Herbicide-resistant sunflower plants, polynucleotides coding herbicide-resistant proteins of large subunit of acetohydroxyacidsynthase, and methods of application

FIELD: agriculture.

SUBSTANCE: plant material is treated with mutagen or is transformed with a polynucleotide structure, containing a sequence of nucleic acid coding mutant polypeptide of large subunit of acetohydroxyacidsynthase (AHASL).

EFFECT: plants acquire tolerance to wide range of herbicides.

74 cl, 2 dwg, 6 tbl, 4 ex

 

The SCOPE of the INVENTION

The invention relates to the field of agricultural biotechnology, in particular to the herbicide-resistant sunflower plants and new polynucleotide sequences coding for proteins of the large subunit acetohydroxyacid sunflower wild-type, resistant to imidazolinones.

The technical FIELD

Acetohydroxyacid (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS) is the first enzyme that catalyzes the biochemical synthesis of branched chain amino acids such as valine, leucine and isoleucine (Singh (1999) "Biosynthesis of valine, leucine and isoleucine", inPlant Amino Acid, Singh, B.K., ed., Marcel Dekker Inc. New York, New York, pp.227-247). AHAS is the object of the actions of five structurally distinct families of herbicides, including sulfanilamide (Tanet al. (2005)Pest Manag. Sci.61:246-57; Mallory-Smith and Retzinger (2003)Weed Technology17:620-626; 'LaRossa and Falco (1984)Trends Biotechnol.2:158-161), imidazolinone (Shaneret al. (1984)Plant Physiol.76:545-546), triazolopyrimidine (Subramanian and Gerwick (1989) "Inhibition of acetolactate synthase by triazolopyrimidines", inBiocatalysis in Agricultural Biotechnology, Whitaker, J.R. and Sonnet, P.E., eds., ACS Symposium Series, American Chemical Society, Washington, D.C., pp. 277-288), Tanet al. (2005)Pest Manag. Sci.61:246-57; Mallory-Smith and Retzinger (2003)Weed Technology17:620-626), sulfonamidophenylhydrazine (Tanet al. (2005)Pest Manag. Sci.61:246-57; Mallory-Smith and Retzinger (2003)Weed Technology17:620-626). Them is gasolinevin and sulfanilamide herbicides are widely used in modern agriculture due to their effectiveness at very low application rate and the relative nontoxicity for animals. The inhibition of AHAS activity, these families of herbicides inhibit further growth and development of sensitive plants, including many weeds. Some examples of commercially available imidazolinone herbicides are PURSUIT® (imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples sulphonylcarbamide herbicides are chlorsulfuron, metsulfuron, sulfometuron, chloroaromatic, thifensulfuron, tribenuronmethyl, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron and halogenation.

Due to its high efficiency and low toxicity imidazolinone herbicides are preferred for application by spraying over large areas of plant growth. The ability to spray the herbicide over a wide range of plants reduces the costs associated with the development and maintenance of plantations, and reduces the need for site preparation prior to the application of such chemicals. Spray on desired stable species also gives the possibility of obtaining the maximum possible harvest yield desirable species due to the lack of competitive VI is impressive. However, the possibility of using such methods of spraying depends on the presence of resistant imidazolinone kinds of desirable plants in the field of spray.

Among the main agricultural crops of some legumes, such as soybeans, have a natural resistance to imidazolinone herbicides due to their ability to metabolize the herbicide compounds (Shaner and Robinson (1985)Weed Sci.33:469-471). Other grains, such as corn (Newhouseet al. (1992)Plant Physiol.100:882886) and rice (Barrettet al. (1989)Crop Safeners for Herbicides, Academic Press, New York, pp.195-220), to some extent sensitive to imidazolinone herbicides. Different sensitivity to imidazolinone herbicides depends on the chemical nature of the specific herbicide and various metabolic compounds from toxic to non-toxic forms in each plant (Shaneret al. (1984)Plant Physiol.76:545-546; Brownet al. (1987)Pestic. Biochem. Physiol.27:24-29). An important role in the sensitivity also play other physiological differences between plants, such as the absorption and movement of substances (Shaner and Robinson (1985),Weed Sci.33:469-471).

Resistant to imidazolinones, sulfanilamides and triazolopyrimidines plants were successfully obtained by using mutagenesis of seeds, microspores, pollen and callus fromZea mays,Arabidopsis thaliana,Brassica napus(i.e. canola),Glycine max,Nicotiana tabacum andOryza sativa(Sebastianet al. (1989)Crop Sci.29:1403-1408; Swansonet al.1989Theor. Appl. Genet.78:525-530; Newhouseet al.(1991)Theor. Appl Genet.83:65-70; Sathasivanet al. (1991)Plant Physiol.97:1044-1050; Mourandet al. (1993)J. Heredity84:91-96; U.S. patent No. 5545822). In all cases, the resistance gave specific dominant nuclear gene. Previously also identified four resistant imidazolinone wheat plants after mutagenesis of seeds ofTriticum aestivumL. cv. Fidel (Newhouseet al.(1992)Plant Physiol.100:882-886). The study of heredity was confirmed that the resistance is due to a specific partially dominant gene. Based on the study of the allele, the authors concluded that mutations in the four identified lines were located in the same locus. One of the resistance genes varieties Fidel identified as FS-4 (Newhouseet al. (1992)Plant Physiol.100:882-886).

Natural populations of plants which have been found to be resistant to imidazolinone and/or sulphonylcarbamide herbicides also were used to produce herbicide-tolerant breeding lines of sunflower. Recently created two lines that are resistant to sulphonylcarbamide herbicide, using as the source of the trait of resistance to the herbicide genetic material originating from populations of sunflower ordinary wild-type (Helianthus annuus) (Miller and Al-Khatib (2004),CropSci. 44:1037-1038). Previously Whiteet al.((2002)Weed Sci.50:432-437) reported that individual members of the population sunflower ordinary wild-type from South Dakota, USA, were cross-resistant to imidazolinones and sulphonylcarbamide herbicide. The analysis part of the coding region of genes of large subunit acetohydroxyacid (AHASL) individual members of a specified population revealed a point mutation leading to substitution in the protein sunflower AHASL amino acids Ala, which corresponds Ala205in AHASL proteinArabidopsis thalianawild-type, Val (Whiteet al. (2003)Weed Sci.51:845-853).

Using computer modeling three-dimensional conformation of the complex AHAS-inhibitor predicted several amino acids in the proposed binding pocket inhibitor as areas where induced mutations, probably, could provide a selective resistance to imidazolinones (Ottet al. (1996)J. Mol. Biol.263:359-368). Wheat plants obtained with some mutations, created on the basis of these conclusions in the proposed binding sites of the enzyme AHAS, really showed selective resistance to one of the classes of herbicides (Ottet al. (1996)J. Mol. Biol.263:359-368).

About plant resistance to imidazolinone herbicides has also been reported in several patents. In U.S. patent No. 4761373, 5331107, 530732, 6211438, 6211439 and 6222100, in General, described the use of the altered AHAS gene to obtain plants herbicide resistance, and, in particular, described is resistant to some imidazolinone maize line. In U.S. patent No. 5013659 described plants exhibiting resistance to herbicides due to mutations in at least one amino acid in one or more conservative areas. Described in these documents mutations encode or cross-resistance to imidazolinone and sulfanilamides, or resistance, is specific to sulfanilamides, but the sustainability of specific in relation to imidazolinones was not described. In U.S. patent No. 5731180 and U.S. patent No. 5767361 described selected gene with a single amino acid substitution in the amino acid sequence of the AHAS monocots wild type, leading to specific towards imidazolinone sustainability. In addition, through mutation breeding and selection of herbicide-tolerant plants from the group of rice plants obtained through anther culture were created rice plants resistant to herbicides that inhibit AHAS (see U.S. patent№№ 5545822, 5736629, 5773703, 5773704, 5952553 and 6274796).

Plants, like all other studied organisms, the AHAS enzyme consists of two subunits: a large subunit (kataliticheski the I function) and small subunit (regulatory function) (Duggleby and Pang (2000) J. Biochem. Mol. Biol.33:1-36). Large subunit of AHAS (also referred to herein as AHASL) can be encoded by a single gene, as in the case ofArabidopsisand rice, or more representatives of a family of genes, as in maize, canola and cotton. Specific single nucleotide substitutions in the large subunit give the enzyme a certain degree of resistance to one or several classes of herbicides (Chang and Duggleby (1998),Biochem J.333:765-777).

For example, soft wheat,Triticum aestivumL., contains three homologous gene of the large subunit acetohydroxyacid. Each of these genes showing significant expression, based on the response to herbicides and biochemical data of the mutants for each of the three genes (Ascenziet al. (2003) International Society of Plant Molecular Biologists Congress, Barcelona, Spain, Ref. No. S10-17). Coding sequences of all three genes have a high homology at the nucleotide level (WO 03/014357). With the sequencing of the AHASL genes of several varieties ofTriticum aestivumit was found that the molecular basis of herbicide resistance in most IMI tolerant (tolerant imidazolinones) lines lies mutation S653(At)N denoting the substitution of serine for asparagine at the position equivalent serine at amino acid position 653 inArabidopsis thaliana(WO 03/01436; WO 03/014357). This mutation is the result of tonuclear the aqueous polymorphism (SNP) in DNA sequences coding AHASL protein.

Given the high efficacy and low toxicity imidazolinone herbicides, they are preferred for use in agriculture. However, the ability to use imidazolinone herbicides in a specific area of crop production depends on the availability of resistant imidazolinone varieties of interest crops. To obtain such resistant imidazolinone varieties breeders need to get a breeding line from the property of resistance to imidazolinones. Thus, more resistant to imidazolinones breeding lines and varieties of crops, as well as methods and compositions for the production and application resistant to imidazolinones breeding lines and varieties.

The INVENTION

The present invention relates to sunflower plants with increased compared to plant sunflower wild-type resistance to herbicides. In particular, the sunflower plants according to the invention have increased compared to plant sunflower wild-type resistance to at least one herbicide that prevents the activity of the AHAS enzyme. Herbicide-resistant sunflower plants according to the invention contain at least one copy of the gene or poly is ucleotide, which encodes a herbicide-tolerant large subunit acetohydroxyacid 1 (AHASL1). Such herbicide-resistant AHASL1 protein contains the amino acid position 182 or equivalent position leucine, alanine, threonine, histidine, arginine, or isoleucine. Herbicide-resistant plant of the sunflower according to the invention may contain one, two, three, four or more copies of a gene or polynucleotide encoding herbicide-resistant AHASL1 protein of the invention. Sunflower plants according to the invention also include seeds and progeny that contain at least one copy of the gene or polynucleotide encoding herbicide-resistant AHASL1 protein of the invention.

In one of the embodiments the present invention relates to herbicide-resistant sunflower plants originating from lines of sunflower, which is designated as MUT28. A sample of seed of the line MUT28 deposited in the American Type Culture Collection (ATCC) patent Deposit ATCC No. PTA-6084. These sunflower plants MUT28 contain AHASL1 gene containing the nucleotide sequence of SEQ ID NO: 1, and encoding AHASL1 protein containing the amino acid sequence of SEQ ID NO: 2. Compared to the amino acid sequence AHASL1 protein (SEQ ID NO: 4)encoded AHASL1 gene (SEQ ID NO: 3) of sunflower plants wild-type amino acid at sledovatelnot SEQ ID NO: 2 is different from the amino acid sequence of the wild type by a single amino acid. In the amino acid sequence of SEQ ID NO: 2 amino acid at position 182 is a leucine. In the amino acid sequence of wild-type SEQ ID NO: 4 amino acid in this position is a Proline.

The present invention also relates to the selected polynucleotides and selected polypeptides AHASL1 proteins of sunflower (Helianthus annuus). Polynucleotide according to the invention include the nucleotide sequence encoding herbicide-resistant AHASL1 proteins and AHASL1 proteins of the wild type. Herbicide-resistant AHASL1 proteins of the invention are herbicide-resistant AHASL1 proteins, supporting compared to the corresponding amino acid sequence of the wild type at position 128 in their respective amino acid sequences, the substitution of Proline for leucine. Polynucleotide according to the invention include the nucleotide sequence of SEQ ID NO: 1 and 3, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 and 4, and fragments and variants of these nucleotide sequences encoding proteins having the activity of AHAS. Polynucleotide according to the invention further include a nucleotide sequence encoding a Mature form described above AHASL1 proteins, in particular the nucleotide sequence of SEQ ID NO: 5 and 7, a nucleotide in which sledovatelnot, encoding the amino acid sequence of SEQ ID NO: 6 and 8, and fragments and variants of these nucleotide sequences encoding proteins having the activity of AHAS. These Mature forms AHASL1 proteins lacking the transit peptide of chloroplasts, which is part of a full-sized AHASL1 proteins.

The present invention relates to expressing the cassettes for the expression of polynucleotides according to the invention in plants, plant cells and other cells of the host non-human cells. Expressing cassette containing the promoter, expressed in a plant, plant cell or other interest cells masters, functionally associated with polynucleotides according to the invention, encoding or AHASL1 protein of the wild type or herbicide-resistant AHASL1 protein. If it is necessary for directed expression in chloroplasts expressing cassette may also contain functionally related sequence for directional transport in chloroplasts encoding a transit peptide chloroplast direction expressed AHASL1 protein in the chloroplast. Expressing cassette according to the invention can be used in the method of increasing tolerance of a plant and the host cell to herbicides. The method involves transforming a plant or host cell expressing cassete is according to the invention, where expressing cassette contains a promoter that can be expressed in interest in the plant or cell host and the specified promoter functionally linked to polynucleotides according to the invention, encoding herbicide-resistant AHASL1 protein of the invention. The method also includes the regeneration of transformed plants from the transformed plant cell.

The present invention relates to a method of increasing the activity of AHAS in a plant, comprising transforming the plant cell a polynucleotide construct containing the nucleotide sequence is functionally linked with a promoter directing expression in a plant cell, and regenerating plants from the transformed plant cell. Nucleotide sequence selected from those nucleotide sequences that encode herbicide-resistant AHASL1 proteins or proteins of wild-type AHASL1 according to the invention, in particular the nucleotide sequences SEQ ID NO: 1, 3, 5 and 7, the nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and their fragments and variants. The resulting plant has increased compared to the untransformed plant AHAS activity.

The present invention relates to a method of producing ustoichivogo the herbicides to the plants, which includes the transformation of the plant cell a polynucleotide construct containing the nucleotide sequence is functionally linked with a promoter directing expression in a plant cell, and regenerating transformed plants from the selected transformed plant cells. Nucleotide sequence selected from those nucleotide sequences that encode herbicide-resistant AHASL1 proteins of the invention, in particular the nucleotide sequences SEQ ID NO: 1 and 5, the nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 2 and 6, and their fragments and variants, including as non-limiting examples of Mature forms of herbicide-resistant AHASL1 proteins of the invention. Received by the specified method of herbicide-tolerant plant has compared to nontransgenic plants increased resistance to at least one herbicide, particularly herbicide inhibiting the activity of the AHAS enzyme, such as, for example, imidazolinone herbicide or sulfanilamides herbicide.

The present invention relates to a method of increasing tolerance to the herbicides we are tolerant to the herbicides plants. This method can be used to enhance the stability of the plant that already the mouth of Ichigo to the level of herbicide, which could destroy or cause significant harm to the wild type plant. Such tolerant to herbicides plant can be a tolerant to herbicides plant, which is to impart tolerance to herbicides changed the methods of genetic engineering, or herbicide-resistant plant, obtained by methods that do not involve recombinant DNA, such as sunflower plants MUT28 of the present invention. The method involves the transformation tolerant to herbicides plants polynucleotide construct containing the nucleotide sequence is functionally linked with a promoter directing expression in a plant cell, and regenerating transformed plants from the transformed plant cell. Nucleotide sequence selected from those nucleotide sequences that encode herbicide-resistant AHASL1 proteins of the invention, in particular, the nucleotide sequences SEQ ID NO: 1 and 5, the nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 2 and 6, and their fragments and variants.

The present invention relates to transforming vectors containing the gene for selective marker according to the invention. Gene selective marker contains a promoter that directs expression in a cell-Ho is aine, functionally associated with polynucleotide containing the nucleotide sequence encoding a herbicide-resistant AHASL1 protein of the invention. Transforming the vector may contain a gene of interest for expression in the cell host and may also, if desired, include a sequence for directed transport in chloroplasts, functionally associated with polynucleotides according to the invention.

The present invention also relates to methods of applying the transforming vectors according to the invention for selection of cells transformed by the gene of interest. Such methods include the transformation of the host cell transformation vector, the impact on the cell that level imidazolinones or sulfanilamides herbicide, which could destroy the normal cells of the host or to inhibit its growth, and identification of transformed host cell for its ability to grow in the presence of the herbicide. In one of the embodiments of the invention a host cell is a plant cell and gene selective marker contains a promoter that directs expression in a plant cell.

The present invention relates to a method of controlling weeds in the vicinity of herbicide-tolerant plants according to the invention, in which including the above herbicide-resistant sunflower plants and plants transformed polynucleotide herbicide-resistant AHASL1 according to the invention. Such transformed plant containing in its genome at least one expressing cassette containing a promoter that directs gene expression in a plant cell, where the promoter is functionally linked to polynucleotides AHASL1 according to the invention. The method involves applying an effective amount of a herbicide to the weeds and herbicide tolerant plant, where herbicide-tolerant plant compared to a plant of wild-type or untransformed plant has increased resistance to at least one herbicide, particularly imidazolinone or sulphonylcarbamide herbicide.

Plants of the present invention can be transgenic or necroshine. Example nedrencheskogo sunflower plants with increased resistance to imidazolinone and/or sulphonylcarbamide herbicides includes plant sunflower (MUT28) patent of ATCC Deposit PTA-6084; or a mutant, recombinant, or genetically engineered derivative of the plant with patent Deposit ATCC PTA-6084; or plant, which is a descendant of any of these plants or plant having resistance to herbicides plant patent number deponirawe the Oia ATCC PTA-6084.

The present invention also relates to plants, plant organs, plant tissues, plant cells, seeds and cells of the host non-human cells, transformed with at least one polynucleotide, one expressing cassette or transforming one vector according to the invention. Such transformed plants, plant organs, plant tissues, plant cells, seeds and cells of the host non-human cells, have a high tolerance or resistance to at least one herbicide at levels of herbicide that respectively destroy nontransgenic plants, plant tissues, plant cell or cell-host non-human cells or inhibit their growth. Preferably, transgenic plants, plant tissues, plant cells and seeds according to the invention areArabidopsis thalianaand crops.

The present invention also relates to the selected polypeptides, including resistant imidazolinone AHASL1 proteins of the sunflower AHASL1 proteins of sunflower wild type. The selected polypeptides contain amino acid sequence of SEQ ID NO: 2 and 4, the amino acid sequence encoded by the nucleotide sequences SEQ ID NO: 1 and 3, and fragments and cook the options specified amino acid sequence, in which encoded proteins with the activity of the AHAS, including as non-limiting examples of Mature forms AHASL1 proteins of the invention, representing proteins of SEQ ID nos: 6 and 8 and the proteins encoded by nucleotide sequences SEQ ID NO: 5 and 7.

BRIEF DESCRIPTION of DRAWINGS

Fig. 1 is an alignment of the nucleotide sequences of the full coding sequences of the herbicide-resistant AHASL1 gene of sunflower (SEQ ID NO: 1), AHASL1 gene sunflower wild-type (SEQ ID NO: 3) and herbicide-resistant AHASL1 geneXanthium sp.(SEQ ID NO: 9, GenBank access number U16280). In the drawing 1248-3, HA89 andXanthiumcorrespond to SEQ ID NO: 1, 3, and 9, respectively. The asterisk indicates the site of a single of the mutations found in the coding sequence of the herbicide-resistant AHASL1 sunflower. Mutation is a transition of C to T at nucleotide 545 (codon 182 in SEQ ID NO: 1. Light shaded areas indicate that the nucleotide at this position is conservative for three aligned sequences. Dark areas indicate that the nucleotide at this position is conservative for two of the three sequences.

Fig. 2 is an alignment of amino acid sequences of the herbicide-resistant AHASL1 protein of sunflower (SEQ ID NO: 2), AHASL1 protein of sunflower wild-type SEQ ID NO: 4) and herbicide-resistant AHASL1 protein Xanthium sp.(SEQ ID NO: 10, the access number in GenBank U16280). In the drawing 1248-3, HA89 andXanthiumcorrespond to SEQ ID NO: 2, 4, and 10, respectively. The asterisk indicates the site of a single amino acid substitutions found in the herbicide-resistant AHASL1 protein of sunflower. In herbicide-resistant protein (SEQ ID NO: 2) Proline at amino acid position 182 wild-type protein (SEQ ID NO: 4) is replaced by leucine. Light shaded areas indicate that the amino acid in this position is conservative for three aligned sequences. Dark areas indicate that the amino acid in this position is conservative for two of the three sequences. Amino acids are presented in bold, are conservative amino acid substitutions.

The LIST of SEQUENCES

Nucleotide and amino acid sequences listed in the attached list of sequences shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Nucleotide sequence located in the standard order, starting from the 5'end of the sequence, and continue (i.e. from left to right in each row) to the 3'-end. Given only one strand of each nucleic acid sequence, but means enabling complementary to the ETUI with any indication given circuit. Amino acid sequences are located in the standard order, starting with the N-end of the sequence, and continue (i.e. from left to right in each row) to the C-Terminus.

In SEQ ID NO: 1 shows the nucleotide sequence encoding a herbicide-resistant AHASL1 protein of sunflower. When compared with the sequence with access number in GenBank U16280 Mature form AHASL1 protein is encoded by a nucleotide sequence that corresponds to nucleotides 253-1965 SEQ ID NO: 1, and the transit peptide is encoded by nucleotides 1-252.

In SEQ ID NO: 2 shows the amino acid sequence of herbicide-resistant AHASL1 protein of sunflower. When compared with the sequence with access number in GenBank U16280 amino acid sequence of the Mature form AHASL1 protein corresponds to amino acids 85-655 SEQ ID NO: 2, and the transit peptide corresponds to amino acids 1-84.

In SEQ ID NO: 3 shows the nucleotide sequence encoding a protein of sunflower AHASL1. When compared with the sequence with access number in GenBank U16280 Mature form AHASL1 protein is encoded by a nucleotide sequence that corresponds to nucleotides 253-1965 SEQ ID NO: 3, and the transit peptide is encoded by nucleotides 1-252.

In SEQ ID NO: 4 shows the amino acid sequence of the protein of sunflower AHASL1. When compared with the sequence number DOS the UPA in GenBank U16280 amino acid sequence of the Mature form AHASL1 protein corresponds to amino acids 85-655 SEQ ID NO: 4, and the transit peptide corresponds to amino acids 1-84.

In SEQ ID NO: 5 shows the nucleotide sequence encoding a Mature herbicide-resistant AHASL1 protein of sunflower. This nucleotide sequence corresponds to nucleotides 253-1965 SEQ ID NO: 1.

In SEQ ID NO: 6 shows the amino acid sequence of the Mature herbicide-resistant AHASL1 protein of sunflower. This amino acid sequence corresponds to amino acids 85-655 SEQ ID NO: 2.

In SEQ ID NO: 7 shows the nucleotide sequence encoding the Mature protein of sunflower AHASL1. This nucleotide sequence corresponds to nucleotides 253-1965 SEQ ID NO: 3.

In SEQ ID NO: 8 shows the amino acid sequence of a Mature protein of sunflower AHASL1. This amino acid sequence corresponds to amino acids 85-655 SEQ ID NO: 4.

In SEQ ID NO: 9 shows the nucleotide sequence deposited under access number in GenBank U16280.

In SEQ ID NO: 10 shows the amino acid sequence deposited under access number in GenBank U16280.

In SEQ ID NO: 11 shows the nucleotide sequence of primer ALS1-1F described in example 2.

In SEQ ID NO: 12 shows the nucleotide sequence of primer ALS1-1R, described in example 2.

In SEQ ID NO: 13 shows the nucleotide sequence of primer ALS1-2F, described in example 2.

SEQ ID NO: 14 shows the nucleotide sequence of primer ALS1-2R, described in example 2.

In SEQ ID NO: 15 shows the nucleotide sequence of primer ALS1-3F, described in example 2.

In SEQ ID NO: 16 shows the nucleotide sequence of primer ALS1-3R described in example 2.

In SEQ ID NO: 17 shows the nucleotide sequence of primer ALS-3F, described in example 2.

In SEQ ID NO: 18 shows the nucleotide sequence of primer SUNALS1F described in example 2.

In SEQ ID NO: 19 shows the nucleotide sequence of primer ALS-6R described in example 2.

DETAILED description of the INVENTION

The present invention relates to sunflower plants with increased compared to plant sunflower wild-type resistance to herbicides. Herbicide-resistant sunflower plants were obtained as described herein below, by affecting the sunflower plants of the wild type (in relation to herbicide resistance) mutagen, allowing the plants to Mature and reproduce and selection of plants-descendants, showing increased resistance to imidazolinone herbicide compared to the resistant sunflower plants of the wild type. The invention relates to herbicide-tolerant lines of sunflower, which is herein designated as MUT28.

Of herbicide-resistant sunflower plants MUT28 and plants is of podsolnechnika wild type were isolated by amplification using the polymerase chain reaction (PCR) and sequenced the coding region of the gene of the large subunit acetohydroxyacid (labeled AHASL1). When comparing the polynucleotide sequences of the herbicide-resistant sunflower plants and plant sunflower wild type, it was found that the coding region of the polynucleotide sequence AHASL1 herbicide-resistant sunflower plants differ from the polynucleotide sequences AHASL1 plants of the wild type by one nucleotide, the transition of C to T at nucleotide 545 (Fig. 1). This transition of C to T in the polynucleotide sequence AHASL1 leads to the substitution of Pro for Leu at amino acid 182 in a conservative region of the predicted amino acid sequence AHASL1 protein compared to the amino acid sequence AHASL1 protein of the wild type (Fig. 2). It is established that a series of substitutions of Proline to another amino acid, including the substitution of Pro for Leu, in this conservative area of vegetable proteins AHASL give the plant containing such AHASL protein, resistance to imidazolinone and/or sulphonylcarbamide herbicides (see Boutsaliset al. (1999) Pestic. Sci. 55:507-516; Guttieriet al. (1992)Weed Sci.40:670-678; Guttieriet al. (1995)Weed Sci.43:143-178 and U.S. patent No. 5141870; each of these publications are incorporated herein by reference. Cm. also example 3 below).

Used in this document, unless otherwise specified or is not clear from context, the term "plant" includes as neoprene ivalsa examples of plant cells. the protoplasts of plants, tissue culture of plant cells from which it is possible to regenerate plants, kalucy plant, group of plants with a common root system, plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovule, seed, cotyledons, leaves, stems, flowers, branches, petioles, fruits, roots, root tips, anthers, etc.

The invention also relates to selected molecules polynucleotide containing the nucleotide sequence encoding the protein of the large subunit acetohydroxyacid (AHASL), and such AHASL proteins. The invention relates to the isolation and nucleotide sequence of polynucleotide encoding herbicide-resistant AHASL1 protein of sunflower seed from herbicide-resistant sunflower plants, obtained by chemical mutagenesis in sunflower plants of the wild type. Herbicide-resistant AHASL1 proteins of the invention are compared with the corresponding amino acid sequence of the wild type in the corresponding amino acid sequences, the substitution of Proline for leucine at position 182. The invention also relates to the isolation and nucleotide sequence of the molecule polynucleotide that encodes a protein of sunflower AHASL1 wild-type.

The present invention relates to vydeleny the molecules of polynucleotides, protein coding genes sunflower AHASL1 (Helianthus annuusL.). In particular, the invention relates to selected molecules polynucleotide containing the nucleotide sequence of SEQ ID NO: 1 and 3, the nucleotide sequence encoding the AHASL1 proteins containing the amino acid sequence of SEQ ID NO: 2 and 4, and fragments and variants of these nucleotide sequences that encode functional proteins AHASL1.

In addition, the present invention relates to selected polynucleotide coding for Mature AHASL1 proteins. The Mature AHASL1 proteins of the invention lacks a transit peptide chloroplast located on the N-end of each AHASL1 protein, but is active AHAS. In particular, polynucleotide according to the invention contain a nucleotide sequence selected from the group consisting of nucleotide sequences SEQ ID NO: 5 and 7, the nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 6 and 8, and fragments and variants of these nucleotide sequences that encode the Mature polypeptide AHASL1 with AHAS activity.

Selected molecules polynucleotide herbicide-resistant AHASL1 according to the invention include nucleotide sequences that encode herbicide-resistant AHASL1 protein. Such polynucleotide molecules can be used in Poliny etigny constructs for plant transformation, in particular crops, with the aim of increasing the resistance of plants to herbicides, in particular herbicides, for which it is established that they inhibit the activity of AHAS, and more particularly to imidazolinone herbicides. Such polynucleotide constructs can be used in expressing cassettes expressing vectors, transforming vectors, plasmids, etc. Transgenic plants obtained after transformation such polynucleotide constructs demonstrate increased resistance to AHAS inhibiting herbicides, such as imidazolinone and sulfanilamide herbicides.

The composition of the invention include the nucleotide sequence encoding the AHASL1 proteins. In particular, the present invention relates to the selected polynucleotide molecules containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and their fragments and variants, encoding polypeptides having the activity of AHAS. Also included are polypeptides with the amino acid sequence encoded by a polynucleotide molecule described herein, for example polynucleotide SEQ ID NO: 1, 3, 5 and 7 and their fragments and variants, encoding the polypeptide having the activity of AHAS.

The present invention relates quidelines or essentially purified compositions of nucleic acid or protein. "Isolated" or "purified" polynucleotide molecule, or protein, or biologically active portion substantially or essentially free from components that normally accompany a polynucleotide molecule or protein in their natural environment, or interact with them. Thus, isolated or purified polynucleotide molecule or protein is essentially free of other cellular components or environment for the cultivation of when they are received by recombinant methods or substantially free from chemical precursors or other chemicals when their chemical synthesis. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein coding sequences), which in natural conditions flank the nucleic acid (i.e. sequences located at the 5'- and 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the obtained nucleic acid. For example, in various embodiments, implementation of the selected polynucleotide molecule can contain less than about 5 TPN, 4 TBN, 3 TBN, 2 TBN, 1 TPN, 0,5 TPN or 0.1 TPN nucleotide sequences, which in nature flank the polynucleotide molecule in genomic DNA of the cell from which the nucleic acid. Protein, to the which is essentially free from cellular components, includes preparations of protein, containing less than approximately 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion receive recombinant method, preferably, the medium for the cultivation presents less than approximately 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or chemical substances which are not of interest protein.

The present invention relates to the selected polypeptides containing AHASL1 proteins. The selected polypeptides contain an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 2 and 4, the amino acid sequences encoded by nucleotide sequences SEQ ID NO: 1 and 3, and functional fragments and variants of these amino acid sequences that encode the polypeptide AHASL1 with AHAS activity. Under the "functional fragments and variants" refers to fragments and variants are given as examples of polypeptides that possess the activity of AHAS.

Additionally, provided the selected polypeptides containing Mature forms AHASL1 proteins of the invention. Such selected polypeptides contain an amino acid sequence selected from the group consisting of the amino acid is different sequences of SEQ ID nos: 6 and 8, the amino acid sequences encoded by nucleotide sequences SEQ ID NO: 5 and 7, and functional fragments and variants of these amino acid sequences, which encode polypeptides with the activity of AHAS.

In specific embodiments of the invention methods include the use of tolerant to herbicides or herbicide-resistant plants. Under the "tolerant to herbicides or herbicide-tolerant" plants mean a plant that is tolerant or resistant to at least one herbicide at levels which normally can destroy or inhibit the growth of normal plants or plants of the wild type. In one of the embodiments of the invention are tolerant to the herbicides plants according to the invention contain tolerant to herbicides or herbicide-resistant AHASL protein. Under the "tolerant to herbicides AHASL protein" or "herbicide-resistant AHASL protein" mean AHASL protein, which exhibits a higher AHAS activity compared with the activity of AHAS AHASL protein of the wild type in the presence of at least one herbicide, for which it is established, that it prevents the activity of AHAS, and at a concentration or level of the herbicide for which it is established that they inhibit the activity of the AHAS protein wild-type AHASL. In addition, the activity of AHAS such tolerant to herbicides or herbicide-resistant AHASL protein may be referred to in this document as "tolerant to herbicides or herbicide-tolerant" AHAS activity.

In the present invention, the term "tolerant to herbicides and herbicide-resistant" are used interchangeably, and it is implied that they have equivalent value and an equivalent amount. Similarly, the terms "herbicide tolerance and resistance to herbicides" are used interchangeably and mean that they are of equivalent value and an equivalent amount. Similarly, the "resistant imidazolinone" and "resistance to imidazolinone" are used interchangeably and mean that the meaning and scope equivalent to the value and volume terms "tolerant imidazolinone" and "tolerance imidazolinone" respectively.

The present invention relates to polynucleotides herbicide-resistant AHASL1 and herbicide-resistant AHASL1 protein. The term "polynucleotide herbicide-resistant AHASL1" mean polynucleotide that encodes a protein having herbicide-resistant AHAS activity. The term "herbicide-resistant AHASL1 protein" means a protein or polypeptide possessing a herbicide-resistant AHAS activity.

In addition, it is obvious that tolerant to herbicides or herbicide-resistant AHASL protein can be introduced into a plant by transforming the plant or its predecessor nucleotide sequence bodyrowstart to herbicides or herbicide-resistant AHASL protein. Such tolerant to herbicides or herbicide-resistant AHASL proteins encoded by polynucleotide tolerant to herbicides or herbicide-resistant AHASL. Alternatively, tolerant to herbicides or herbicide-resistant AHASL protein may be in the plant as a result of natural or induced mutation in endogenous AHASL gene in the genome of the plant or its predecessor.

The present invention relates to plants, plant tissues, plant cells and cells of the host with increased resistance or tolerance to at least one herbicide, particularly herbicide inhibiting the activity of the AHAS enzyme, more specifically imidazolidinone or sulphonylcarbamide herbicide. The preferred amount or concentration of herbicide is an "effective amount" or "effective concentration". By "effective amount" and "effective concentration" mean respectively the quantity or concentration that is sufficient to destroy or inhibit the growth of similar plants of the wild type, plant tissue, plant cell, or host cell, but which does not destroy herbicide-resistant plants, plant tissues, plant cells and cells are the masters of the present invention or not so strongly inhibits their growth. As a rule, most effective the top the amount of herbicide is a number, commonly used in agricultural production systems for the destruction of interest weeds. This number is known to specialists in this field.

The herbicides of the present invention are herbicides inhibiting the activity of the AHAS enzyme in such a way that the AHAS activity in the presence of herbicides is reduced. Such herbicides in this document also referred to as "any abscopal AHAS herbicides" or simply "AHAS inhibitors". Used herein, the terms "AHAS inhibiting herbicide or AHAS inhibitor" imply a limitation to one herbicide that prevents the activity of the AHAS enzyme. Thus, unless otherwise indicated or is not clear from context, "AHAS inhibiting herbicide or AHAS inhibitor" can be a single herbicide or a mixture of two, three, four or more herbicides, each of which inhibits the activity of the AHAS enzyme.

Under "similar to the wild type plant, textile plant, plant cell, or the host-cell" refers to a plant, plant tissue, plant cell or a cell of the host, respectively, lacking the properties of resistance to herbicides, and/or specific polynucleotide according to the invention that are described herein. Thus, the use of the term "wild type" is not intended DL the mark in the genome of plants, plant tissues, plant cell, or other cell-host is missing recombinant DNA and/or they do not possess the properties of resistance to herbicides other than as described in this document properties.

As used herein, unless explicitly indicated otherwise, the term "plant" is intended to refer to plants at any stage of development, as well as any part or parts of plants that can be attached to the whole intact plant or separated from him. Such parts of the plant include as non-limiting examples of organs, tissues and cells of plants. Examples of specific parts of the plant include the stem, leaf, root, inflorescence, flower, flower buds, fruit, stem, stamen, anther, stigma, pistil, ovary, petals, sepals, carpel, the tip of the root, the root cahlik, root hair, the hair of the sheet, the beard of grain, pollen grain, microspora, sameday, hypocotyl, epicotyl, xylem, phloem, parenchyma tissue, the endosperm, cell, satellite, closing the cage and any other known organs, tissues and cells of plants. In addition, it is clear that the seed is a plant.

Plants of the present invention include both necroshine and transgenic plants. Under "nereshennymi plant" means a plant that is in which the genome is missing recombinant DNA. By "transgenic plant" means a plant that contains in its genome the recombinant DNA. Such transgenic plants can be obtained by introducing into the genome of a plant a recombinant DNA. When this recombinant DNA is introduced into the genome of transgenic plants, progeny plants may also contain recombinant DNA. Plant-descendant that contains at least part of the recombinant DNA of at least one transgenic plants predecessor, is a transgenic plant.

The present invention relates to herbicide-tolerant lines of sunflower, which is herein referred to as MUT28. Was made a Deposit of at least 650 sunflower seed line MUT28 with the patent Depository of the American Type Culture Collection (ATCC), Mansassas, VA 20110, USA, June 18, 2004 and assigned patent Deposit ATCC PTA-6084. Have been deposited in the ATCC on July 15, 2005 additional seeds line MUT28 with more than 2500 seeds of the patent ATCC Deposit PTA-6084. The Deposit shall retain in accordance with the rules of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Deposit lines of sunflower MUT28 carried out for a period of at least 30 years and at least 5 years from the most recent request for the supply of the sample ATCC Deposit has been accepted. In addition, the applicants have complied with all the requirements of §§ 1.01-1.809 37 C.F.R., including the provision of data about the viability of the sample.

The present invention relates to herbicide-tolerant plants of sunflower lines MUT28 obtained through mutation breeding. The sunflower plants of the wild type was subjected to mutagenesis by influencing plant mutagen, in particular chemical mutagen, more specifically ethylmethanesulfonate (EMS). However, the present invention is not limited to herbicide-resistant sunflower plants obtained by the method of mutagenesis with chemical mutagen EMS. To obtain the herbicide-resistant sunflower plants of the present invention can use any known in the field method of mutagenesis. For example, such methods of mutagenesis can include any one or more of the following mutagens: radiation, such as x-rays, gamma rays (e.g., cobalt 60 or cesium 137), neutrons (for example, the product of the fusion of nuclei of uranium 235 in an atomic reactor), beta radiation (e.g., emitted by radioactive isotopes such as phosphorus 32 or carbon 14), ultraviolet radiation (preferably from 2500 to 2900 nm) and chemical mutagens, such as analogs of nucleotides (for example, 5-bromouracil), related compounds (for example, 8-toxication), antibiotics (e.g. who, streptonigrin), alkylating means (for example, sulfur mustards, nitrogen mustards, epoxides, ethylenimine, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid or acridine. Herbicide-tolerant plants can also be obtained by methods using tissue culture for selection of plant cells containing mutations herbicide resistance, and then regeneration of these herbicide-resistant plants (see, for example, U.S. patent No. 5773702 and 5859348, which are incorporated herein by reference in full). Additional details of mutation breeding can be found in the publication "Principals of Cultivar Development", Fehr, 1993, Macmillan Publishing Company, the description of which is incorporated herein by reference.

When analyzing the AHASL1 gene of sunflower plants line MUT28 it was found that the mutation leads to the substitution of Proline in the 182 amino acids in the amino acid sequence of wild-type AHASL1 SEQ ID NO: 4, leucine. Thus, in the present invention revealed that the substitution of Proline at position 182 to another amino acid can result in sunflower plants increased resistance to herbicides, in particular to imidazolinone and/or sulphonylcarbamide herbicide. As described in example 3 below, Proline 182 is in a conservative area of proteins AHSL and the other amino acid substitutions, which was found to give the plant containing such AHASL protein, resistance to herbicides. Thus, the herbicide-resistant sunflower plants according to the invention include as non-limiting examples of such plants sunflower, containing in its genome at least one copy of polynucleotide AHASL1 encoding herbicide-resistant AHASL1 protein containing leucine, alanine, threonine, histidine, arginine, or isoleucine at amino acid position 182 or equivalent position.

Sunflower plants according to the invention also include plants containing compared with the AHASL1 protein of the wild type leucine, alanine, threonine, histidine, arginine, or isoleucine at amino acid position 182 or equivalent position and one or more additional amino acid substitutions in the AHASL1 protein compared to the protein of wild-type AHASL1, where the plant sunflower has compared to plant sunflower wild-type high resistance to at least one herbicide. Such plants contain sunflower AHASL1 proteins containing at least one member selected from the group consisting of threonine at amino acid position 107 or equivalent position; aspartate or valine at amino acid position 190 or equivalent position; leucine in position the AI amino acids 559 or equivalent position and asparagine, threonine, phenylalanine, or valine at amino acid position 638 or equivalent position.

The present invention relates to AHASL1 proteins with amino acid substitutions at specific positions of amino acids in conservative areas AHASL1 proteins of sunflower described in this document. Unless otherwise specified in the present description, each specific position of the amino acids correspond to the position of amino acids in the full amino acid sequences of sunflower AHASL1 SEQ ID NO: 2 and 4. In addition, experts will be clear that such provisions amino acids can vary depending on, added or deleted amino acids, for example, N-end amino acid sequence. Thus, the present invention relates to the replacement amino acid at the specified position or an equivalent position (for example, amino acid position 182 or equivalent position). Under the "equivalent position" means a position that is in the same conservative region that cited as an example the position of the amino acids. For example, equivalent position for Proline in the amino acid 182 in SEQ ID NO: 4, SEQ ID NO: 8 is an amino acid 98.

In addition, the present invention relates to polypeptides AHASL1 containing amino acid substitutions, the La are installed, they give stability to plant at least one herbicide, particularly the inhibitory AHAS herbicide, more specifically imidazolinone herbicide and/or sulphonylcarbamide herbicide. For example, such polypeptides AHASL1 include polypeptides that contain at least one member selected from the group consisting of: leucine, alanine, threonine, histidine, arginine, or isoleucine at amino acid position 182 or equivalent position; threonine at amino acid position 107 or equivalent position; aspartate or valine at amino acid position 190 or equivalent position; leucine at amino acid position 559 or equivalent position and asparagine, threonine, phenylalanine, or valine at amino acid position 638 or equivalent position. The invention also relates to the selected polynucleotide coding for the polypeptides AHASL1, as well as expressing tapes, transforming vectors, transformed cells-owners, transformed plants and methods comprising such polynucleotide.

The present invention relates to methods for increasing the tolerance or resistance of plants, plant tissues, plant cell or other host cell at least one herbicide that prevents the activity of the AHAS enzyme. Predpochtite is) such an AHAS inhibiting herbicide is imidazolinone herbicide, sulfanilamides herbicide, triazolopyrimidine herbicide, pyrimidinecarbonitrile herbicide, sulfonamidophenylhydrazine herbicide or a mixture. More preferably, the herbicide is imidazolinone herbicide, sulfanilamides herbicide or a mixture. In accordance with the present invention imidazolinone herbicides include as non-limiting examples of PURSUIT® (imazethapyr), CADRE® (imazapic), RAPTOR® (imazamox), SCEPTER® (imazaquin), ASSERT® (kesetevent), ARSENAL® (imazapyr), a derivative of any of the above herbicides and mixture of two or more of the above herbicides, such as imazapyr/imazamox (ODYSSEY®). More specifically, imidazolinone herbicide can be selected from, but not limited to, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, [2-(4-isopropyl)-4-][methyl-5-oxo-2-imidazolin-2-yl)-3-quinoline-carboxylic]acid [5-ethyl-2-(4-isopropyl-]4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid, [2-(4-isopropyl-4-methyl-5-oxo-2-]imidazolin-2-yl)-5-methylnicotinic acid and a mixture of methyl ester [6-(4-isopropyl-4-]methyl-5-oxo-2-imidazolin-2-yl)-m-toluene acid and methyl ester [2-(4-isopropy the-4-methyl-5-]oxo-2-imidazolin-2-yl)-p-toluene acid. Preferably the use of 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid and [2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-]yl)-5-(methoxymethyl)nicotinic acid. Especially preferably the use of [2-(4-isopropyl-4-]methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid.

In accordance with the present invention sulfanilamide herbicides include as non-limiting examples chlorsulfuron, metsulfuron, sulfometuron, chloroaromatic, thifensulfuron, tribenuronmethyl, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron, halogenation, azimsulfuron, ciclosporin, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, iodosulfuron, oxasulfuron, mesosulfuron, prosulfuron, sulfosulfuron, trifloxysulfuron, tritosulfuron, a derivative of any of the above herbicides and mixture of two or more of the above herbicides. Triazolopyrimidine herbicides according to the invention include as non-limiting examples of florasulam, dicloflam, florasulam, flumetsulam, metosulam and penoxsulam. Pyrimidinecarbonitrile of gerbic the water according to the invention include as non-limiting examples bispyribac, pyrithiobac, Perminova, perbenzoic and piritramid. Sulfonamidophenylhydrazine herbicides include as non-limiting examples flucarbazone and propoxycarbazone.

It is established that pyrimidinecarbonitrile herbicides are closely associated with pyrimidinediamine herbicides, therefore, Weed Science Society of America their generalize under the latter name. Thus, the herbicides of the present invention also include pyrimidinediamine herbicides, including as non-limiting examples described above pyrimidinecarbonitrile herbicides.

The present invention relates to methods for increasing the activity of the AHAS in plants, including the transformation of plants polynucleotide construct containing a promoter functionally linked to a nucleotide sequence AHASL1 according to the invention. The methods include the introduction of the polynucleotide constructs of the invention at least one plant cell and regeneration from her transformed plants. Methods include the use of a promoter capable of directing gene expression in a plant cell. Preferably, the promoter is a constitutive promoter or a tissue-specific promoter. Methods used to enhance or improve the sustainability of plants is at least one herbicide, inhibiting the catalytic activity of the AHAS enzyme, in particular to imidazolinone herbicide.

The present invention relates to expressing the cassettes for the expression of polynucleotides according to the invention in plants, plant tissues, plant cells, and other cells of the host. Expressing cassette contains a promoter expressed in plants, plant tissues, plant cell, or other interest cells masters, functionally associated with polynucleotides according to the invention, containing the nucleotide sequence encoding either full (i.e. including the transit peptide chloroplast)or Mature AHASL1 protein (i.e., not containing the transit peptide of chloroplasts). If expression is desired in plastids or chloroplasts of plants or plant cells expressing cassette may also contain functionally related sequence for directional transport in chloroplasts encoding a transit peptide chloroplast.

Expressing cassette according to the invention can be used in the method of increasing tolerance of a plant or host cell to herbicide. The method involves the transformation of the plant or host cell expressing cassette according to the invention, where expressing cassette contains a promoter, expressing the I in the plant or of interest to the cell-master, and a promoter functionally linked to polynucleotides according to the invention, containing a nucleotide sequence encoding a resistant imidazolinone AHASL1 protein of the invention.

The use of the term "polynucleotide constructs" in the present invention is not intended to limit the present invention polynucleotide constructs containing DNA. Specialists in this field it is clear that the polynucleotide constructs, in particular polynucleotides and oligonucleotides containing ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the ways described here. Thus, the polynucleotide constructs of the present invention include all of polynucleotide constructs that can be used in the methods of the present invention for transformation of plants, including as non-limiting examples of polynucleotide constructs that contain deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both natural molecules, and synthetic analogs. Polynucleotide constructs of the invention also include all forms of polynucleotide constructs, including as non-limiting examples of single-stranded forms, double-stranded forms, hairpins structure of the stem and loop, etc. In addition, professionals in this field it is clear that each described in this document, the nucleotide sequence includes a sequence complementary specified is given as an example of the nucleotide sequence.

In addition, it was found that in the methods according to the invention can be applied polynucleotide construct, capable of directing, in a transformed plant, the expression of at least one protein or at least one RNA, such as antisense RNA, complementary to at least part of an mRNA. Typically, such polynucleotide construction consists of the coding sequence for a portion of the protein or RNA operatively linked with regulatory transcription 5'- and 3'-regions. Alternatively, it is also known that in the methods according to the invention can be applied polynucleotide construct, not capable of directing, in a transformed plant, the expression of a protein or RNA.

In addition, it is known that the expression of polynucleotides according to the invention in interest cage-host polynucleotide, as a rule, functionally linked to a promoter capable of directing gene expression in the interest of the cell host. The methods according to the invention for the expression of polynucleotides in cells-the owners do not depend on Conques is to maintain promoter. Methods include the use of any known in the field of promoter capable of directing gene expression in interest cage-master.

The present invention relates to polynucleotide molecules AHASL1 and their fragments and variants. Polynucleotide molecules, which represent fragments of the above nucleotide sequences, are also included in the present invention. By "fragment" means a portion of the nucleotide sequence encoding AHASL1 protein of the invention. Fragment AHASL1 nucleotide sequence according to the invention may encode a biologically active portion of the AHASL1 protein, or it may be a fragment that can be used as a probe for hybridization or PCR primer using the following methods. Biologically active part of the AHASL1 protein can be obtained by selection of one of the AHASL1 nucleotide sequences according to the invention, the implementation of the expression of the encoded part of the AHASL1 protein (e.g., by recombinant expressionin vitro) and assessing the activity of the encoded portion AHASL1 protein. Polynucleotide molecules that are fragments of the AHASL1 nucleotide sequence contain 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, 1200, 1250, 1300, 1350, 1400, 1500, 1600 1700, 1800, 1900 or 1950 nucleotides or up to the number of nucleotides described here are presented in full nucleotide sequence (e.g., 1968, 1968, 1716 1716 and nucleotides in the case of SEQ ID NO: 1, 3, 5 and 7, respectively), depending on the intended application.

Fragment AHASL1 nucleotide sequence that encodes a biologically active portion of the AHASL1 protein of the invention encodes at least approximately 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 650 contiguous amino acids, or up to the total number of amino acids that are in full AHASL1 protein of the invention (for example, 655, 655, 571 and 571 amino acids for SEQ ID NO: 2, 4, 6 and 8, respectively). Fragments AHASL1 nucleotide sequence that can be used as probes for hybridization or PCR primers generally need not encode a biologically active portion of the AHASL1 protein.

The present invention also relates to polynucleotide molecules representing the options described in this document nucleotide sequences. "Options" AHASL1 nucleotide sequences according to the invention include those sequences that encode described in this document AHASL1 proteins, but that differ conservatively because of the degeneracy of the genetic code. Shown is natural allelic variants can be identified using well known methods of molecular biology, such as polymerase chain reaction (PCR) and hybridization methods, as outlined below. Variants of the nucleotide sequences also include synthetically derived nucleotide sequences, which are obtained, for example, by using site-specific mutagenesis but which still encode a AHASL1 protein described in the present invention, as discussed below. Generally, variants of a nucleotide sequence according to the invention is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the specific described here the nucleotide sequence. Option AHASL1 nucleotide sequence will encode the AHASL1 protein, respectively, which has an amino acid sequence at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to those described in this document amino acid sequence AHASL1 protein.

In addition, specialists in this area will be further understood that changes can be made by mutation into the nucleotide sequences according to the invention thereby leading to changes in the amino acid sequence encoded AHASL1 protein without altering the biological activity AHASL1 protein. Thus, the selected polynucleotide molecule, AHASL1 protein-coding sequence, the best is considered from the sequence SEQ ID NO: 1, 3, 5 or 7, respectively, can be obtained by implementing one or more substitutions, insertions or deletions of nucleotides in the corresponding described in this document nucleotide sequence, so that the encoded protein will be one or more amino acid substitutions, insertions or deletions. Mutations can be made by standard methods, such as site-specific mutagenesis and mediated PCR mutagenesis. Such variants of the nucleotide sequences are also covered by the present invention.

For example, conservative amino acid substitutions, preferably, can be implemented as one or more predicted, preferably, nonessential amino acid residues. "Nonessential" amino acid residue is a residue that can be altered protein sequence of wild-type AHASL1 (for example, the sequence SEQ ID NO: 2, 4, 6 and 8, respectively) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. "Conservative amino acid substitution" is a substitution in which the amino acid residue is substituted with amino acid residue with a similar side chain. Family amino acid residue with a similar side chains are defined in this area is. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), branched at the beta-position side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such replacement cannot be made to conservative amino acid residues, or for amino acid residues residing in the conservative motives.

Proteins according to the invention can be modified in various ways including amino acid substitutions, deletions, shortening and paste. Methods for such manipulation, generally known in this field. For example, amino acid sequence variants of the AHASL1 proteins can be obtained through mutations in DNA. Methods mutagenesis and changes in the nucleotide sequences are well known in this field. (see, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA82:488-492; Kunkelet al. (1987)Methods in Enzymol.154:367-382; U.S. patent No. 4873192; Walker and Gaastra, eds. (1983)Techniques in Molecular Biology(MacMillan Publishing Company, NewYork) and cited in these references). Guide to amino acid substitutions so that you do not change the biological activity of the protein of interest can be found in the model of Dayhoffet al. (1978)Atlas of Protein Sequence and Structure(Natl. Biomed. Res. Found., Washington, D.C.), incorporated herein by reference. Preferred may be conservative substitutions, such as replacing one acid for another with similar properties.

Alternative options AHASL1 nucleotide sequences can be obtained by introducing random mutations in the whole or part of the coding sequence AHASL1, for example, using saturating mutagenesis, and the resulting mutants can be screened for AHAS activity to identify mutants that retain activity of AHAS, including herbicide-resistant AHAS activity. After mutagenesis, the encoded protein can Express the recombinant method and to determine the activity of the protein using standard methods of analysis.

Thus, the nucleotide sequences of the invention include the sequences described herein as well as their fragments and variants. AHASL1 nucleotide sequence according to the invention, and fragments and variants can be used as probes and/or primers to identify and/or clone homologues AHASL in other plants. Such zones is s can be used to detect transcripts or genomic sequences encoding the same or identical proteins.

Thus, for the identification of such sequences, to a large extent identical to the sequences according to the invention, it is possible to apply techniques such as PCR, hybridization, and the like (see, for example, Sambrooket al. (1989)Molecular Cloning: a Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and Inniset al.(1990)PCR Protocols: A Guide to Methods and Applications(Academic Press, NY). AHASL nucleotide sequence, selected based on their identity to the sequence specified in the present description the nucleotide sequences AHASL1 or their fragments and variants, are included in the scope of the present invention.

In the method of hybridization with all or part of the known nucleotide sequence of the AHASL1 can be used for screening of cDNA libraries or genomic libraries. The methods of constructing such cDNA libraries and genomic libraries are generally known in this field and are described in Sambrooket al.(1989)Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY). The so-called probes for hybridization can represent fragments of genomic DNA, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with detectable group, such as32P, or any other detektivami marker, such as other radioactive isotopes, fluorescent compound, f is rment or cofactor of the enzyme. Probes for hybridization can be obtained by labeling synthetic oligonucleotides were obtained based on the known nucleotide sequence of the AHASL1 described in this document. Additionally, you can use degenerate primers designed on the basis of conservative nucleotides or amino acid residues in a known AHASL1 nucleotide sequence or encoded amino acid sequence. The probe typically includes a region of nucleotide sequence that hybridizes in stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600 or 1800 consecutive nucleotides AHASL1 nucleotide sequence according to the invention or its fragment or variant. Ways to generate probes for hybridization, generally known in this field and are described in Sambrooket al. (1989)Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), incorporated herein by reference.

For example, as a probe capable of specifically gibridizatsiya with the corresponding sequences AHASL1 and messenger RNA, you can use described in this document the full sequence AHASL1 or one or a few is to its parts. Methods of hybridization include hybridization screening deposited on the floor of library DNA in the form of patches or colonies; see, for example, Sambrooket al. (1989)Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

The hybridization of these two sequences can be carried out in strict conditions. Under "stringent conditions" or "stringent hybridization conditions" refers to conditions under which a probe will be gibridizatsiya with its sequence targeted to a significantly greater degree than to other sequences (e.g., at least 2 times compared with the background level). Strict conditions depend on the sequence and will be different in different circumstances.

Generally, stringent conditions are conditions where the salt concentration is less than about 1.5 M Na ions, as a rule, the Na ion concentration (or other salts) is from about 0.01 to 1.0 M at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least approximately 60°C for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing substances, such as formamide. Illustrative conditions of low stringency include hybridization in BU what NRN solution with a content of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C and washing in a solution with a content of from 1× to 2× SSC (20× SSC = 3.0 M NaCl/0.3m of trinacria citrate) at 50-55°C. Illustrative conditions of medium stringency include hybridization in solution with the content of 40-45% formamide with 1.0 M NaCl, 1% SDS at 37°C and washing in a solution with a content of from 0.5× to 1× SSC at a temperature of 55-60°C. Illustrative conditions of high stringency include yourself hybridization in solution with a content of 50% formamide, 1 M NaCl, 1% SDS at 37°C and washing in a solution containing 0.1× SSC at a temperature of 60-65°C. Optionally, a wash buffer may contain from about 0.1% to about 1% SDS. The duration of hybridization, as a rule, is less than about 24 hours, typically from about 4 to about 12 hours.

Specificity, as a rule, is a function of washing after hybridization, where the decisive factors are ionic strength and temperature of the final solution for cleaning. For hybrids, DNA-DNA Tmyou can approximately be calculated using the equation of Meinkoth and Wahl (1984),Anal. Biochem.138:267-284: Tm= 81,5°C + 16,6(log M) + 0,41 (%GC) - and 0.61 (% form) - 500/L; where M represents both molarity of monovalent cations, %GC is the percentage of guanosine and casinovip nucleotides in the DNA, % form is the percentage of formamide in the solution for g is britishly and L represents the length of the hybrid in pairs of bases. Tmis the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes with exactly the same probe. Tmis reduced by approximately 1°C for each 1% mismatch; thus, Tmthe conditions of hybridization and/or washing can be adjusted to hybridisable sequence with the desired degree of identity. For example, if the desired sequences with ≥90% identity, Tmcan be reduced by 10°C. generally, stringent conditions are chosen so that they were approximately 5°C below the melting temperature (Tm) for the specific sequence and she complementary sequence at a defined ionic strength and pH. However, under very strict conditions, you can use the hybridization and/or washing at a temperature of 1, 2, 3 or 4°C below the melting temperature (Tm); moderately stringent conditions can utilize a hybridization and/or washing at a temperature of 6, 7, 8, 9, or 10°C below the melting temperature (Tm); in conditions of low stringency, you can use the hybridization and/or washing at a temperature of 11, 12, 13, 14, 15, or 20°C lower than the point of the melting temperature (Tm). When using equation formulations for hybridization and washing and desirable Tmwhen ecialists will be clear, that changes the severity of hybridization and/or solutions for cleaning are essentially described. If desired degree of mismatch leads to Tmless than 45°C (aqueous solution) or 32°C (solution with formamide), it is preferable to increase the SSC concentration so that it is possible to use a higher temperature. A detailed guide to the hybridization of nucleic acids is found in Tijssen (1993),Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York), and Ausubel et al, eds. (1995)Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York), (see Sambrook (1989),Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Established that the polynucleotide molecules and proteins of the invention include polynucleotide molecules and proteins containing nucleotide or amino acid sequence that is substantially identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5 and/or 7 or the amino acid sequence of SEQ ID NO: 2, 4, 6 and/or 8. The term "substantially identical" is herein used to refer to a first amino acid or nucleotide sequence that contains a sufficient or maximum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides with the second amino acid is Oh or nucleotide sequence so that the first and second amino acid or nucleotide sequences have a common structural domain and/or functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain with an identity of at least about 45%, 55% or 65%, preferably identity 75%, more preferably identity, 85%, 95% or 98%, defined herein as identical to a considerable extent.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences align with the purpose of better comparison. The percent identity of two sequences is a function of the number of identical positions in the sequences (i.e., the percent identity = number of identical positions/total number of positions (for example, overlapping - positions) × 100). In one embodiment, the implementation of the two sequences are sequences of the same length. The percent identity of two sequences can be determined using methods similar to those described below ways with tolerance gaps or without. When calculating percent identity, as a rule, considered to be an exact match.

Determination of percent identity of two members is of telestai can be performed using a mathematical algorithm. Preferred non-limiting example of a mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264, modified by Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is included in the algorithms of the programs NBLAST and XBLAST Altschul et al. (1990) J. Mol. Biol. 215:403. Search nucleotides in the BLAST can be performed using the NBLAST program, index = 100, word length = 12, to obtain nucleotide sequences homologous polynucleotide molecules according to the invention. The search for proteins in the BLAST can be performed with the XBLAST program, figure = 50, word length = 3, to obtain amino acid sequences homologous to protein molecules according to the invention. To obtain alignments with gaps in order to compare, you can use Gapped BLAST, as described in Altschulet al.(1997)Nucleic Acids Res.25:3389. Alternatively, for carrying out an iterative search that detects distant affinity molecules can be used PSI-Blast (see Altschulet al. (1997), supra). When using the programs BLAST, Gapped BLAST and PSI-Blast can be used the parameters of the respective programs (e.g., XBLAST and NBLAST) by default (see http://www.ncbi.nlm.nih.gov). Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences represents lgorithm Myers and Miller (1988), CABIOS4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the software package for sequence alignment GCG. When using the ALIGN program for comparing amino acid sequences, you can use the table of weights residues PAM120, the penalty on the length of the gap 12 and the penalty for gap 4. The alignment can also be carried out manually during the inspection.

Unless otherwise provided herein, the values of the identity/similarity of the sequences correspond to the value obtained using full-length sequences according to the invention and with the use of multiple alignment by means of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA)using default parameters or any equivalent program. By "equivalent program" means any program of sequence comparison, which for any two of the considered sequences, creates alignment with matches identical nucleotide and amino acid residues and an identical percent sequence identity when compared to the corresponding alignment generated by AlignX in the package programmatically what about the software Vector NTI Suite Version 7.

AHASL1 nucleotide sequence according to the invention include natural sequence, as well as mutant forms, in particular mutant form encoding AHASL1 proteins with herbicide-resistant AHAS activity. Similarly, proteins of the invention include natural proteins, and variants and modified forms. Such variants will continue to possess the desired activity of AHAS. Obviously, the mutations that produce DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see patent application EP No. 75444).

Assume that deletions, insertions and substitutions in the protein sequence covered by this description, do not lead to radical changes in the properties of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion prior to their implementation, the person skilled in the art it will be clear that the effect should be assessed using the normal screening studies (see, for example, the publication Singhet al. (1988)Anal. Biochem.171:173-179 included in this description by reference).

Variants of the nucleotide sequences and proteins also include sequences and proteins obtained by OSU mutagenesis and recombinant method, such as the rearrangement of DNA. Using this method create a new AHASL protein possessing the desired properties can be manipulated by one or more different coding sequences AHASL. Thus, from a group of polynucleotides with similar sequences that contain region sequences with a high degree of sequence identity and which can be subjected to homologous recombinationin vitroorin vivoreceive a library of recombinant polynucleotides. For example, in this approach, the motives of the sequences encoding the interest domain, you can swap between the AHASL1 gene according to the invention and other known AHASL genes to obtain a new gene encoding a protein with superior interest in the property, such as increased Kmin the case of the enzyme. Strategy for the rearrangement of DNA known in the art (see, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994)Nature370:389-391; Crameriet al. (1997)Nature Biotech.15:436-438; Mooreet al. (1997) J Mol. Biol. 272:336-347; Zhanget al. (1997)Proc. Natl. Acad. Sci. USA94:4504-4509; Crameriet al. (1998)Nature391:288-291 and U.S. patent No. 5605793 and 5837458).

The nucleotide sequences according to the invention can be used to highlight the corresponding sequences from other organisms, in chastest the other plants, more specifically from other dicotyledonous plants. Thus, to identify such sequences based on homology of these sequences provided by the present document sequences, it is possible to apply techniques such as PCR, hybridization, etc. Sequence selected taking into account the identity of these sequences full sequences AHASL1 according to the invention or their fragments, are included in the scope of the present invention. Thus, isolated sequences that encode a protein AHASL and hybridization in stringent conditions described herein by sequence or its fragments are included in the scope of the present invention.

When you approach with the use of PCR can be designed oligonucleotide primers for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA isolated from interest plants. Methods of designing primers for PCR and cloning using PCR is widely known in this field and are described in Sambrooket al. (1989)Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), (see also Inniset al., eds. (1990)PCR Protocols: A Guide to Methods and Applications(Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies(Academic Press, New York); Innis and Gelfand, eds. (1999)PCR Methods Manual/i> (Academic Press, New York)). Known methods of PCR include as non-limiting examples of methods using paired primers, nested primers, one specific primers, degenerate primers, gene-specific primers specific for the vector primers, partially mismatched primers, etc.

Polynucleotide sequence AHASL1 according to the invention can be incorporated in expressing cassettes for expression of interest the plant. The cassette will include 5' and 3' regulatory sequence functionally linked to a polynucleotide sequence AHASL1 according to the invention. By "operatively linked" refers to functional linkage between a promoter sequence and the second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Usually functionally related means related nucleic acid sequence are contiguous and, when it is necessary to link the two protein-coding region are adjacent and are in the same reading frame. The cassette may additionally contain at least one additional gene for joint transformation in the body. Alternatively, the additional gene(s) is activated in several expressing cassettes.

This expressing cassette contains multiple restriction sites for insertion of the polynucleotide sequence AHASL1 for the transcriptional regulation of the regulatory regions. Expressing cassette may additionally contain genes selective markers.

Expressing cassette comprises in the direction of transcription of the 5'-3' region of transcription initiation and translation (i.e., the promoter), a polynucleotide sequence AHASL1 according to the invention and the scope termination of transcription and translation (i.e., termination), functioning in plants. The promoter for the host plant and/or polynucleotide sequence AHASL1 according to the invention may be native or analogous or foreign or heterologous. In addition, the promoter may be the natural sequence or, alternatively, a synthetic sequence. When the promoter is "foreign" or "heterologous" to the host plant, imply that this promoter is not in the natural plant, which enter this promoter. When the promoter is "foreign" or "heterologous" to the polynucleotide sequence AHASL1 according to the invention, with the implication that the promoter is not native or natural promoter for operatively linked polynucleotide sequence AHASL1 from which bretania. As used herein, a chimeric gene contains the coding sequence functionally linked to the region of transcription initiation, which is heterologous to the coding sequence.

Although it may be preferred expression of polynucleotides AHASL1 according to the invention with the use of heterologous promoters, you can use the native promoter sequence. Such constructs can change expression levels AHASL1 protein in the plant or plant cell. Thus, the phenotype of a plant or plant cell is changed.

The termination region may be native to the region of transcription initiation may be own for functionally related interest sequence AHASL1 may be peculiar to the plant host, or may be obtained from another source (i.e. foreign or heterologous to the promoter of interest polynucleotide sequence AHASL1, the plant host, or any combination thereof). Appropriate field termination is available from the Ti-plasmidA. tumefacienssuch as the field termination octopunctata and napadisylate (see also Guerineauet al. (1991)Mol. Gen. Genet.262:141-144; Proudfoot (1991)Cell64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogenet al. (1990)Plant Cell2:1261-1272; Munroeet al. (1990)Gene 91:151-158; Ballaset al. (1989)Nucleic Acids Res.17:7891-7903 and Joshiet al. (1987)Nucleic Acids Res.15:9627-9639).

If appropriate, the gene(s) can be optimized for increased expression in the transformed plant. This means that the genes for enhanced expression can be synthesized with the use of preferred plants codons. Discussion of application of the preferred host codons see, for example, Campbell and Gowri (1990),Plant Physiol.92:1-11. For the synthesis of preferred plant genes in this region have the means available (see, for example, U.S. patent No. 5380831 and 5436391 and Murrayet al. (1989)Nucleic Acids Res.17:477-498, incorporated herein by reference).

Known additional modifications of the sequences that increase gene expression in the cell host. They affect the deletion of sequences encoding spurious polyadenylation signals, signals splicing of exons-introns-like transposons replays and other well-studied sequence, which can reduce gene expression. The content of G-C in the sequence can be adjusted to levels average for a given host cell, which is calculated on the basis of known genes expressed in the cell host. If possible, modify the sequence so as to avoid theoretically expected secondary when ructur mRNA in the form of a stiletto.

In expressing the vectors of plants can also be applied to the nucleotide sequence to increased gene expression. They include the introns of the maize AdhI gene intron1 (Calliset al. (1987)Genes and Development1:1183-1200) and leader sequence (W-sequence) of tobacco mosaic virus (TMV), virus pale craptastic and maize mosaic virus alfalfa (Gallieet al. (1987)Nucleic Acids Res.15:8693-8711 and Skuzeskiet al. (1990)Plant Mol. Biol.15:65-79). It is shown that the first intron locus rugosity-1 corn increases the expression of genes in the construction of chimeric genes. In U.S. patent No. 5424412 and 5593874 described the application of specific introns in genetic expressing designs, and Gallieet al. (Plant Physiol.106:929-939, 1994) also shows that introns can be used for regulation of gene expression, based on tissue specificity. To further improve or to optimize expression of the small subunit of AHAS gene expressing vectors of plants according to the invention can also contain DNA sequences that includes the area of attachment to the matrix (MAR). Then plant cell transformed by such modified expressing systems can demonstrate the overexpression or constitutive expression of a nucleotide sequence according to the invention.

Ek is pressious cassette may also contain a 5'-leader sequence in the design of expressing cassette. Such leader sequences can act in the direction of strengthening the broadcast. Translation leader sequences known in the field and include a leader sequence of picornaviruses, such as a leader sequence EMCV (5'-non-coding region of the virus encephalomyocarditis) (Elroy-Steinet al. (1989)Proc. Natl. Acad. Sci. USA86:6126-6130); leader sequence potyviruses, for example, a leader sequence TEV (virus engraving tobacco) (Gallieet al. (1995)Gene165(2):233-238), leader sequence MDMV (the virus of the mosaic of dwarf maize) (Virology154:9-20), and linking the heavy chain of human immunoglobulin protein (BiP) (Macejaket al. (1991)Nature353:90-94); untranslated leader sequence of the mRNA of the protein shell of the virus alfalfa mosaic (AMV RNA 4) (Joblinget al. (1987)Nature325:622-625); leader sequence of tobacco mosaic virus (TMV) (Gallieet al. (1989) inMolecular Biology of RNA,, ed. Cech (Liss, New York), pp. 237-256) and leader sequence of the virus pale craptastic corn (MCMV) (Lommelet al. (1991)Virology81:382-385) (see also Della-Cioppaet al. (1987)Plant Physiol.84:965-968). It is also possible to use other known methods of strengthening the broadcast, such as introns, etc.

When receiving expressing cassettes can be manipulated by various DNA fragments to obtain the settlement is egovernance DNA in the proper orientation and, as appropriate, in the correct reading frame. For this purpose you can use adapters or linkers for linking DNA fragments, or you can apply other procedures for obtaining the appropriate restriction sites, removal of superfluous DNA, removal of restriction sites, etc. With this purpose you can use mutagenesisin vitro, restore primers, restriction, annealing, reverse the substitution, for example the transitions and transverse.

In the practical implementation of the invention can be used a number of promoters. Promoters can be selected on the basis of the desired result. Nucleic acids can be combined with constitutive, tissue-specific, or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters described in WO 99/43838 and U.S. patent No. 6072050; a core promoter, CaMV 35S (Odellet al. (1985)Nature313:810-812); the promoter of the rice actin (McElroyet al. (1990)Plant Cell2:163-171); the promoter of ubiquitin (Christensenet al. (1989)Plant Mol. Biol.12:619-632 and Christensenet al. (1992)Plant Mol. Biol.18:675-689); pEMU promoter (Lastet al. (1991)Theor. Appl. Genet.81:581-588); the MAS promoter (Veltenet al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. patent No. 5659026), etc. Other constitutive promoters include the promoters are described, for example, in U.S. patents№№ 5608149; 5608144; 5604121; 5569597; 5466785; 5399680; 5268463; 5608142 and 6177611.

Danes Titicaca promoters can be used to improve the AHASL1 gene expression in specific plant tissues. Such tissue-specific promoters include as non-limiting examples of specific leaf specific promoters root specific promoters of seed-specific promoters and stems promoters. Tissue-specific promoters include promoters, described by Yamamotoet al.(1997)Plant J.12(2):255-265; Kawamataet al. (1997)Plant Cell Physiol.38(7):792-803; Hansenet al. (1997) Mol. Gen Genet. 254(3):337-343; Russellet al. (1997)Transgenic Res.6(2): 157-168; Rinehartet al. (1996)Plant Physiol.112(3):1331-1341; Van Campet al. (1996)Plant Physiol.112(2): 525-535; Canevasciniet al. (1996)Plant Physiol.112(2):513-524; Yamamotoet al. (1994)Plant Cell Physiol.35(5):773-778; Lam (1994)Results Probl. Cell Differ.20:181-196; Orozcoet al. (1993)Plant Mol. Biol.23(6): 1129-1138; Matsuokaet al. (1993)Proc. Natl. Acad. Sci. USA90(20):9586-9590 and Guevara-Garciaet al. (1993)Plant J.4(3):495-505. Such promoters, if necessary, can be modified for weak expression.

In one of the embodiments of interest, the nucleic acid is directed to expression in chloroplasts. Thus, when the interest of the nucleic acid does not enter directly into the chloroplast, expressing cassette will also contain the sequence for the directed transport in chloroplasts, containing the nucleotide sequence encoding the transit peptide of chloroplasts to the direction of the product represent the interest of the gene in the chloroplast. Such transit peptides are known in this field. In the case of sequences for directed transport in chloroplasts "functionally linked" means that the nucleic acid sequence encoding a transit peptide (i.e. the sequence for directed transport in chloroplasts), associated with polynucleotides AHASL according to the invention so that the two sequences are contiguous and are located in the same reading frame (see, for example, Von Heijneet al. (1991)Plant Mol. Biol.Rep. 9:104-126; Clarket al. (1989)J. Biol. Chem.264:17544-17550; Della-Cioppaet al. (1987)Plant Physiol.84:965-968; Romeret al.(1993)Biochem. Biophys. Res. Commun.196:1414-1421 and Shahet al. (1986)Science233:478-481). Although AHASL1 proteins of the invention include native transit peptide chloroplast, with the amino acid sequence of the Mature AHASL1 protein of the invention can be drained any known in this area transit peptide chloroplast through functional binding guide in the chloroplast sequence with the 5'-end a nucleotide sequence that encodes a Mature AHASL1 protein of the invention.

Sequence for directed transport in chloroplasts is known in this area and include a small unit of ribulose-1,5-bestofferbuy chloroplasts (Rubisco) (de Castro Silva Filhoet al.(1996)Plant Mol. Biol.30:769-780; Schnellet al. (1991)i> J. Biol. Chem.266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-fosfolinazou (EPSPS protein) (Archeret al. (199O) J. Bioenerg. Biomemb. 22(6):789-810); cryptophycins (Zhaoet al. (1995)J. Biol. Chem.270(11):6081-6087); plastocyanin (Lawrenceet al. (1997)J. Biol. Chem.272(33):20357-20363); charismaticism (Schmidtet al. (1993)J. Biol. Chem.268(36):27447-27457) and connecting the light absorbing chlorophyll a/b protein (LHCP) (Lamppaet al. (1988)J. Biol. Chem.263:14996-14999), (see also Von Heijneet al.(1991)Plant Mol. Biol Rep.9:104-126; Clarket al.(1989)J. Biol. Chem.264:17544-17550; Della-Cioppaet al. (1987)Plant Physiol.84:965-968; Romeret al.(1993)Biochem. Biophys. Res. Commun.196:1414-1421 and Shahet al. (1986)Science233:478-481).

Methods of transformation of chloroplasts is known in the art (see, for example, Svabet al.(1990)Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga (1993)Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga (1993)EMBO J.12:601-606). The method is based on the delivery of DNA containing a selective marker, using bombardment by particles and DNA integration into the genome of plastids via homologous recombination. In addition, the transformation of plastids can be accompanied by a transactivation originating from plastids silent transgene by tissue-specific expression of the encoded in the nucleus and exported to the plastids RNA polymerase. This system published in McBrideet al.(1994)Proc. Natl. Acad. Sci. USA91:7301-7305.

Interest of nucleic acids for directed transport in chlorops the texts can be optimized for expression in chloroplasts taking into account differences in the use of codons between the nucleus of plants and these organelles. Thus, the interest of the nucleic acid can be synthesized using preferred for chloroplast codons (see, for example, U.S. patent No. 5380831, incorporated herein by reference).

As described herein, AHASL1 nucleotide sequence according to the invention find application in improving tolerance to the herbicide plant containing in its genome a gene encoding tolerant to herbicides AHASL1 protein. Such a gene can be an endogenous gene or a transgene. In addition, in particular embodiments, the implementation of the nucleic acid sequence of the present invention can be combined with any combination of interest polynucleotide sequences to obtain plants with a desired phenotype. For example, polynucleotide of the present invention can be combined with any other polynucleotide encoding the polypeptide with pesticidal and/or insecticidal activity, for example, such as toxic proteinsBacillus thuringiensis(described in U.S. patent№№ 5366892; 5747450; 5737514; 5723756; 5593881 and Geiseret al. (1986)Gene48:109). The resulting combination can also include multiple copies of any interest of polynucleotides.

It is known that on the basis of these nucleotide sequences can konstruirovanie the antisense constructs, complementary to at least part of the messenger RNA (mRNA) polynucleotide sequences AHASL1. Antisense nucleotides design for hybridization with the corresponding mRNA. Modifications of the antisense sequences can be performed up until the sequence hybridize with the corresponding mRNA and inhibit its expression. Thus, it is possible to use antisense constructs with a sequence identical to 70%, preferably 80%, more preferably 85%, corresponding antimuslim sequences. In addition, you can use part of the antisense nucleotides for violations of expression of the target genes. Usually, you can use a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more.

The nucleotide sequence of the present invention can also be used in the sense orientation to suppress the expression in plants of endogenous genes. Methods of suppressing gene expression in plants using nucleotide sequences in the sense orientation known in the field. The methods generally include the transformation of plant DNA constructs containing the promoter that directs expression in the plant, functionally associated with at least part of the nucleotide PEFC is the sequences, corresponding to the transcript of the endogenous gene. Typically, such a nucleotide sequence has a high degree of sequence identity with the sequence of the transcript of the endogenous gene, preferably the degree of sequence identity of more than about 65%, more preferably the degree of sequence identity of more than about 85%, most preferably the degree of sequence identity of more than 95% (see U.S. patent No. 5283184 and 5034323, incorporated herein by reference).

Although polynucleotide herbicide-resistant AHASL1 according to the invention can be used genes as selective markers for transformation of plants expressing cassette according to the invention may include other gene selective marker for selection of transformed cells. Genes selective markers, including genes are selective markers of the present invention, is used for selection of transformed cells or tissues. Marker genes include as non-limiting examples of genes encoding antibiotic resistance, such as genes encoding neomycinphosphotransferase II (NEO) and GigabitEthernet (HPT), as well as genes that give resistance to herbicide compounds such as glufosinate ammonium, b is amoxicil, imidazolinone and 2,4-dichlorophenoxyacetate (2,4-D) (mostly, see Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christophersonet al. (1992)Proc. Natl. Acad. Sci. USA89:6314-6318; Yaoet al. (1992)Cell71:pp.63-72; Reznikoff (1992)Mol. Environ.6:2419-2422; Barkleyet al. (1980) inThe Operon, RR-220; Huet al. (1987)Cell48:555-566; Brownet al. (1987)Cell49:603-612; Figgeet al. (1988)Cell52:713-722; Deuschleet al. (1989)Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerstet al. (1989)Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschleet al. (1990)Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reineset al. (1993)Proc. Natl. Acad. Sci. USA90:1917-1921; Labowet al. (1990)Mol. Cell. Biol.10:3343-3356; Zambrettiet al. (1992)Proc. Natl. Acad. Sci. USA89:3952-3956; Baimet al. (1991)Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborskiet al. (1991)Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989)Topics Mol. Struc. Biol.10:143-162; Degenkolbet al.(1991)Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidtet al.(1988)Biochemistry27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossenet al. (1992)Proc. Natl. Acad. Sci. USA89:5547-5551; Olivaet al.(1992)Antimicrob. Agents Chemother.36:913-919; Hlavkaet al. (1985)Handbook of Experimental PharmacologyVol. 78 (Springer-Verlag, Berlin); Gillet al. (1988)Nature334:721-724, which are incorporated herein by reference).

The above list of genes selective markers is not a limitation. You can use any selective gene marker in the present invention.

Selected molecules polynucleotide containing the nucleotide sequence encoding the tree AHASL1 according to the invention, you can use vectors for plant transformation, so that the resulting plants had increased resistance to herbicides, in particular to imidazolinone herbicides. When making plant resistance to herbicides selected polynucleotide molecules AHASL1 according to the invention can be used in the vectors alone or in combination with nucleotide sequence that encodes the small subunit of AHAS enzyme (AHASS) (see U.S. patent No. 6348643, which is incorporated herein by reference).

The invention also relates to expressing the vector of plants containing the promoter that directs expression in the plant, functionally associated with the selected polynucleotide molecule according to the invention. The selected polynucleotide molecule contains a nucleotide sequence encoding a AHASL1 protein, in particular AHASL1 protein containing the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or a functional fragment and variant. Expressing the vector of plants according to the invention does not depend on the specific promoter except that such a promoter capable of directing gene expression in a plant cell. Preferred promoters include constitutive promoters and tissue specific promoters.

Transforming the vectors according to the invention can be used for the teachings of plants transformed gene of interest. Transforming a vector containing a gene selective marker according to the invention and a gene of interest for introduction in the transformed plant and, as a rule, for expression in it. Such gene selective marker contains polynucleotide herbicide-resistant AHASL1 according to the invention, is functionally associated with a promoter directing expression in a cell host. For use in plants and plant cells transforming a vector containing a gene selective marker containing polynucleotide herbicide-resistant AHASL1 according to the invention, is functionally associated with a promoter directing expression in a plant cell.

Interest genes according to the invention vary depending on the desired result. For example, may be of interest to different changes of the phenotype, including the modification of the content in a plant fatty acid modifying content in plant amino acids, the change in defense mechanisms of plants against insects and/or pathogens, etc. Data results can be achieved when the condition of expression of heterologous products or increased expression of endogenous products in plants. Alternatively, these results can be achieved under the condition of reduction of expression in the plant of one or NESCO is gcih endogenous products, particularly enzymes or cofactors. These changes lead to a change in phenotype of the transformed plant.

In one of the embodiments of the invention of interest genes include genes for resistance to insects, such as genes toxic proteinsBacillus thuringiensis(U.S. patent№№ 5366892; 5747450; 5736514; 5723756; 5593881 and Geiseret al. (1986)Gene48:109).

AHASL1 proteins or polypeptides according to the invention can be selected, for example, from sunflower plants and can be used in the compositions. Also isolated polynucleotide molecule encoding a AHASL1 protein of the invention can be used for expression AHASL1 protein of the invention in a microorganism, such asE. colior yeast. Expressed AHASL1 protein can be isolated from extracts ofE. colior any yeast known to specialists in this field by the way.

The invention also relates to a method for producing herbicide-resistant transgenic plants, providing for the transformation of plants expressing vector plants containing the promoter that directs expression in the plant, functionally associated with the selected polynucleotide molecule according to the invention. The selected polynucleotide molecule contains a nucleotide sequence encoding a AHASL1 protein of the invention, in particular AHASL1 protein containing aminoxy the pilot sequence SEQ ID NO: 2 or 6, amino acid sequence encoded by SEQ ID NO: 1, or 5, or a functional fragment and variant specified amino acid sequence.

The invention also relates to nereshennymi the sunflower plants, transgenic plants obtained by the methods according to the invention, and the offspring and other relatives such nereshennyh and transgenic plants, where plants show an increased resistance to herbicides inhibiting the enzyme AHAS, in particular to imidazolinones and sulphonylcarbamide herbicides.

Polynucleotide AHASL1 according to the invention, in particular polynucleotide encoding herbicide-resistant AHASL1 proteins, find use in the methods of increasing the resistance tolerant to herbicides plants. In one of the embodiments of the invention are tolerant to the herbicides plants contain tolerance to herbicides or herbicide-resistant AHASL protein. Tolerant to herbicides plants include plants, transformed with nucleotide sequences are tolerant to the herbicides AHASL and plants containing the genomes of the endogenous gene encoding tolerant to herbicides AHASL protein. The nucleotide sequence encoding tolerant to herbicides AHASL proteins, and herbicide tolerant plants, containing an endogenous gene that encodes a tolerant GE is beldam AHASL protein, include polynucleotide and plants according to the present invention and polynucleotide and plants, which are known in the art (see, for example, U.S. patents№№ 5013659, 5731180, 5767361, 5545822, 5736629, 5773703, 5773704, 5952553 and 6274796; each of which is incorporated herein by reference). Such methods of increasing resistance tolerant to herbicides plants include transformation tolerant to herbicides plant at least one polynucleotide construct containing a promoter that directs expression in a plant cell, functionally associated with polynucleotides herbicide-resistant AHASL1 according to the invention, in particular with polynucleotides coding for herbicide-resistant AHASL1 protein of SEQ ID NO: 1, or 5, with polynucleotide, encoding the amino acid sequence of SEQ ID NO: 2 or 6, and fragments and variants of these polynucleotides that encode polypeptides with herbicide-resistant AHAS activity.

There are many transforming vectors of plants and methods of plant transformation (see, for example, An G.et al. (1986)Plant Pysiol.81:301 to 305; Fry J.et al.(1987)Plant Cell Rep.6:321-325; Block, M. (1988)Theor. Appl Genet.76:767-774; Hincheeet al.(1990)Stadler. Genet. Symp.203212. 203-212; Cousinset al.(1991)Aust. J. Plant Physiol.18:481-494; Chee P.P. and Slightom J.L. (1992)Gene. 118:255-260; Christouet al.(1992)Trends. Biotechnol.10:239-246; D Halluinet al (1992)Bio/Technol.10:309-314; Dhiret al.(1992)Plant Physiol.99:81-88; Casaset al. (1993)Proc. Natl. Acad. Sci. USA90:11212-11216; Christou P. (1993)In Vitro Cell. Dev. Biol.-Plant,29P:119-124; Davieset al.(1993)Plant Cell Rep.12:180-183; Dong J.A. and Mchughen, A. (1993)Plant Sci.91:139-148; C.I. Franklin and Trieu T.N. (1993)Plant. Physiol.102:167; Golovkinet al.(1993)Plant Sci.90:41 to 52;Guo Chin Sci. Bull.38:2072-2078; Asanoet al.(1994)Plant Cell Rep.13; Ayeres N.M. and Park, W.D. (1994)Crit. Rev. Plant. Sci.13:219-239; Barceloet al.(1994)Plant. J.5:583-592; Beckeret al.(1994)Plant. J.5:299-307; Borkowskaet al. (1994)Acta. Physiol. Plant.16:225-230; Christou P. (1994)Agro. Food. Ind. Hi Tech.5: 17-27; Eapenet al. (1994)Plant Cell Rep.13:582-586; Hartmanet al.(1994)Bio-Technology12: 919923; Ritalaet al.(1994)Plant. Mol. Biol. 24:317-325 and Y.C. Wan and Lemaux P.G. (1994)Plant Physiol.104:3748).

The methods of the invention include the introduction of a polynucleotide constructs into the plant. Under the "introduction" means the presentation of the plant a polynucleotide constructs in such a way that the construct gains access to the inside of the plant cells. The methods according to the invention do not depend on a particular method of introducing a polynucleotide constructs into plants except that the polynucleotide construct gains access to the inside of at least one cell of the plant. Methods of introducing a polynucleotide constructs into plants are known in this area, including as non-limiting examples of methods for stable transformation methods is tranzitornoe transformation mediated by viruses methods.

Under "stable transformation" refers to that introduced into the plant a polynucleotide construct integrated into the genome of plants and able to be inherited by his offspring. Under "transient transformation" implies that the introduced polynucleotide in the plant design does not integrate into the genome of a plant.

For the transformation of plants and plant cell a nucleotide sequence according to the invention is inserted using standard methods, as any known in the field a vector suitable for expression of the nucleotide sequences in a plant or plant cell. The choice of vector depends on your preferred method of transformation and plant species targeted for transformation. In one of the embodiments of the invention AHASL1 nucleotide sequence functionally linked to a promoter of a plant, which is known as the promoter, providing a high level of expression in a plant cell, and then the construct introduced into a plant sensitive to imidazolinone herbicide, and transformed plants regenerate. The transformed plant is tolerant to the effects of this level imidazolinone herbicide that can kill or cause significant harm to the untransformed plant. This method can be applied for any the IDA plants; however, the most beneficial when it is used for crops, particularly for crops that are typically grown in the presence of at least one herbicide, particularly imidazolinone herbicide.

The methods of constructing expressing cassettes plants and the introduction of foreign nucleic acids in plants are widely known in this field and described previously. For example, the foreign DNA can be introduced in plants with application of inducing tumors (Ti) plasmid vectors. Other methods used to deliver foreign DNA, include the use of PEG mediated transformation of protoplasts, electroporation, microinjection hairs and biolistic or bombing microparticles for direct capture DNA. Such methods are known in the art (U.S. patent No. 5405765 issued by Vasilet al.; Bilanget al. (1991)Gene100:247-250; Scheidet al.(1991)Mol. Gen. Genet.228:104-112; Guercheet al.(1987)Plant Science52:111-116; Neuhauseet al.(1987)Theor. Appl Genet.75:30-36; Kleinet al. (1987)Nature327:70-73; Howellet al.(1980) Science 208:1265; Horschet al.(1985)Science227:1229-1231; DeBlocket al.(1989)Plant discrimination91: 694-701;Methods for Plant Molecular Biology(Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) andMethods in Plant Molecular Biology(Schuler and Zielinski, eds.) Academic Press, Inc. (1989)). The way of transformation depends on the transformed plant cells, Stabi is a major applied vector, the level of expression of gene products, and other parameters.

Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the genome of plants include microinjection in accordance with Crosswayet al. (1986)Biotechniques4:320-334, electroporation, as described by Riggset al. (1986)Proc. Natl. Acad. Sci. USA83:5602-5606, mediated byAgrobacteriumtransformation, as described by Townsendet al., U.S. patent No. 5563055, Zhaoet al., U.S. patent No. 5981840, direct gene transfer, as described by Paszkowskiet al. (1984)EMBO J.3:2717-2722, and ballistic particle acceleration, for example, as described by Sanfordet al., U.S. patent No. 4945050; Tomeset al. U.S. patent No. 5879918; Tomeset al., U.S. patent No. 5886244; Bidneyet al., U.S. patent No. 5932782; Tomeset al.(1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabeet al.(1988)Biotechnology6:923-926); and the transformation of the Lec1 (WO 00/2808) (also see publications Weissingeret al. (1988)Ann. Rev. Genet.22:421-477; Sanfordet al.(1987)Particulate Science and Technology5:27-37 (onion); Christouet al.(1988)Plant Physiol.87:671-614 (soybean); McCabeet al. (1988)Bio/Technology6:923-926 (soybean); Finer and McMullen (1991)In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singhet al.(1998)Theor. Appl. Genet.96:319-324 (soybean); Dattaet al.(1990)Biotechnology8:736-740 (rice); Kleinet al.(1988)Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Kleinet al.(1988)Biotechnology6:559-563 (corn); Tomes, atent U.S. No. 5240855; Buisinget al., U.S. patent No. 5322783 and 5324646; Tomeset al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Kleinet al. (1988)Plant Physiol.91:440-444 (maize); Frommet al. (1990)Biotechnology8:833-839 (corn); Hooykaas-Van Slogterenet al. (1984)Nature (London)311:763-764; Bowenet al., U.S. patent No. 5736369 (cereals); Bytebieret al.(1987)Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wetet al.(1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapmanet al.(Longman, New York), RR-209 (pollen); Kaeppleret al.(1990)Plant Cell Reports9:415-418 and Kaeppleret al.(1992)Theor. Appl. Genet.84:560-566 (indirect hairs transformation); D Halluinet al. (1992)Plant Cell4:1495-1505 (electroporation); Liet al.(1993)Plant Cell Reports12:250-255 and Christou and Ford (1995),Annals of Botany75:407-413 (rice); Osjodaet al.(1996)NatureBiotechnology14:745-750 (maize viaAgrobacterium tumefaciens); each of which is incorporated herein by reference).

Polynucleotide according to the invention can be introduced in plants by contact of plants with virus or viral nucleic acids. Typically, such methods include the introduction of the polynucleotide constructs of the invention into a molecule of viral DNA or RNA. It is established that AHASL1 protein of the invention can first be obtained as part of a viral polyprotein, which can later be processional by proteolysisin vivoorn vitro to produce the desired recombinant protein. In addition, it was found that the promoters of the invention include promoters used for transcription using the viral RNA polymerase. Methods of introducing a polynucleotide constructs into plants and expression of the encoded proteins they, including molecules of viral DNA and RNA are known in the art (see, for example, U.S. patent No. 5889191, 5889190, 5866785, 5589367 and 5316931, incorporated herein by reference).

Transformed cells can be grown in plants by traditional methods (see, for example, McCormicket al. (1986)Plant Cell Reports5:81-84). These plants can then grow and pollinate or pollen from the same transformed lines, or other lines and to identify the resulting hybrid with constitutive expression of the desired phenotypic characteristics. It is possible to grow two or more generations to confirm that the expression of the desired phenotypic characteristic is stably maintained and inherited and then collect the seeds to confirm that achieved the expression of the desired phenotypic characteristics. Thus, the present invention relates to a transformed seed (also referred to as "transgenic seed") with the polynucleotide construct according to the invention, for example expressing to what Setai according to the invention, stably integrated into the genome.

The present invention can be used for transformation of any plant species, including as non-limiting examples of monocots and dicots. Examples of interest plant species include as non-limiting examples of corn (Zea mays),Brassica spp.(for example,B. napus,B. rapa,B. juncea), especially those speciesBrassicasuitable as a source of oil from the seed, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor,Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), common millet (Panicum miliaceum), Italian millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorins), wheat (Triticum aestivumthe types of solidT. Turgidum), soybean (Glycine max), tobacco (Nicotiana tabacum), potatoes (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), Yam (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa), avocado (Persea americana), figs (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olives (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), min is al ( Prunus amygdalus), beet sugar (Beta vulgaris), sugar cane (Saccharum), oats, barley, vegetables, ornamental plants and conifers. Preferably, plants of the present invention are crops (e.g. sunflower,Brassica spp., cotton, sugar beets, soybeans, peanuts, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley, triticale, sorghum, millet etc).

Herbicide-tolerant plants of the invention can be used in the methods of weed control. Thus, the present invention also relates to a method of controlling weeds in the vicinity of herbicide-tolerant plants according to the invention. The method involves applying an effective amount of a herbicide to the weeds and herbicide-tolerant plant, where the plant compared to a wild type plant has increased resistance to at least one herbicide, particularly imidazolinone or sulphonylcarbamide herbicide. In this method of weed control herbicide-tolerant plants according to the invention preferably represent crops, including as non-limiting examples of sunflower, alfalfa,Brassica spp., soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, corn, barley, rye, millet and sorghum.

When polucheniyami with increased resistance to herbicides, in particular imidazolinones and sulphonylcarbamide herbicides, can be applied to a wide range of compositions for protecting plants from weeds to enhance plant growth and reduce competition for nutrients. Herbicide can be applied alone for weed control to shoot after shoot, before sowing and weed control at sowing in the fields, others described herein plants, or you can apply the composition imidazolinone herbicide containing other additives. Herbicide can also be used for seed treatment. Thus, the effective concentration or an effective amount of a herbicide or a composition comprising an effective concentration or an effective amount of herbicide that can be applied directly to the seeds before planting or during sowing of seeds. Being part with imidazolinone or sulphonylcarbamide herbicide additives include other herbicides, detergents, adjuvants, lyophilizers means, adhesive means, stabilizers, etc. Herbicide composition may be a wet or dry product, and may include as non-limiting examples of bulk powders, emulsifiable concentrates and liquid concentrates. Herbicide and herbicide compositions can be used in accordance with traditional JV the ways, for example, by spraying, watering, obsypaniya, coatings, etc.

The present invention relates to nereshennymi and transgenic seeds with increased resistance to at least one herbicide, particularly the inhibitory AHAS herbicide. Such seeds include, for example, necroshine sunflower seeds, having properties of resistance to herbicides of the plant with patent Deposit ATCC PTA-6084, and transgenic seeds containing the polynucleotide molecule of the invention encoding a herbicide-resistant AHASL1 protein.

The present invention relates to methods of producing herbicide-tolerant plants, particularly herbicide-resistant sunflower plants, using traditional plant breeding, including sexual reproduction. The methods include the crossing of the first herbicide-resistant plant with a second plant resistant to herbicides. The first plant can be any of herbicide-resistant plants of the present invention, including, for example, a transgenic plant containing at least one of polynucleotides of the present invention, encoding herbicide-resistant AHASL, and necroshine sunflower plants having resistance to herbicides sunflower plants with the number of the patent is th ATCC Deposit PTA-6084. The second plant can be any plant that is able to give viable plants-descendants (i.e. seeds), when crossed with the first plant. Typically, but not necessarily, the first and second plants belong to the same species. The methods of the invention also relate to one or several generations of back-crossing plants of the descendants of the first crossing with a plant of the same line or genotype as the first or second plants. Alternatively, the first offspring of crosses or any subsequent interbreeding could be crossed with a third plant, which is a plant of another line or genotype in contrast to the first or second plants. The methods according to the invention can also include the selection of plants having resistance to herbicides of the first plants.

The present invention also relates to methods for increasing the resistance of plants to herbicides, particularly herbicide-resistant sunflower plants, using traditional plant breeding, including sexual reproduction. The methods include the crossing of the first herbicide-resistant plant with a second plant, which may be resistant to herbicides, or may not be resistant to herbicides, or may be resistant to other herbicide or herbicides than the first plant. PE is the first plant can be any of herbicide-resistant plants of the present invention, including, for example, a transgenic plant containing at least one of polynucleotides of the present invention, encoding herbicide-resistant AHASL, and necroshine sunflower plants having resistance to herbicides sunflower plants with the patent number of ATCC Deposit PTA-6084. The second plant can be any plant that is able to give viable plants-descendants (i.e. seeds), when crossed with the first plant. Typically, but not necessarily, the first and second plants belong to the same species. Obtained by this method according to the present invention the plants are descendants have increased compared with the first or second plant, or with both resistance to herbicides. When the first and second plants resistant to various herbicides, plant-descendants have properties combined herbicide resistance of the first and second plants. The methods of the invention also relate to one or several generations of back-crossing plants of the descendants of the first crossing with a plant of the same line or genotype as the first or second plants. Alternatively, the first offspring of crosses or any subsequent interbreeding could be crossed with a third plant, which is a plant of another line or genotype than the first or second is e plant. The methods according to the invention may also include the selection of plants having resistance to herbicides of the first plant, the second plant or both of the first and second plants.

The present invention relates to methods, involving inhibition of AHAS herbicide. In these ways AHAS inhibiting herbicide can be applied by any known in this field way, including as non-limiting examples of seed treatment, soil treatment and processing of the leaves.

Before using AHAS inhibiting herbicide can be obtained in the form of the usual formulations, such as solutions, emulsions, suspensions, powders, powders, pastes and granules. The application depends on the specific purpose; in each case it is necessary to provide the best and uniform distribution of the compounds according to the invention.

Trains get in a known manner (for example, refer to review US 3060084, EP-A 707445 (for liquid concentrate), Browning, "Agglomeration", Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical Engineer''s Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and following: WO 91/13546, US 4172714, US 4144050, US 3920442, US 5180587, US 5232701, US 5208030, GB 2095558, US 3299566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hanceet al., Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN-7514-0443-8)), as an example, in addition to the active compound additives suitable for the composition of chemical fertilizers, such as solvents and/or carriers, if desired, emulsifiers, surfactants and dispersing funds, preservatives, protivovspenivayushchie tools, anti-freezing, for a composition for seed treatment also optionally colorants and/or binders and/or gelling tools.

Examples of suitable solvents include water, aromatic solvents (for example, products of aromatic oil fractions, xylan), saturated hydrocarbons (e.g. petroleum fractions), alcohols (e.g. methanol, butanol, pentanol, benzyl alcohol), ketones (for example cyclohexanone, gamma-butyrolactone), pyrrolidone (NMP, NOP), acetates (glycolized), glycols, dimethylamide fatty acids, fatty acids and esters of fatty acids. In principle, it is also possible to use mixtures of solvents.

Examples of suitable carriers are soil natural minerals (e.g. kaolins, clays, mica, chalk) and soil synthetic minerals (e.g. highly dispersed silica, silicates).

Suitable emulsifiers are non-ionic and anionic emulsifiers (for example, polyoxyethylene ethers of fatty alcohols, alkylsulfate the ATA and arylsulfonate).

Examples of dispersing funds are lignosulfate liquor and methylcellulose.

Suitable used surfactants are alkali metal salts, alkaline earth metal and ammonium salts of lignosulfonic acid, naphtalenesulfonic acid, phenolsulfonic acid, dibutylaminoethanol acid, alkylarylsulfonates, alkyl sulphates, alkyl sulphonates, sulphates of fatty alcohols, glycol esters of fatty acids and sulfated fatty alcohols, furthermore condensates of sulfonated naphthalene and derivatives of naphthalene with formaldehyde, condensates of naphthalene or naphtalenesulfonic acid with phenol and formaldehyde, polyoxyethyleneglycol simple ester, an ethoxylated isooctylphenol, op, Nonylphenol, alkylphenol ethers, tributyltinchloride simple ether, criteriological simple ether, alkylarylsulfonate alcohols, condensation products alcohols and fatty alcohols with ethylene oxide, ethoxylated castor oil, polyoxyethylenesorbitan esters, ethoxylated, polyoxypropylene, acetal lauralanthalasa ether, esters of sorbitol, lignosulfate liquor and methylcellulose.

Substances suitable for obtaining directly sprayable p the cross-sections, emulsions, pastes or oil dispersions, are oil fractions of medium to high boiling point, such as kerosene or diesel oil, furthermore coal tar and resin of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example toluene, xylene, paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, methanol, ethanol, propanol, butanol, cyclohexanol, cyclohexanone, isophorone, highly polar solvents such as dimethylsulfoxide, N is an organic or water.

Also in the structure it is possible to add anti-freeze, such as glycerin, ethylene glycol, propylene glycol and bactericides.

Suitable protivovspenivayushchie means, for example, represent protivovspenivayushchie funds on the basis of silicon, or magnesium stearate.

Suitable preservatives are, for example, are dichlorophen and antialkoholiker.

Compositions for seed treatment may also contain a binder, and optionally dyes.

Binders can be added to improve the adhesion of the active substances to the seed after processing. Suitable binders are copolymers EO/PO surfactants, as well as polyvinyl alcohols, polyvinylpyrrolidone the polyacrylates, the polymethacrylates, polybutenes, polyisobutylene, polystyrenes, polyethylenimine, polyethylenimine, polyethylenimine (Lupasol®, Polymin®), polyethers, polyurethanes, polyvinyl acetate, Tilos and copolymers derived from these polymers.

Optionally the composition can include dyes. Suitable dyes or pigments to the composition for seed treatment are rhodamine B, C.I. Pigment Red 112, C.I. Solvent Red 1, blue pigment 15:4, blue pigment 15:3, blue pigment 15:2, blue pigment 15:1, blue pigment 80, yellow pigment 1, yellow 13 pigment, pigment red 112, pigment red 48:2, pigment red 48:1, pigment red 57:1, pigment red 53:1, orange pigment 43, orange pigment 34, orange pigment 5 green pigment 36, the green pigment 7, white pigment 6, brown pigment 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108.

Examples of suitable gelling funds are carragan (Satiagel®).

Powders, materials for dispersion and spray products can be obtained by mixing or concomitant grinding the active substances with a solid carrier.

Granules, for example coated granules, impregnated granules and homogeneous granules, can be obtained by hydrogen bonds the project active compounds with solid media. Examples of solid carriers are mineral substances, such as silica gels, silicates, mica, kaolin, AttaClay, limestone, lime, chalk, marl, loess soil, clay, dolomite, hard-shelled soil, calcium sulfate, magnesium sulfate, magnesium oxide, soil synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and products of vegetable origin, such as oatmeal, flour, tree bark, wood flour and flour from walnut shells, cellulose powders and other solid carriers.

Basically, the compositions contain from 0.01 to 95 wt.%, preferably from 0.1 to 90 wt.% inhibition of AHAS herbicide. In this case, use of AHAS inhibiting herbicides with a purity of from 90 to 100 wt.%, preferably from 95 to 100 wt.% (according to NMR spectrum). For seed treatment appropriate formulations can be diluted 2-10 times, leading to a concentration in ready for use drugs from 0.01 to 60 wt.% active compounds, preferably from 0.1 to 40 wt.%.

AHAS inhibiting herbicide can be used as such, in the form of its formulations or the use forms derived from them, for example in the form of directly sprayable solutions, powders, suspensions or dispersions, emulsions, oil dispersions, pastes, spray products, substances to spray or granules, the village is edstam spray, comminution, dusting, scattering or watering. Forms of application depend entirely on the purpose; some are intended to ensure in each case the best allocation inhibition of AHAS herbicide according to the invention.

Aquatic forms for use can be obtained from concentrates of emulsions, pastes or wettable powders (sprayable powders, oil dispersions) by adding water. To obtain emulsions, pastes or oil dispersions, the substances themselves or dissolved in an oil or solvent, can be homogenized in water by means of wetting means, which imparts stickiness means dispersing means or emulsifier. However, it is also possible to obtain concentrates consisting of active substance, wetting means, which imparts stickiness means dispersing means or emulsifier and, if appropriate, solvent or oil, and such concentrates are suitable for dilution with water.

The concentration of active compounds in ready-to-use preparations can be varied within relatively wide limits. Mostly, they are from 0.0001 to 10 wt.%, preferably from 0.01 to 1 wt.%.

AHAS inhibiting herbicide can also be applied successfully in the way of ultra low volume (ULV)under which it is possible to apply formulations containing more than 95 wt.% sports the connection, or even to apply the active compound without additives.

Examples of the compounds.

1. Products for dilution with water for use on the leaves. For seed treatment such products can be used diluted or undiluted.

A) water-Soluble concentrates (SL, LS)

Ten mass parts inhibition of AHAS herbicide dissolved in 90 mass parts of water or a water-soluble solvent. Alternatively, add a wetting tools or other AIDS. AHAS inhibiting herbicide when breeding is dissolved in water to form a composition containing 10% (wt./wt.) inhibition of AHAS herbicide.

B) Dispersible concentrates (DC)

Twenty-mass parts of the inhibition of AHAS herbicide dissolved in 70 mass parts of cyclohexanone with addition of 10 mass parts of the dispersing means, for example polyvinylpyrrolidone. When diluted with water to obtain a dispersion, thereby obtaining a composition with 20% (wt./wt.) inhibition of AHAS herbicide.

C) Emulsifiable concentrates (EC)

Fifteen mass parts inhibition of AHAS herbicide dissolved in 7 mass parts of xylene with addition of dodecylbenzenesulfonate calcium and ethoxylate of castor oil (5 mass parts, in each case). When diluted with water to obtain an emulsion, thereby obtaining a composition with 15% (m is S./wt.) inhibition of AHAS herbicide.

D) Emulsions (EW, EO, ES)

Twenty-five mass parts inhibition of AHAS herbicide dissolved in 35 mass parts of xylene with addition of dodecylbenzenesulfonate calcium and ethoxylate of castor oil (5 mass parts, in each case). This mixture is added to 30 mass parts of water, using the device for emulsification (e.g., Ultraturrax), and transformed into a homogeneous emulsion. When diluted with water to obtain an emulsion, thereby obtaining a composition with 25% (wt./wt.) inhibition of AHAS herbicide.

E) Suspensions (SC, OD, FS)

In the granulator when mixing the pulverized 20 mass parts inhibition of AHAS herbicide with the addition of 10 mass parts of the dispersing means, the wetting means and 70 mass parts of water or organic solvent, receiving fine suspension inhibition of AHAS herbicide. When diluted with water to obtain a suspension, thereby obtaining a composition with 20% (wt./wt.) inhibition of AHAS herbicide.

F) Dispersible in water granules and water-soluble granules (WG, SG)

Fifty-mass parts of the inhibition of AHAS herbicide finely milled with the addition of 50 mass parts of the dispersing means and the wetting means and form, in the form of dispersible or water-soluble granules by means of technical appliances (for example extrusion, irrigation columns, fluidized bed). If R is the. water gives a stable dispersion or solution of inhibition of AHAS herbicide, thereby obtaining a composition with 50% (wt./wt.) inhibition of AHAS herbicide.

G) Dispersible powders in water and water-soluble powders (WP, SP, SS, WS)

Seventy-five mass parts inhibition of AHAS herbicide crushed in a crusher rotor and stator with the addition of 25 mass parts of the dispersing means, the wetting means and silica gel. Dilution with water gives a stable dispersion or solution of inhibition of AHAS herbicide, thereby obtaining a composition with 75% (wt./wt.) inhibition of AHAS herbicide.

I) a Gel composition (GF)

Stir in the crushed pellet 20 mass parts inhibition of AHAS herbicide with the addition of 10 mass parts of the dispersing means, 1 mass part of the wetting means with a gelling agent and 70 mass parts of water or an organic solvent to obtain micronized suspension inhibition of AHAS herbicide. Dilution with water gives a stable suspension of inhibition of AHAS herbicide with the formation of the composition with 20% (wt./wt.) inhibition of AHAS herbicide. This gel composition suitable for the treatment of seeds.

2. Products for use in undiluted form on the leaves. For seed treatment, such products can be applied in diluted form.

A) Sprayable powders (DP, DS)

Five mass parts inhibition of AHAS herbicide finely pulverized and thoroughly smeshivayte 95 mass parts of fine kaolin. This gives the sprayed product with 5% (wt./wt.) inhibition of AHAS herbicide.

(B) Granules (GR, FG, GG, MG)

Half mass parts inhibition of AHAS herbicide finely pulverized and mixed with 95,5 mass parts of the media with the formation of a composition containing 0.5% (wt./wt.) inhibition of AHAS herbicide. Used methods are extrusion, spray drying or the use of a fluidized bed. The resulting granules, which are used undiluted for the leaves.

Traditional formulations for seed treatment include, for example, bulk concentrates FS, LS solutions, powders for dry treatment DS, dispersible powders in water for processing suspension WS, water-soluble powders SS and emulsion ES and EC and gel composition GF. These compositions may be applied to seeds diluted or undiluted. Processing in the case of seeds is carried out before sowing or treated seeds directly.

In the preferred embodiment, for seed treatment use of FS. Typically, the composition of FS may contain 1-800 g/l of active ingredient, 1-200 g/l surface-active products, from 0 to 200 g/l anti-freezing, from 0 to 400 g/l connecting means, from 0 to 200 g/l of dye and up to 1 liter of solvent, preferably water.

The present invention relates to netrange the first and transgenic seeds of herbicide-tolerant plants of the present invention. Such seeds include necroshine sunflower seeds, having properties of resistance to herbicides of the plant with patent Deposit ATCC PTA-6084, and transgenic seeds containing the polynucleotide molecule of the invention encoding a herbicide-resistant AHASL1 protein.

In the case of seed treatment seeds of herbicide-tolerant plants of the present invention is treated with herbicides, preferably herbicides selected from the group consisting of AHAS inhibiting herbicides, such as amidosulfuron, azimsulfuron, encultured, chlorimuron, chlorsulfuron, chinaculture, cycloaliphatic, atomiculture, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, halogenation, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, florasulam, dicloflam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac, Perminova, propoxycarbazone, flucarbazone, perbenzoic, piritramid, pyrithiobac, and mixtures thereof, or a composition containing inhibited the speaker AHAS herbicide.

The term "seed treatment" includes all appropriate methods of seed treatment, known in this area, such as the disinfection of seeds, drazhirovanie seeds, dusting the seeds, soaking of seeds and fertilizer seeds.

One of the variants of the present invention further object of the invention is a method of soil treatment by making, in particular, in the ordinary planter, granular composition containing AHAS inhibiting herbicide in the form of a composition/composition (e.g., granular composition, optionally with one or more solid or liquid applicable for agriculture by native and/or optionally with one or more applicable for agriculture surfactants). This method is successfully applied, for example, for a soil prepared for planting cereals, maize, cotton and sunflower.

The present invention also relates to seeds, coated with the composition for seed treatment or containing, where the composition contains at least one ALS inhibitor selected from the group consisting of amidosulfuron, azimsulfuron, benalouane, chlorimuron, chlorsulfuron, chinaculture, cyclomethicone, ethanesulfonic, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, halogenation, imazosulfuron, iodosulfuron is on, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, florasulam, deloulme, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac, peremenovana, propoxycarbazone, flucarbazone, perisesarma, piantanida and pyrithiobac.

The term "seed" includes the seeds and shoots of plants of all types, including as non-limiting examples of true seed, part seed, sprouts, globalwave, bulbs, fruits, tubers, grains, layers, sections, etc. and in the preferred embodiment, means true seeds.

The term "coated with and/or containing" usually means that the active ingredient at the time of application is mainly on the surface of cultured product, although depending on the application method, a greater or lesser part of the ingredient can penetrate into the cultured product. When the specified cultured product (re)planted, it can absorb the active ingredient.

The seed treatment application of AHAS inhibiting herbicide or containing AHAS inhibiting herbicide composition carried out by spraying or dusting the seeds before sowing of the plants and before germination of plants.

During seed processing corresponding formulations are applied by treating the seeds with an effective amount of inhibition of AHAS herbicide or composition containing AHAS inhibiting herbicide. In accordance with this application application rates are generally from 0.1 g to 10 kg A.I. (or a mixture of AI or formulated) per 100 kg of seed, preferably from 1 g to 5 kg per 100 kg of seed, more preferably from 1 g to 2.5 kg per 100 kg of seeds. For specific crops, such as lettuce, the number may be large.

The present invention relates to a method of controlling undesirable vegetation or to method of weed control, which includes bringing the seeds of resistant plants of the present invention before sowing and/or after pre-germination in contact with AHAS inhibiting herbicide. The method may also include sowing seeds, for example, in soil, or in soil in the greenhouse. The method finds particular use for controlling undesirable growth or to control weeds in the immediate vicinity of the seed.

Assume that the control unwanted growth means the destruction of weeds and/or other inhibition or inhibition of normal growth of weeds. Assume that the weeds in the broadest sense refers to all the plants that grow in those areas where they are unwanted.

SOR the yaks of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include as non-limiting examples of the weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum. Monocotyledonous weeds include as non-limiting examples of the weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera.

In addition, the weeds of the present invention may include, for example, agricultural crops, growing in an undesirable place. For example, samsennai plant corn, on the field, which mainly contains soybean plants can be considered as a weed, if you plant corn undesirable on the field with soybean.

As used herein, the designation "include" or variations such as "comprises" or "comprising"should be understood as an expression of inclusion where any specified item, number, or stage or group of elements, integers or steps, but not the exclusion of any other element, number, or stage or group of elements, integers or steps.

The following at the minimum level proposed for the purpose of illustration and do not limit the scope of the present invention.

EXAMPLE 1: Mutagenesis lineHelianthus annuusHA89 and selection of resistant imidazolinone plants

At the end of the vegetation period 1 sunflower plants (Helianthus annuussupport line HA89 were treated with ethylmethanesulfonate (EMS, also called ethyl ester methanesulfonic acid). EMS is a known mutagen, which usually induces DNA transition G-C to A-T (Janderet al. (2003)Plant Physiol.131:139-146). Conducted two separate experiments. In the first experiment used three concentrations of EMS. Plants were treated with a solution containing 0.1%, 1% or 10% (wt./about.) The EMS. For each treatment EMS were seeded 14 rows in the open ground research station Advanta Semillas Biotech Research Station in Balcarce, BsAs, Argentina.

In the second experiment, in the open ground in Advanta Winter Nursery in Oran, Salta, Argentina, seeded 25 rows of seeds of sunflower line HA89. Of these 25 rows 8 rows was treated with 5% EMS, as described above. The remaining 17 rows left untreated.

In each experiment to ensure that the resulting seeds M1are the product of selfing, all plants M0before flowering covered bags. The seed heads after each treatment EMS collected and harvested grains throughout a large party. In the following vegetation period mutant seeds M1plants treated with 0.1%, and 1.0%, to 5.0% or 10.0% EMS, visaria and outdoors, each processed batch in a separate patch. After twelve days, when the plants were under development, with 2-4 pairs of leaves, all EMS treated plants were sprayed with 2× Sweeper 70DG (100 g A.I/ha). The active ingredient in the Sweeper is imazamox. After spraying with herbicide survived all 53 plants, and they were selected as presumably stable. The distribution of resistant plants for processing EMS are presented in table 1.

Table 1
The number of resistant imidazolinone sunflower plants M1obtained after each treatment EMS
The concentration of EMS (%)Qty received resistant plants
0,114
118
55
1016

From each individual surviving plants M1took tissue samples from each sample for research by amplification using PCR and sequencing, as described below in example 2, was isolated DNA.

53 supposedly resistant plants (table 1) allowed coservative field. Of these 53 plants 29 formed seeds M2and these seeds were collected. Soon after every one of these families of M1:2were sown in separate rows (i.e. 29 the row number of rows from 1 to 3 each in Fargo, North Dakota, USA. These families M1:2and sensitive (wild-type) control plants HA89 were sprayed with 0.5 × Sweeper (25 g A.I/ha). Eleven days after herbicide treatment identified three families, who after herbicide treatment survived more than 50% of the plants. Before flowering surviving plants in each of these three families M1:2covered with bags to get resulting from self-pollinated seed M3. Separate the seed heads of each plant M1:2collected and harvested grains throughout. Collected individual tissues of plants M2selected collections.

EXAMPLE 2: PCR Amplification and sequencing of polynucleotides sunflower coding-resistant imidazolinones and wild-type AHASL1 proteins

DNA was isolated from tissue M1one of the three families of M1:2that were described above in example 1. DNA plants M1were subjected to amplification using the polymerase chain reaction (PCR) and sequenced to determine the source of tolerance to imidazolinones that are described in detail below.

Plant M1the specified site collection identified MUT28. Fabric MUT28, as well as from plant tissues HA89 wild type were isolated genomic DNA. Samples selected DNA from MUT28 and HA89 was diluted to the initial concentration 100 ng/μl for use as DNA templates for PCR amplification. For DNA samples from MUT28 and HA89 amplified the complete coding region of the gene of sunflower AHASL1. Specific primers used to obtain each amplicon, are shown in table 2.

Table 2
The PCR primers for amplification of the coding region of the gene of sunflower AHASL1
Region AHASL1Name of primerSequence primer
1st amplicon (843 BP)ALS1-1FCATCATCATTAAATAACCAGAC (SEQ ID NO: 11)
ALS1-1RAACCCGGTAACCTCATCGGTTC (SEQ ID NO: 12)
2nd amplicon (739 BP)ALS1-2FCCCGGTTTTGATAGATGTACCG (SEQ ID NO: 13)
ALS1-2RCTGAGCAGCCCACATCTGATGT (SEQ ID NO: 14)
3rd amplicon (674 BP)ALS1-3FCTGAGCAGCCCACATCTATGT (SEQ ID NO: 15)
ALS1-3RAATTACACAACAAAACATTAAC (SEQ ID NO:16)

On the basis of comparisons of the nucleotide sequences of known genes AHASL1, AHASL2 and AHASL3 were designed PCR primers for specific amplification of the gene of sunflower AHASL1. Used the following PCR conditions for a total reaction mixture of 25 μl: 1× buffer (Invitrogen Corp., Carlsbad, CA, USA), 0.2 mm dNTP (Invitrogen), 2.5 mm MgCl2(Invitrogen), 0.2 μm each primer, and 0.5 μl of platinum Taq (5 U/μl) (Invitrogen) and 100 ng genomic DNA. The PCR reactions were carried out in the device for PCR GeneAmp PCR System 9700 (PerkinElmer, Inc., Boston, MA, USA). Conditions cycles were as follows: stage initial denaturation at 94°C for 1 min, followed by 35 cycles consisting of 94°C for 45 seconds, 52°C for 45 seconds, and 72°C for 70 seconds, and the stage of final elongation at 72°C for 10 minutes. Then two microliters of each PCR product were analyzed by electrophoresis in agarose gel and the expected concentration of DNA in comparison with low molecular weight marker DNA Low DNA Mass Ladder (Invitrogen Corp., Carlsbad, CA, USA). The remaining PCR product was purified using Wizard® SV Gel and PCR Clean-Up System (Promega Corp., Madison, WI, USA). Then purified PCR products were subjected to cycle sequencing using the kit BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in accordance with instructions what s the manufacturer. In addition to the primers used in the PCR amplification (table 2), for carrying out the sequencing of the full coding region of the gene of sunflower AHASL1 were used additional primers listed in table 3.

Table 3
Additional primers for sequencing the coding region of the gene of sunflower AHASL1
Region AHASL1Name of primerSequence primer
1st ampliconALS-3FGCGCTGTTAGACAGTGTCC (SEQ ID NO: 17)
2nd ampliconSUNALS1F1ACTAATCTTGATTTTTCG (SEQ ID NO: 18)
3rd ampliconALS-6RCGGCAGATTTTCAACACGGA (SEQ ID NO:19)

Fluorescently-labeled products after the sequencing reactions were resolved by capillary electrophoresis on the device ABI Prism 310 Genetic Analyzer (Applied Biosystems) and analyzed using the software ABI Prism DNA Sequencing Analysis version 3.7. Files with the results of the sequencing of the sunflower AHASL1 obtained for each amplicon, combined with the use of about the testing the software Vector NTI Suite-Contig Express, version 7.0 (InforMax, Frederick, MD, USA). The obtained DNA sequences were aligned with polynucleotide sequences AHASL1 lines of sunflower HA89 andXanthium sp.(Fig. 1). The predicted amino acid sequence of a new mutant gene sunflower AHASL1 was aligned with the amino acid sequences AHASL1 HA89 andXanthium sp.(Fig. 2) using the software Vector NTI Suite-AlignX, version 7.0 (InforMax), using the default settings. Then identified single nucleotide polymorphisms and single amino acid substitutions.

EXAMPLE 3: Resistance to herbicides sunflower plants MUT28

To assess the stability of sunflower plants MUT28 to imidazolinone herbicides sunflower plants HA89 (wild-type), MUT28 (homozygotes) and HA89/MUT28 (heterozygotes) were sown in the open ground in Balcarce, Argentina, during the growing period in the field trial, a randomized scheme pornoblonde experiment (RCBD)with two replications for assessing the tolerance of plants MUT28 and HA89/MUT28 to three levels Sweeper 70DG: 1×, 2× and 3×. The active ingredient in the Sweeper is imazamox, and 1× dose of 50 g A.I/ha the Results are shown in table 4.

Table 4
Tolerance of sunflower plants MUT28 to imidazolinones (levels damage is possible herbicide)
LineLevel
HA890*7575
MUT28 within families033-
HA89/MUT2802845
IMISUN-1049
* No damage = 0

Compared to HA89 wild-type lines of sunflower MUT28 felt less damage at 1× the level of the Sweeper. Line HA89/MUT28 in this test was also rendered less harm than line HA89, in the case of both levels Sweeper: 1× and 2×. The test results show that both lines: MUT28 (heterozygote) and HA89/MUT28 (heterozygote)had increased tolerance to imidazolinone herbicide, in particular imazamox. However, neither MUT28 or HA89/MUT28 not demonstrated the level of tolerance of sunflower lines IMISUN-1, for which it is known that they nd is Sigaty AHASL1 gene, codereuse AHASL1 protein with the substitution of Ala190on Val.

In a separate test in Balcarce, similar to the one described just test line MUT28 not demonstrated increased tolerance to Sweeper compared to HA89. However, in another separate test conducted in Fargo, ND, USA, 52% of plants M2MUT28 were tolerant, but showed a lower level of tolerance than line SURES-1. SURES-1 is resistant to sulfanilamides line derived from F3 supports oilseeds line F4, which is obtained from plants of the wild populationHelianthus annuuscollected in Kansas, USA (Al-Khatibet al. (1999) "Survey of common sunflower (Helianthus annuus) resistance to ALS-inhibiting herbicides in northeast Kansas, inProceedings of 21th Sunflower Research Workshop, National Sunflower Association, Bismarck, N.D., RR-215).

To assess the tolerance of sunflower plants MUT28 to sulphonylcarbamide herbicides lines of sunflower HA89 (wild-type), MUT28, IMISUN-1 and SURES-1 were planted in the open ground in Balcarce, Argentina during the growing period in the field trial RCBD with two replications for assessing the tolerance of plants MUT28 to sulphonylcarbamide herbicide thifensulfuron (TFS) at 1× and 2× levels. 1× level for TFS is 4.4 g A.I./ha the Results are presented in table 5.

Table 5
Tolerance of plants MUT28 to sulfanilamides (levels of damage by the herbicide)
LineLevel
HA890*7575
MUT2803042
IMISUN-102075
SURES-1053
* No damage = 0

Line MUT28 showed a greater tolerance to TFS than HA89 at 1× and 2× levels, demonstrating that plants MUT28 have greater tolerance to sulphonylcarbamide herbicide compared with sunflower plants of the wild type.

EXAMPLE 4: herbicide-Resistant AHASL1 proteins of sunflower

The present invention relates to nucleotide and amino acid sequences of the polypeptides of the sunflower AHASL1 wild-type and herbicide-resistant. Plants with herbicide-resistant polypeptides AHASL1 been identified is anee, and was described by a number of conserved regions of polypeptides AHASL1, which plots substitutions of amino acids that cause resistance to herbicides (see Devine and Eberlein (1997), "Physiological, biochemical and molecular aspects of herbicide resistance based on altered target sites" inHerbicide Activity: Toxicology, Biochemistry and Molecular Biology, Roe (eds.), RR-185, IOS Press, Amsterdam and Devine and Shukla, (2000)Crop Protection19:881-889).

Using sequences AHASL1 according to the invention and known to experts in the field of the ways you can get additional polynucleotide encoding herbicide-resistant polypeptides AHASL1 with one, two, three or more amino acid substitutions identified in the plots in these conservative areas. Table 6 lists the conservative region AHASL1 protein, amino acid substitutions in these areas, for which it is known that they give resistance to herbicides, and the corresponding amino acids in the protein sunflower AHASL1 SEQ ID NO: 4.

Table 6
Mutations in the conservative regions of polypeptides AHASL1 for which it is known that they give resistance to herbicides, and their equivalent position in the sunflower AHASL1
Conservative region1Mutation2Ref is and The position of amino acids in sunflower
VFAYPGGASMEIHQALTRS3Ala122on ThrRelaxet al.4Ala107
Wright & Penner14
AITGQVPRRMIGT3Pro197on AlaBoutsaliset al.6Pro18213
Pro197on ThrGuttieriet al.7
Pro197on HisGuttieriet al.8
Pro197on LeuGuttieriet al.7
Kolkmanet al.15
Pro197for ArgGuttieriet al.7
Pro197on IleBoutsaliset al.6
Pro197on GlnGuttieriet al.7
Pro197on SerGuttieriet al.7
AFQET 3Ala205AspHartnettet al.9Ala190
Ala205on ValSimpson10
Kolkmanet al.15
Whiteet al.16
QWED3Trp574on LeuBriniard11Trp559
Boutsaliset al.6
IPSGG4Ser653on AsnDevine & Eberlein12
Leeet al.17Ala638
Ser653on ThrChang & Duggleby18
Ser653on Phe
1Conservative region of Devine and Eberlein (1997), "Physiological, biochemical and molecular aspects of herbicide resistance based on altered target sites" inHerbicide Activity: Toxicology, Biochemistry and Molecular Biology, Roeet al. (eds.), RR-185, IOS Press, Amsterdam, and Devine and Shukla, (2000)Crop Protection19:881-889.
2The numbering of amino acids according to the corresponds to the amino acid sequence of the polypeptide AHASL1 Arabidopsis thaliana.
3Sunflower AHASL1 (SEQ ID NO: 4) is the same conserved region.
4The scope of the sunflower AHASL1 (SEQ ID NO: 4)corresponding to the specified conservative region has the sequence IPAGG.
5Relaxet al. (1995)J. Biol. Chem.270(29):17381-17385.
6Boutsaliset al. (1999) Pestic. Sci. 55:507-516.
7Guttieriet al. (1995)Weed Sci.43:143-178.
8Guttieriet al. (1992)Weed Sci.40:670-678.
9Hartnettet al. (1990) "Herbicide-resistant plants carrying mutated acetolactate synthase genes" inManaging Resistance to Agrochemicals: Fundamental Research to Practical StrategiesGreenet al. (eds.), American Chemical Soc. Symp., Series No. 421, Washington, DC, USA.
10Simpson (1998), Down to Earth 53(1):26-35.
11Bruniard (2001), Inheritance of imidazolinone resistance, characterization of cross-resistance pattern, and identification of molecular markers in sunflower (Helianthus annuusL.). Ph.D. Thesis, North Dakota State University, Fargo, ND, USA, RR-78.
12Devine and Eberlein (1997), "Physiological, biochemical and molecular aspects of herbicide resistance based on altered target sites" inHerbicide Activity: Toxicology, Biochemistry and Molecular Biology, Roeet al. (eds.), RR-185, IOS Press, Amsterdam.
13The present invention relates to amino acid sequences of herbicide-resistant AHASL1 with replacement Pro182on Leu (SEQ ID NO: 2) and polynucleotide sequence that encodes this herbicide-resistant AHASL1 (SEQ ID NO: 1).
14Wright and Penner (1998),Theor. Appl. Genet.96:612-620.
15Kolkmanet al.(2004)Theor. Appl. Genet.109: 1147-1159.
16Whiteet al. (2003)Weed Sci.51:845-853.
Leeet al. (1999)FEBS Lett.452:341-345.
18Chang and Duggleby (1998),Biochem J.333:765-777.

All publications and patent applications mentioned in this description, indicative of specialists in this field that applies the present invention. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was directly and individually listed for inclusion as a reference.

Although the above description for a better understanding of the invention is quite detailed with the use of illustrations and examples, it is obvious that you can make certain changes and modifications within the scope of the submitted claims.

1. Plant sunflower with properties of herbicidetolerant plants line MUT28, and a sample of the seed of the specified line deposited under the number of ATSS MOUTH-6084, where this plant sunflower contains at least one copy of polynucleotide encoding herbicidetolerant protein large subunit acetohydroxyacid 1 (AHASL1).

2. Plant sunflower according to claim 1, where the protein of the large subunit acetohydroxyacid 1 (AHASL1) contains a leucine at amino acid position 182 or equivalent position.

3. Plant sunflower according to claim 1 or 2, where the be is OK AHASL1 contains the amino acid sequence of SEQ ID NO: 2 or 6.

4. Plant sunflower according to claim 2, where the AHASL1 gene contains a nucleotide sequence of SEQ ID NO: 1 or 5.

5. Plant sunflower according to claim 1, where this plant has increased tolerance to at least one herbicide selected from the group consisting of imidazolinone herbicides, sulphonylcarbamide herbicides, triazolopyrimidine herbicides, pyrimidinylpiperazine herbicides and sulfonamidophenylhydrazine herbicides and their combinations.

6. Plant sunflower according to claim 1, where the specified plant sunflower is a plant line MUT28, and a sample of the seed deposited under the number of ATSS MOUTH-6084.

7. Plant sunflower according to claim 1, where this plant is transgenic for the specified polynucleotide encoding AHASL1.

8. The seed of the sunflower plants according to any one of claims 1 to 7, where the seed has a high tolerance to at least one herbicide selected from the group consisting of imidazolinone herbicides, sulphonylcarbamide herbicides, triazolopyrimidine herbicides, pyrimidinylpiperazine herbicides and sulfonamidophenylhydrazine herbicides, or their combinations, and where the seed has a high tolerance compared with the tolerance of the seed of the sunflower plants of the wild type NA.

9. The control method sorne the Cove near sunflower plants according to any one of claims 1 to 7, where this method involves the application of an effective amount of a herbicide selected from the group consisting of imidazolinone herbicides, sulphonylcarbamide herbicides, triazolopyrimidine herbicides, pyrimidinylpiperazine herbicides and sulfonamidophenylhydrazine herbicides or mixtures thereof, weeds and plant sunflower.

10. The method according to claim 9, where the specified imidazolinone herbicide selected from the group consisting of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinoline-carboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid and a mixture of methyl ester of 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-Truelove acid and methyl ester of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-Truelove acid and combinations thereof.

11. The method according to claim 9, where the specified sulfanilamides herbicide selected from the group consisting of chlorsulfuron, metsulfuron, sulfometuron, chloroaromatics, thifensulfuron, tribenuronmethyl, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron is methyl, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron, halogenation and mixtures thereof.

12. Selected recombinant or mutated polynucleotide molecule encoding a protein with the activity of AHAS where the specified polynucleotide molecule contains a nucleotide sequence selected from the group consisting of
(a) any of the nucleotide sequences SEQ ID NO: 1 and 5;
(b) nucleotide sequences encoding any amino acid sequence of SEQ ID NO: 2 or 6;
(c) nucleotide sequences that are at least 85% identical to any of the nucleotide sequences selected from the group consisting of SEQ ID NO: 1 and 5;
(d) a nucleotide sequence encoding a protein sequence which is at least 95% identical to any amino acid sequence selected from the group consisting of SEQ ID nos: 2 and 6;
(e) nucleotide sequences that hybridize under tough conditions with any nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and 5;
(f) nucleotide sequences that are fully complementary to any of nucleotide sequences specified in (a)-(e).

13. Selected recombinant or mutated polynucleotide mole is ula indicated in paragraph 12, where specified the protein encoded by the nucleotide sequence specified in (C) or (e), further comprises at least one amino acid selected from the group consisting of
(a) leucine, alanine, threonine, histidine, arginine, or isoleucine at amino acid position 182 or equivalent position;
(b) a threonine at amino acid position 107 and the equivalent provision;
(c) aspartate or valine at amino acid position 190 and the equivalent provision;
(d) leucine at amino acid position 559 and the equivalent provision; and
(e) asparagine, threonine, phenylalanine, or valine at amino acid position 638 and the equivalent provision.

14. The expression cassette containing a promoter functionally linked to a polynucleotide molecule according to item 12 or 13.

15. The expression cassette according to 14, where the specified promoter capable of directing gene expression in the bacterium, a fungal cell, an animal cell or plant cell.

16. A host cell, non-human cell transformed with the expression cassette according to 14 or 15.

17. A host cell according to item 16, where specified a host cell selected from the group consisting of bacteria, fungal cells, animal cells and plant cells.

18. Transforming a vector containing a gene of interest and a gene selective marker, where the specified gene selective mA is Kera contains a promoter, functionally associated with the polynucleotide molecule according to item 12.

19. Transforming a vector by p where specified promoter capable of expression in a plant cell.

20. Transforming a vector by p or 19, where the specified promoter is a constitutive promoter.

21. Transforming a vector by p where the specified gene selective marker further comprises a functionally cohesive sequence for directed transport in chloroplasts.

22. Transforming a vector by p where specified promoter capable of expression in bacteria or yeast.

23. A host cell, non-human cell containing transforming the vector according to any one of p-22.

24. Transformed plant containing in its genome is stably integrated polynucleotide construct containing a nucleotide molecule according to item 12, functionally associated with a promoter directing expression in a plant cell, where the AHAS activity of the indicated transgenic plants compared with nontransgenic plant is increased, and where the specified tolerance transgenic plants with at least one herbicide compared with nontransgenic plants raised.

25. The transformed plant according to paragraph 24, where the specified promoter selected from the group consisting of a constitutive is momotarou and tissue-specific promoters.

26. The transformed plant according to paragraph 24 or 25, where the specified polynucleotide structure further comprises a functionally cohesive sequence for directed transport in chloroplasts.

27. The transformed plant according to paragraph 24, where the specified herbicide is imidazolinone herbicide.

28. The transformed plant according to item 27, where the specified imidazolinone herbicide selected from the group consisting of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinoline-carboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, mixtures of methyl ester of 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-Truelove acid and methyl ester of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-Truelove acid and combinations thereof.

29. The transformed plant according to paragraph 24, where the specified herbicide is sulfanilamides herbicide.

30. The transformed plant according to clause 29, where the specified sulfanilamides herbicide selected from the group consisting of chlorsulfuron, metsulfuron, sulfometuron, chloroaromatics, thifensulfuron, three is eurometal, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron, halogenation and mixtures thereof.

31. The transformed plant according to paragraph 24, where the aforementioned transformed plant is a dicotyledonous or monocotyledonous.

32. Transformed plant p, where the specified dicotyledonous plant selected from the group consisting of sunflower, soybean, cotton, Brassica spp., Arabidopsis thaliana, tobacco, potatoes, sugar beets, alfalfa, safflower and peanuts.

33. Transformed plant p, where the specified monocotyledonous plant selected from the group consisting of wheat, rice, maize, barley, rye, oats, triticale, millet, and sorghum.

34. The seed of the transformed plant according to any one of p-33, where the specified seed contains the specified polynucleotide structure.

35. Transformed plant cell containing in its genome is stably integrated polynucleotide construct containing a nucleotide molecule according to item 12, functionally associated with a promoter directing expression in a plant cell.

36. Method of increasing the activity of AHAS in plants, involving the transformation of a plant cell a polynucleotide construct, the soda is containing a nucleotide molecule according to item 12, functionally associated with a promoter directing expression in a plant cell, and regenerating transformed plants from the selected transformed plant cells.

37. The method of obtaining herbicidetolerant plants, providing
the transformation of the plant cell a polynucleotide construct containing a polynucleotide molecule according to item 12, functionally associated with a promoter directing expression in a plant cell; and
regeneration of transgenic plants from the selected transformed plant cells, where the aforementioned transformed plant has increased tolerance to at least one herbicide compared with the tolerance nontransgenic plants to a specific herbicide.

38. The method according to clause 37, where the specified promoter selected from the group consisting of constitutive promoters and tissue specific promoters.

39. The method according to clause 37 or 38, where the specified polynucleotide structure further comprises a functionally cohesive sequence for directed transport in chloroplasts.

40. The method according to clause 37 or 38, where the AHAS activity of the indicated transgenic plants increased compared to the transformed plant.

41. The method according to clause 37 or 38, where the specified herbicide is imidazoline the inhibiting herbicide.

42. The method according to paragraph 41, where the specified imidazolinone herbicide selected from the group consisting of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinoline-carboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, mixtures of methyl ester of 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-Truelove acid and methyl ester of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-Truelove acid and combinations thereof.

43. The method according to clause 37 or 38, where the specified herbicide is sulfanilamides herbicide.

44. The method according to item 43, specified sulfanilamides herbicide selected from the group consisting of chlorsulfuron, metsulfuron, sulfometuron, chloroaromatics, thifensulfuron, tribenuronmethyl, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron, halogenation and mixtures thereof.

45. The method according to clause 37 or 38, where the aforementioned transformed plant is a dicotyledonous or Oded is determined as being.

46. The method according to item 45, where the specified dicotyledonous plant selected from the group consisting of sunflower, soybean, cotton, Brassica spp., Arabidopsis thaliana, tobacco, potatoes, sugar beets, alfalfa, safflower and peanuts.

47. The method according to item 45, where the specified monocotyledonous plant selected from the group consisting of wheat, rice, maize, barley, rye, oats, triticale, millet, and sorghum.

48. The method according to clause 37 or 38, where this plant cell has a tolerance to at least one herbicide to the specified stage of transformation.

49. The way to increase herbicidetolerant at herbaceuticals plants, providing a stage of transformation herbaceuticals plants polynucleotide construct containing a nucleotide molecule, functionally associated with a promoter directing expression in a plant cell, and regenerating transformed plants from the selected transformed plant cells, where the specified herbaceuticals plant has increased tolerance compared with transformed herbaceuticals plant; where the polynucleotide molecule contains a nucleotide sequence selected from the group consisting of
(a) any of the nucleotide sequences SEQ ID NO: 1 or 5;
(b) nucleotide sequences encoding any amino acid sequence of alnost SEQ ID NO: 2 or 6;
(c) nucleotide sequences that are at least 85% identical to any of the nucleotide sequences selected from the group consisting of nucleotide sequences SEQ ID NO: 1 and 5;
(d) a nucleotide sequence encoding a protein sequence which is at least 95% identical to any amino acid sequence selected from the group consisting of SEQ ID nos: 2 and 6.

50. The method according to § 49, where the specified herbaceuticals plant, up to the specified stage of transformation contains herbicidally AHASL protein.

51. The method according to § 49 or 50, where the specified herbaceuticals plant is not genetically engineered for the expression of the specified herbaceuticals AHASL protein.

52. The method according to § 49 or 50, where the specified herbaceuticals plant is genetically engineered for the expression of the specified herbaceuticals AHASL protein.

53. The method according to § 49 or 50, where the specified herbaceuticals plant, up to the specified stage of transformation is imidazolidinethione plant.

54. The method of selection of transformed plant cells, providing for transforming plant cells transforming vector plants, impacts on specified transformed plant cell at least one herbicide concentration, and generouse growth of the untransformed plant cell, and identification of the specified transformed plant cell by its ability to grow in the presence of a specified herbicide; where specified transforming a vector of plant gene contains a selective marker containing a promoter functionally linked to a polynucleotide molecule according to item 12, where specified, the promoter directs expression in a plant cell.

55. The method according to item 54, where the specified herbicide is imidazolinone herbicide, sulfanilamides herbicide or a mixture.

56. The method according to item 54, where specified transforming the vector plant further comprises at least one gene of interest.

57. The method according to any of PP-56, additionally providing for the stage of regeneration of transformed plants from the selected transformed plant cells.

58. The method of controlling weeds in the vicinity of the transformed plants, where the method involves the application of an effective amount of imidazolinone herbicide, sulfanilamides herbicide, triazolopyrimidine herbicide, pyrimidinemethanol herbicide and sulfonamidophenylhydrazine herbicide, or a mixture thereof to the weeds and to the transformed plant, where the aforementioned transformed plant has increased tolerance to the herbicide compared the structure with nontransgenic plant, and the transformed plant contains in its genome at least one expression cassette containing a polynucleotide molecule according to item 12, functionally associated with the promoter, directing gene expression in a plant cell.

59. The method according to § 58, where the specified imidazolinone herbicide selected from the group consisting of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinoline-carboxylic acid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)nicotinic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid and a mixture of methyl ester of 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-Truelove acid and methyl ester of 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-Truelove acid and combinations thereof.

60. The method according to § 58, where the specified sulfanilamides herbicide selected from the group consisting of chlorsulfuron, metsulfuron, sulfometuron, chloroaromatics, thifensulfuron, tribenuronmethyl, benzylbromide, nicosulfuron, ethanesulfonate, rimsulfuron, triflusulfuron, triasulfuron, primisulfuron, chinaculture, amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron is netila, halogenation and their combinations.

61. The method according to any of PP-60, where this plant is a dicotyledonous or monocotyledonous.

62. The method according to PP, where the specified dicotyledonous plant selected from the group consisting of sunflower, soybean, cotton, Brassica spp., Arabidopsis thaliana, tobacco, potatoes, sugar beets, alfalfa, safflower and peanuts.

63. The method according to p, where the specified monocotyledonous plant selected from the group consisting of wheat, triticale, maize, rice, sorghum, rye, millet and barley.

64. The selected polypeptide encoded by the polynucleotide molecule according to item 12.

65. The method of obtaining herbicidetolerant plants, involving crossing a first plant that is tolerant to the herbicide, with a second plant that is tolerant to the herbicide, where the first plant is a plant according to any one of claims 1 to 7 and 24-33.

66. The method according to p, additionally providing for the selection of plants descendant, tolerant to the herbicide.

67. Herbicidetolerant plant obtained by the method according to p or 66.

68. Seed plants on p, where the specified seed has a high tolerance to at least one herbicide selected from the group consisting of imidazolinone herbicides, sulphonylcarbamide herbicides, triazolopyrimidine herbicides, pyrimidinylpiperazine herbicides and
sulfone aminocarbonylmethyl herbicides, or their combinations, and where the seed has a high tolerance compared with the tolerance of the seed of the sunflower plants of the wild type NA.

69. The way to increase herbicidetolerant plants, involving crossing the first plant with a second plant, where the first plant is a plant according to any one of claims 1 to 5, 24-33 and 67.

70. The method according to p, additionally providing for the selection of plants descendant, with increased herbicidetolerant compared with herbicidetolerant specified second plant.

71. The plant obtained by the method according to p or 70.

72. Seed plants on p, where the specified seed has increased herbicide-tolerance to at least one herbicide selected from the group consisting of imidazolinone herbicides, sulphonylcarbamide herbicides, triazolopyrimidine herbicides, pyrimidinylpiperazine herbicides and sulfonamidophenylhydrazine herbicides, or their combinations, and where the seed has a high tolerance compared with the tolerance of the seed of the sunflower plants of the wild type NA.

73. The seed of a plant according to any one of claims 1 to 5, 24-33, 67 and 71, where the specified seed treated with herbicide, AHAS inhibiting.

74. The method of controlling undesirable vegetation, providing reduction of plant seeds for pleasure is in one of claims 1 to 5, 24-33, 67 and 71 before sowing and/or after pre-germination in contact with the herbicide, AHAS inhibiting.



 

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21 cl, 2 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of producing (meth)acrylates of N-hydroxy-alkylated amides, in which cyclic N-hydroxyalkylated amides (C): , where Z1 denotes unsubstituted or mono-substituted nitrogen (N-R3); R1 and R2, in each case independently denote an alkylene with 2-20 carbon atoms, cycloalkylene with 5-12 carbon atoms, arydene with 6-12 carbon atoms; R3 denotes hydrogen, alkyl with 1-18 carbon atoms, alkenyl with 2-18 carbon atoms, aryl with 6-12 carbon atoms or cycloalkyl with 5-12 carbon atoms; etherified with methacrylic acid or reesterified with at least one (meth)acrylic ester (D) in the presence of at least one heterogeneous catalyst selected from a group consisting of inorganic salts (Co), having pKb not higher than 7.0 and not lower than 1.0, and its solubility in the reaction medium at 25°C is not more than 1 g/l, and enzymes (F) selected from a group comprising esterase (E.C.3.1.-.-), lipase (E.C.3.1.1.3), glycosylase (E.C.3.2. -.-) and protease (E.C.3.4. -.-), where the heterogeneous catalyst is separated from the reaction mixture through filtration, electrofiltration, absorption, centrifuging or decantation.

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FIELD: medicine.

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4 ex

FIELD: chemistry.

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4 ex

Butinol 1 esterase // 2412996

FIELD: medicine.

SUBSTANCE: cultivated is microorganism, which produces specified protein or is transformed with recombinant vector for transformation of eukaryotic or prokaryotic host organism, containing polynucleotide, encoding said protein, as well as complementary to it polynucleotides, and hybridised with it nucleotide sequences, or expression cassette, which contains, at least, one said polynucleotide operatively bound with, at least, one regulatory nucleotide sequence, protein is isolated from cell culture. Invention also relates to method of enantio-selective enzymatic ester hydrolysis and method of enantio-selective re-etherification with application of obtained protein.

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26 cl, 1 dwg, 3 tbl, 7 ex

FIELD: medicine.

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2 dwg, 5 tbl, 5 ex

FIELD: medicine.

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30 cl, 34 dwg, 23 tbl, 13 ex

FIELD: medicine.

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16 cl, 6 tbl, 7 ex

FIELD: medicine.

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8 cl, 3 ex, 1 tbl

FIELD: chemistry.

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2 cl, 2 dwg, 3 tbl, 1 ex

FIELD: medicine.

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EFFECT: invention makes it possible to obtain increased output of highly purified human TβRII protein with native N-end.

3 cl, 3 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to construction of transposon-induced mutant of melioidosis causative agent Burkholderia pseudomallei, bearing inactivated sequences of genes of drug efflux (multidrug efflux pumps) and chromosomal class A β-lactamases. The strain is obtained as a result of directed genetic construction, including stages of insertion mutagenesis of cells of polyresistant strain B. pseudomallei 57576 SMCP with high level of resistance to cephalosporins and fluoro-quinolones. The strain is deposited in SC "Microbe" under No KM31. In accordance with the invention the strain is a stable mutant, bearing insertions Tn9 in sequences of genes blaD, omp27 and omp71, preserving its properties during storage, cultivation in nutrient media and during passages through organism of laboratory animals, and intended for investigation of role of said R-determinant in development of drug resistance of multiple type in melioidosis causative agent and closely related microorganisms.

EFFECT: strain KM 31 can be used for investigation of functional role of genes of antibiotic efflux and determinants of functional modification, inactivation of antimicrobial drugs in pathogenic species of genus Burkholderia.

3 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: micropowder, obtained by crushing and dispersion of ordered packings of microspheres SiO2, is subjected to preliminary thermal processing at temperature 750-850°C for 40-60 min, with rate of temperature change within interval 150-200°C/hour and further thermal processing in vacuum at temperature 300-350°C and pressure 0.1-0.5 Pa for 80-100 min, with application as ordered packings of microspheres of synthetic opal matrices with size of microspheres 0.25-0.4 mcm and micropores 0.15-0.3 mcm or natural mineral from silica with opal-like structure with size of microspheres 0.3-4 mcm and micropores 0.5-4 mcm.

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6 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: plasmid vector pE-Trx-Aur is constructed for expression of aurelin in cells of Escherichia coli in composition of hybrid protein Trx-Aur, consisting of two DNA fragments, whose nucleotide sequence is given in description. By means of said vector parent strain of Escherichia coli is transformed, obtaining strain-producent of hybrid protein Trx-Aur. In order to obtain peptide aurilin cultivation of cells of obtained strain-producent is carried out, after that, performed are: cell lysis, affine purification of hybrid protein Trx-Aur on metal-chelate carrier, decomposition of hybrid protein Trx-Aur with bromine cyan by residue of methionine, introduced between sequences of aurelin and thioredoxin, and purification of target peptide by method of reversed-phase HPLC.

EFFECT: invention makes it possible to obtain biologically active aurelin by simplified technology and without application of hard-to-obtain natural raw material.

3 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: claimed is novel mutant oxidase of D-amino acids with higher temperature stability. Amino acid sequence of enzyme corresponds to amino acid sequence of oxidase of D-amino acids from Trigonopsis variabilis of wild type, in which tryptophan residue in position 46 is substituted with phenylalanine residue and cysteine residue in position 298 is substituted with alanine residue, and 6 last amino acids on C-end are replaced by novel sequence of 28 residues.

EFFECT: invention makes it possible to increase temperature stability of obtained mutant oxidase of D-amino acids in comparison with thermal stability of wild type enzyme.

2 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method related to field of medicine, namely to transfusiology. Method includes collecting umbilical cord blood (UCB) into container, weighing it with calculation of its volume, determining type of human leukocyte antigen for comparing hypocompatibility, presence or absence of infection, introduction into packet with UCB, anticoagulant and hydroxyethyl starch, mechanical mixing of packet content in various planes, incubation after mixing at room temperature with separation of erythrocyte mass (EM) by sedimentation, placement of centrifuge chamber in separator, connecting to it on symmetry axis main pipeline, the latter being connected via distribution unit via pipelines to sacks for collection of blood components and to packet with mixture of UCB, anticoagulant and hydroxyethyl starch. After that, chamber is filled with packet content, and mixture is subjected to separation, with its separation into EM, plasma and leukocyte concentrate with their distribution into corresponding sacks and into empty packet. After separation, viability and amount of SC are determined, they are checked on biological inoculation of sample of plasma and EM mixture, dose of plasma is tested for detection of antibodies, hemotransmissive infections, dose of EM is used to determine blood group and Rhesus factor.

EFFECT: method application makes it possible to increase quality and reliability of determining SM from various biological contaminations.

1 dwg

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

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EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

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