029311, for obtaining plants resistant to GOST inhibitor herbicides. Here and in the following, the terms "herbicide-coumarone derivative" or "herbicide-inhibitor of GOST" cover compounds that fall under the ISRAS nomenclature 5Hn-thiopyrano(4,3-bB|Ipyridin-8-ol, / 5H-thiopyranoY3,4-b| Ipyrazin-8-ol, oxathiino|5,6-b|Ipyridin-4-ol.KCh/ and oxathiino|5, 6-b|pyrazin-4-ol.

Thus, a gene for "resistance to a GFAP inhibitor" according to the invention should be understood as a gene encoding a protein that gives a cell or organism the ability to tolerate higher concentrations of a GFAP inhibitor than the same cell or organism that does not express the protein, or tolerate some concentration of a GFAP inhibitor for a longer period of time than the same cell or organism that does not express the protein, or that provides the cell or organism with the ability to perform photosynthesis, grow and/or reproduce with less damage or inhibition of growth than observed in the same cell or organism that do not express a similar protein. A "GFAP inhibitor resistance protein" includes a protein that confers on a cell or organism the ability to tolerate higher concentrations of a GFAP inhibitor than the same cell or organism that does not express the protein, or to tolerate some concentration of a GFAP inhibitor for a longer period of time than the same cell or organism that does not express the protein a cell or organism that does not express the protein, or that confers on the cell or organism the ability to perform photosynthesis, grow and/or reproduce with less damage or inhibition of growth than is observed in the same cell or organism that does not express a similar protein. By "resistant" or "persistence" is meant either the ability to survive the application of a particular GFAP inhibitor herbicide, or the ability to perform essential cellular functions such as photosynthesis, protein synthesis, respiration, or reproduction in a manner that is not markedly different from that present in untreated cells, or organisms, or the ability of plants treated with a GFPD inhibitor herbicide to have no significant difference in yield or even to have an improved yield compared to the same

With plants not treated with the same herbicide (but for which weeds were removed or prevented by a mechanism other than the application of a GFAP inhibitor herbicide, such as the methods described in M/O2011/100302, which is incorporated herein in its entirety in its entirety by reference).

In addition to rendering the cell resistant to HFPD inhibitors, the HFPD nucleic acid sequences according to the invention also encode polypeptides having HFPDdD activity, i.e., which catalyze the reaction in which para-hydroxyphenylpyruvate (HFP) is converted into a homogenizer.

The catalytic activity of the GFPD enzyme can be determined by various methods known in the field. M/O2009/144079 describes various suitable screening methods.

Enzymatic activity of GFIPD proteins can be measured by any method that allows determining either a decrease in the amount of GFP or O»2 substrates, or the accumulation of any products of the enzymatic reaction, i.e. homogenized or CO». In particular, the activity of GFPD can be measured using the method described in M/O2009/144079; Sagsia and others. (1997),

Viospet. 4). 325, 761-769; Sagsia and others. (1999), Riiapi Rnusioi. 119, 1507-1516; or in UUMO2012/021785, which are included in this description in their entirety by reference.

For the purposes of this invention, a "reference" GFPD protein (or GFPD gene) is any GFPD protein or nucleic acid to which the GFPD protein or GFPD nucleic acid according to the invention is compared. For the purpose of describing the GFPD proteins according to the proposed invention, the terms "protein" and "polypeptide" are used interchangeably. The reference HFPD can be a natural plant, bacterial or animal HFPD, or a mutant HFPD known from the state of the art, such as mutants РИР215И and РИСЗ3З36Е, described in the Publication of the international application M/O2009/144079 and set forth here as respectively ZEO IU MO:20 and 2, or it can be any of the proteins RINRROEmo33,

RINRROEmo3b, RINRROEmo37, RINRROEmo40 or RINRROEmo41, set forth herein as respectively

ZBEO IU MO:6 and 34-37, which are also described in International Patent Application PCT/O52013/59598, filed September 2013 and incorporated herein by reference. Similar standard

GFAP can be used to determine whether a GFAP protein or nucleic acid of the invention has a particular property of interest (e.g., improved, comparable, or decreased resistance to a GFAP inhibitor herbicide or activity of the GFAP enzyme; improved, comparable, or decreased expression in the host cell; improved, comparable, or reduced stability of the protein, etc.) 60 In various embodiments of the invention, a protein encoded by nucleic acid (including its isolated, recombinant and chimeric genes, vectors, cell- hosts, plants, plant parts and seeds containing nucleic acids, HFPD polypeptides and nucleic acid-encoded compositions thereof, as well as methods of using the nucleic acid-encoded protein to increase resistance to HFPD inhibitor herbicides, especially to increase resistance to herbicides-inhibitors of the GFPD class

M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)y- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole) -5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide; M-(1,3,4- oxadiazol-2-yl)/benzamides, preferably such as 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (Spol 1); 1H-tetrazol-5-yl/benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3- (methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (M/O2013/050421 and

UMO2013/083774); substituted 1,2,5-oxadiazoles (М/О2013/072300 and УМО2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, such as isoxaflutol; or the class of pyrazolinates, such as pyrasulfotol and topramesone) contains proline at position corresponding to position 335 5EBO IJUO MO and phenylalanine or tyrosine at position corresponding to position 336 5EO IJU MO and, optionally, one or more amino acid substitutions at positions, corresponding to provisions 188, 189, 200, 215, 226, 339 and 340 of the 5EO IU MO:1, including the GFPD protein set forth in any of the ZEO IU MO:7-33.

By "corresponding" should be understood the position of a nucleotide or amino acid relative to such a position in 5EO IJOO MO:1 when two (or more) sequences are aligned using the standard alignment algorithms described in other sections herein. Characteristic alignment

ZEO IU MO:1 with the amino acid sequences of GFPD of different species of microbes and plants is shown in Figure 1. For example, the positions of amino acids 188, 215, 335, 336, 339 and 340 of 5EO IU MO 1 correspond to the positions of amino acids 241, 271, 412, 413, 416 ii 417, respectively, GFPD Amepa occupies (ZEO IO MO:38); by the position of amino acids 235, 265, 406, 407, 410 and 411, respectively, GPA

Nogdesht myYidage (ZEO IU MO:42) and the position of amino acids 242, 272, 413, 414, 417 and 418, respectively, GFPD ea tauhye (ZEBEO OO MO:40). Alignment of multiple amino acid sequences of GFPD of different species can also be found in Tables 2a and 265 of European Patent Publication Mo EP2453012, which is incorporated herein by reference. Accordingly, depending on the length of the considered amino acid sequence of HFOPD, which has more or fewer residues than the sequence of ZEO IU MO:1, the corresponding position may be located in a position different from positions 188, 189, 200, 215, 226, 335, 336, 339 and 340 in this considered GFPD protein.

In one embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing proline in the position corresponding to the position of amino acid 335 ZEO IU MO:1, and phenylalanine or tyrosine in the position corresponding to the position of amino acid 336 5EO IU MO:1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

GFPD according to the invention) consists of an amino acid sequence containing both proline in the position corresponding to the position of the amino acid 335 5EBEO IU MO, and phenylalanine or tyrosine in the position corresponding to the position of the amino acid 336 ZEO IU MO:1 and ii. alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 188 5EO IO MO :1; and II. alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 189 5EO IU MO:1 ; and them isoleucine, leucine, or methionine at the position corresponding to amino acid position 200

ZEO IU MO:1; and 60 m alanine, leucine, proline or asparagine in the position corresponding to the position of the amino acid

215 ZE IU MO:1; and mi. histidine or glutamine in the position corresponding to the position of amino acid 226 ZEO IU

MO:1; and mii. histidine, isoleucine, leucine, methionine, glutamine, arginine, alanine, lysine, serine, threonine, or valine in a position corresponding to the position of amino acid 339 5EO IU MO:1; and mine alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 340 5EO ІЮ MO :1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing both proline in the position corresponding to the position of the amino acid 335 5EO IUO MOL, and phenylalanine or tyrosine in the position corresponding to the position of the amino acid 336 ZEO IU MO :1 and i. alanine, glycine, histidine, serine or tryptophan in the position corresponding to the position of amino acid 188 5EO IU MO-:1; and her arginine, cysteine, glutamine, glutamic acid, aspartic acid, glycine, histidine, phenylalanine or serine in a position corresponding to the position of amino acid 189 5EO ІЮ MO:1; and them isoleucine, leucine, or methionine at the position corresponding to amino acid position 200

ZEO IU MO:1; and m. alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 ZE IU MO:1; and mi. histidine or glutamine in the position corresponding to the position of amino acid 226 ZEO IU

MO:1; and mi. serine, alanine, threonine, glutamine or lysine in the position corresponding to the position of amino acid 339 5EO IU MO:1; and mine alanine, arginine, aspartic acid, glutamic acid, glutamine, glycine or leucine in a position corresponding to the position of amino acid 340 ZEO 10 MO 1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing both proline in the position corresponding to the position of the amino acid 335 5EBO IU MO/1, and phenylalanine or tyrosine in the position corresponding to the position of the amino acid 336 EC 10 MO: and ii. alanine, or glycine in the position corresponding to the position of amino acid 188 5EO IO MO:1; and II. arginine, cysteine, glutamic acid, aspartic acid or glycine in the position corresponding to the position of amino acid 189 5EO IU MO:1; and them isoleucine, leucine, or methionine at the position corresponding to amino acid position 200

ZEO IU MO:1; and m. alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 ZE IU MO:1; and mi. histidine or glutamine in the position corresponding to the position of amino acid 226 5EO IU

MO:1; and mii. alanine or lysine in the position corresponding to the position of amino acid 339 ZEO IO MO:1; and mine alanine, glutamic acid, glutamine or glycine in a position corresponding to the position of amino acid 340 5EO I MO:1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. alanine in the position corresponding to the position of amino acid 339 5EO IU MO:1; and her glutamine in the position corresponding to the position of amino acid 340 5BEO IO MO:1.

In another embodiment, the implementation of HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing proline in a position corresponding to the position of amino acid 335 ZEO IO MO:1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5BEO IO MO:1; and glutamic acid in a position corresponding to the position of amino acid 340 5EO IU MO:1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO IU MO:1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and and leucine in the position corresponding to the position of amino acid 215 5EO I MO1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 ZEO IU MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and and leucine in a position that corresponds to the position of amino acid 215 5EO IO MO-:1; and her glutamic acid in a position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 ZEO IU MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. glycine in the position corresponding to the position of amino acid 188 5EO IYUO MO:1; and her cysteine in the position corresponding to the position of amino acid 189 5EO IYUO MO:1; and named after leucine in the position corresponding to the position of amino acid 215 5EO IU MO:1; and m. glutamic acid in the position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IYUO MO:1; and II. glycine in the position corresponding to the position of amino acid 188 5EO IYUO MO:1; and her cysteine in the position that corresponds to the position of amino acid 189 5EO IO MO:1; and named after glutamic acid in a position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 ZEO IU MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. asparagine in the position corresponding to the position of amino acid 215 5EO IU MO:1; and her glutamic acid in a position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding 60 HFPD according to invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 ZEO IU MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. alanine in the position corresponding to the position of amino acid 215 5EO IU MO:1; and her glutamic acid in a position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 ZEO IU MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and and leucine in a position that corresponds to the position of amino acid 215 5EO IO MO-:1; and her glycine in the position corresponding to the position of amino acid 340 5EO IU MO:1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. isoleucine in a position that corresponds to the position of amino acid 200 5EO IO MO-:1; and her leucine in the position corresponding to the position of amino acid 215 5BEO IO MO-1; and named after glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. isoleucine in a position that corresponds to the position of amino acid 200 5EO IO MO-:1; and her glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of amino acid 336 5EO I MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. methionine in the position corresponding to the position of amino acid 200 5EO IU MO:1; and her glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. methionine in the position corresponding to the position of amino acid 200 5EO IU MO:1; and her leucine in a position that corresponds to the position of amino acid 215 5EO IO MO-1; and named after glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

MO-1.

In another embodiment, the implementation of HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and and leucine in a position that corresponds to the position of amino acid 215 5EO IO MO-:1; and her histidine in the position corresponding to the position of amino acid 226 5EO IO MO-1; and named after glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO ИО МО-1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5BEO IO MO:1; and II. glycine in the position corresponding to the position of amino acid 188 5EO IYUO MO:1; and her cysteine in the position that corresponds to the position of amino acid 189 5EO IO MO:1; and named after asparagine in the position corresponding to the position of amino acid 215 5BEO ИО MO-:1; and m. glutamic acid in the position corresponding to the position of amino acid 340 5EO IU

MO-1.

In another embodiment, the HFPD according to the proposed invention (including the nucleotide sequence encoding it and its recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing the nucleotide sequence encoding

HFPD according to the invention) consists of an amino acid sequence containing and. proline in the position corresponding to the position of amino acid 335 5EO IU MO:1; and a. phenylalanine in the position corresponding to the position of the amino acid 336 5EO IO MO:1; or p. tyrosine in a position corresponding to the position of amino acid 336 5EO IU MO:1; and II. histidine in the position corresponding to the position of amino acid 226 ZEO IO MO:1; and her glutamic acid in the position corresponding to the position of amino acid 340 of ZEO IU

From MO-1.

The corresponding amino acid positions of reference GFPD proteins and GFPD proteins according to the invention containing one or more amino acid substitutions are summarized in Table 1.

Table 1

Substitution of amino acids of reference proteins of HFPD and proteins of HFPD according to the invention, relative to 5EO

IO MO/:1, also contains column 1 with the clone identification number and the corresponding ZEO IU MO according to the list of sequences and references in the description. In the case of an empty cell, this position contains the wild-type amino acid sequence (RINRRO).

RINRRO | (1 A |in | R (0 in | (ik A rROZZVE |! 7772 11117171 TT

DISSOLVE! 77 of 7117 G17111111ГМ

ROZM |! 4 11717171 mmm1 6959... | ..5 |! GG her R

REMZ3Z3 | 6 | / / GG RM 1

REMAZ | 7 2 Sh | Her 1 her P in her

Rhema. | (FV HER 11711711 RM 1 her

RIO! 779 CYU 1 1 1 R |v A OO vRIZO HER 77lo 17 Г171717717171111711Р (is | 1

RIZ Her 11711171 R is Her

RO | 7712 | 17 GG R (is | EE

Kizi Г777711377 ГГ Г1117171771717171111711Р DM FA 0 kIz2 7 Г7771714 17171171 Р1М

КкИ37 7 Г77711115 1717 Г171711111ГР M EU 1

Kgoz 7777716 11711711 R YIM | g go | 717 (a |sS | 1-4 Her R (is | p

Table 1

Kg55 | 777718 17117117 lA ІІ R (is | EE

Kg58 HER 77777197 17111711 GR (is | EE

Кг3я/ Ї777712го 11711111 ko Г777717211 11711171 Р is Her s kzo3 7 Г77771122 | 11711111 GR (is | 0 kzo4 7 Ї77777723 117111 1мМ 1 Her R (is | EE kzob6 7 І 77777724 17 І171171717711111- 13. R (is | EE kah | 725 (a |S | m Her R | | GE kz57 | 26 ( A | C | 171 P | | He K Ї7777111127 17111711 1r (there are | 1 kV 77771728 117117 1mm 17717 its p (there is | it | EE KV3 7 Ї777771729 117. Р | GE

In another embodiment, the sequence of GFPD proteins according to the invention is at least 53 95, at least 60 95, at least 65 95, at least 70 95, at least 75 95, at least 80 95, at least 85 95, at least 90 95, at least 91 95, at least 92 95, to a lesser extent 93 95, at least 94 95, at least 95 95, at least 96 95, at least 97 95, at least 98 95, or at least 99 95 identical to the amino acid sequence , presented here as BEO IU MO:1.

Typical sequences of GFPD that can be modified according to the proposed invention include such bacteria, for example, of the type Rheipdotopavkh 5r., or cyanobacteria, for example, of the genus Zupesposubiya5. The sequence may also be of plant origin, particularly from monocotyledonous or dicotyledonous plants. As useful examples, we can cite such plants as tobacco, Agaridorvzi5 Ipaiyapa (M/096/38567), Yubaisiv sagoia (M/096/38567), 7e6ea tauz (maize, UMO2012/021785), wheat (Tiyysit aevzimit, UMO2002/046387) , barley (ER2453012), Amepa bvaiima (MMO2002/046387LMO2011/068567), Vgaspiaga riadzurpuia (MO2002/046387),

Sepsngiv espipai5 (MO2002/046387), Iot hydidit (M/02002/046387), Reviisa appaipasea (MMO2002/046387), Beiagia ttabegi (M/O2002/046387), Eivizipe ipaisa (M/O2002/046387), or sorghum ( MIO2002/046387, UMO2012/021785). In a specific embodiment of the invention, the GFPD, which can be modified according to the proposed invention, has a bacterial or protist origin, in particular from Rzepdotop5 5r., more specifically from Rheidotop5 Pyogezsepv,

Reepdotopaz riyida, Rvepdotopaz aegidipozva, Rvbecydotopavz ieviovivgopi (Sotatopav

Ievioviegopi), NPodosossiv v5r. (UMMO2011/076892), Viernagvta |aropisit (MO2011/076882), from Zupesposossi5 5 years. (ММО2011/076877), Kogaia aidisida (М/02011/076889), from Euryarchaeota

Risgorpiyiv5 yoggidib5 (ММО2011/076885), or plant origin, including from Agabridorvy

Taizhapa, Zogopyit bBisoiog, Ogula 5aihima, Tgyisit aeviimit, Nogdesht msidage, Goiisht gidiysht or

Auepa is busy.

For purposes of the present invention, the GFPD of the invention may also include further modifications, for example, in which certain amino acids (eg, from 1 to 10 amino acids) are replaced, added or removed for purposes of cloning, transit peptide fusion, etc., while maintaining

From this, the activity of HFPD, that is, the ability to catalyze the transformation of para-hydroxyphenylpyruvate into homogenizate, or it can be any other HFPD, which can be further improved.

For example, a GFPD that can be further improved by the modifications described herein can be a variant of the GFPD obtained from Rzhendotopes PYpyogezsep5, set forth herein as any of the ZEO IU

MO:34-37, a variant of the GFPD derived from Amepa loan, listed here as 5ZEO IU MO:38, a variant of the GFPD sequences listed here as any of the BEO IU MO:3-326, 383-389, 393, 395 and 397 -459 in UMO2012/021785, which is incorporated into this description in its entirety by reference; by sequences of GFPD, laid out as any of ZEO IU MO:2-14 and 20-50 in

МОО2011/068567, which is incorporated in this description in its entirety by reference; GFPD sequences set forth as any of 5EO IU MO:15-26 in UMO2010/08570, which is incorporated herein by reference in its entirety; GFPD having one or more substitutions described in UMO2009/144079 or US Patent 6,245,968, each of which is incorporated herein by reference in its entirety; GFPD having one or more substitutions described in Tables 1, 2, 5 or 6 of UMO2010/085705; and/or GFPD having one or more substitutions described in Table 1 of M/O2011/068567.

In some embodiments, the nucleotide sequence of the invention (including its isolated, recombinant and chimeric genes, vectors, host cells, plants, plant parts and seeds containing nucleic acid sequences, amino acid sequences and compositions thereof encoded by the nucleic acid sequence, as well as methods of using a nucleic acid sequence to increase resistance to HFPD inhibitor herbicides, especially increasing resistance to HFPD inhibitor herbicides of the class M-(1,2,5-oxadiazol-3-yl)/benzamides; M-(tetrazol-4 -yl)- or M-(triazol-3-yl"arylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide;N-(1,3,4-oxadiazol-2-yl) benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-"(trifluoromethyl)benzamide (Col. 1); M- (tetrazol-5-yl)y- or M-(triazol-3-ylaryl). arboxamides, preferably such as 2-chloro-3-ethoxy-4--"methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl/benzamide (Spol. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro-3-(methylsulfanyl) -M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H - tetrazol-5-yl)-4-trifluoromethyl)benzamide (Spol. 5); pyridazinone derivatives (M/O2013/050421 and

MO2013/083774); substituted 1,2,5-oxadiazoles (ММО2013/072300 and ММО2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, such as isoxaflutol; or of the pyrazolinates class, such as pyrasulfotol and topramesone) encodes the amino acid sequence set forth in any of ZEO IU MO:7-33, as well as its fragments and variants encoding the GFPD herbicide resistance polypeptide.

A. Methods of measuring resistance to the HFPD inhibitor

Any suitable method for measuring resistance to GFPD inhibitor herbicides can be used to evaluate GFPD sequences according to the invention. Persistence can be measured by monitoring the ability of a cell or organism to survive application of a particular GFAP inhibitor herbicide, or the ability to perform essential cellular functions such as photosynthesis, protein synthesis, respiration, or reproduction in a manner that does not differ markedly from that present in untreated cells or organisms. or the ability of plants treated with a GFAP inhibitor herbicide to have no significant difference in yield or even improved yield compared to the same plants not treated with the same herbicide (but for which weeds were removed or prevented by some other mechanism, than the use of herbicide-inhibitor GFPD). In some embodiments, resistance can be measured by a visible indicator phenotype of a cell or organism transformed with a nucleic acid containing a gene encoding a corresponding HFPD protein, or in an ip migo assay of the HFPD protein, in the presence of various concentrations of various HFPD inhibitors. Dose responses and relative shifts in dose responses associated with such indicator phenotypes (browning, growth inhibition, discoloration, herbicidal effect, etc.) are conveniently expressed, for example, as SC50 values (concentration causing 50 95th growth inhibition) or MIC (minimum inhibitory concentration), where increasing values correspond to increasing resistance inherent to the expressed GFPD, in the usual way, based on plant damage, symptoms of meristem discoloration, etc. in the range of different concentrations of herbicides. These data can be expressed, for example, as SC50 values obtained from dose/response curves, where "dose" is plotted on the x-axis and "percentage killed", "herbicidal effect", "number of green plants emerged" and etc., where the increased values of ZK50 correspond to an increase in the level of resistance characteristic of the expressed HFPD. Herbicides can be applied appropriately pre-emergence or post-emergence.

In various embodiments, the level of stability of the nucleic acid or protein-encoding gene

A GFPD according to the invention, or a GFPD protein according to the invention, can be tested by gene transfer, regeneration, propagation and spray tests of a test plant, such as tobacco, or a crop plant, such as soybean, corn or cotton. According to the results of such testing, such plants are more resistant and preferably tolerate a dose of at least 2 times the normal dose recommended for field use, even more preferably tolerate a dose of up to 4 times the normal dose recommended for field use, of inhibitor herbicides GFPD (e.g., GFPD inhibitor herbicides of the M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides), preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)- M-(1-methyl-1H-tetrazol-5-yl)benzamide; M-(1,3,4-

oxadiazol-2-yl)/benzamides, preferably such as 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-"(trifluoromethyl)benzamide ( Commun. 1); M-(tetrazol-b-yl)y- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-"methylsulfonyl)-M-(1-methyl-1H-tetrazol- 5-yl/benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2 -chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3-(methylsulfinyl)- M-(1-methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (M/O2013/050421 and

UMO2013/083774); substituted 1,2,5-oxadiazoles (ММО2013/072300 and ММО2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, such as isoxaflutol; or of the pyrazolinate class, such as pyrasulfotol and topramesone) than similar plants containing no exogenous gene encoding a GFPD protein, or than plants containing a gene that includes a reference GFPD-encoding DNA, such as the HFPD-encoding DNA of Rzeidotopav5 Pyogesep5, under the control of the same promoter as the nucleic acid encoding the GFPD protein according to the invention. Accordingly, the term "capable of increasing the resistance of a plant to at least one herbicide acting on GFPD" means the resistance of a plant expressing GFPD according to the invention to a dose of at least 1, 2, or 3, or 4 or more times the normal of the field dose of the HFPD inhibitor herbicide compared to a plant expressing only its own endogenous HFPD or a plant expressing a reference enzyme

GFPD In this regard, the term "herbicide that acts on GFPD" is not limited to substances known and/or used as herbicides, but also refers to any substance that inhibits the catalytic activity of GFPD proteins.

Alternatively, on a quantitative level, data similar to Rigo (the value of Rigo means the logarithm of the concentration of inhibitor required to inhibit 50 95 enzymatic activity, in molar concentration) can be obtained for the GFPD protein of the invention and compared to the reference sequence of GFPD in the presence or absence of any - what appropriate inhibitor herbicides

GFPD

A specific, although not limiting, type of assay that can be used to evaluate GFPD sequences according to the invention is a colorimetric assay. In this assay, a tryptone-yeast broth-type culture medium (U1-UOgoi) with 1 95 agarose, 5 mM /1-

From tyrosine and 42 mM succinate, containing a selective agent for the vector ro5E420 (Ipmigodep,

Karlerue, Germany) or a modified version of roE420 (p5E429(EI)MX), poured into tablets with deep wells. Into each well is introduced a culture of E. soya in the exponential phase of growth containing the p5E420-HRPOX vector (HRPOX means any gene encoding a putative GFID enzyme/protein). After 16 hours at 37 °C, wells containing no culture medium, those inoculated with E. soya culture containing the empty rZE420 vector are clear, or those inoculated with E. soya culture containing the RZE420 vector -HRROX, which includes the gene that encodes an inactive GFPD, are clear, while wells inoculated with E. soya culture containing the RZE420-HRROX vector, which encodes an active GFPD, are brown.This test has previously been shown to reflect GFPD activity, whatever the source of this activity may be, and allows identification of GFPD activity (05 6,768,044), that is, at a qualitative level.

B. Methods of introduction of mutations in the sequence of HFPD

In the mutant white GFPD encoded by a nucleic acid according to the invention, at least one amino acid has been replaced as defined above.

The replacement can be carried out in the nucleic acid sequence encoding the reference GFPD, as defined above, by any means suitable for replacing in the indicated sequence the codon encoding the replacement amino acid with the codon corresponding to the replacement amino acid; the indicated codons are widely described in the literature and are well known to those skilled in the art.

Several methods of molecular biology can be used to achieve replacement.

A practical method of preparing a mutant nucleic acid sequence according to the invention and a corresponding protein involves performing site-directed mutagenesis of codons encoding one or more preselected amino acids. The methods of obtaining such site-directed mutations are well known to the expert and are widely described in the literature (in particular: Oigesivei Miiadepeviv: A

Rgasiisa! Arrgoasi, 1991, Edye Bu MO). Merpegzop, IR RKE55), or are methods for which it is possible to use commercial kits (for example, the kit for mutagenesis OSHIKSNAMOE M

Idychepipd from Oiadepe or Zigaiadepe). After site-directed mutagenesis, it is not superfluous to also select cells containing a mutant HFPD, which is less sensitive to the HFPD inhibitor, using an appropriate selection mechanism. Appropriate selection methods to achieve this goal have been described above.

Alternatively, the DNA sequence encoding the reference GFPD can be modified by ip 60 ziso to encode a GFPD protein containing one or more of the substitutions listed here, and then synthesized de pomo. The nucleotide sequence encoding the mutant GFPD protein can be introduced into the host cell as described herein in other sections.

B. Isolated polynucleotides, their variants and fragments

In some embodiments, the present invention includes isolated or recombinant polynucleotides. A "recombinant" polynucleotide or polypeptide/protein, or a biologically active portion thereof, is defined herein as no longer present in its original, native organism, such as found in a heterologous host cell or in a transgenic plant cell, seed, or plant. In one embodiment, the recombinant polynucleotide is free of sequences (eg, protein-coding or regulatory sequences) that naturally frame the nucleic acid (ie, sequences located at the 5" and 3" ends of the nucleic acid) in the genomic DNA of the organism from which taken polynucleotide. The term "recombinant" refers to polynucleotides or polypeptides that have been manipulated with respect to a naturally occurring polynucleotide or polypeptide such that the polynucleotide or polypeptide differs (eg, in chemical composition or structure) from that found in nature. In another embodiment, the "recombinant" polynucleotide is free of internal sequences (ie, introns) that occur naturally in the genomic DNA of the organism from which the polynucleotide is derived. A typical example of such a polynucleotide is the so-called complementary DNA (cDNA). For example, in various embodiments, an isolated polynucleotide encoding GFPD inhibitor herbicide resistance may comprise less than 5 kb, 4 kb,

With kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotide sequence that naturally frames the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Nucleic acid molecules of the invention include those that encode the GFPD of the invention. In some embodiments, a nucleic acid molecule of the invention is operably linked to a promoter capable of directing expression of the nucleic acid molecule in a host cell (eg, a plant host cell or a bacterial host cell).

The present invention also provides for variants and fragments of any nucleic acid sequence encoding amino acid sequences set forth in any of 5EO IU MO:7-33. A "fragment" of a polynucleotide can encode a biologically active part of a polypeptide, or it can be a fragment that can be used as a hybridization probe or PCR primer,

Using the methods described in other sections here. Polynucleotides that are polynucleotide fragments include at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 contiguous nucleotides, or up to the number of nucleotides present in the full-length polynucleotide described herein, depending on the intended purpose (eg, the HFPD nucleic acid described herein). "Contiguous" nucleotides should be understood as nucleotide residues located directly next to each other.

Fragments of polynucleotides according to the proposed invention generally encode fragments of a polypeptide that preserve the biological activity of the full-length protein of resistance to the herbicide-inhibitor GFPD; ie, herbicide resistance activity. By "retaining herbicide resistance activity" is meant that the fragment has at least about 30 95, at least about 50 95, at least about 70 95, at least about 80 95, 8595, 90 95, 95 95, 100 95, 110965, 125965, 15095 , 17595, 200 95, 250 95, at least about 300 95 or more of the herbicide resistance activity of the full-length herbicide resistance protein GFPD inhibitor set forth herein as ZEO IU MO:7-33. Methods for measuring herbicide resistance activity are well known in the art, and illustrative methods are described herein. In a non-limiting example, a fragment according to the invention will be resistant to the same dose of the herbicide-GPD inhibitor, or will be resistant to a dose of 1, 2, 3, 4 times higher or even higher dose of the herbicide-GPD inhibitor, or the fragments will be the same or more stable based on ribo or Ki between the fragment and ZEO IU

MO:7-33.

A polynucleotide fragment encoding a biologically active part of a polypeptide according to the invention encodes at least about 150, 175, 200, 250, 300, 350 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide according to the invention. In a non-limiting example, a fragment of a polynucleotide encoding a biologically active part of a GFPD protein containing a proline at a position corresponding to position 335 5EO IU MO:1 and a phenylalanine or tyrosine at a position corresponding to position 336 5EO IU MO: and, optionally, one or more amino acid substitutions in positions corresponding to positions 188, 189, 200, 215, 226, 339 and 340 of 5EO IU MO:1, including the GFPD protein set forth in any of BEO IU MO:7-33.

The invention also encompasses variants of polynucleotides as described above. "Variants" of the 60 polynucleotide also include sequences encoding HFPD according to the invention, but conservatively different due to degeneracy of the genetic code, as well as those that are sufficiently identical. Variants of the present invention retain GFPD enzyme activity and resistance to the GFPD inhibitor herbicide. The term "sufficiently identical" means that the polypeptide or polynucleotide sequence is at least about 53 95, at least about 60 95 or 65 95 identical, about 70 95 or 75 95 identical, about 80 95 or 85 95 identical, about 90 95, 91 95, 92 95, 93 o, 94 95, 95 95, 96 95, 97 Fo, 98 95 or 99 95 identical compared to the reference sequence, when using one of the alignment programs with standard parameters. One skilled in the art will appreciate that these values can be suitably adjusted to determine the respective identity of the polypeptides encoded by the two polynucleotides, taking into account codon degeneracy, amino acid similarity, reading frame position, and the like.

Bacterial genes very often have multiple methionine initiation codons close to the start of the open reading frame. Often, initiation of translation at one or more of these start codons results in the formation of a functional protein. Such start codons may include ATO codons. However, bacteria such as Bacilli5 5r also recognize as a start codon

STO, and proteins that initiate translation at STO codons, contain methionine as the first amino acid.

Moreover, it is often impossible to determine which of these codons is used by bacteria in nature. Therefore, it should be understood that the use of one of the alternative methionine codons can lead to the formation of variants that confer resistance to herbicides. These herbicide resistance proteins are encompassed by the present invention and may be used in the methods of the present invention. Naturally occurring allelic variants can be determined using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization, as described below. Alternative polynucleotides also include polynucleotides of synthetic origin, generated, for example, using site-directed or other mutagenesis strategies, but which still encode a polypeptide having the desired biological activity.

It is also clear to an expert that further mutation of polynucleotides according to the invention can make changes, thus leading to further changes in the amino acid sequence of the encoded polypeptides, without changing the biological activity of the polypeptides. Thus, variants of isolated polynucleotides can be created by introducing one or more additional nucleotide substitutions, additions or deletions into the corresponding polynucleotide encoding HFPD according to the invention, so that 1-5, 1-10 or 1-15 substitutions are introduced into the encoded polypeptide, additions or deletions of amino acids, or 1,2, 3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, additions or deletions of amino acids. Further mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis, or by gene rearrangement techniques. Similar variants of polynucleotides are also covered by the invention.

Variants of polynucleotides can be created by randomly introducing mutations in all or part of the coding sequence, such as saturation mutagenesis or permutation mutagenesis, and the resulting mutants can be screened for the ability to confer herbicide resistance activity to identify mutants that retain the activity.

Additional ways of generating variants include subjecting a cell that expresses the protein described here (or a library thereof) to special conditions that create a stress on the activity of the protein. Special conditions may include (but are not limited to) changes in temperature, changes in pH, and changes in substrate or inhibitor concentrations. The protein library can be subjected to these conditions during the time of expression of the protein (eg, in E. soybean or another host) or after the generation of the protein extract, or after purification of the protein.

The functional or enzymatic activity of a protein library exposed to stress conditions can then be compared to a reference protein to identify proteins with improved properties. This comparison of activities can be performed as part of a growth screen or alternatively as part of an enzymatic assay that quantifies protein activity.

Properties that may be identified as improved may include resistance to GFAP inhibitor herbicides, changes in kinetic constants (including Kt, Ki, Ks), protein stability, protein thermal stability, or temperature and pH optimum for the protein.

D. Isolated proteins, their variants and fragments

Herbicide resistance polypeptides are also encompassed by the present invention. A herbicide resistance polypeptide includes polypeptide preparations containing less than about 30 95 , 20 96 , 10 95 , or 5 95 (by dry weight) of a non-herbicide resistance polypeptide (also hereinafter referred to as "contaminant protein"). As used herein, "herbicide resistance protein" 60 refers to the GFPD polypeptide described herein. Also provided are its fragments, biologically active parts and variants that can be used in the practical embodiment of the methods according to the present invention. "Fragments" or "biologically active parts" include fragments of a polypeptide containing a portion of the amino acid sequence that encodes a herbicide resistance protein and that retains herbicide resistance activity. The biologically active part of the herbicide resistance protein can be a polypeptide with a length of, for example, 10, 25, 50, 100 or more amino acids. Similar biologically active parts can be prepared by recombination techniques and evaluated for herbicide resistance activity.

By "variants" should be understood proteins or polypeptides whose amino acid sequence is at least about 53 95, 60 95, 65 95, about 70 95, 75 95, about 80 95, 85 Ov, 90 9, 91 Fo, 92 95, 93 o , 94 Fo, 95 Fo, 96 95, 97 U, 98 95 or 99 95 is identical to any of the ZEO IU MO:7- 33, while the specified variant has GFPD enzymatic activity and resistance to GFPD inhibitor herbicides. One skilled in the art will appreciate that these values can be suitably adjusted to determine the respective identity of the polypeptides encoded by the two polynucleotides, taking into account codon degeneracy, amino acid similarity, reading frame position, and the like.

For example, conservative amino acid substitutions can be made in one or more non-essential amino acid residues. A "non-essential" amino acid residue is a residue that can be changed in the reference sequence of a polypeptide without changing biological activity, while an "essential" amino acid residue is required for biological activity. "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by a residue having a similar side chain. Families of amino acids with similar side chains are defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine , tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains ( e.g., tyrosine, phenylalanine, tryptophan, histidine).

Substitutions of amino acids can be made at non-conservative sites that retain their function. In general, such substitutions are not made for conserved amino acid residues or for amino acid residues within a conserved motif when such residues are essential for the activity of the polypeptide. However, one skilled in the art will appreciate that functional variants may have minor conservative or non-conservative changes in conserved residues.

Antibodies to GFPD according to the present invention, or to variants or fragments thereof, are also encompassed. Methods for obtaining antibodies are well known in the art (see, e.g., Nagyom/apa Gape (1988) Apiirodiev: A Iarogayutu Mapiai, So briipud Nagbog Iarogaiogu, So 5riipd

Nagbog, MU; US Patent Mo 4,196,265).

Thus, one aspect of the invention relates to antibodies, single-chain antigen-binding molecules or other proteins that specifically bind to one or more protein or peptide molecules according to the invention and their homologues, chimeras or fragments.

In a particularly preferred embodiment, the antibody specifically binds to the protein, the amino acid sequence of which is set forth in 5EBEO IU MO:7-33, or its fragment. In another embodiment, the antibody specifically binds to a chimeric protein containing an amino acid sequence selected from the sequences set forth in ZEO IU MO:7-33, or its fragment. In some embodiments, the antibody specifically binds to a region of the protein corresponding to amino acid positions 188 and 189 of 5EO IU MO:1, or to a region of the protein corresponding to amino acid position 200 5EO IU MO:1, or to a region of the protein corresponding to the position

BO amino acids 215 ZEO IU MO:1, or a section of the protein corresponding to the position of amino acids 335-340 5EO I MO.

Antibodies according to the invention can be used for quantitative or qualitative determination of peptide or protein molecules according to the invention, or for detection of post-translational modifications of proteins. Hereinafter, an antibody or peptide is considered to "specifically bind" to a protein or peptide molecule according to the invention, if such binding is not competitively inhibited by the presence of unrelated molecules.

D. Accumulation of genes

In commercial crop production, it is desirable to eliminate unwanted plants (i.e., "weeds" in a crop field through reliable pesticide regulation. An ideal treatment would be one that could be applied to an entire field,

but which would destroy only unwanted plants without affecting cultivated plants.

One such treatment system involves the use of herbicide-resistant crops, so that when the herbicide is sprayed over a field of herbicide-resistant crops, they continue to grow, while herbicide-resistant weeds are killed or severely damaged. Ideally, such treatment systems should take advantage of the variation in herbicide properties so that weed control is the best possible combination of flexibility and economy. For example, individual herbicides have varying longevity in the field, and some herbicides persist and are effective for a relatively long time after field application, while other herbicides rapidly degrade into other and/or inactive compounds. An ideal treatment system would allow the application of different herbicides, so that growers could tailor the choice of herbicides to the specific situation.

While a number of herbicide-resistant crops are commercially available today, a problem with the use of many commercial herbicides and herbicide/crop combinations is that individual herbicides usually have an incomplete spectrum of activity against common weed species. For most of the individual herbicides that have been used for some time, populations of herbicide-resistant weed species and biotypes have come to predominate (see, e.g., Tgape!Yi apa Uugidni (2002) M/eyed 5siepse 50: 700-712; Omep apa 7eiIaua (2005 )

Rezi Mapad. 5 61: 301-311). Transgenic plants resistant to more than one herbicide have been described (see, e.g., M/02005/012515). However, improvements are constantly needed in every aspect of crop production, weed control capabilities, continued residual weed control, and improved grain yields.

The GFPD protein or the nucleotide sequence according to the invention is preferably combined in plants with other genes encoding proteins or RNAs, which give such plants useful agronomic properties. Among the genes that encode proteins or RNAs that provide useful agronomic properties to transformed plants, we can mention DNA sequences that encode proteins that confer resistance to one or more herbicides that differ in their chemical structure from GFPD-inhibiting herbicides, etc. that provide resistance to some insects, that provide resistance to some diseases, DNA that encodes RNA, that provide the fight against

With nematodes or insects, etc.

Similar genes, in particular, are described in published PCT applications UMO91/02071 and M/O95/06128, as well as in US Patent 7,923,602 and US Patent Application Publication No. 20100166723, each of which is incorporated herein by reference in its entirety.

Among the DNA sequences that encode proteins that confer resistance to certain herbicides in transformed plant cells and plants, we can mention the Bag or PAT genes, or the Zygeriotus soeiissog gene described in U/O2009/152359 that confers resistance to glufosinate herbicides, a gene that encodes a suitable ERBR5 conferring resistance to targeted herbicides

ЕРБ5БРЗ, таким як гліфозат і його солі (05 4,535,060, 05 4,769,061, 05 5,094,945, 05 4,940,835, 5 5,188,642, 05 4,971,908, 005 5,145,783, Ш5 5,310,667, 05 5,312,910, 005 5,627,061, 5 5,33,435), ген, що кодує гліфозат -n-acetyltransferase (eg, 05 8,222,489, 05 8,088,972, 005 8,044,261, 005 8,021,857, 05 8,008,547, 005 7,999,152, 05 7,998,703, 05 7,863,503,

OB 7,714,188, 005 7,709,702, 5 7,666,644, OB 7,666,643, 05 7,531,339, OB 7,527,955, and 05 7,405,074), a gene encoding glyphosate oxidoreductase (eg, О5 5,463,175).

Among the DNA sequences encoding suitable ERBR5 conferring resistance to herbicides targeting ERBR5, more specifically mention can be made of the gene encoding the plant

ERBR5, in particular ERBRBZ of maize, in particular ERBR5 of maize, containing two mutations, in particular a mutation at amino acid position 102 and a mutation at amino acid position 106 (M/O2004/074443), and which is described in Patent Application 05 6566587, is hereinafter referred to as double mutant EPZR5 of maize or 2pERB5BR5B, or the gene encoding ERBOR5, isolated from Adhorasiegisht and described by IU Mo sequences. 2 and IU Mo. From US Patent 5,633,435, also referred to as SRA.

Among the DNA sequences encoding suitable ERBR5 conferring resistance to herbicides targeting ERBR5, one can more specifically mention the gene encoding ERBRZ SKO23 with

Ahiphorasieg diobitogitih5, but also mutant OKO23 ACE1, SKO23 ACE2, or SKS23 ACEZ, especially mutants or variants of SKOS23 as described in UUO2008/100353, such as oKOo23(ase3)K17ZK 5EO IU Mo. 29 in IMO2008/100353.

In the case of DNA sequences encoding ERBR5B, and which in particular encode the genes listed above, the sequence encoding these enzymes is preferably preceded by a sequence encoding a transit peptide, in particular an "optimized transit peptide" described in US Patent 60 5,510,471 or 5,633,448.

Typical herbicide resistance traits that can be combined with a nucleic acid sequence of the invention also include at least one ALS (acetolactate synthase) inhibitor. (M/O02007/024782); mutant gene Agaridorziz ALS/AGKS (US Patent 6b,855,533); genes that encode 2,4-d-monooxygenases that confer resistance to 2,4-H0 (2,4-dichlorophenoxyacetic acid) by metabolism (US Patent 6,153,401); and genes encoding Juisatra monooxygenases that confer resistance to dicamba (3,6b-dichloro-2-methoxybenzoic acid) by metabolism (5 2008/0119361 and 05 2008/0120739).

In various embodiments, the GFAP according to the invention is assembled with one or more herbicide resistance genes, including one or more additional genes for resistance to GFAP inhibitor herbicides, and/or one or more genes resistant to glyphosate and/or glufosinate. In one variant implementation of HFPD according to the invention is combined with 2ptERZRZ and Bag.

Among the DNA sequences encoding proteins related to insect resistance properties, it is worth mentioning the Vi proteins, widely described in the literature and well known to those skilled in the art. It is also worth mentioning the proteins isolated from bacteria, such as Rpoiotptaraiv (M/097/17432 8

MO98/08932).

Among such DNA sequences encoding target proteins conferring novel insect resistance properties, it is worth mentioning more specifically the Wi Stu or MIR proteins, widely described in the literature and well known to those skilled in the art. They include Stu1E protein or hybrids derived from it (e.g. Stu1tA-Sgu1!E hybrid proteins described in 05 6,326,169; 05 6,281,016; 5 6,218,188, or their toxic fragments), StutA-type proteins or their toxic fragments, preferably Stu1Ac protein or hybrids obtained from it (e.g., StutAb-sguTAs hybrid protein described in O5 5,880,275), or StgutAB or VI2 protein or their insecticidal fragments as described in EP451878, Stu2Ae, Stu2Ai or Stgu2Ad proteins as described in UMO2002/057664 or their toxic fragments, protein

StutA.105, described in UUO 2007/140256 (5EO IO Mo. 7) or its toxic fragment, protein

MIRZAa19 under the catalog number AVO20428, MIRZAag0 protein under the catalog number of the Ministry of Internal Affairs and Communications

AVOS20429 (5EO IYU Mo. 2 in UMO 2007/142840), MIRZA proteins produced in cotton events

СОТ202 or СОТ203 (МО2005/054479 and ММО2005/054480, respectively), white Sgu as described in

UO2001/47952, MIRZAa protein or its toxic fragment as described in Evbigis et al. (1996), Rhos

Mai! Asai 5si 0 5 A. 28:93(11):5389-94 and 05 6,291,156, insecticidal proteins from Hepoparadiv strains

Zo (as described in M/O98/50427), Zeitain (especially from 5. epiothorpia) or RNOiignoraiv, such as Tobelki from Ripoiogparda5 as described in UUO98/08932 (e.g., Umayiegiy et al., 2001, Arry Epmigop

Mistobrio!. 67(11):5017-24; Eptepsp-sopviapi apa Woep, 2000, Seiyi Moi Me z5si.; 57(5):828-33).

Also included here are any variants and mutants of any of these proteins that differ by several (1-40, preferably 1-5) amino acids from any of the above sequences, especially the sequence of their toxic fragment, or that are fused to the transit a peptide such as a plastid transit peptide or any other protein or peptide.

In various embodiments, the GFID sequence of the invention can be combined in plants with one or more genes conferring a desired trait, such as herbicide resistance, insect resistance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency , improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress resistance, improved reproduction, etc.

Particularly useful transgenic events that can be combined with genes according to the proposed invention in plants of the same species (eg, by crossing or retransforming a plant containing another transgenic event with a chimeric gene according to the invention) include event 531/ RU-СНВКО4 (cotton, insect control, described in

UMO2002/040677), event 1143-14A (cotton, insect control, not deposited, described in

MO2006/128569); event 1143-5188 (cotton, insect control, not deposited, described in

UMO2006/128570); event 1445 (cotton, resistance to herbicides, not deposited, described in Sh5-A 2002-120964 or UMO2002/034946; event 17053 (rice, resistance to herbicides, deposited as PTA-9843, described in UMO2010/117737); event 17314 ( rice, resistance to herbicides, deposited as

PTA-9844, described in UMO2010/117735); event 281-24-236 (cotton, insect control - resistance to herbicides, deposited as PTA-6233, described in UMO2005/103266 or I5-A 2005-216969); event 3006-210-23 (cotton, insect control - resistance to herbicides, deposited as PTA-6233, described in Sh5-A 2007-143876 or UMO2005/103266); event 3272 (maize, quality property deposited as PTA-9972, described in MUMO2006/098952 or О5-А 2006-230473); event 33391 (wheat, resistance to herbicides, deposited as PTA-2347, described in MO2002/027004), event 40416 (corn, insect control - resistance to herbicides, deposited as ATSS PTA-11508, described in UMO 11/075593); event 43A47 (maize, control of 60 insects - resistance to herbicides, deposited as ATCC PTA-11509, described in MLO2011/075595);

event 5307 (maize, insect control, deposited as ATCC PTA-9561, described in

MMO2010/077816); event A5K-368 (tonconig, resistance to herbicides, deposited as ATCC PTA-4816, described in ShSh5-A 2006-162007 or M/O2004/053062); event B16 (maize, resistance to herbicides, not deposited, described in O5-A 2003-126634); event VRUZ-SM127-9 (soybean, resistance to herbicides, deposited as MSIMV Mo. 41603, described in M/O2010/080829); event VI K1 (rapeseed oil, recovery of male sterility, deposited as МСИМВ 41193, described in

UMMO2005/074671), event СЕ43-67В (cotton, insect control, deposited as Ю5М

ACC2724, described in Sh5-A 2009-217423 or UMO2006/128573); event CE44-690 (cotton, insect control, not deposited, described in I5-A 2010-0024077); event CE44-690 (cotton, insect control, not deposited, described in UMO2006/128571); event СЕ46-0О2А (cotton, insect control, not deposited, described in UMO2006/128572); event SOT102 (cotton, insect control, not deposited, described in Ш5-А 2006-130175 or

UO2004/039986); event SOT202 (cotton, insect control, not deposited, described in O5-

А 2007-067868 or UMO2005/054479); event SOT203 (cotton, insect control, not deposited, described in UMO2005/054480);); event OA521606-3 / 1606 (soy, resistance to herbicides, deposited as PTA-11028, described in UMO2012/033794), event OA540278 (maize, resistance to herbicides, deposited as ATSS PTA-10244, described in M/O2011/022469) ; event OA5Z-44406-6 / рорАВ8264.44.06.1 (soybean, resistance to herbicides, deposited as PTA-11336, described in

ММО2012/075426), event ЮМАБ5Б-14536-7 /прАВ8291.45.36.2 (soybeans, resistance to herbicides deposited as PTA-11335, described in ММО2012/075429), event OA5-59122-7 (corn, insect control - resistance to herbicides, deposited as ATCC PTA 11384, described in O5-A 2006-070139); event YuAB-59132 (maize, insect control - resistance to herbicides, not deposited, described in UMO2009/100188); event RBABZBb8416 (soybean, herbicide resistance, deposited as ATCC PTA-10442, described in IMO2011/066384 or IMO2011/066360); event ОР-098140-6 (maize, resistance to herbicides, deposited as ATSS PTA-8296, described in О5-А 2009-137395 or UMO 08/112019); event ОР-305423-1 (soy, quality property, not deposited, described in Sh5Z-A 2008-312082 or U/O2008/054747); event YuR-32138-1 (maize, hybridization system deposited as ATSS PTA-9158, described in I5B-A 2009-0210970 or

UMO2009/103049); event YOR-356043-5 (soybean, resistance to herbicides, deposited as ATCC PTA-

Ko) 8287, described in Sh5-A 2010-0184079 or UMO2008/002872); event EE-1 (eggplant, insect control, not deposited, described in MO 07/091277); event RI117 (maize, resistance to herbicides, deposited as ATSS 209031, described in O5-A 2006-059581 or MO 98/044140); the event

EC72 (soybean, herbicide resistance, deposited as PTA-11041, described in MMO2011/063413), event

CA21 (maize, resistance to herbicides, deposited as ATS 209033, described in ShZ-A 2005-086719 or UMO 98/044140); event 5525 (maize, resistance to herbicides, deposited as ATCC 209032, described in O5-A 2005-188434 or UMO 98/044140); event START119 (cotton, insect control - resistance to herbicides, deposited as ATCC PTA-8398, described in ММО2008/151780); event ЗНВО614 (cotton, resistance to herbicides, deposited as ATCC PTA-6878, described in 05-

А 2010-050282 or ММО2007/017186); event 511 (maize, resistance to herbicides, deposited as ATCC 209030, described in Sh5-A 2005-188434 or M/098/044140); event SM K213 (sugar beet, virus resistance, deposited as МСИМВ-41601, described in UMO2010/076212); event H7-1 (sugar beet, resistance to herbicides, deposited as МСИМВ 41158 or МСИМВ 41159, described in О5-А 2004-172669 or UMO 2004/074492); event ZORI IM1 (wheat, disease resistance, not deposited, described in O5-A 2008-064032); event GI 27 (soybean, resistance to herbicides, deposited as МСИМВ41658, described in М/О2006/108674 or Ш5-А 2008-320616); event GI 55 (soybean, resistance to herbicides, deposited as МСИМВ 41660, described in UMO 2006/108675 or О5-А 2008-196127); event GIsoyop25 (cotton, herbicide resistance, deposited as ATCC PTA-3343, described in

UMO2003/013224 or I5-A 2003-097687); event GI KISEOb (rice, herbicide resistance, deposited as ATCC 203353, described in 5 6,468,747 or M/O2000/026345); event I Igiseb2 (mal, resistance to herbicides, deposited as ATSS 203352, described in UMO2000/026345), event

HEKISEbOT1 (rice, resistance to herbicides, deposited as ATSS PTA-2600, described in Sh5-A 2008-2289060 or UMO2000/026356); event I 038 (corn, quality property, deposited as ATSS

PTA-5623, described in I5-A 2007-028322 or UMO2005/061720); event MIK162 (maize, insect control, deposited as PTA-816b6, described in I5-A 2009-300784 or

UMO2007/142840); event MIKbO4 (maize, insect control, not deposited, described in O5-

A 2008-167456 or UMO2005/103301); event IOM15985 (cotton, insect control, deposited as ATSS PTA-2516, described in SHV-A 2004-250317 or UMO2002/100163); the event

IOM810 (corn, insect control, not deposited, described in О5З-А 2002-102582); the event

IOM863 (maize, insect control, deposited as ATSS PTA-2605, described in bo UMO2004/011601 or O5-A 2006-095986); event IOM87427 (maize, pollination control,

deposited as ATS PTA-7899, described in UMO2011/062904); event МОМ87460 (maize, stress tolerance, deposited as ATSS PTA-8910, described in MUMO2009/111263 or 0О5-А 2011-0138504); event IOM87701 (soybean, insect control, deposited as ATCC PTA-8194, described in O5-A 2009-130071 or M/O2009/064652); event MOM87705 (soybean, quality trait - resistance to herbicides, deposited as ATCC PTA-9241, described in I5-A 2010-0080887 or

MO2010/037016); event IOM87708 (soybean, herbicide resistance, deposited as ATCC PTA-9670, described in UMO2011/034704); event IOM87712 (soybean, yield, deposited as PTA-10296, described in UMO2012/051199), event IOM87754 (soybean, quality trait, deposited as ATOS

PTA-9385, described in UMO2010/024976); event IOM87 769 (soybean quality property deposited as

ATOSS PTA-8911, described in I5-A 2011-0067141 or M/02009/102873); event IOM88017 (maize, insect control - resistance to herbicides, deposited as ATCC PTA-5582, described in SHV-A 2008-028482 or M/O2005/059103); event IOM88913 (cotton, resistance to herbicides, deposited as ATCC PTA-4854, described in UMO2004/072235 or O5-A 2006-059590); event MOM88302 (canola oil, herbicide resistance, deposited as PTA-10955, described in

МО2011/153186), event IOM88701 (cotton, herbicide resistance, deposited as PTA-11754, described in M/O2012/134808), event IOM89034 (maize, insect control, deposited as

ATOSS RTA-7455, described in UMO 07/140256 or 05-А 2008-260932); event IOM89788 (soybean, herbicide resistance, deposited as ATS PTA-6708, described in O5-A 2006-282915 or

MO2006/130436); event M511 (oil rape, pollination control - resistance to herbicides, deposited as ATS PTA-850 or PTA-2485, described in M/O2001/031042); event M58 (canola oil, pollination control - herbicide resistance, deposited as ATCC PTA-730, described in

UMO2001/041558 or 05-A 2003-188347); the MKbOZ event (maize, resistance to herbicides, deposited as ATCC PTA-2478, described in O5-A 2007-292854); event RE-7 (rice, insect control, not deposited, described in M/O2008/114282); KEZ event (oil rape, pollination control - resistance to herbicides, deposited as ATS PTA-730, described in M/O2001/041558 or 0О5-А 2003-188347); event KT7Z3 (oil rape, resistance to herbicides, not deposited, described in UMO2002/036831 or I5-A 2008-070260); event ZUNTON2 / 5MM-000Н2-5 (soybean, resistance to herbicides, deposited as PTA-11226, described in M/O2012/082548), event T227-1 (sugar beet, resistance to herbicides, not deposited, described in UMO2002/44407 or Sh5-A

From 2009-265817); event T25 (maize, resistance to herbicides, not deposited, described in O5-A 2001-029014 or UMO2001/051654); event 7304-40 (cotton, insect control - resistance to herbicides, deposited as ATCC PTA-8171, described in SHV-A 2010-077501 or ММО2008/122406); event T342-142 (cotton, insect control, not deposited, described in M/O2006/1285658); event TC1507 (corn, insect control - resistance to herbicides, not deposited, described in 0О5-А 2005-039226 or UMO2004/099447); event MIR1034 (maize, insect control - herbicide resistance, deposited as ATCC PTA-3925. described in M/O2003/052073), event 32316 (maize, insect control - herbicide resistance, deposited as PTA-11507, described in UMO2011/084632), event 4114 (maize, insect control - resistance to herbicides, deposited as PTA-11506, described in MMO2011/084621), event EE-СМ3З / ЕС72 (soybean, resistance to herbicides, ATS registration number PTA- 11041) optionally collected from: event EE-

СМ1/Л1 27 or event EE-СМ2/Л/И.55 (М/О2011/063413А2), event ЯА5З-68416-4 (soybean, resistance to herbicides, ATS registration number РТА-10442, УМО2011/066360А1), event ЯА5З -68416-4 (soybean, resistance to herbicides, ATCC registration number PTA-10442, UMO2011/066384А1), ОРР event- 040416-8 (corn, insect control, ATCC registration number PTA-11508,

UMO2011/075593А1), event ЮР-043А47-3 (corn, insect control, registration number

ATOSS РТА-11509, ММО2011/075595А1), event ЮР-004114-3 (maize, insect control, registration number АТСС РТА-11506, УМО2011/084621А1), event ОР-032316-8 (corn, insect control, registration number ATS PTA-11507, UMO2011/084632А1), the event. IOM-88302-9 (rapeseed oil, resistance to herbicides, ATCC registration number PTA-10955,

МО2011/153186А1), event ЯА5-21606-3 (soybean, resistance to herbicides, registration number ATSS

РТА-11028, УМО2012/033794А2), event IOM-87712-4 (soybean, qualitative property, registration number

ATOSS РТА-10296, M/О2012/051199А2), event OBAB5Z-44406-6 (soybean, accumulated resistance to herbicides, registration number ATS РТА-11336, УМО2012/075426А1), event ЯА5-14536-7 (soybean, accumulated resistance to herbicides, ATCC registration number PTA-11335, МО2012/075429А1), event ZUM-000Н2-5 (soy, resistance to herbicides, ATCC registration number PTA-11226,

UMMO2012/082548А2), event ЮР-061061-7 (oilseed rape, resistance to herbicides, deposit number unavailable, УМО2012071039А1), event ОР-073496-4 (oilseed rape, resistance to herbicides, deposit number unavailable, И52012131692), event 8264.44. 06.1 (soybean, accumulated resistance to herbicides, registration number PTA-11336, UMO2012075426А2), event 60 8291.45.36.2 (soybean, accumulated resistance to herbicides, registration number PTA-11335,

UMO2012075429А2), event 5MUNTON2 (soybean, registration number ATSS /РТА-11226,

UMO2012/082548A2), event МОМ88701 (cotton plant, ATCC registration number РТА-11754,

UMO2012/134808А1), event KK179-2 (alfalfa, registration number ATSS RTA-11833,

МО2013/003558А1), event прАВ8264.42.32.1 (soybean, accumulated resistance to herbicides, registration number ATS РТА-11993, МУО2013/010094А1), event М2ОТО9У (corn, registration number

ATCC RTA-13025, ММО2013/012775А1).

E. Polynucleotide constructions

Polynucleotide polypeptides encoding GFPD according to the proposed invention can be modified to obtain or enhance expression in plant cells.

Polynucleotides encoding the polypeptides identified here can be provided in expression cassettes for expression in a plant of interest. "Plant expression cassette" includes a DNA construct, including a recombinant DNA construct, capable of leading to the expression of a polynucleotide in a plant cell. The cassette may include, in the 5'-3' direction of transcription, a transcription initiation site (ie, a promoter, particularly a heterologous promoter) operably linked to one or more required polynucleotides, and/or a translation and transcription termination site (ie, a termination site), which function in plants. The cassette may also additionally contain at least one additional polynucleotide introduced into the organism, such as a selectable marker gene. Alternatively, additional polynucleotide(s) may be provided in the multiple expression cassette. Such an expression cassette is provided with many restriction sites for the insertion of polynucleotide(s) so that it is subject to transcriptional regulation in the regulatory regions.

In a further embodiment, the present invention relates to a chimeric gene containing a coding sequence that includes a heterologous nucleic acid according to the invention, functionally linked to a plant-expressed promoter and optionally a transcription termination and polyadenylation site. "Heterologous" generally refers to a polynucleotide or polypeptide that is not endogenous to the cell or that is not endogenous to the location in the natural genome in which it is present and that is added to the cell by infection, transfection, microinjection, electroporation, microprojections or the like By "functional linkage" is meant a functional linkage between two polynucleotides. Example,

Since the promoter is functionally linked to the DNA sequence, the promoter sequence initiates and acts as an intermediate link in the transcription of the DNA sequence. It should be understood that functionally linked polynucleotides may or may not be contiguous, and where this term is used to denote the joining of two regions encoding a polypeptide, the polypeptides are expressed in the same reading frame.

The promoter can be any polynucleotide sequence that exhibits transcriptional activity in selected plant cells, plant parts, or plants.

The promoter can be natural or analogous, or foreign and heterologous, in relation to the host plant and/or DNA sequence according to the invention. If a promoter is "natural" or "analogous" in relation to the host plant, it is meant that the promoter is present in the native plant into which it is introduced. When a promoter is "foreign" or "heterologous" to a DNA sequence of the invention, it is meant that the promoter is not the natural or naturally occurring promoter of the DNA sequence of the invention operably linked thereto. A promoter can be inducible or constitutive. It may occur in nature, be composed of parts of various naturally occurring promoters, or may be partially or fully synthetic. A guide to promoter design provides studies on the structure of promoters such as in Nagyeu apa Keupoid5 (1987) Misieis Asiib5 Nev5. 15:2343-2361. In addition, the location of the promoter relative to the start of transcription can be optimized. See, for example, Kobegiz and others. (1979) Rhos. May). Asai. si. OB5A, 76:760-764. Many suitable promoters are well known in the art for use in plants.

For example, suitable constitutive promoters for use in plants include: promoters of plant viruses, such as the promoter of peanut chlorotic stripe caulimovirus (Rsibyem) (US Patent Mo 5,850,019); promoter 355 of cauliflower mosaic virus (Satu) (Oaei et al. (1985) Maishge 313:810-812); the promoters of the methyltransferase genes of SpPiogepa virus (US Patent No. 5,563,328) and the full-length transcriptional promoter of morning mosaic virus (EMU) (US Patent No. 5,378,619); promoters of such genes as rice actin (Mseigou et al. (1990)

Reapi Sci. 2:163-171 and US Patent No. 5,641,876); ubiquitin (Spgistepzep et al. (1989) Riapi Moi. Vioi. 12:619-632 and Spgiztepzep et al. (1992) Riapi Moi. Vioi. 18:675-689); rety (Gavi et al. (1991) Tpeog. Arri.

Sepoys 81:581-588); MAZ (Makep et al. (1984) EMVO 3. 3:2723-2730 and US Patent Mo 5,510,474); for histone NZ of maize (I ereii et al. (1992) Moi. Sep. Sepei. 231:276-285 and Agapazzoma et al. (1992) Riapi

U. 2(3):291-300); Vgazzisa pair AI 53 (PCT application MUO97/41228); the gene for the small subunit of plant ribulose-biscarboxylase/oxygenase (Kibibhs; circovirus (A 689 311) or cassava vein mosaic virus (Svmitpm, 05 7,053,205); as well as the promoters of various Adgobasiegisht genes (see

US Patent Mo 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

Suitable inducible promoters for use in plants include: a promoter from the copper-responsive ASET system (May et al. (1993) RMAB 90:4567-4571); the promoter of the Ip2 gene of corn, which responds to benzenesulfonamide herbicides (NegePeu et al. (1991) My. Sep. Sep. Sep. 227:229-237 and Sai" et al. (1994) My. Sep. Sep. Sep. 5 243:32-38) ; as well as the promoter of the repressor Tei from Tp10 (Sai; et al. (1991) Moi. Sbep. Sepei. 227:229-237). Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An example of an inducible promoter of this type is the inducible promoter of a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (5spepa et al. (1991) Rhos. Mai!. Asai. 5si. OBA 88:10421), or the recent use of a chimeric transcription activator, HME , for use in an estrogen receptor-based inducible expression system in plants activated by estradiol (2tso et al. (2000) Riapi 9., 24:265-273). Other inducible promoters for use in plants are described in EP 332104, PCT UMO 93/21334 and PCT UMO 97/06269, which are incorporated herein in their entirety by reference. Promoters composed of parts of other promoters and partially or fully synthetic promoters may also be used. See, for example, Mi et al. (1995) Riapi 9. 7:661-676 and PCT UMO 95/14098 describing such promoters for use in plants.

In one embodiment of the present invention, a promoter sequence specific to specific regions or plant tissues, such as seed-specific promoters (Oaya, K. et al., 1997, Vibiespoiodu) may be used to express the GFPD proteins of the invention

App. Vem. 3, 269-296), especially napin promoter (ER 255 378 A1), phaseolin promoter, glutenin promoter, gelatininin promoter (М/092/17580), albumin promoter (МУО98/45460), oleosin promoter (ММО98/45461), promoter 5ATI1 or ZATZ promoter (PCT/O598/06978).

It is also possible to use an inducible promoter, preferably selected from phenylalanine ammonia-lyase (FAL), NMO-CoA reductase (NM), chitinase, glucanase, inhibitor

From proteinase (IP), RK1 family gene, nopaline synthase (po5) and merB promoters (005 5 670 349,

Table 3), the promoter of HMO2 (05 5 670 349), the promoter of apple beta-galactosidase (YABG1) and the promoter of apple aminocyclopropanecarboxylate synthase (Ack-synthase) (M/098/45445).. In the constructions according to the invention, numerous promoters can be used, including the following one after the other.

A promoter may include, or be modified to include, one or more enhancer elements. In some embodiments, a promoter may include multiple enhancer elements. Promoters containing enhancer elements provide higher levels of transcription compared to promoters that do not contain them. Suitable enhancer elements for use in plants include the Resism enhancer element (US Patent No. 5,850,019), the Satm 355 enhancer element (US Patent Nos. 5,106,739 and 5,164,316), and the EMM enhancer element (Mayi et al. (1997) Tgapzdepis Ke5. 6 :143-156); activator of translation of tobacco mosaic virus (TMV), described in the application UUO87/07644, or tobacco engraving virus (TEM), described

Satipdiop 4. Egevey 1990, 9. Migoi. 64: 1590-1597, for example, or introns such as the maize adnp1 intron or rice actin intron 1. See also PCT UUO96/23898, MO2012/021794,

UMO2012/021797, MMO2011/084370 and UMO2011/028914.

Often, similar structures can contain 5' and 3' untranslated regions. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide in question to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum or Golgi apparatus, or for secretion. For example, the construct can be designed to contain a signal peptide to facilitate translocation of the peptide into the endoplasmic reticulum. "Signal sequence" should be understood as a sequence known or assumed to result in co-translational or post-translational transport of a peptide across a cell membrane. In eukaryotes this usually involves secretion into the Golgi apparatus, with some resulting glycosylation. "Leader sequence" should be understood as any sequence that, upon translation, provides an amino acid sequence sufficient to initiate co-translational transport of a peptide chain into a subcellular organelle. Thus, this includes leader sequences targeted for transport and/or glycosylated through endoplasmic reticulum transition, vacuole transition, plastids including chloroplasts, mitochondria, etc. It may also be advantageous to construct a plant expression cassette that

containing an intron, so that expression requires the processing of this intron in mRNA.

By "3" untranslated region" should be understood the polynucleotide located below the coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of influencing the addition of polyadenylic acid regions to the 3'-end of the mRNA precursor are 3'-untranslated regions. By "5" untranslated region" should be understood the polynucleotide located above the coding sequence.

Other nontranslated elements upstream or downstream include enhancers. Enhancers are polynucleotides whose action enhances the expression of the promoter region. Enhancers are well known in the art and include, but are not limited to, the 5M40 enhancer site and the 355 enhancer site.

The interruption site may be inherent to the transcription initiation site, inherent to the sequence according to the present invention, or may be derived from another source. Suitable termination sites are available in A. yshtegiasiep5 Ti-plasmids, such as octopine synthase and nopaline synthase termination sites. See also Siegipeai and others. (1991) Mine. Sept. Sepoys 262:141-144;

Rgotsagooi (1991) Sei! 64:671-674; Zapiasop and others. (1991) Sepe5 Oem. 5:141-149; Modep and others. (1990)

Riapi SeiI 2:1261-1272; Mypgoe and others. (1990) Sep 91:151-158; Vaiazh and others. (1989) Mysiewis Asid5 Kezv. 17:7891-7903; dov5pPi, etc. (1987) Mysieis Asia Kez. 15:9627-9639; and the European patent application EP 0 633 317 JSC.

In one aspect of the invention, synthetic DNA sequences are designed for a given polypeptide, such as the polypeptides of the invention. Expression of the open reading frame of the synthetic DNA sequence in the cell leads to the production of the polypeptide according to the invention. Synthetic DNA sequences may be useful for simple elimination of unwanted restriction endonuclease sites, for facilitating DNA cloning strategies, for altering or eliminating any potential codon shift, for altering or improving GC content, for eliminating or altering alternative reading frames, and/or for elimination or changes of intron/exon splicing recognition sites, polyadenylation sites, Shine sequences

Dalgarno, unwanted promoter elements, etc., which may be present in the native DNA sequence. It is also possible to use synthetic DNA sequences to introduce other improvements into the DNA sequence, such as introducing an intronic sequence,

From the creation of a DNA sequence that is expressed as a fusion protein to organelle-targeted sequences such as chloroplast transit peptides, apoplast/vacuole-targeted peptides, or peptide sequences that result in retention of the resulting peptide in the endoplasmic reticulum. Synthetic genes can also be synthesized using host-preferred codons for improved expression, or can be synthesized using host-preferred codons. See, for example, Satrbeiyi apa Szom/ugi (1990) Riapi RpuzhioY. 92:1-141; US Patent Mo 6,320,100; 6,075,185; 5,380,831; and 5,436,391, published US patent applications Moe 20040005600 and 20010003849, and Mygau et al. (1989) Mysivys Asiyd5 Kev5. 17:477-498, incorporated herein by reference.

In one embodiment, the target polynucleotides for expression are targeted to the chloroplast. In this method, if the target polynucleotide is not directly introduced into the chloroplast, the expression cassette additionally contains a polynucleotide encoding a transit peptide to direct the target nucleotide into the chloroplast. Similar transit peptides are known in the art. See, for example, Mop Neii/|pe and others. (1991) Riapi Moi. Cheers! Ner. 9:104-126; SiagkK and others. (1989) 9.

VioI. Snet 264:17544-17550; Reiia-siorra and others. (1987) Riapi Rpuzio!. 84:965-968; Kopteg and others. (1993)

Viospet. Viorpuz. Be5. Sottype 196:1414-1421; and Pan and others. (1986) Zsiepse 233:478-481.

Target polynucleotides directed to the chloroplast can be optimized for expression in the chloroplast, taking into account the difference in codon usage in the plant nucleus and this organelle. In this way, target polynucleotides can be synthesized using codons preferred for chloroplasts. See, for example, US Patent No. 5,380,831, incorporated herein by reference.

Such a plant expression cassette can be introduced into a plant transformation vector. A "transformation vector" should be understood as a DNA molecule that makes cell transformation possible. Such a molecule may consist of one or more expression cassettes and may be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors to encode all the necessary cis- and trans-acting functions for the transformation of plant cells (NeiYep5 apa

MiiPpeaih (2000) Ttepav ip Riapi Osiepse 5:446-451). "Vector" means a polynucleotide construct designed for transfer between different host cells. "Expression vector" means a vector capable of including, integrating and expressing a heterologous DNA sequence or its fragments in a foreign cell.

A plant transformation vector contains one or more DNA vectors to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors that contain more than one contiguous segment

DNA. These vectors are often denoted as binary vectors. Binary vectors, like vectors with helper plasmids, are most often used for Adgobasiegiit-mediated transformation, where the size and complexity of the DNA segments required to achieve efficient transformation is very large and it is advantageous to separate the functions into individual DNA molecules. Binary vectors typically contain a plasmid vector containing cis-acting sequences required for T-DNA transfer (such as a left border region and a right border region), a selectable marker designed to be expressed in a plant cell, and a "targeting polynucleotide" (polynucleotide , designed with the possibility of expression in a plant cell, for which it is desirable to create transgenic plants). This plasmid vector also contains sequences necessary for bacterial replication. The cis-acting sequences are arranged to allow efficient transfer into and expression in plant cells. For example, the selectable marker sequence and the target sequence are located between the left and right border regions. Often, the second plasmid vector contains trans-acting factors that play the role of an intermediate link in the transfer of T-DNA from Adhorasiegisht to plant cells. This plasmid often contains virulence functions (Mig genes) that make it possible to infect plant cells

Adhorasiegisht, and DNA transfer by cleavage at border sequences and mig-mediated DNA transfer, as understood in the art (NeiPep5 apa MiiPpeaih (2000) Tgepavz ip Riapi bsiepse, 5:446-451). Several types of Adgobasiegisht strains can be used for plant transformation (eg, I BA4404, 5MUZ3101, ЕНА1ТО1, ЕНАТО5, etc.) The second plasmid vector is not necessary for the introduction of polynucleotides into plants by other methods, such as microprojection, microinjection , electroporation, polyethylene glycol, etc.

E. Transformation of plants

Methods according to the invention include introducing a nucleotide construct into a plant. By "introduction" is meant the presentation of a nucleotide construct to a plant in such a way that said construct gains access to the interior of a plant cell. The methods according to the invention do not

These require the use of a special method for introducing the nucleotide construct into the plant, only that the nucleotide construct gains access to at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods. See, for example, methods of transformation of plant cells and plant regeneration, described in: 05 4,459,355, 5 4,536,475, 5 5,464,763, 05 5,177 , 005 5,100,792, 05 5,371,014, 005 5,478,744, 005 5,179,022, 05 5,565,346, 05 5,484,956, 05 5,508,468, 05 5,538,877, 05 5,554,798, 05 5,489,520, 05 5,510,318, 05 5,204,253, 05 5,405,765, ЕР 442 174 АТ, ЕР 486 233 АТ, ER 486 234 JSC, ER 539 563 JSC, ER 674 725 A1,

MO91/02071, M/O95/06128 and M/O2011/095460, each of which is incorporated herein by reference, particularly with respect to the described transformation methods.

In general, methods of plant transformation include the transfer of heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of the maximum threshold level of a suitable selector (depending on the selective gene marker) to isolate transformed plant cells from untransformed cell mass. Explants are usually transferred to the same fresh medium and cultured as planned. After that, the transformed cells are differentiated into sprouts after being placed in a regeneration medium with the addition of the maximum limiting level of the selective agent. The sprouts are then transferred to a selective rooting medium to produce a rooted sprout or seedling. The transgenic seedlings are then grown to mature plants and similar seeds are harvested (eg, Nyei et al. (1994) Te Riapi doigpa! 6:271-282; Ibpida et al. (1996) Maishge

Viotesppoiodu 14:745-750). Explants are usually transferred to the same fresh medium and cultured as planned. A general description of the techniques and methods of creating transgenic plants can be found in Auge5 apa Ragk (1994) Styisa! Kemiemu5 ip Riapi Zsiepse 13:219-239 apa

Wottipepi apa dashnag (1997) Maudisa 42:107-120. Since the transformed material contains many cells, both transformed and non-transformed cells are present in any part of the target callus to be treated, or tissue or group of cells. The ability to kill untransformed cells and allow transformed cells to grow leads to transformed plant cultures. Often, the ability to harvest untransformed cells is a limitation to the rapid isolation of transformed cells and the successful creation of transgenic plants. Molecular and biochemical methods can be used to confirm the presence of an integrated heterologous target gene in the genome of a transgenic plant.

Generation of transgenic plants can be accomplished by one or more methods, including, but not limited to, introducing heterologous DNA into plant cells using

Adgorasiegiist (Agorasiegiit-mediated transformation), bombardment of plant cells with heterologous foreign DNA attached to particles, and various other direct or mediated methods of DNA transfer without the participation of particles (eg, Nieei et al. (1994) Tpe Riapi ota! 6:271- 282; IzPpida et al. (1996) Maishge Vioyesppoiodo 14:745-750; Auge5 apa Raik (1994)

Stisa! Vemivemuv ip Riapi 5siepse 13:219-239; Vottipepi apa dashiNag (1997) Maudisa 42:107-120)

Methods of chloroplast transformation are known in the art. See, for example, zmab, etc. (1990) Rhos. Mai! Asad si. BOTH 87:8526-8530; 5mab apa Mayida (1993) Rhos. Mai). Asad bsi BOTH 90:913-917; had apa Mayida (1993) EMVO 4). 12:601-606. The method is based on the delivery of DNA containing a selectable marker using a gene gun and the targeting of the DNA to the plastid genome through homologous recombination. In addition, plastid transformation can be performed by transactivation of a silent plastid-resistant transgene by preferential tissue expression of a nuclear-encoded plastid-directed RNA polymerase. Such a system was reported in Mebgiae et al. (1994) Rgos. May Asai. 5 BOTH 91:7301-7305.

Transformed plant cells can be grown into plants according to standard techniques. See, for example, MeosoptiskK and others. (1986) Riapi Sei! Kerogov 5:81-84. These plants can be grown and pollinated with the same transformed strain or other strains to produce a hybrid that has constitutive expression of the desired phenotypic characteristic.

Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds may be harvested to ensure that expression of the desired phenotypic characteristic is achieved. In this way, the present invention provides a transformed seed (also referred to as a "transgenic seed") containing a nucleotide construct according to the invention, for example, an expression cassette according to the invention, stably incorporated into the genome. In various embodiments, the seed may be coated with at least one fungicide and/or at least one insecticide,

Co) with at least one herbicide, and/or at least one preservative, or any combination thereof.

Zh. Assessment of plant transformation

After the introduction of heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as the analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

A quick method of screening transformed cells, tissues or sprouts for the presence of the included gene at an early stage before transplanting into the soil is PCR analysis (Zatbgook apa

Wizzy! (2001). PCR is performed using oligonucleotide primers specific for the target gene, or the vector background Adgobrasiegist, etc.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (ZatrgooK apa Kizzei! (2001) zirga). In general, total DNA is extracted from the transformant, cleaved with suitable restriction enzymes, fractionated in an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" can then be probed, for example, with a radioactive ZR-labeled fragment of the target DNA to confirm the incorporation of the introduced gene into the plant genome according to standard techniques (ZatbgookK apa Rizzei, 2001, zirga).

In Northern analysis, RNA is isolated from specific tissues of the transformant, fractionated in a formaldehyde-agarose gel, and applied to a nylon filter according to standard procedures commonly used in this field (ZatrgookK apa KizzeiYi, 2001 zirga).

The expression of the RNA encoded by the nucleotide sequences according to the invention is then verified by hybridization of the filter with a radioactive probe obtained from BOS by known methods (ZatrgooK apa Kyvkhzeii (2001) virga)" RNA can also be detected and/or quantified using reverse transcriptase PCR, as known in the art (eg, Sgeep apa Zbatgook (2012) MyIesshag SiIopipd: A Garogaiogu Mapiai, 4 Eyop,

Soa 5rgipd Natog I aroghaiogu Rgez55, MMoodrigu, MU).

Transgenic plants can be analyzed by Western blotting, EHISA, liquid drop assays, and biochemical assays for the presence of the protein encoded by the herbicide resistance gene by standard procedures 60 (bBatrgooK apa Vizzei! (2001) zirga) , using antibodies that bind to one or more epitopes present on the herbicide resistance protein.

In one aspect of the invention, the GFPD genes described herein are useful as markers for assessing the transformation of bacterial or plant cells. 3. Use as a transformation marker

The invention also relates to the use in a method of plant transformation of a nucleic acid encoding GFPD according to the invention, as a marker gene or coding sequence, which makes it possible to confer resistance to GFPD inhibitor herbicides in a plant, and the use of one or more GFPD inhibitor(s) in plants , containing the nucleic acid sequence encoding HFPD according to the invention. See, e.g., US Patent 6,791,014, incorporated herein by reference in its entirety.

In this embodiment, a GFAP inhibitor can be introduced into the culture medium of competent plant cells to decolorize said cells prior to transformation.

The depigmented competent cells are then transformed with a gene for resistance to GFAP inhibitors as a selectable marker, and transformed cells that have incorporated this marker into their genome turn green, making it possible to isolate them. Such a process can reduce the time required to select transformed cells.

Thus, one of the variants of the implementation of this invention consists in the method of transforming plant cells by introducing a heterologous gene into the specified plant cells with a gene for resistance to HFOD inhibitors as a selective marker, while the method includes the preparation and cultivation of competent plant cells capable of accepting the heterologous gene on a suitable medium medium, and introducing a sufficient amount of GFAP inhibitor into a suitable culture medium with competent plant cells. Competent cells are then transformed with a heterologous gene and a selectable marker, and the transformed cells containing the heterologous gene are grown in a suitable medium and transformants are selected. The transformed cells can then be regenerated into a fruiting transformed plant.

I. Plants and plant parts

"Plant" should be understood as the whole plant, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos, and their offspring. Plant cells can be

With differentiated or undifferentiated (eg, callus, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen). The present invention can be used to introduce polynucleotides into any type of plant, including, but not limited to, monocotyledons and dicotyledons. Examples of target plants include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, oilseed rape, Vgazzis species , alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cacas, tea, bananas, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond , oats, vegetables, ornamental plants and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, green peas, lima beans, peas, and species of the Sigsit family such as cucumber, cantaloupe, and cantaloupe. Ornamental plants include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. Agricultural crops may also be of interest, including, for example, corn, sorghum, sunflower, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, oilseed rape, etc.

This invention is suitable for any member of the monocot plant family, including but not limited to corn, rice, barley, oats, wheat, sorghum, rye, sugar cane, pineapple, yam, onion, bananas, coconut and dates.

I. Methods of increasing plant productivity

Ways to increase plant productivity are provided. The methods include providing a plant containing a polynucleotide, or introducing into a plant or a plant cell a polynucleotide comprising a nucleotide sequence encoding a GFPD according to the invention, growing the plant or its seeds in the field, and harvesting these plants or seeds. Here and further, the "yield" of a plant refers to the quality and/or quantity of biomass produced by the plant. "Biomass" should be understood as any measurable plant product. An increase in biomass production is any improvement in the yield of a measurable plant product. Increasing plant yield has several commercial applications.

For example, increasing the biomass of plant leaves can increase the yield of leafy vegetables for human and animal consumption. In addition, the increase in leaf biomass can be used to increase the production of pharmaceutical or industrial products from bo plants. An increase in yield may include any statistically significant increase,

including, but not limited to, an increase of at least 1 95, an increase of at least

From 96, an increase of at least 5 95, an increase of at least 10 95, an increase of at least 2095, an increase of at least 30 95, an increase of at least 50 95, an increase of at least 70 95, an increase of at least 100 95 or more.

In specific methods, a plant comprising a HFPD nucleotide sequence of the invention is treated with an effective concentration of a HFPD inhibitor herbicide, such as one or more HFPD inhibitor herbicides selected from the group consisting of Class M-(1,2) HFPD inhibitor herbicides ,5-oxadiazole-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-methylsulfonyl)-M-(1-methyl-1H-tetrazol-5 -yl/benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole-

B-yl) benzamide; M-(1,3,4-oxadiazol-2-yl)benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-methylsulfonyl)-4 -(trifluoromethyl)benzamide (Spol. 1); M-(tetrazol-5-yl)- or

M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 2), 4--difluoromethyl)-2-methoxy-3-(methylsulfonyl)-

M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro-3--"methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4 --trifluoromethyl)benzamide (Sol. 4), 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethylubinzamide (Sol. 5); pyridazinone derivatives (M/O2013/050421 and UO2013/083774); substituted 1,2,5-oxadiazoles (M/O02013/072300 and

MO2013/072402); and oxoprazine derivatives (M/02013/054495); triketones, mainly such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, mainly such as isoxaflutol; or the class of pyrazolinates, mainly such as pyrasulfotol and topramezone, where the use of the herbicide leads to an increase in the yield of the plant.

Also provided are methods of imparting herbicide resistance to a plant or part of a plant.

In similar methods, a nucleotide sequence encoding HFPD according to the invention is introduced into the plant, where the expression of the polynucleotide leads to resistance to HFPD inhibitor herbicides.

Plants generated in this manner may be treated with an effective concentration of a GFAP inhibitor herbicide (such as one or more GFAP inhibitor herbicides selected from the group consisting of M-(1,2,5-oxadiazole-Z -yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-yl)arylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-

Zo (methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole -5-yl)benzamide; M-(1,3,4-oxadiazol-2-yl)benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)- 4-(trifluoromethyl)benzamide (Spol. 1); M-(tetrazol-b5-yl- or M-(triazol-3-ylarylcarboxamides), preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5- yl)benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl/benzamide (Col. C), 2- chloro-3-"("methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3-(methylsulfinyl) -M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (M/O2013/050421 and

UMO2013/083774); substituted 1,2,5-oxadiazoles (ММО2013/072300 and УМО2013/072402); and oxoprazine derivatives (UMMO2013/054495); triketones, mainly such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, mainly such as isoxaflutol; or the class of pyrazolinates, mainly such as pyrasulphotol and topramesone), and show increased resistance to the herbicide. "Effective concentration" of a herbicide as used herein means an amount sufficient to slow or stop the growth of plants or plant parts that do not have innate or acquired resistance to the herbicide.

Y. Methods of weed control in field conditions

The present invention also relates to a method of controlling unwanted plants or regulating plant growth in plant crops comprising a GFPD nucleotide sequence according to the invention, wherein one or more GFPD inhibitor herbicides, for example, one or more

BO herbicides-inhibitors of GFPD selected from the group consisting of the class M-(1,2,5-oxadiazol-3-yl)/benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-yl"arylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole) -5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide; M-(1,3,4- oxadiazol-2-yl)benzamides, preferably such as 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-"-trifluoromethyl)benzamide (Spol 1); 1H-tetrazol-5-yl/benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Col. 3), 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3- (methylsulfinyl)-M-(1-methyl-1H-bo tetrazol-5-yl)-4-trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (M/O2013/050421 and

UMO2013/083774); substituted 1,2,5-oxadiazoles (ММО2013/072300 and ММО2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, mainly such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, mainly such as isoxaflutol; or of the pyrazolinate class, preferably such as pyrasulphotol and topramesone, are applied to plants (e.g. plant pests such as monocotyledonous or dicotyledonous weeds or unwanted crop plants), to seeds (e.g. grain, seeds or vegetative shoots such as tubers or sprouting parts with buds) or to the plant growing area (e.g. cultivation area). In this context, an effective concentration of one or more GFAP inhibitor herbicides, e.g., one or more GFAP inhibitor herbicides selected from the group consisting of class M-(1,2,5-oxadiazol-3-yl) GFAP inhibitor herbicides / benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-yl)yaurylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol- 5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)/benzamide; M-(1,3,4-oxadiazol-2-yl/benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)- 4-(trifluoromethylubenzamide (Col. 1); M-(tetrazol-5-yl)- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M -(1-methyl-1 H-tetrazol-5-yl)benzamide (Spol. 2), 4--(difluoromethyl)-2-methoxy-

3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide (Spol. C), 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazole -5-yl)-4--trifluoromethyl)benzamide (Spol. 4), -2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1 H-tetrazol-5-yl)-4- -trifluoromethyl)benzamide (Spol. 5); pyridazinone derivatives (M/O02013/050421 and UMO2013/083774); substituted 1,2,5-oxadiazoles (MUMO2013/072300 and MO2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, mainly such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, mainly such as isoxaflutol; or the class of pyrazolinates, preferably such as pyrasulphotol and topramesone), can be applied, for example, before planting (if necessary, then incorporated into the soil), before emergence or after emergence, and can be applied in combination with other herbicides to which the culture is innately resistant or resistant due to the expression of one or more herbicide resistance transgenes. See, for example, Publ. stalemate. US application Mo. 2004/0058427 and Publ. stalemate. application PCT Mo M/O98/20144. "Effective concentration" shall be understood as the concentration which controls the growth or spread of weeds or other non-transformed plants without significant

From the effect on a plant or seed resistant to the HFPD inhibitor. Those skilled in the art will appreciate that herbicide applications can take many different forms and can be made at various times prior to and/or during the seeding and growth process. "Pre-emergent" application - the application of herbicide in the desired area (eg, a field or cultivation area) before the visible emergence of the plant from the soil. "Post-emergence" application - the application of the herbicide in the desired area after the visible emergence of the plant from the soil. In some cases, the terms "pre-emergent" and "post-emergent" are used in relation to weeds in the desired zone, and in some cases these terms are used in relation to the cultivated plant in the desired zone. When used in relation to weeds, these terms may refer to a specific type or species of weed present or anticipated in the target area. "Preplant incorporation" of herbicide involves mixing the compounds into the soil prior to planting.

Thus, the present invention includes a method of controlling weeds in a field, comprising planting in a field a plant or its seed containing a GFPD according to the invention, and applying to said plant or an area surrounding it an effective concentration of one or more herbicides - GFPD inhibitors.

In one embodiment of the present invention, a field to be planted with plants (such as soybeans, cotton, corn or wheat, etc.) containing a nucleotide sequence

GFPD according to the invention can be treated with a GFPD inhibitor herbicide, such as isoxaflutol (IFT), before planting or seeding, which clears the field of weeds killed by the GFPD inhibitor and allows for zero-tillage practices, followed by planting or by sowing plants on the same pre-treated field (contact application of the GFPD inhibitor herbicide). The residual activity of IFT will also protect seedlings and growing plants from competition with weeds in the early stages of growth. When plants grow to a certain size and weeds tend to re-emerge, glufosinate or glyphosate, or a GFAP inhibitor or a mixture of a GFAP inhibitor with another herbicide, such as glyphosate, can be applied over the plants if those plants are resistant to those herbicides.

In another embodiment of the invention, a field sown with seeds containing the nucleotide sequence of GFPD according to the invention can be treated with a GFPD inhibitor herbicide, such as isoxaflutol (IFT), before plant emergence, but after sowing the seeds (the field can be pre-cleaned of borers 60 by other means, usually standard tillage practices such as plowing, deep plowing, or seedbed preparation), while the residual activity will keep the field free of herbicide-killed weeds, so that emerging and growing plants do not have competition with weeds (pre-emergence application of herbicide-inhibitor GFPD). When plants grow to a certain size and weeds tend to re-emerge, glufosinate or glyphosate, or a GFAP inhibitor or a mixture of a GFAP inhibitor with another herbicide, such as glyphosate, can be applied over the plants if those plants are resistant to those herbicides.

In another embodiment of the invention, plants containing a nucleotide sequence

GFPDs according to the invention can be treated with a GFPD inhibitor herbicide on top of plants emerging from sown seed, which clears the field of weeds killed by the GFPD inhibitor, some treatment can be carried out together with (e.g., in a sprayer tank mix), with subsequent or prior treatment with glyphosate or glufosinate as a post-emergence herbicide over plants (post-emergence application of a GFPD inhibitor herbicide (with or without glyphosate)), if such plants are resistant to these herbicides.

Examples of selected monocotyledonous and dicotyledonous weeds that can be controlled with GFAP inhibitor herbicides include:

Monocot plants-pests of genera: Aediior5, Adgorugop, Adgovii5, AIoresygi5, Arega, Amepa,

Vgasniapa, Vgotiv, Sepspgyb5, Sotteiipa, Supodop, Suregiz5, Oasiuiosiepisht, Oidnapa,

EsPipospiIoa, EiIeosNagi5, EIvivipe, Egadgobii5, Epospioa, Reviisa, Ritbiiziui5, Neiegapipega,

Itregaia, Izspaeetit, I eryuospisa, I ot, Mopospogia, Rapisit, Razra!sht, RNaiagi5, Rpieit, Roa,

Voiboeia, Zzadinata, warmed, Zeiagia, 5ogapyt.

Dicotyledonous plants-pests of the genera: Arshiop, Atagapiyi5, Atrbgozia, Apoda, Apipetiv,

Arpapez, Apetivia, Airiech, Veiii5, Videp5, SarzeiPa, Sagdtsi5, Savvzia, Sepiastea, Speporodiit,

Siquisht, Sopmoimiiiv, Oaishta, JuOevtodiit, Etech, Eguzitit, Eirpogbia, SaiIeorziv5, Saiiipzoda,

Saiisht, Nirizsiv, Irotoea, Cospia, Iatiit, I eriaiisht, I ipdetia, Maisagia, Mepia, Megsitpiaiv5,

Miiiishdo, Muozoiii5, Rarameg, RNagbiii5, Riapiado, Rouudopit, Royshiasa, Napipsshi5, Narpapyv,

Vohirra, Roiaia, Vitekh, zZaivoia, Zepesio, Zezbrapia, zida, b5ipari5, Zoiapit, 5opspyv5, Zrpeposiva, zieiYagia, Tagahasit, TNIazri, Titoiisht, Opisa, Megopisa, Mioiia, Hapipit.

GFIPD inhibitor herbicides suitable for this invention, including, but not limited to, GFIPD class M-(1,2,5-oxadiazol-3-yl)benzamide herbicides; M-

Zo (tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides, preferably such as 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol- 5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide; M-(1,3,4-oxadiazole -2-yl)benzamides, preferably such as 2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (Col. 1 ); 5-yl)benzamide (Col. 2), 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl/benzamide (Col. C), 2-chloro-3-"("methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4), 2-(methoxymethyl)-3-( methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide (Col. 5); pyridazinone derivatives (M/O2013/050421 and

UMMO2013/083774); substituted 1,2,5-oxadiazoles (МИО2013/072300 and МОО2013/072402); and oxoprazine derivatives (M/O2013/054495); triketones, mainly such as tembotrione, sulcotrione and mesotrione; class of isoxazoles, mainly such as isoxaflutol; or a class of pyrazolinates, preferably such as pyrasulphotol and topramesone, can be made in the form of different formulations, depending on the prevailing biological and/or physicochemical parameters. Examples of possible formulations are: wettable powders (SP), water-soluble powders (WP), water-soluble concentrates, emulsifying concentrates (EC), emulsions (EV), such as oil-in-water or water-in-oil emulsions , sprayable solutions, suspension concentrates (CS), oil- or water-based dispersions, oil-mixable solutions, capsule suspensions (SC), powders (PP), products for seed treatment, granules for spreading and on the ground, granules (GR) in the form of microgranules, sprayable granules, coated granules and adsorption granules, water-dispersible granules (VG), water-soluble granules (RG), ultra-low volume formulations (OLM), microcapsules and waxes.

These individual forms of recipes are in principle known and described, for example, in: Umippaskeg- kaspieg, "Spetisspe Tesppoiodije" |Spetisa! Tesppoiodu|, moishte 7, S. Napseg Mepyad Mypisn, At

Eh. 1986; U/ade map MaIKeprigu, "Reviiside Eoptiiaiopv", Magse! Oekkeg, M.U., 1973; K. Mapepv, "Brgau Oguipa" Naparook, Zga Ea. 1979, Sh. Xuoodm/ip Tsa. And opdop.

Additional substances required in formulations, such as inert substances, surface-active substances, solvents and other additives, are also known and described, for example, in: Umaikip5, "Napabook oi Ipsesiiside Bivi Oiishepiv apa Sapiegv", 2pa Ea., bapapa VookK5, Saydmzhey! M.9)., N.m. Oirnep, 60 0 "Ppshtodysiop o Siau SoPoid Spetivigu"; 2pa Ea., 9). M/Peu b 5op5, M.U.; b. Magzayep, "Zoimepiv

Zo

Spcyde"; 2pa Ea., Ipiegesiepse, M.M. 1963; MeSshspeopye "Oeviveyegdepiv apa Etyiviyegtv Appia!", MO

Fish Sogr., Viddezhmosa M.).; 5izvieu apa M/osa, "Epsusioreedia oi Zipase Asiime Adepiv", Spet. Fish

Co. Ips., M.M. 1964; bspbpgeidi, "Sgeplpaspepakiime Atuiepokhidaadikie" HIpiegase-asiime eipuiieepe ohide addisiv|, Mi. Mepadzaezei!., eiindapy 1976; MippasKeg-kaspeg, "Spetibzspe Tesppoiodie"

ISpetisa! Tesppoiodu), moishte 7, S. Napseg Mepyad Mypisn, 4 Ea. 1986.

Based on these formulations, it is also possible to prepare combinations with other pesticidally active substances, such as, for example, insecticides, acaricides, herbicides, fungicides, and with preservatives, fertilizers and/or growth regulators, for example, in the form of a ready mix or a tank mix.

J. Methods of introducing a gene according to the invention into another plant

Methods of introducing the nucleotide sequence of HFPD according to the invention into another plant are also provided here. The nucleotide sequence of GFIPD according to the invention or its fragment can be introduced into another plant by repeated selection, backcrossing, varietal breeding, linear selection, mass selection, mutational breeding and/or selection enhanced by a genetic marker.

Thus, in one embodiment, the methods of the invention include crossing a first plant containing a GFPD nucleotide sequence according to the invention with a second plant to produce ET progeny plants, and selecting E1 progeny plants resistant to a GFPD inhibitor herbicide or containing the nucleotide sequence of HFPD according to the invention.

The methods may also include crossing the selected progeny plants with a first plant containing the GFPD nucleotide sequence of the invention to produce backcross progeny plants, and selecting backcross progeny plants resistant to the GFPD inhibitor herbicide or containing the GFPD nucleotide sequence according to the invention. Methods for assessing resistance to the GFPD inhibitor herbicide are described in other sections here.

The methods may also include repeating these steps one or more times in succession to obtain selected progeny plants of the second generation or higher backcross, resistant to the GFPD inhibitor herbicide or containing the GFPD nucleotide sequence according to the invention.

In the method according to the present invention, any dilution method can be used,

Which includes the selection of plants according to the desired phenotype. In some embodiments, the plant

E1s can self-pollinate to produce a selected generation 2. Then, in each generation (EZ, E4, E5, etc.), individual plants representing the desired phenotype (eg, resistance to a GFAP inhibitor herbicide) can be selected until the traits will not become homozygous or fixed in the breeding population.

The second plant may be a plant having a desired property such as herbicide resistance, insect resistance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved quality fibers, stress resistance, improved reproduction, etc. The second plant can be an elite event, as described in other sections here.

In various embodiments, parts of plants resulting from crosses (whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos, etc.) can be collected for further propagation or processing (such as production of food, feed, biofuel, oil, flour, consumption, etc.)

K. Methods of obtaining a plant product

The present invention also relates to the process of obtaining a commercial product, which includes the collection and/or grinding of a culture grain, which includes the nucleotide sequence of GFPD according to the invention, to obtain a commercial product. Agronomically and commercially available products and/or compositions, including but not limited to animal feed, commodities, and plant products and by-products, which are intended for use as food for human consumption or for use in compositions and commodities, intended for human consumption, especially non-viable seeds / grain products, including (semi-) processed products obtained from such grains / seeds, where the specified product either contains whole or processed seeds or grain, animal feed, corn or soybean meal coarsely ground, corn or soy flour, corn, corn starch, soy meal, soy flour, flakes, soy protein concentrate, soy protein isolate, textured soy protein concentrate, cosmetics, hair care products, soybean oil, natto, tempeh, hydrolyzed soy protein, whipped cream, shortening, lecithin, whole edible soybeans (raw, roasted, or k edamame), soy yogurt, soy cheese, tofu, fuchu, as well as cooked, polished, scalded, baked or steamed grains, etc., should be understood as covered by the essence of this invention, if these products and compositions contain,

number of detectable nucleotide and/or amino acid sequences set forth herein hereinafter as diagnostic of any plant containing such nucleotide sequences.

The following examples are provided for illustrative purposes and are not intended to be limiting.

Experimental part

Example 1. Library of point mutants of the first generation.

Mutant GFPD RIEMMo33 (described in International Patent Application Me PCT/O52013/59598 (filed September 13, 2013) and set forth herein as 5BEO IU MO:5) was mutated at positions 334 and 336. Saturated mutagenesis of these positions was performed using the OSHIKSNAMOSEF kit

Idylip The mutants were accumulated and transformed into ОНзЗайрна E. soybean cells. Six hundred individual clones were tested for resistance to the GFAP inhibitor tembotrione (TBT). Clones were grown on IV medium plus kanamycin at 37 degrees C in a shaker until reaching OGbOOnm of 0.3. The cultures were then transferred to 30 degrees C and incubated for an additional 17 hours. Cultures were centrifuged and cell pellets were resuspended in 10 mM Nerez/KOH r 7.6, 4 mM Moaosi", 1

MM OTT. Cells were lysed by pelleting, and soluble cell extracts were obtained after centrifugation.

Mutants were analyzed by the brown color method. More specifically, HFPD extracts were assayed in a 9b-well format for resistance to HFPD inhibitor by plating on solid medium containing I Br agar, kanamycin, 5 mM tyrosine, 42 mM succinate, and inhibitor

GFPD In the main screening, 20 μl of the extract was applied in triplicate to plates containing 250, 500, 1000 or 2000 μM tembotrione. The tablets were covered with an air-permeable tape and incubated at 37 degrees C. After 24 hours, the formation of brown pigment was visually compared with samples containing RINRRO and RIEmo33. Variants showing a darker brown color in the presence of TBT than RIEmo33 were re-assayed at 0, 500, 1000, 2000 μM TBT and 500, 1000, 2000 μM diketonitrile (DKN, the active compound of isoxaflutol (IPT)), and 500, 1000, 2000 μM 1000, 2000 μM mesotrione. Variants that again showed a darker brown color were reexpressed and the extract titrated against all GFAP inhibitors tested to determine the degree of improvement.

The darkest color was shown by the double mutants Riez35R-336E and Riez35R-336U (which are hereafter denoted as RIEmo43 (5EO IUO MO:7) and RIEmo44 (ZEO IU MO:8), respectively). These proteins

Zo was produced in E. soya), purified and tested for their activity using the assays described below

GFPD

Example 2: Production and purification of HFPD proteins and determination of HFPD activity in the presence of HFPD inhibitor herbicides

GFPD proteins were produced and purified as described in M/O2011/076882. The activity of GFAP proteins in the absence or presence of GFAP inhibitors was determined using either the so-called OGD-assay or OGD-assay. a) Over-analysis: The activity of HFPD was determined colorimetrically by measuring the quantities

HFP remaining in the analytical mixture at the end of the incubation period after derivatization

HFP with 2,4-dinitrophenylhydrazine (DNF) for the formation of amber-brown 2,4-dinitrophenylhydrazone under alkaline conditions. The analysis was carried out at room temperature in 96b-well plates of Segiipeg with an E-shaped bottom profile. The analytical mixture contained 50 mM Ttgi5-NOI pH 7.8, 0.5 mM GFP, 10 mM ascorbate, 650 units of catalase, and appropriate amounts of the purified enzyme

HFPD with a total analysis volume of 100 μl. The reaction was started with the addition of an enzyme. After an incubation period of 24 minutes, the reaction was stopped by adding 50 μl of a solution of 0.04 95 DNF in 3.8 N NOI. After a further incubation period of 15 minutes, 100 μl of 5 N KOH solution was added to the reaction mixture, and the amount of unreacted GFP was measured photometrically at 405 nm.

Enzyme activity was calculated as the difference in E405 between the analysis without enzyme (negative control) and the analysis containing the enzyme (DE405-E405(negative control) - E405).

Enzyme activity (nmol of processed substrate per minute) was calculated according to the calibration curve made on GFP. The value of ri5o (the negative logarithm of the concentration of the inhibitor required to inhibit 50 96 enzymatic activity, in molar terms) was determined by the dose-response graph of GFPD activity against the tested concentration of the inhibitor (5.0 x 10-6.1.0 x 10-5.2 .5 x 10-5, 4.0 x 10-5, 7.0 x 10-5, 1.0 x 10-4, 2.0 x 10-4 and 5.0 x 10-4M), using Logistic 4-variable regression or the Sigmoid dose-response model from the software package KhNI IU Vyvzipe55 ZoiIshiop5 TSY. The result "5.6" means that the inhibition at the lowest tested concentration was greater than 85 95, and therefore the value of ri50 could not be accurately determined over the entire range of tested concentrations. A result of "2.5" means that the inhibition at the highest tested concentration was less than 25 95, and therefore the ribo value could not be accurately determined over the entire range of tested concentrations. "don't admit it." means "undefined".

p) HnaO-analysis: In the Nob-analysis, the activity of HFPD was measured at room temperature by adding the appropriate amounts of HFPD to a solution of 200 mM Ttgi5-HCII pH 7.6, 10 mM ascorbate, 20

MKM RezO4, 650 units of catalase, 8 μg of HGA-dioxygenase (HGA: homogentisate) and 600 μm of HFpP in a total volume of 1 ml. Initial reaction rates in the absence or presence of inhibitors were determined by the increase in absorbance at 318 nm due to the formation of maleyl acetoacetate (e318-11,900 M-1 cm-1). The ribo value (the negative logarithm of the inhibitor concentration required to inhibit 50 95 enzymatic activity, in molar terms) was determined from a dose-response plot of GFAP activity against the tested inhibitor concentration using 4-variable Logistic Regression or the Sigmoid Dose-Response Model from the software package provision of ХЙЙ ЙУ Vyvzipe55 Boishiop5 4. Due to the UV absorption of the tested HFPD inhibitors, concentrations of inhibitors above 100 µM could not be tested.

The result ""4" means that the inhibition at the inhibitor concentration of 100 μM was less than 25 95, and therefore the value of ri5bO could not be accurately determined over the entire range of tested concentrations. "not recognized." means "undefined".

Nar-assay allows continuous monitoring of HFPA-catalyzed formation of HGA over a certain period of time and was therefore used to determine the type of inhibition of the tested HFPA inhibitors. When, in the presence of an inhibitor, the activity of HFPD decreased in a time-dependent manner, characteristic of slow-binding or slow-binding inhibitors (for definition, see Moitisop (1982) Tgepaz Viospet. Ssbi. 7, 102-105), the inhibitor was called time-dependent (abbreviated as "zv"). When, in the presence of an inhibitor, the activity of HFPD was inhibited, but the inhibition did not decrease in a time-dependent manner, the inhibitor was called reversible (abbreviated as "reversible").

Abbreviation "po-ip" means that the type of inhibition could not be determined due to the fact that no inhibition of the corresponding variant of GFPD was observed at a concentration of 100 μM inhibitor.

When the resistance of the HFPD proteins RiIEmo43 and RIiemo44 to diketonitrile (DKN) and mesotrione (MZT) was determined using the OI analysis, it became obvious that their resistance was improved in comparison with the reference HFPD proteins RINRRO, РИСЗбЕ, РИСЗ3З3бУ, РИZZЗбУМ, С959 and РИемоЗ3З3 (see . Table 2).

Table 2

Evaluation of the resistance (riI5O) of the first generation HFPD proteins to diketonitrile (DKN) and mesotrione (MZT) using OO-analysis

FEOJ Regulations

Clone MO amino acid Activity in RINRRO

Rekag/24 min ICG 711" | in reve zno 0

RIZU 2 |E | | 066 | 53 | 56

RIZO! WITH |E HIM | 066 9 Sh | 52 | 55 seba | 5 | P | 6 (| 009 2 | 55 | »56

REMOZZI 6 |R | | 025 | 43 | 5

REMAZ 7 |P | | 039 | 4 | 46

BELT 4|, 8 |. to them 01 9 Sh | 40 | 46

Coo)

When the resistance of the GFPD proteins RiEEmo43 and RiEmo44 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT) was determined using the HOoU assay, it was found that their resistance was significantly improved compared to the reference GFPD proteins (details in Table 7 lower). More importantly, analysis of the time dynamics of inhibition by diketonitrile (DKT), tembotrione (TBT), and mesotrione (MZT) showed that the mechanism of inhibition of the RiEmo43 and RiiEmo44 GFAP proteins was significantly different from that of the reference GFAP proteins in that it changed from a time-dependent from the inhibition time characteristic of slow binding or slow, strong binding inhibitors to fully reversible inhibition (details in Table 11 below).

Example 3: Library of point mutants of the second generation

RIEmo43 and RIEmo44, obtained as described in Example 1, were used to create proteins

GFPDs containing further amino acid substitutions at positions 339 and 340 using a set

OSHIKSNAMSEF Idnpipipod. Their resistance to HFPD inhibitors, determined by OO-analysis, is given in

Tables 3.

Table C

Evaluation of the resistance (riI5O) of second-generation GFPD proteins to diketonitrile (DKN), tembotrione (TBT), mesotrione (MZT) and Spol. 2 (2-chloro-3-ethoxy-4-(methylsulfonyl)-N-(1-methyl-1H-tetrazol-5-yl)benzamide) using OD analysis. yayu ee De oyu anbeee r Tag et (ee. mcg Protein 2

REMAZ | 7 | R | E | Kk | And | 039 | 41 |556| 46 | 556 shRIgO /i 9 | R | E | And | 0 | 036 33 | 48 | 37 | 54

ESRIZO | lo | R | in | Kk | E | 039 / ze | 48 | 37 | 55 tent ik yayu rvvi zvzn. "11151815 15581 recognized se ||| kt syu 11 recognized

The resistance of HFPD proteins RIH/RI29, RIGR1IZ30, K131 and K132 to diketonitrile (DKT) and/or tembotrione (TBT) and/or mesotrione (MZT) has significantly improved in comparison with reference HFPD proteins RINRRO, РИСЗБЕ, РИЗЗ33б6У, РИЗЗбУУ, С959, and REEEEmo33. When the stability of proteins

HFPD BHRI129 and RI RI130 to diketonitrile (DKT) and/or tembotrione (TBT) and/or mesotrione (MZ3T) were determined using HOoO analysis, it was found that their resistance was also significantly improved in comparison with reference proteins HFPD RINRRO, РИСЗЗбЕ, RISZZBU, RISZZBMU,

С959, and РІЕемо33 (details in Table 7 below). Analysis of the temporal dynamics of inhibition showed that diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT) were fully reversible inhibitors of HFPD proteins RIH P129 and RI P130 (details in Table 11 below). Analysis of temporal dynamics of inhibition

Spol. 2 GFPD proteins K131 and K1I32 showed that Spol. 2 was a fully reversible protein inhibitor

HFPD КИ131 and К132 (details in Table 12 below).

Example 4: Library of point mutants of the third generation

Variants of GFPD, obtained as described in Example 3, were used to create proteins

GFPDs containing further amino acid substitutions at position 215 using a kit

OSHIKSNAMSEF Iidnipipod. Their resistance to HFPD inhibitors, determined by OO-analysis, is given in

Table 4. K299, which carries only the P2151I mutation, was used as a second standard in addition to RINRRO.

Table 4

Evaluation of resistance (pI5O) of third-generation HFPD proteins to diketonitrile (DKN), tembotrione (TBT), mesotrione (M3ZT) and Spol. 2 (2-chloro-3-ethoxy-4-"methylsulfonyl)-M-(1-methyl-1H-tetrazole-

B-yl)benzamide) using Ob-analysis. I roar | heTview threads Tag t |. in µg Protein 2

RINRRO in 1 / R | E | and | Kk | And | 0.76 | 556 | 55.6 | 55.6 | 55.6

REMYAZ | 7 | R | R | E | Kk | And | 039, | 41 |556.| 46 | 556

ESHR2O2 | 12 1 | R | is | Kk | E | 0.09. | "25 36 | "25 45

ESRIZ6 | 11 1 2 HER R | is | to | A / 009 / 26 | 47 | 33 | 48

ERIg3 | 9 | R | R | E | And | 0 | 0.936 / 33 | 48 | 37 | 54

ESRIZO | lo | R | R | is | to | E | 039 | ze | 48 | 37 | 55

Table 4

ZEO I Amino acid position in . dk 215 | 335 | 336 | 339 z4o |SeyaB/ayah!dkn o |tBto|mzto|Spol. in μg Protein 2 ter vi i|y 1 ki |ovvrav in rezn.

Feb | in: | and quarter | 25 | with recognition

SEZNY NE NN SNY STAGE NU NSHU NI PEUCHISSI recognized.

SEN NESTI NS S BUT NOT OST NOSA VE NOT NESLO recognized. che ||| i1kTk syu |i recognized.

The resistance of HFPD proteins with the P215Y mutation to diketonitrile (DKT), tembotrione (TBT), and mesotrione (MZ3T) was significantly improved compared to reference HFPD proteins. When the resistance of HFPD proteins RI P136 and RIH P202 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZ3T) was determined using the HOoU assay, it was found that their resistance was significantly improved compared to the reference HFPD proteins (details in Table 7 lower). Analysis of the temporal dynamics of inhibition showed that diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT) were fully reversible inhibitors of HFPD proteins RI /RI136 and RI P202 (details in Table 11 below). Analysis of the temporal dynamics of inhibition of Spol. 2 GFPD proteins K137 and K2O3 showed that Spol. 2 was a fully reversible inhibitor of GFPD proteins K137 and K20O3 (details in Table 12 below).

Example 5: Library of point mutants of the fourth generation

The HFPD variant EGR202, obtained as described in Example 4, was used to generate HFPD proteins containing further amino acid substitutions at position 215 using a kit

OSHIKSNAMSEEF Iopipipod. Their resistance to HFPD inhibitors, determined by UR-analysis, is given in

Tables 5.

Table 5

Evaluation of the resistance (pI5O) of fourth-generation HFPD proteins to diketonitrile (DKN), tembotrione (TBT), mesotrione (MZT) and Spol. 2 (2-chloro-3-ethoxy-4-"methylsulfonyl)-M-(1-methyl-1H-tetrazole-

B-yl)benzamide) using Ob-analysis.

We CARRY NNYA in µg Protein 2

RSRIZO | 10 | R | R | R | E | 039 | 32 | 48 3/7 | 55

РР2О2 | 12 | and | R | in | E | 009 |-«25)| 36 "25| 45

Kab | 18 | And | R | E | E | 009 | 30 45 | 37 | 50

Kka; in | 19 | m | R | ER | E | 05 |-25) 44 | 34 | 54

Resistance of GFPD proteins K255 and K258 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (M3ZT) was significantly improved compared to reference GFPD proteins. Analysis of the temporal dynamics of inhibition of Spol. 2 GFPD proteins K255 and K258 showed that Spol. 2 was a fully reversible inhibitor of GFAP proteins K255 and K258 (details in Table 12 below).

Example 6: Library of point mutants of the fifth generation

Variants of HFPD RGGR130, obtained as described in Example 3, and EGR202, obtained as described in Example 4, were used to create HFPD proteins containing further amino acid substitutions at positions 226 and 340 using the OSHIKSNAMSEF Idnipipd kit. Their resistance to HFPD inhibitors, determined by Or-analysis, is shown in Table 6.

Table 6

Evaluation of resistance (pI50O) of fourth-generation HFPD enzymes to diketonitrile (DKN), tembotrione (TBT), mesotrione (MZT) and Spol. 2 (2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide) using OO analysis.

Repay/24x Spol.

Pere reeroe r r rnesvvya Ge et ek.

Р2О2 | 12 | | ORE | KE 009 / "25 36 | "25 45 kzoo her 21 | ORI EC || 001 / "25| ZL | "25 38 kzob 1 24 | | 3. RE | KE 001 / "251 48 | 37 | 5 kz63 | 29 | R | 3. | R | is | K | E | 020 | 38 | 55 | 43 | 54

The resistance of HFPD proteins KZO0 and K306 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT) improved in comparison with reference HFPD proteins. Analysis of the temporal dynamics of inhibition of Spol. 2 GFPD proteins KZO0, KZ306 and KZ63 showed that Spol. 2 was a fully reversible inhibitor of HFPD proteins KZO0, KZ306 and KZ363 (details in Table 12 below).

Example 7: Resistance of GFPD variants generated as described in Examples 1-6 to inhibitors

GFPD determined by HOoO analysis

The resistance of HFPD variants created in Examples 1-6 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT), determined by HOb analysis, is shown in Table 7.

Table 7

Evaluation of the resistance (ri5O) of GFPD variants created in Examples 1-6 to diketonitrile (DKT), tembotrione (TBT) and mesotrione (MZT) using the NoO analysis 7711... |2151|3935| 336 | 339 | from 40 DdKn. tBT //- |m3t

RINRRO in 1 | R | E | and | Kk | And | 58 | 64 | 58 rRIZZ3bMI 4 | R | E | M'| Kk | And | 53 | 6 | 55 /

RIZO! 2 | R | E | is | Kk | And | 51 | 59 | 51 rRIZZvMI Z | R | E | M | Kk | And | 49 | 55 | 50

REMOZZ | 6 | R | R | M | Kk | And | 46 | 62 | 55

REMAZ | 7 | R | R | is | Kk | And I 40 | 60 | 51

REMYAA4 | 8 | R | R | M | Kk | A Y 41 | 59 | 52

RIgO i 9 | R | R | E | A. | 2 | 46 | 62 | 50

ESRIZO | 10 | R | R | is | Kk | EE Her 43 | 62 | 53

CUT | 11 | 4 | R | E | Kk | And she is 40 53 | 45

Р2О2g | 12 | Her | R | is | Kk |! EI "4 | 42 | "4

Stability of the GFPD options created in Examples 1-6 to Spol. 1 (2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-methylsulfonyl)-4-(trifluoromethyl)benzamide), Spol. 2 (2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide), Spol. C (4-(difluoromethyl)-2-methoxy-

3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide), Spol. 4 (2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)benzamide) and Spol. 5 (2-(Methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide), determined by HOoO analysis, is given in Table 8.

Table 8

Evaluation of the stability (pi5O) of the GFPD variants created in Examples 1-6, using HOr-analysis

ZEO And not. hours

Clone MO Position of mutations in RINRRO Herbicides-inhibitors of GFPD 71 ..yu.yuyu |215|226 | 335 | 336 | 339 | 340 | Spol. 2| Spol. 1| Spol. Z|Spol. 4| Spol. 5

RINRRO | 1 |P OO |E |c0 |kK A | 67 | 63 | 66 | 70 | b riazz3bu| 4 |R |O |E |M |K A | 68 | be | 64 | 67 | b

R.RI2gU | 9 |R OO |R |E TA (0 | 58 | b | 59 66 | b

RSRIZO | lo |P 0 |P |E |K is | 62 | be | 64 | 66 | 60 se Gete ke ee tn (tn (Mn Hn recognized | recognized | recognized | recognized

SEZNN NESLO STI S STVO SUNOSUNO VEST PSY NSB recognized. recognized

SEASONAL KNOWN DECLINE OF PINE SUS EN recognized. | recognized | recognized | recognized se and GE E UE ze t» b b b recognized. | recognized | recognized | recognized

R. RIZB | 11 |- TO |R |E |K A | 59 | 56 | 56 | 60 | 56

РрР2гоО2г | 12 | 0 |P | |K E | 47 | 41 | 43 | 57 | 43 st Ge er yeu en (chn (tn (tn recognized | recognized nn recognition |recognition |recognition |recognition se Ge ЕЮ ze rn Gb» Hn recognition |recognition |recognition |recognition ee Ge ЕЮ t er Te Te recognition |recognition |recognition |recognition

Example 8: Library of point mutants of the sixth generation

The HFPD variants RGR130, obtained as described in Example 3, EGR202, obtained as described in Example 4, and K255 and K258, obtained as described in Example 5, were used to generate HFPD proteins containing further amino acid substitutions at positions 188 and 189 using set OSHIKSNAMOEF IIpipipu. Their resistance to GFAP inhibitors, determined by OOR analysis, is shown in Table 9.

Table 9

Evaluation of the resistance (ri5O) of the sixth-generation GFPD proteins to diketonitrile (DKN), tembotrione (TBT), mesotrione (MZT) and Spol. 2 (2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide) using OD analysis

ZEO I. . south:

Clone MO: Amino acid position in RINRRO | Activity Inhibitors of GFPD 1 re rea oo vu revsova mcg Protein 2

РР2О2 | 12 | And | in | SG | R | E | E | 009 |-25)/36 25 45

Ka58 | 19 | And | In | M | R | E | E | 05 |-25)| 44 | 34 | 54 kab7 | 26 | and | with | R | RE |E | 047 |35|53| And | 5A4

KkagBbo | 17 | JSC | RI |E | 007 |-25)| 38 30 | 46 kzagh; | 25 | and | with | m /|RIE|E | 041 |-25)|46| 36 | 56 ko5 | 30 | and | C | And | R | EE | E | 06 | 33 | 48 | 37 | 49 kob | 31 | and sa | m | RI | E | 052 /|-25)|45| 35| 56 ko7 | 32 | and E | M | R | E | EE | 041 |«25)| 46 | 36 | 57 kov | with 3 | with | 0 | mm R | ER | E | 056 |«25) 46) 365

The combination of mutations at positions 188 and 189 with mutations at positions 215, 335, 336, and 340 positively affects the activity level of enzymes that still show a high level of inhibitor resistance. Analysis of the temporal dynamics of inhibition of Spol. 2 GFPD proteins K250, KZ25 and

KZ357 showed that Spol. 2 was a fully reversible inhibitor of HFPD proteins K250, KZ325 and K357.

The value of ribo Spol. 2 against GFPD proteins K250, KZ325 and KZ3Z57 of the past defined as "4, 5.51 5.1, respectively.

Example 9: Library of point mutants of the seventh generation

Variants of HFPD RGGR130, obtained as described in Example 3, and EGR202, obtained as described in Example 4, were used to create HFPD proteins containing further amino acid substitutions at position 200 using the OSHIXNAMSEF Idpippd kit. Their resistance to HFPD inhibitors, determined by OO analysis, is shown in Table 10.

Table 10

Evaluation of the resistance (pI50) of the seventh-generation HFPD enzymes to diketonitrile (DKN), tembotrione (TBT), mesotrione (M3ZT) and Spol. 2 (2-chloro-3-ethoxy-4-"methylsulfonyl)-M-(1-methyl-1H-tetrazole-

B-yl)benzamide) using Ob-analysis.

ZEO I. . skjav re v rvevu Ievoe le Teg reg ety m g Protein 2

SHR2O2 | 12 | 4 | 1 7 R | is | E | 009 /|25)136 25|45 kzo3 1 22 | AND! | 4 1 R | is | EE | 0008 |25142 32 | 46 kzo4 1 23 | M | her R | is | E | 001 //25142 32| 46 kZzbo 1 27 | And | R | R | E | EE | 028 //36153 43|53 zbi | 28 | M | R | R | There is | E | 021 |z34/| 51140 | 52

The combination of mutations at position 200 with mutations at positions 215, 335, 336, and 340 results in GFAP variants that still show high levels of resistance to GFAP inhibitors. Analysis of the temporal dynamics of inhibition of Spol. 2 GFPD proteins KZ303, K304, KZ360 and KZ61 showed that Spol. 2 was a fully reversible inhibitor of HFPD proteins KZ303, K304, KZ60 and KZ61 (details in Table 12 below). The meaning of ri5Bo Spol. 2 against GFPD proteins KZO03, K304, KZ360 and KZ61 of the past were determined as 5.0, 4.7, 5.5 and 5.3, respectively.

Example 10: Analysis of the type of inhibition of HFPD variants by HFOD inhibitors

The type of inhibition of the HFPD variants obtained as described in Examples 1-9 by different inhibitors

The GFPD determined using the above-described NOA analysis is shown in Tables 11 and 12.

Table 11

The type of inhibition of HFPD variants by DKN (diketonitrile), TBT (tembotrione) and MZT (mesotrione) was determined using the HOoU analysis.

ZEO I g. from 777 ..yu.yuyuyu | 215| 335 | 336 | 339 | z40 |DKN |tBT (mzt

RINRRO | 1 | R | E | b | Kk | And | M | Kk | A zv (zv (zv

REMOZZ | 6 | R | R | M | Kk | And zv (zv (zv

REMAZ | 7 | R | R | E | Kk | A (revolution (|revolution |revolution,//

Rhema. | 8 | R | R / U | Kk | A (revolution (|revolution |revolution,//

EshRIgO | 9 | R | R | E | And | OO (revolution (|revolution |revolution,//

ESHRIZO | lo | R | R / E | CC | There is Tsobor (|obor, |obor,/7/

ESHRIZ6 | 711 | E Y R in 1 KO A (turn, |turn |turn,//

ESHR2O2 | 712 | | R | in | Kk | is Tspo-n // |obor,(|po-n/Z

Table 12

Type of inhibition of variants of GFPD Spol. 1 (2-methyl-M-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide), Spol. 2 (2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide), Spol. C (4--difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide), Spol. 4 (2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide) and Spol. 5 (2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl)-4--trifluoromethyl)benzamide), determined by HOr analysis.

ZEO

Clone IO The position of mutations in RINRRO Herbicides-inhibitors of GFPD

Mo

. Tgo0|215|226|335|336| 3391340 | Spol. 2 | Spol. 1 | Spol. With | Spol. 4 | Spol. 5

RINRRO | 1 | (R TO |E (s |kK TA (zv. zv. (zv, |zv (zv rRISHZZZ6MU| 4 | (R TO (EE |M/ |K TA (zv. zv. (zv (zv)

EshRI2go | 9 | R TO |Р R TA WHAT Tsobor |obor )|obor |obor |obor

EshRIZO | lo | R TO |Р R |K TE Cathedral | 14 (S (R TO (R YIM |K TE Cathedral |obor )|unrecognized obor |not recognized kiz7 |15|- | To (R YIM |K TA Oobor |/not recognized|not recognized| not recognized | not recognized

Kgoz | 16 | o (R YIM |K TE Oobor |/|not recognised|not recognised| not recognised| not recognised

EshRIZb6 | 1117

ESHR2Og | 12 |. (S To (Р R |K TE Cathedral |turn)|turn |turn |turn

Kgo5 |18 | TA TO |R |E |K THIS Oobor |/|not recognized|not recognized| not recognized not recognized

Kg58 | 19| |m TO (Р R |K CE Oobor |/|not recognized|not recognized| not recognized| not recognized kz0o |2g1 | | To (Р R |kK (b Oobor |/not recognized|not recognized .| not recognized| not recognized kz63 | 29| |R 3. (R |E |K ČE Tsobor |/|not recognized|not recognized| not recognized| not recognized kzo6 | 24 | | (3 . | not recognized| not recognized kzo4 | 2gz|Mm | To (Р R |K TE Oobor |/|not recognized|not recognized| not recognized| not recognized kzbo | 27 |! |Р TO |Р |E |K THIS Oobor |/|not recognized| not recognized| not recognized| not recognized because |28|m (TO |R |is |K THIS Oobor not recognized| not recognized| not recognized | not recognized

All variants of HFPD obtained as described in Examples 1-9 have no or significantly reduced affinity for HFPD inhibitors, and at the same time the rate of dissociation of HFPD inhibitors from the enzyme is increased to such an extent that the HFPD inhibitors no longer act as slow-binding or slowly, tightly binding inhibitors, but become fully reversible inhibitors.

Example 11. Transformation of soybeans

Transformation of soybeans is achieved using well-known methods, such as those described for the transformation of semi-seed explants of soybeans, mediated by Adgobasiegisht ishptegasiepv, using mainly the method described by Ra; etc. (2006), Riapi se Ver. 25:206.

Transformants were identified using tembotrione or isoxaflutol as a selective marker. Green sprouting was observed, documented as an indicator of resistance to the herbicides isoxaflutol or tembotrione. Resistant transgenic sprouts showed normal greening compared to wild-type soybean sprouts not treated with isoxaflutol or tembotrione, while wild-type soybean sprouts treated with the same amounts of isoxaflutol or tembotrione were completely decolored. This indicates that the presence of the HFPD protein creates the possibility of resistance to HFPD inhibitor herbicides such as isoxaflutol or tembotrione.

Resistant green sprouts were transferred to a medium for rooting or grafted.

The rooted seedlings were placed in the soil and transferred to the greenhouse after a period of acclimatization. Then, the plants containing the transgene were sprayed with GFPD inhibitor herbicides, for example, tembotrione in the amount of 100 g of Aga or mesotrione in the amount of 300 g of AI/ha with the addition of ammonium sulfate and rapeseed oil methyl ether. Ten days after the application of the herbicide, the symptoms that appeared were evaluated and compared with the symptoms observed in wild-type plants under the same conditions.

Example 12. Resistance of soybean plants to mesotrione

TO soybean plants that express a GFAP inhibitor-resistant enzyme according to the present invention together with a gene conferring resistance to glyphosate and a gene conferring resistance to glufosinate or having a "plant expression cassette"? containing only the enzyme resistant to GFAP inhibitors, was tested for resistance to mesotrione. Prior to greenhouse trials of transgenic plants, soybean transformants were routinely screened for the expression and presence of transgenes using the EG I5A protein detection method (see Sections G and E for a detailed description).

For the analysis of resistance to herbicides, only plants isolated on a selective medium and having the expression of the transgenic protein GFPD, which is detectable, were used. Sprayer

Oemgieee TgasKkeg Zrgaueg was calibrated before each spraying. The chemical formulation used in the test for mesotrione (MZT) was the formulation of SaiysioF 4 KS with the addition of ammonium sulfate and methylated rapeseed oil (Asiyhor). Spray tests were conducted using a 3X field rate (equivalent to 9 fluid ounces per acre of the same herbicide formulation containing 40 95 active ingredient (AI) mesotrione), which equates to 316 grams AI per acre. Resistance was assessed one week after spraying. Wild-type soybean plants sprayed with the same herbicide formulation were completely discolored and showed 100 95 damaged leaves. The resistance rating "O" was assigned to plants in which the tops of sprouts, newly released shamrocks and some axillary buds were completely discolored. A rating of "1" was assigned to plants with low resistance, meaning the youngest shoot tissues had some green leaves and were not completely discolored. The rating "2" was assigned to plants with medium resistance, that is, more than 50 95 of the leaf area of the three upper shamrocks did not show chlorosis or discoloring damage. A rating of "3" was assigned to plants with almost complete resistance, that is, less than 10 95 of the leaf area showed chlorosis or discoloration. The results are shown in Table 13.

Table 13

Evaluation of damage to the leaf area of transgenic TO soybean events expressing different variants of GFPD after seven days of treatment with mesotrione at an amount of ZX from the field, equivalent to 316 grams of AiY per hectare. A rating of "1" was assigned to plants with low resistance, meaning the youngest shoot tissues had some green leaves and were not completely discolored. The rating "2" was assigned to plants with medium resistance, that is, more than 50 95 of the leaf area of the three upper shamrocks did not show chlorosis or discoloring damage. A rating of "3" was assigned to plants with almost complete resistance, that is, less than 10 95 of the leaf area showed chlorosis or discoloration. Different plant expression cassettes were constructed either with enzyme(s) conferring resistance only to GFPD-inhibiting herbicides (Ht-single) or conferring resistance to three herbicides with different mechanisms of action (Ht-triple), i.e., inhibitors

GFPD, glyphosate and glufosinate. gFPD MO: expression 0 | 1 | 2 | 3 | plants

RIEMO43 | 7 |NTtriple// | 6 | 718 | 26 | 20 | 7

RIEMOD4D | 8 |NTtriple// | 4 | 22 | 19 | 26 | l

RIEMO44 | 8 |NTodinarly.iu/ | 7 | 714 | 43 | 21 | 85

ESHRI2 | 19 |NTtriple// | 13 | 718 35 | 12 | 78

SHR2O2 | 12 |NTpotrynyi// | 7 | 8 | lo | 14 | 39

Of the 713 regenerated and selected transgenic transformants, more than 25 95 showed a high level of resistance with less than 10 95 lesions per total leaf area. A total of -70,95 tested transgenic plants were visually rated as having a moderate to high resistance rating.

In addition, the expression of three different enzymes conferring resistance to three different classes of mechanisms of action (GFPD inhibitor compounds, glyphosate and glufosinate) did not interfere with the manifestation of resistance to the GFPD inhibitor. Transgenic plants transformed only with resistance to the GFPD inhibitor

With the enzyme variant according to the present invention, they showed a distribution of ratings similar to that of accumulated events.

Example 13: Creation and selection of TO cotton plants.

Cotton transformations were achieved using well-known methods, giving particular preference to the method described in patent publication MUO 00/71733. The regenerated plants were transferred to the greenhouse. After the acclimatization period, sufficiently grown plants were sprayed with GFPD inhibitor herbicides, for example, the equivalent of tembotrione from 100 to 200 g AI/ha with the addition of ammonium sulfate and rapeseed oil methyl ether. Seven days after the application of the spray, the symptoms that appeared were evaluated and compared with those observed in wild-type cotton plants subjected to the same treatment under the same conditions.

Example 14. Transformation of corn plant cells by Adgobasiegiite-mediated transformation

The construction of a plant expression cassette for stable expression in a maize plant and the transformation of maize are well known in the art, and this particular example describes and utilizes methods from patent publications UU02014/043435 and UM/O2008/100353.

The polynucleotide sequences encoding the GFPD variants in this application were fused to the DNA sequence encoding the ERBR5 pain variant to confer resistance to ERBR5-targeting herbicides. The ERBR5 gene was isolated and mutated from Agipgobasieg dioritogts (MUO2008/100353) and joined in-frame to a transit peptide sequence for the direction of translocation of the translated protein to the chloroplast. Stable expression was achieved using a common promoter (ubiquitin-4 promoter from sugar cane, Patent

US Mo 6,638,766) and the interruption sequence 355 from the cauliflower mosaic virus, which were respectively cloned upstream and downstream of the ERBR5 gene.

The corresponding variants of HFPD were cloned with the same promoter, chloroplast transit peptide and interruption sequence as described for the ERBR5 gene expression cassette. The coding sequences of both genes were codon-optimized for expression in corn. For corn transformation, the cobs are best harvested 8-12 days after pollination. Embryos were isolated from cobs, and embryos 0.8-1.5 mm in size were mainly used for transformation.

Embryos were placed shield-side up on a suitable incubation medium and incubated overnight at 25 °C in the dark.

However, it was not mandatory to incubate the embryos overnight. Embryos were contacted with Adgobasiegisht strain containing the desired vectors comprising the nucleotide sequence of the present invention for T-plasmid-mediated transfer,

Zo for about 5-10 min, and then put on the medium for co-cultivation for about 3 days (at 25 "C in the dark). After co-cultivation, the explants were transferred to the medium of the recovery period for about 5 days (at 25 "C in the dark) .

Explants were incubated on selective medium with glyphosate for up to 8 weeks, depending on the nature and characteristics of the specific selective medium used.

After the selection period, the resulting callus was transferred to the embryo maturation medium until the formation of mature somatic embryos was observed. The resulting mature somatic embryos were then placed under low light and initiated the regeneration process as is known in the art.

The resulting sprouts were allowed to root in a rooting medium, and the resulting plants were transferred to seedling pots and propagated as transgenics. Plants were regularly analyzed for the expression and presence of transgenes using the protein detection method

EGIB. For the analysis of resistance to herbicides, only plants isolated on a selective medium and having a detectable expression of the transgenic protein GFPD were used.

Example 15. Resistance of TO plants of corn to GFPD herbicides in greenhouse studies

Regenerated TO events from cell culture were transplanted into 2-inch square pots with synthetic soil (Raiagaf Mich) and a controlled-release fertilizer (Naiiia

Miyisoieit; polymer-coated fertilizer with controlled release, MRK Rgo 18-6-12 micronutrients) and grown in a greenhouse (TP) under additional high-pressure sodium lamps for 12 days at a maximum temperature of 30 "C during the day and a minimum of 22 "C at night.

Fully recovered plants were transferred to 5-inch square pots with synthetic soil and controlled-release fertilizer under the same environmental conditions. After the end of seven days, TO-plants were sprayed with 2-chloro-3-(methylsulfanyl)-

M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Col. 4); in an agronomically significant concentration of 100 g AI/ha (while "g Aha" means "gram of active ingredient per hectare", prepared from the recipe SP20 (wettable powder 2095), with the addition of a mixture of esterified vegetable oils (Naziepit adjuvants for sprays, 0.578 95 vol./06.) and ammonium sulfate (АМС, 0.97 95 wt/vol). Herbicide treatment was carried out in the UOemgie5 TgasKeg 5rgaueg system according to standard application protocols well known in this field. As a control event

TO was sprayed with a mixture of adjuvants without herbicide. All MOT events sprayed with this mixture had no discolored sheets. bo Unless otherwise specified, six days after treatment (DPO) 2-chloro-3-(methylsulfanyl)-M-

(1-methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)/benzamide was used to assess damage to transgenic TO events.

TO events that express the ERBR5 selectable marker gene and that do not have a type variant

GFPD, were used as control corn plants and showed 100 Uo-e of leaf damage already at 25 g of Aiga 2-chloro-3-methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl ) of benzamide.

Non-transformed corn plants also showed 100 U5-e leaf damage already at 25 g AI/ha of 2-chloro-3-(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-trifluoromethyl)benzamide .

Table 14 summarizes the transgenic maize plants expressing the mutants

EGR2O2 and BG/P136 of the protein HFPD Rzeiidotopakh Psiogesep5, obtained as described in Example 14.

Plants rated "0" showed severe leaf discoloration ranging from 41 95 to 100 95 damage per total leaf area. The rating "1" was assigned to plants with average resistance with 16-40 95 injuries from the total leaf area. A rating of "2" was assigned to plants with good resistance with 6-15,906 lesions per total leaf area. Plants assigned a rating of "3" showed almost no discoloration, with 5 95 or less leaf area damaged by the herbicide treatment.

The results in Table 14 demonstrate that a significant part of the independent events of TO of corn are resistant to the herbicide GFPD /2-chloro-3-(methylsulfanyl)-M-(1-methyl-1 H-tetrazol-5-yl)-4-(trifluoromethyl) benzamide in agronomically significant doses of 100 g AI-ha in comparison with control plants.

Approximately 40 95 tested events expressing RI P136 (p-84) showed good or high resistance with 15 95 or less discolored leaf area after treatment with 100 g AI/ha Spol. 4.

Of all EGR136-expressing corn plants tested, 15,95 show less than 5 Fo or no visible leaf damage.

The same picture is valid for the TO of corn plants that express RI P202. More than 30 95 of the total number of events tested (p-89) also showed good or high resistance with 15 95 or less discolored leaf area after treatment with 100 g AI/ha herbicide, and 13 95 showed less than 5 95 or none at all. showed visible leaf damage.

Table 14

Assessment of damaged leaf area in transgenic maize TO events six days after application of 2-chloro-3-("methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide ( Society 4) in the amount of 100 g AI/ha. Transgenic corn plants that express variants RI P202 or RI P136 of the GFPD protein of Rzeidotopavh Pyogessepo were obtained as described in Example 4. The following classes of resistance to herbicides were determined: "0": slight resistance; 41 95 - 100 95 areas of leaves are damaged; "17: medium resistance; 16 95 - 41 95 leaf areas damaged; "2": good resistance; 6 95 - 15 95 leaf areas damaged; "3" - high resistance; 0 9 - 5 95 leaf areas damaged.

ESHR2O2 77777771 171712 | 49 | 16, 1712 | 7777778984Thank you:

Coo)

All publications and patent applications mentioned in the description indicate the level of skill of those skilled in the art to which this invention belongs. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually designated as being incorporated by reference.

Although the disclosed invention has been described in some degree of detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended formulae.

Claims (30)

FORMULA OF THE INVENTION 40 1. Recombinant nucleic acid molecule encoding the 4-hydroxyphenylpyruvate dioxygenase (HFPD) protein, which consists of an amino acid sequence containing proline in a position corresponding to the position of the amino acid 335 5EO IYUO MO", and phenylalanine or tyrosine in a position corresponding to the position of the amino acid 336 5EO IU MO, 45 at the same time the specified GFPD protein is resistant to the GFPD inhibitor herbicide. 2. Recombinant nucleic acid molecule according to claim 1, which is characterized by the fact that the specified coded protein GFPD consists of an amino acid sequence that also contains: i) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 188 5EO IU MO:1; and i) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 189 5EO IO MO:1; and ii) isoleucine, leucine or methionine in the position corresponding to the position of amino acid 200 5EO IO MO:1; and them) alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 5EO IU MO1; and m) histidine or glutamine in the position corresponding to the position of amino acid 226 5EO IYUO MO:1; and mi) histidine, isoleucine, leucine, methionine, glutamine, arginine, alanine, lysine, serine, threonine or valine in the position corresponding to the position of the amino acid 339 5EO IO MO:1; and mii) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine or valine at a position corresponding to the position of amino acid 340 5EO IU MO:1. 3. Recombinant nucleic acid molecule according to claim 1, which is characterized by the fact that the specified coded protein GFPD consists of an amino acid sequence that also contains: i) alanine, glycine, histidine, serine or tryptophan in a position corresponding to the position of amino acid 188 5EO ИО МО :1; and ii) arginine, cysteine, glutamine, glutamic acid, aspartic acid, glycine, histidine, phenylalanine, or serine in a position corresponding to the position of amino acid 189 5EO IO MO:1; and ii) isoleucine, leucine or methionine in the position corresponding to the position of amino acid 200 5EO IO MO:1; and them) alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 5EO IU MO1; and m) histidine or glutamine in the position corresponding to the position of amino acid 226 5EO IYUO MO:1; and mi) serine, alanine, threonine, glutamine or lysine in the position corresponding to the position of the amino acid 339 5EO IO MO:1; and mii) alanine, arginine, aspartic acid, glutamic acid, glutamine, glycine or leucine in a position corresponding to the position of amino acid 340 5EO 10 MO 1. 4. Recombinant nucleic acid molecule according to claims 1, 2 or 3, which is characterized by the fact that the indicated GFPD protein includes an amino acid sequence that is at least 53 95 identical to the amino acid sequence set forth here as HEO IU MO:1. 5. Recombinant nucleic acid molecule according to any of claims 1-4, which is characterized in that its nucleotide sequence is a synthetic sequence intended for expression in a plant. 6. Recombinant nucleic acid molecule according to any of claims 1-4, which is characterized by the fact that its nucleotide sequence is functionally linked to a promoter capable of directing the expression of the nucleotide sequence in a plant cell. 7. Recombinant nucleic acid molecule according to claim 1, which is characterized by the fact that the indicated herbicide-inhibitor GFPD is selected from the group consisting of M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides, M-(1,3,4-oxadiazol-2-yl)benzamides, M-(tetrazol-5-yl)- or M- (triazol-3-ylaluarylcarboxamides, pyridazinone derivatives, substituted 1,2,5-oxadiazoles, oxoprazine derivatives, triketones, isoxazoles and pyrazolinates. 8. Recombinant nucleic acid molecule according to claim 7, which is characterized by the fact that the indicated herbicide-inhibitor of GFPD is selected from the group consisting of 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1 H -tetrazol-5-yl)benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-methyl-M-( 5-methyl-1,3,4-oxadiazol-2-yl)-3-methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1- methyl -1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro-3- "(methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazole -5-yl)-4-(trifluoromethyl)benzamide, tembotrione, sulcotrione, mesotrione, isoxaflutol, pyrasulphotol and topramesone. 9. A host cell containing a recombinant nucleic acid molecule according to claims 1, 2 or 3. 10. The host cell according to claim 9, which is a bacterial host cell. 60 11. The host cell according to claim 9, which is a plant host cell. 12. A transgenic plant that includes a recombinant nucleic acid molecule according to claims 1, 2 or 3. 13. The plant according to claim 12, which is characterized by the fact that the specified plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco , barley and oilseed rape. 14. Transgenic seed, including a recombinant nucleic acid molecule according to claims 1, 2 or 3. 15. Recombinant polypeptide comprising a GFPD protein, while the specified GFPD protein is resistant to the GFPD inhibitor herbicide, and the indicated GFPD protein contains proline at a position corresponding to the position of amino acid 335 5EO IU MO:1, and phenylalanine or tyrosine at the position , which corresponds to the position of amino acid 336 5EO IU MO:1, 16. Recombinant polypeptide according to claim 15, which is characterized by the fact that the specified GFPD protein also contains: i) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, serine , threonine, tryptophan, tyrosine or valine in a position corresponding to the position of amino acid 188 5EO I1O MO:1; and i) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine in a position corresponding to the position of amino acid 189 5EO IO MO:1; and ii) isoleucine, leucine or methionine in the position corresponding to the position of amino acid 200 5EO IO MO:1; and them) alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 5EO IYUO MO:1; and m) histidine or glutamine in the position corresponding to the position of amino acid 226 5EO IYUO MO:1; and mi) histidine, isoleucine, leucine, methionine, glutamine, arginine, alanine, lysine, serine, threonine or valine in the position corresponding to the position of the amino acid 339 5EO IO MO:1; and mii) alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine or valine at a position corresponding to the position of amino acid 340 5EO IU MO:1. 17. Recombinant polypeptide according to claim 15, which is characterized by the fact that the indicated protein GFPD also contains: i) alanine, glycine, histidine, serine or tryptophan in a position corresponding to the position of amino acid 188 5EO IO MO:1; and ii) arginine, cysteine, glutamine, glutamic acid, aspartic acid, glycine, histidine, phenylalanine, or serine in a position corresponding to the position of amino acid 189 5EO IO MO:1; and ii) isoleucine, leucine or methionine in the position corresponding to the position of amino acid 200 5EO IO MO:1; and them). alanine, leucine, proline or asparagine in the position corresponding to the position of amino acid 215 5EO IYUO MO:1; and m) histidine or glutamine in the position corresponding to the position of amino acid 226 5EO IU MO; and mi) serine, alanine, threonine, glutamine or lysine in the position corresponding to the position of the amino acid 339 5EO IO MO:1; and mii) alanine, arginine, aspartic acid, glutamic acid, glutamine, glycine or leucine in a position corresponding to the position of amino acid 340 5EO 10 MO 1. 18. Recombinant polypeptide according to claims 15, 16 or 17, which is characterized by the fact that the specified GFPD protein includes an amino acid sequence that is at least 53 95 identical to the amino acid sequence set forth here as ZEO IU MO-1. 19. Recombinant polypeptide according to claims 15, 16 or 17, which is characterized by the fact that the indicated herbicide-inhibitor of GFPD is selected from the group consisting of M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-ylarylcarboxamides, M-(1,3,4-oxadiazol-2-yl)benzamides, M-(tetrazol-5-yl)- or M- (triazol-3-ylaluarylcarboxamides, pyridazinone derivatives, substituted 1,2,5-oxadiazoles, oxoprazine derivatives, triketones, isoxazoles and pyrazolinates. 20. Recombinant polypeptide according to claim 19, which is characterized by the fact that the indicated GFPD inhibitor herbicide is selected from the group consisting of 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole) -5-yl)/benzamide and 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-methyl-M-(5 -methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl- 1H-tetrazol-5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl)benzamide, 2-chloro-3- ((methylsulfanyl)-M-(1-methyl-1H-tetrazol-5-yl)-4-bo (trifluoromethyl)benzamide, 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H -tetrazol-5-yl)- 4-(trifluoromethyl)benzamide, tembotrione, sulcotrione, mesotrione, isoxaflutol, pyrasulphotol and topramesone. 21. A method of producing a polypeptide with activity of resistance to GFPD inhibitor herbicides, which includes culturing the host cell according to claim 9 under conditions in which the nucleic acid molecule encoding the polypeptide is expressed. 22. A plant having a DNA construct stably incorporated into the genome, wherein the construct includes a promoter operably linked to the nucleic acid according to any one of claims 1, 2 or 3. 23. The plant according to claim 22, characterized in that said plant is selected from the group consisting of a plant cell, a plant tissue, and a plant seed. 24. The plant according to claim 22, which is characterized in that said plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomatoes, cruciferous vegetables, peppers, potatoes, cotton, rice, soybeans, sugar beet, sugar cane, tobacco , barley and oilseed rape. 25. Transgenic plant seed according to claim 22. 26. A method of weed control in a field, which includes planting a plant according to claim 22 or sowing its seeds in a field and applying to the specified field an effective concentration of the GFPD inhibitor herbicide. 27. The method according to claim 26, which is characterized by the fact that the indicated herbicide-inhibitor GFPD is selected from the group consisting of M-(1,2,5-oxadiazol-3-yl)benzamides; M-(tetrazol-4-yl)- or M-(triazol-3-ylluarylcarboxamides, M-(1,3,4-oxadiazol-2-yl)benzamides, M-(tetrazol-5-yl)- or M- (triazol-3-yl)arylcarboxamides, pyridazinone derivatives, substituted 1,2,5-oxadiazoles, oxoprazine derivatives, triketones, isoxazoles, and pyrazolinates. 28. The method according to claim 27, which is characterized by the fact that the indicated herbicide-inhibitor of GFPD is selected from the group consisting of 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole-5 - yl/benzamide Y 2-chloro-3-(methoxymethyl)-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl/benzamide, 2-methyl-M-(5-methyl-1 ,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide, 2-chloro-3-ethoxy-4-(methylsulfonyl)-M-(1-methyl-1H-tetrazole- 5-yl)benzamide, 4-(difluoromethyl)-2-methoxy-3-(methylsulfonyl)-M-(1-methyl-1H-tetrazol-5-yl/benzamide, 2-chloro-3-"(methylsulfanyl)- M-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide, 2-(methoxymethyl)-3-(methylsulfinyl)-M-(1-methyl-1H-tetrazol-5-yl) - Zo 4-(trifluoromethyl)benzamide, tembotrione, sulcotrione, mesotrione, isoxaflutol, pyrasulphotol and topramesone. 29. Use of the nucleic acid according to any one of claims 1-3 for rendering a plant resistant to one or more herbicide(s)-inhibitor(s) of GFPD. 30. A commercial product comprising a nucleic acid molecule according to any of claims 1-3 or a protein according to any of claims 15-17, which is characterized in that the specified product is selected from the group containing whole or processed seeds or grains , animal feed, corn or soybean meal, corn or soybean meal, corn, corn starch, soybean meal, soybean meal, flakes, soy protein concentrate, soy protein isolate, textured soy protein concentrate, cosmetics, hair care products , soybean oil, natto, tempeh, hydrolyzed soy protein, whipped cream, shortenings, lecithin, whole edible soybeans, soy yogurt, soy cheese, tofu, fuchu, and cooked, polished, scalded, baked, or steamed grains. РІНРЕр МАПЬУЕМРМОЇМО-------- я ЕЕ 16 Ауепа вабіхта МРЕТРАТ-АТОАААААУТРЕНААКО----КРЕУУВУМРЕКООВЕРУТЕЕНН 45 Абепа вабіча де1 МЕРТРАТ-АТОАААААУТРЕНААКО----КРЕУУВУМРЕКООВЕРУТЕЕНН 45 зеа маув МОРТРТААААБААУАААКААКОААКНГУЧЗНРМЕУВЕМРЕВОВЕНТСАЕННО ЗО АхавіЗор:ів патїіапа МОНОМААУБЕМВОООААВЕРОСЕКРУС-КЗККУВККМРеКООКЕКУККЕНН.О 45 Nnokavyu upidatga MERTRTTRAATSAAAAUTRENAVR-- - "NE MUKEMNREEVOVKENTSENN o 44 ailiv sakoba MOKK-OBEAETROVMOBMMTVRATEKIUO-KNMHKUKANEKVOVEAUKKENN 4 pEgersoshtshusei aueyusvis1i1i5 MTOTTENTRIITAKOABRRER------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------AAGUUsSEUMUVOtTNN 44 bSassisoidZvbe ip siv MARAAABORTIORAOREPIM-------ttttta nn 5 yu OUBOHON OS Ye x . RINEVB TIEKAZRTIROTIERIEBIMOVETKUATNVOkM-------UBIUROSKIMEYIT. 58 Aepa zaiiuav UKIMSARAAZAAOVKEZKAGSARVRNAANOPYOTOMOANAVGBPVESAGAVTE 55 Atepa zatsiua che1. УКІМСАБААЗААОБЕЗКАТЗАРЬААКОПЬСТОМБАНАБІТТВСОАТАВТЕ 55 дев щшаув УКІМСАБААЗААОБЕЗЕКОТЗАРЬААКОПЬОТОМБАНАЗТЬТБЕОВІБЗЕТЕ 100 АкаріЗорвія спвітапа ТЕВКМСОБАТМУАВБЕВМСТЗМЕЕВАКОСЬВТОММУНАВУТТТБОВЕТЕ 59 Нотовит спідЧате УККМСАПААБААСКЕАКАГЛАРЬААВВОЬБТОМОАНАСОТЬЬВЕОБТАВБЕ 54 Пасив савроба ІТЕВМСОБАТМТЕЕКЕБМСЬЧМРЕУАКОрЬТМОУНАБУБУВКАМНІОВУК 5 Збуертоптусев аусттівії18 УУКАУЗМАКОАА-НУВТАВІМО1 МАХ ВІРЕМОЗВЕТАВУУТЕМОБАВЕУТ 7А Мусоврпаеггеїїа цтатіпіссіа АЕМУУЗМАКОУАОКТІТАМОКЕРУАНКОБЕТООЗВЕКАЗНУУСММИОСУВЕУВ 54 Соссісоїдев інтісів УБМУУЗМАКОААТУУУТРЕМОВЕКУАХУРВЬЕТООКАУЗАЗНУУВМОМІТЕІІ, 76 РЕНЕРр ММЕБЕМЕ------------- --------1АБУКААБрЕРБУССМАЕКУКІОВЇ 88 Ачтепа ватіча ТАРТАРРРОБА-АТАААТАБІРБЕБАРААВТЕВААБСЬАУВБУСУКУАрА 144 Ачепа затіуа деї ТАРТАЕРРОЕА-АТ-ААТАВІРОЕБАРААВТЕАААБСТАУВБУСУКУАРА 143 йеа маух ТАРТАН-- - 5 А А ЗА РА АТААБРЕКБААААВНЕААПОЕСЬАУВАМАБКУАГА 144 Ахтарідсрзів Ена1іапа ТАРТВРЗІВАСЗЕРТКРЕТТТАВІРЕЕСНОгСВЕКЕВосНсЬОУВАМАІВУЮІА 149 Ногавиют учічаге ТАБТАМ----- -SPSPAATAZROBEBABAAVKEBVALESOTAUVB УАТЕУАПА 138 ансив сагога ТАБУБРЕтТТТ----ВБОБААТРОЕБАВОЕНВКЕААКЕСЬАУВАТАТЕУАПУ тай ЗЕгерсотусев ауектісіїї158 ТЕБУІКРАТРЯСОС---БЕГА------------ПНУАЕБСОСУУ.АТЕУРрА 105 Мусовопаєкеї1їа згатіпісоїа ТЕРУКОВАКОТ---БКААРГАЛОАКОРЛЕМУрНІ ОК ОСУКОУАЕБУТОУ 141 Собслооїйез івлітіх тТЕРІКЗУКОАЗ--- TAKES---PEATIKEINANIEVBSTUKOUAKEMIOM 19 . . ярих РЕНРРЕ ОКАХМКАБЕТСсАОР----ІНІПТСРМЕБМІБАТКОТІСОАВЬУТІСОВЕСВО 134 Ахепв ваціхча АБАКЕКУБУАЧОАНКРАКАРАГІЄ---НОСКСГАБУКТТСПУЧУОКЕУЄУРОВТ 191 Ахепв ваціха бв1 АБАКЕКУБУАЧНОАНРАКАРАПІЄЇ---НОСКСГАВБУКТІУСПУУЦКЕУВУРрОВтТ 190 -еа таув еПАЕКАБУААСАВКРАКОРУрІО---БОСКБГАБУКТУСОГУЧЬКУУВУРОСА 151 Ахарійсоювів їпаїіїапа ЕБЕАЕБІВУАМОАТРОВРРІЗІМ---КАМТІАВУКЬХСГУУЬКУУВІКАВГ 195 Ногавот уцпічЧате АБАЕКАБВЕКСАКРАКАРУПІС---БСКАКАБУКБЬУСПУУЧІКЕУВЕРОСТ 185 вас сатоїа КААББАБУАКСАКРАВАРУВІО- --РОАУМТАБУВБЬТСОУУТКЕУВЕСНЕВ 11 кігерсотусевз зуертіціїї5 ВКААНАТАТЕНСАКОУАЕРУВІКРЕБНОТУУБААТАТУОКТЕНТІУПеТОоХВ 153 Мусоврпаетеїіїа сгашіпісоїа ТАУТЕМАУАМОАЕВУВОРНТОБСПЕОПУТКААТКЕТУООТТЕТЕтОВТТУТ 191 Соссісоійев іптітів КВУКВААУКМСАКБУУВОУКТУЮВКОООІКМАТІВТУСЕРТТНТІІЕБсСУК 170 601 ЖЖ «1 х. I. Rinrur D-Vitirkhgeshgesi--Uvvmruvaskutonitemukhvikhmukhmukhmukhmanmeu. ----- Introbiconumoreumoreumauaahuaahuauauai 233 acarios of spaii-texbeebrozheepwauepva-wer-bousovavkayaouma-ultzhutzhkhkauauzhkauzhisauzhisa-nnogayauzhayauzhano 834 пегерсотусех азектіє1115 п---ВУБРСУМАААРРІУКРЕРАНВ---ТКЕОАТОНСУСІУВІЗВММЕМУСКХУ 503 Мусоврпавтетія дкамівпісоіїа сп---ВЕБРЕСУВБСТТУВКАМКЕСРРУМЬЕАТОНСУСМОПЯСЕМИІПАСОКУ ЗВ босстісоідев іштіб18 б5---СЕМРОУРМЕВМАПАТОКЕГСРКУУСЕКТІОНСУСМОПУЯРЕМЕВУСЬУХУ 817 го нини 1 : ФІЛ1А РЕНЕРБ ВКІЕМЕВЕАВУК---РІКОСВБУТОІТЕКАМБАРОЕМІКІРІМЕ--ЕНОКОА Да Ахепа ваціха КОКІСЕНЕБАБЕТАВОУустТтТВО С МОУУТАММОЕАУТЬРІМЕРУНОТККЕ 282 Ачепа ваціув деї КОКРСЕНЕКАКБЕТАВБОУустТтТВО МОУ УТАММОЕАЛРІМЕРУБОСТККВ 9581 пев пауг АсСЕТСЕНЕКАБЕТТЕОУСТАВОНЬМОМУБАММОЕМУТЬРОМЕРУНОСТЕКВВ 283 АкавріЗсрхів Спв1ітарпа АСЕТСЕНОКАБЕТАТОЮМОТАВОНЬМЗАУБАЕМРЕМЛОЬРІМЕРУБИТИВИ 991 Нотовит усічЧата АСЕТСЕНЕКАБЕТАВОУОТТВО НМУ УБАММОБЕСЛОЬРОМЕРУБИТИКЕ ТЕ апсав сакота КаЕТОСЕНЕКАКЕТАВОУСТІВООЬМОУУБАММЕЕМУТЬРМЕрУ У ТКВК ва ЗЕтерсотусев аузктісі115 МКУМСЕТКМКЕВБЕЧЛ ТІ ЮТАТЕУБАБМОКУУАПОСТЬКУКЕРІМЕРАБАКК-К 257 Mustovshopaegei1a dtatipisoia VESTSENVEVMVOKOTSTEEOABKOZIUMBAEMOUUKMRIMERANSKK-K 287 SopsisoiYaee inptisi VKIBSENVEMVEUVIIKOTSTEEBABKOZIUMAPEMOTUKMRIMERAKOKK-O 266 ah. . хі ткож 1: НЕШНИаН их РЕНЕРЕ СОТЕВБКІМОВМСОСЕЗІОНУАКТ ПО УКТИВАЬККІСО----МЕЕМТАВРІТ ит Ахепа вакіча ЗОтОотТуБЕХНОБРІУЗНІАГАСМОУБВТБВЕМВАКТЕКОЧЕВБЕМАРРОАК ОЗ Ачзепа вагіча Зеї1 ЗОХОТУБЕХНООРУЛОНІАГСАЄМЕУТВТІВЕМЕАВТРМОКЗЕВЕМАРРОАК О 331 леза тауУуз ЗОХОТЕБОННОСРААЛОНМАГАБОЕУТВТЬВЕМОАВНАМОУЗЕВЕМАРРІЗЕ 333 Ахаврійсюрзівя сва1іівпа ЗОТОТУБЕНМЕОСАЬОНЬАГМОЕПІКВТЬРЕМРКАВНТОЕОКЕМРОЕвЕт ЗА Нотовиш спідЧате ЗОатОТЕБЕННОСБРІУОНІАМАВЕПУБВТЬВКМВАНКВАМОСЕОКЗ,РЕРЬЕК Зо Гансав сакога ЗОХОТУБЕНМЕСАТЛОІНСЬАТОУБЕБІКЕВТІВЕМЕКВОСТОКЗЕВЕМЕОЗРЕРТ 334 ЗЕгергсотусев атеїхтісіїї:5 ЗОТРЕЖІКБЕХООАЗУОНІАТМТОРІУЕТУВІМВАА- ---УОКІОТР-НВ 297 Кусовопаєкеііїа згашіпіссоїа ЗОТЕБУУПЕУМОРУЛОНІАГЕТЕМНІТЕАУВМЬВОВ----ОУЕКІБУР- ПТ 33 Соссісої9ЦЧез зінщшісів ЗОТЕБУУПЕУМОАЛОНІАГЕТМИІТІПАТТМІЬКАВК--- -ОТЕКІКУР-ЕТ 311 «хжі 1: ж жрикук, її 11, 1 її: жЖрох РБІНЕРЕ ХУЕМПЕСКЬРОВО----БРУПОТОСАВСТРІОСВБЕУКОрККІТЛОтКВЕТІ . 316 Acepa Wagich Hubosuwawawetasomi-Kawkbisugau ---- Ovetricter 376 Achep Pvagita of Zhvouvetasi in Kesokbisutl and---- Vilusliotkra 375 Jea Makhuuwkasimite- . 385 Ногавит Уучідчатге ХЖВОУВЕБАСОМІВЇ-- ВАСКО А ---ВІЛЮОСЛІІОТЕТКРУ 370 Папсав сакога УК ОМІ- ПОТ КЕСЕПТСТІЛА----ВІЛЮСТІТОТЕТКРУ 375 ЗЕгерсотусев аувртісіїї1ї8 УХОТіБЕМУЄОТ----ВУРУПТІВЕЕБКІБАП-- ОБО ОО ІЛОТЕТКРУ 335 Кусовопаєкаї!1а здчратіплосіая УХЕММЕТАГБКААСМКБЕБЕБЕЛІТОКЬМІГІ0----ЕБЕСОТІТОБЕТКРІ 378 Sossisoiyez iivilitih UZHEBMKINKNOSIUBOBPEETKEBOTII0----EBEMOHITOTETKNI. 357 xx. 1 пи хх «І хжкІТІ: : РЕНРРР Мо--РУКЕЕЕТОВК---------------срОСЕСЕСЦЕКАБЕЕЕТЕНОО 345 Ахепа закіха ОПЕРЕТЕКБЕМІОВІСОСМЕКОВУО ВУС КОС ОопЕОоКОМЕОЕСЕКСІВОУЕ 4796 Ахепа пакіха дбеї ОЗПВЕТЕКГЕМІОКІССМЕКрЕУОвУСКОоСОСЕОКОМЕБЕБЕКСТЕПУЄ 455 ва тауд ОЗПВЕТОЕБЕІТОКІССМЕКОЕКоОвУСКОоСОсЕОоКОМЕООБЕКСІВОЕ 477 Атхарісюювія Єпа1їіїапа ОЗПВРЕТІКТБІТОКУуУССММКМЕЕСКАОСБОССОСЕОКОМЕБЕБЕКСІВЕХУЕ 435 Ногасиют УчічЧатге ОПЕРТОЕБЕМТОВІССМЕКОВНОКЕУОКОССОСЗЕОКОМЕЗЕБЕКИІТЕПЕ 49 Оапсав сакога ОБРТІОЕТБІТОКУСЄСМІКОПАСОМ КОСОВО ОЕБЕКІБВУЕ 425 ЗЕгерсотусев аузртіціїї8 ОПЕРЕТУКЕВІТЕКН-О- Я - «З «З 5 5 5 5 « ОМ КОКОМПЕКАБЕЕАТЕНВО 375 Мусовопаєкеіїа згашіпіссоїа МОБРТУКІВІТОВМ---З------------МЕГЗЕСАСЗМНЕКБІТВЕЕАТЕНВО 415. cha khizzhiyih . s SHE ee zhzhtzhk : VENUS UAVEUBTAB-- Z 55 5 -- 358 Ahepa vatsicha KEIEUKOVUUvOKO--- 44 Ahepa shwatsicha bei1i kOTEUKOBUUAOKO--- 439 yea tauv KETKAKOVAAVAAdAOsGIZY 444 Aghariysoryuvuv Spatiiapa EKRTICEAKOT UlkaeKAU------ 445 noteka Uchiko------ - 234 Ozysav sakoga KTIKEAKOTRTOBAAA--- 449 vigersotusev zuzrtisiy 15 VKVOMI.----------- 380 Musovopayekeiiz zrashivisoia PBYAMI.----------- 815 Sossisoi4Yaev ppshsisiv ABRstiI------- ---- 8355 FIG. IV