Barley with low hordein content

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

 

The present invention relates to methods of producing food or drinks on the basis of malt, suitable for consumption by a person suffering from celiac disease. In particular the present invention relates to methods of producing food or drinks on the basis of malt with a low content Gordeyev. Also available barley plants, giving the grain that can be used in the methods of the invention.

Celiac disease (gluten enteropathy) (CD, also called celiac disease-sprue) is mediated T-cell autoimmune disease of the small intestine, which is called in susceptible people ingestion of certain spare proteins, known generally as the prolamins from wheat (gluten, consisting of glutenins and gliadins), barley (Gordeyev) or rye (secalinum). The oat prolamins (Avenida), apparently tolerated by most patients with celiac disease (Hogberg et al., 2004; Peraaho et al, 2004a), but fewer patients with celiac disease they can cause a positive reaction (Lundin et al. 2003; Peraaho et al. 2004b). CD occurs in approximately 0.25 to 1% of the population, at least in Australia, North and South America, Europe, Africa and India (Hovell et al. 2001; Fasano et al. 2003; Treem 2004), however, the disease is likely to be diagnosed at a low level. Increase awareness about the symptoms and consequences of nepodal wsysa treatment of CD cases in Australia leads to an increase in the number of diagnosed cases by 15% annually. Approximately 1-4 Europeans and inhabitants of Western Asia have alleles HLA-DQ8 or-DQ2, which are necessary but not sufficient determinant CD (Treem 2004). However, only about 1 in 20 people with these alleles developing CD. Currently, the only treatment is total abstinence from wheat, barley and rye, as relapses can be caused by the consumption of only 10 milligrams of gluten per day (Biagi et al., 2004).

In case of not detecting disease or lack of treatment, CD leads to serious health consequences that can be life-threatening, especially in children the first year of life. CD causes deformation absorbing villi of the small intestine and can lead to destruction of the villi. In the result, nutrients are absorbed poorly, and it may be associated with weight loss, fatigue, mineral deficiency, dermatitis and loss of night vision, as well as intensive intestinal disorders, which usually includes bloating, diarrhea and cramping. Persons with untreated CD are at increased risk of developing cancer, for example, 10-fold increased risk of carcinoma of the small intestine, 3-6-fold increase in the risk of non-Hodgkin lymphoma and 28-fold increased risk of T-cell lymphoma of the intestine. CD is also 3-fold increased risk times the development of type I diabetes (Peters et al., 2003; Peters et al., 2003; Verkarre et al., 2004). Also reported a five-fold increase in the incidence of mental depression in patients with celiac disease (Pynnonen et al., 2004).

The molecular basis of celiac disease is now fairly well understood (Sollid 2002; Hadjivassiliou et al. 2004) and is a reaction to a specific sequence of amino acids in the prolamins. Poorly digested peptides of prolamins, which are rich in Proline and glutamine, correspond substrate motif, which in the intestinal mucosa interacts tissue transglutaminase (tTG) of a person, which desmidium key residues of glutamine. The resulting negatively charged glutamic acid allows dezaminirovanie the prolamin contact with a specific class of HLA molecules (DQ2 or DQ8) (Kim et al. 2004). Stimulated proliferation of specific clones of T-cells, the so-called DQ2 (8)/CD4+bearing T-cells directed to the endothelial lining of the intestine that secrete lymphokines, causing atrophy of the intestinal villi or antibody production (Hadjivassiliou et al. 2004). These clones of T-cells reach maximum concentration in peripheral blood of patients with celiac disease about six days after the food provocation (Anderson et al. 2000). The toxicity of purified proteins in celiac disease patients, therefore, can be accurately and specific determined by measuring isposable to stimulate T-cells to produce IFN-γ - cytokine, which is essential in the pathogenesis of enteropathy observed in celiac disease. It is therefore likely that the disease is caused by the immune system of the host, which responds to the prolamins, forming a powerful immune response, as if they were more of a pathogen than the allergen.

Gluten wheat consists of many hundreds of different, but related proteins, including Monomeric gliadins and polymeric glutenin. The gliadins comprise approximately half of the gluten fraction, while the α-gliadins comprise more than 50% of gliadins (Wieser et al., 1994; Gellrich et al., 2003). To date, most data on toxicity in celiac disease is focused on the α-gliadin - first cloned and fully tsakonian the prolamin (Kasarda et al., 1984). The toxicity of α-gliadin wheat celiac disease is largely determined by a single glutamine residue within the key epitope length of 17 amino acids (Arentz-Hansen et al., 2000; Anderson et al., 2000; Shan et al., 2002). Were identified natural and synthetic peptides that carry point mutations in this region that were not toxic (Vader et al., 2003). Thus, it seems possible that can be identified other non-toxic, but functional prolamine molecules. At the moment the practical prediction of toxicity in celiac disease limited to the small fractie the prolamins, which were described by the amino acid sequence or nucleotide sequence of the genes encoding them.

Barley is a diploid cereal culture, which is widely grown in areas with cooler climates for the production of food and beverage. Proteins of barley seeds are divided into albumin, globulin, prolamin (hordein) and glutelin depending on their solubility in water, salt solution, an aqueous solution of alcohol, as well as in alkaline or acid solutions, respectively. Approximately half of the spare seed proteins in barley is present in prelaminates faction. These prolamins are primarily spare proteins, which serve as sources of carbon, nitrogen or sulfur for growth and development after germination. Hordein is approximately 40% protein seed, although this depends on the availability of nitrogen to plants in the growth process. The loci encoding the barley prolamins were characterized mainly due to their contribution to the quality barley malt, and foaming and turbidity in beer production. In barley there are four class of prolamins - B-, C-, D - and γ-hordein encoded by loci Hor2, Horl, Hor3 and Hor5 respectively, on chromosome 1H (Shewry et al. 1999). These loci encode proteins that vary from one prolamine (for example, D-hordein) on the families of proteins, containing 20-30 members (for example, B - and C-hordein). B - and C-hordein are relatively more often, including approximately 70% and 24% of the total number Gordeyev respectively. D - and γ-hordein represent minor components of approximately 2-4% each. Molecular weight Gordeyev changes approximately from 35 kDa to 100 kDa. Any barley prolamins, which have close homology with α-gliadins of wheat, no, but it is well known that Gordini toxic to celiacs (Williamson & Marsh 2000). Regarding the extent to which individual Gordini barley stimulate the CD, not reported.

Beer is widely consumed by the product made from malted barley, so beer, as is widely assumed, is not suitable for celiacs and, as a rule, excluded from their diet. Kanerva et al. (2005) were able to identify the prolamins in low levels in all varieties of many Beers, except for one. Doctors and nutritionists usually persuade their patients with CD try to avoid consumption of any food containing wheat, barley or rye, including beer. In the USA the FDA definition of "gluten-free" requires that the product was made from raw materials that do not contain gluten, no wheat, barley or rye. The Codex Alimentarius allows labeled "gluten-free" on food products is the settlement, containing not more than 200 ppm of gluten (0.2 g per kilogram or liter), it also complies with the European standard for products that do not contain gluten". The majority of patients with celiac disease can consume approximately 10 mg of gluten per day without the development of the main effect (Thompson, 2001).

The prolamins, toxic for celiac patients, specific can be detected using immunoassays such as ELISA (Ellis et al., 1990; Sorell et al., 1998). Data analyses are based on a specific interaction between a protein and an antibody. Also used electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (LTOs-PAG) and HPLC (Kanerva et al., 2005; Marchylo et al., 1986; Sheehan and Skerritt, 1997).

Thus, there is a need in the barley with a significantly lower content of the CD-inducing Gordeyev, which can be used in foods and beverages intended for persons at risk of developing CD.

The INVENTION

In barley there are four class of prolamins - B-, C-, D-and γ-hordein encoded by loci Hor2, Horl, Hor3 and Hor5 respectively. The authors of the present invention have found that, at least, B, C and D classes cause unwanted inflammatory responses in persons with celiac disease.

Whereas previously identified various mutants of barley with the products of certain classes Gordeyev reduced in ur is vnah, also watched it at least offset by increased production Gordeyev other classes. This suggests that the seed of barley has compensatory mechanisms that provide certain levels Gordeyev necessary to ensure that the seed was viable. Unexpectedly, the authors of the present invention have determined that most, if not all number, Gordeyev produced in the barley, you can remove and get viable seeds that can germinate and give barley plants in the field, despite the loss of the alternate forms of nitrogen in the seed. These seeds are particularly useful for the production of food and beverages suitable for consumption by persons suffering from celiac disease.

Thus, in one aspect the present invention provides a method of producing food or drink made of malt, including mixing grains of barley or malt, flour or wholemeal flour derived from the specified grain with at least another component of food or drink, and grain, malt, flour or wholemeal flour contains about 25% or less of the content Gordeyev in the corn, the barley plants of the wild type, or malt, flour or wholemeal flour obtained in a similar way from the grain obtained from the corresponding plant barley wild-type, obtaining as a result of food or beverage on the basis of malt.

Preferably, grain, malt, flour or wholemeal flour contains about 15% or less, about 10% or less, about 7.5% or less, about 5% or less or, more preferably about 2.5% or less of the content Gordeyev in the corresponding grain barley plants of the wild type, or malt, flour or wholemeal flour obtained in a similar way from the seeds of wild type.

Examples of plants of barley wild-type and include, among others, Bomi, Sloop, Carlsberg II, K8 or L1.

In another embodiment, the grain contains about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, or more preferably about 2.5% or less of the contents B, C and/or D-Gordeyev or any combination of the above in the corresponding grain barley plants of the wild type. Malt, flour or wholemeal flour can include a similar degree of reduction of the level Gordeyev B, C and/or D, or any combination of the above.

In another embodiment, the flour contains less than about 0.4%, less than about 0.3%, less than about 0.2% and more preferably less than about 0.1% Gordeyev. Levels hordein in flour obtained from the specified corn, can be determined using any method known in the prior art, such as Speer the TV fractionation.

In one embodiment, the implementation of the grain has an average weight (average weight 100 grains), at least about 2.4, Preferably, the grain has an average weight of from about 2.4 to about 6 g, more preferably the average weight of from about 3.5 to about 6,

In another embodiment, the starch content in the grain is at least about 50% (by weight). More preferably, the starch content in the grain is from about 50% to about 70% (by weight). The starch content may be determined using any methods known in the art. For example, there may be used the method described in Example 4.

In the following embodiment, the toxicity in celiac flour produced from grain, is less than about 50%, less than about 25%, more preferably about 10% or less of the relevant indicators of flour obtained from the corresponding grain barley plants of the wild type. Toxicity in celiac disease can be determined using any technique known in the prior art. For example, there may be used the method described in Example 1.

In yet another embodiment, a malt obtained from the grain, contains less than about 200 ppm Gordeyev, less than about 125 ppm Gordeyev, more preferably less than approximately the nutrient 75 ppm Gordeyev. Content hordein can also be determined using any method known in the prior art. For example, there may be used the method described in Example 7.

In another embodiment, at least about 50% of the genome of barley grain is identical to the genome of barley varieties Sloop.

Preferably, the seed obtained from a plant that is homozygous for at least one, at least two, at least three or more loci for genetic variations (variations), which leads to a low content gardein at least one of at least two or all three classes B, C and D, compared with the corresponding plant barley wild-type. More preferably, these genetic variants are alleles, which removed most or all of the genes encoding the B-hordein, locus Hor2, and/or mutant allele in the locus ofLys3barley.

In one embodiment, the grain obtained from nedrencheskogo plants. For example, the grain can be obtained by crossing between Riso 56 and Riso 1508 or their offspring, including mutationhor2andLys3accordingly, present in these parental lines. Preferably, such plants obtained by crossing include genetic background, essentially different from Rio 56 or Riso 1508, for example containing less than about 25% of the genetic background of these parental lines.

In another embodiment, the seed obtained from the transgenic plants. One of the embodiments the transgenic plant is a plant that includes a transgene encoding polynucleotide, which suppresses production of at least one hordein in the grain. Preferably, polynucleotide this option is antimuslim polynucleotides, semantic polynucleotides, catalytic polynucleotide, artificial microRNA or RNA duplex, which inhibit the expression of one or preferably several genes encoding Gordini.

In another embodiment the transgenic plant is a plant that comprises a transgene encoding a prolamin, which is less toxic, or preferably non-toxic, for persons suffering from celiac disease. An example of prolamine, which is non-toxic for individuals suffering from celiac disease, including, without limitation, Avenida oats and Seine corn.

In one of the embodiments the method includes obtaining from grain flour or wholemeal flour.

In a particularly preferred embodiment, the method further includes malt produced from corn. In the embodiment, the method further including the AET fractionation dried, sprouted grains into two or more fractions of endosperm fraction endothelial layer fraction of the husk, the germ fraction and fraction of malt sprouts; and combining and mixing predetermined amounts of two or more fractions.

As for the production of malt and beer, an important component of seed barley is starch. While the levels of starch in barley mutants with reduced Gordeyev, as was shown earlier, is reduced, which may cause the seed to be unsuitable for the production of malt and beer. The inventors have in particular found that the seeds of barley, in which most, if not all the amount produced gardein has been removed, can be used for the production of malt and beer with appropriate properties for commercial production. Thus, in the preferred embodiment, a drink made of malt is to beer or whiskey, and the method includes the germination of grain.

In one embodiment, the implementation of a drink based on the malt is to beer, which contains at least about 2%, more preferably at least about 4% alcohol. Preferably, the alcohol is ethanol.

In yet another embodiment, a drink made of malt is to beer, which contains less than about 1 ppm of the mountains is einav.

In the following embodiment, at least about 50% of the grain germinates within 3 days after swelling in standard conditions used for malting.

Examples of food products that can be produced using the methods of the invention include, among others, flour, starch, yeast or yeast-free bread, pasta, noodles, pet food, ready meals (cereal), snack foods, cakes, malt, flour confectionery or food products containing sauces based flour.

Preferably, the food product or a beverage on the basis of the malt is intended for human consumption. In another preferred embodiment, after the consumption of food or drink, at least one symptom of celiac disease does not develop from a person with the specified condition.

In another aspect the present invention provides a method of producing food or drink made of malt, including the mixing of malt including one or more proteins of barley grain and less than about 200 ppm Gordeyev, and/or flour that includes one or more proteins of barley grain and less than about 0.4% Gordeyev at least one other component of the food product or beverage, obtaining as a result of food or beverage on the basis of olode.

In one of the embodiments, the method comprises the production of malt and/or flour.

In another aspect the present invention provides a method of producing food or drink made of malt, including mixing grains of barley or malt, flour or wholemeal flour derived from the specified grain, at least one other component of the food product or beverage, obtaining as a result of food or beverage on the basis of malt, where flour derived from beans, contains less than about 0.4% Gordeyev, and/or malt obtained from grain, contains less than about 200 ppm Gordeyev.

In one of the embodiments, the method comprises the production of malt and/or flour.

In another aspect the present invention provides a plant of barley, of which the grain, comprising about 25% or fewer Gordeyev, from the level in the grain obtained from the corresponding barley plants of the wild type.

Preferably, the grain contains about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, or more preferably about 2.5% or less Gordeyev, content in grain from the barley plants of the wild type. Examples of plants of barley wild-type and include, among others, Bomi, Sloop, Carlsberg II, K8 or L1.

In another embodiment, the implement is placed grain contains about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, or more preferably about 2.5% or less of the contents B, C and/or D-Gordeyev, or any combination of the above, in the grain from the barley plants of the wild type.

In another embodiment, the flour produced from grain, contains less than about 0.4%, less than about 0.3%, less than about 0.2% and more preferably less than about 0.1% Gordeyev.

In the embodiment, the grain has an average weight (average weight 100 grains), at least about 2.4, Preferably, the grain has an average weight of from about 2.4 g to about 6 g, more preferably the average weight of from about 3.5 g to about 6 g

In another embodiment, the starch content in the grain is at least about 50% (by weight). More preferably, the starch content in the grain is from about 50% to about 70 % (by weight).

In the following embodiment, the toxicity in celiac flour produced from grain, is less than about 50%, less than about 25%, more preferably about 10% or less of the level of toxicity of flour obtained from the corresponding grain barley plants of the wild type.

In yet another embodiment, a malt obtained from the grain, contains less than about 200 ppm Gordeyev, less than OK the lo 125 ppm Gordeyev, more preferably less than about 75 ppm Gordeyev.

In another embodiment, at least about 50% of the genome of barley grain is identical to the genome of barley varieties Sloop.

Preferably, the seed obtained from a plant that is homozygous for at least one, at least two, at least three or more loci for genetic variations (variations), which leads to lower levels gardein at least one of at least two or all three classes B, C and D, compared with the level in the corresponding plant barley wild-type.

In one embodiment, the grain obtained from nedrencheskogo plants. For example, the grain can be obtained by crossing Riso 56 and Riso 1508 or their offspring, including mutationhor2andLys3accordingly, present in these parental lines. Preferably, such grain includes genetic background, essentially different from Riso 56 or Riso 1508, for example, containing less than about 25% of the genetic background of these parental lines.

In another embodiment, the seed obtained from the transgenic plants.

One of the embodiments the transgenic plant is a plant that includes a transgene encoding polynucleotide, which suppresses production of grain, Melsheimer, one gardein. Preferably, polynucleotide this option is antimuslim polynucleotides, semantic polynucleotides, catalytic polynucleotide, artificial microRNA or RNA duplex, which inhibit the expression of one or preferably several genes encoding Gordini.

In another embodiment the transgenic plant is a plant that comprises a transgene encoding a prolamin, which is less toxic, preferably non-toxic to people with celiac disease. An example of prolamine, which is non-toxic to people with celiac disease, includes, among other things, Avenyn oats.

In the following embodiment, at least about 50% of the grain germinates within 3 days after swelling in standard conditions used for malting.

In another aspect the present invention provides a plant of barley, of which the grain, where the flour is obtained from the grain comprises less than about 0.4% Gordini, and/or malt obtained from the grain comprises less than about 200 ppm Gordeyev.

In another aspect the present invention provides a grain of barley plants according to the invention.

In the following aspect the present invention provides a method of obtaining a grain of barley, including:

a) growing the plant cell is Ana inventions

b) harvesting grain, and

c) perhaps the processing of grain.

Preferably, the plants are grown on a commercial scale in the field. For example, in one embodiment, the method comprises growing at least 1000, more preferably at least 5000 plants in a field on the area of at least one hectare.

Also proposed is a method of obtaining flour, wholemeal flour, starch or other product produced from corn, including:

a) obtaining grain of the invention, and

b) the processing of grain for the production of flour, wholemeal flour, starch or other product.

In another aspect the present invention provides a product obtained from barley plants of the invention or grains of the invention.

In one embodiment, the implementation of the product is a food product or a beverage on the basis of malt.

Preferably based drink malt is to beer or whiskey.

In another embodiment, the product is a food product, preferably containing starch or consisting of at least about 50% starch. Examples include, without limitation, films, coatings, adhesives, paper, building materials and packaging materials, or products that do not contain starch, such as ethanol.

In another aspect of the present invention is about who has food or drink based on malt, produced with the method of the invention.

In one of the embodiments based drink malt is to beer, which includes at least about 2%, more preferably at least about 4% alcohol. Preferably, the alcohol is ethanol.

In yet another embodiment, a drink made of malt is to beer, which includes less than approximately 1 ppm Gordeyev.

In another aspect the present invention provides beer, including one or more proteins of barley grain and less than about 1 ppm Gordeyev. In one of the embodiments beer contains less than about 0.5 ppm Gordeyev.

Preferably beer includes at least about 2%, more preferably at least about 4% alcohol. Preferably the alcohol is ethanol. Examples of proteins of barley grain include, among others, 9 kDa protein transfer of lipids 1 barley (LTP1) and protein Z.

In another aspect the present invention provides a meal that includes one or more proteins of barley grain and less than about 0.4% Gordeyev.

In one embodiment, the implementation of the flour includes less than approximately 0.3%, less than about 0.2% and more preferably less than about 0.1% Gordeyev.

Preferably the flour VK is uchet less than about 7 mg, more preferably less than about 5 mg soluble protein/g dry weight of flour.

In another aspect the present invention provides a malt that includes one or more proteins of barley grain and less than about 200 ppm Gordeyev.

In one embodiment, the implementation of malt includes less than about 125 ppm Gordeyev, more preferably less than about 75 ppm Gordeyev.

In another aspect the present invention provides a method for the identification of barley grain that can be used to obtain food and/or beverage on the basis of malt, suitable for use by a person with celiac disease, including:

a) obtaining one or more of the following materials:

i) a sample from a plant, able to give a specified grain,

ii) grain,

iii) malt produced from corn, and/or

iv) extract the specified grain,

(b) analysis of stage a) in the presence of at least one hordein and/or at least one gene encoding hordein,

where the more Gordeyev produced in the grain, the grain is less suitable for the production of food and/or beverage on the basis of malt, suitable for use by a person with celiac disease.

In the embodiment, the sample is a grain, and stage b) includes the analysis of the material n the presence Gordeyev B and/or C. This can be accomplished using any method known in the prior art, for example, using an immunological method such as ELISA. Can apply the method described in Example 1. In one embodiment, the implementation of stage b) comprises oral administration of the material from stage a) to a person with celiac disease and determination of the immunoreactivity of T cells obtained from individuals in relation to one or more hordeins barley.

In another embodiment, the material of the sample stage (a) includes genomic DNA, and stage b) includes detecting the absence of one or more functional genes Gordeyev. Again, this can be accomplished using any method known in the prior art. For example, performing a stage of amplification of the gene as described in Example 9.

In one of the options for implementing the method includes a step of selection of plants barley, grain or malt according to the invention from a variety of samples of plants, grain or malt for cultivation or use. Such selection is based, directly or indirectly, on the reduced toxicity of the material in celiac disease.

In another aspect the present invention provides a method of preventing or reducing the frequency or severity of celiac disease in persons, including oral administration of food or beverage on the basis of the solo is and inventions, or grains of the invention. The frequency or the severity of the disease in this context, as I understand, is reduced relative to the introduction of equal amounts of food or drink made from barley wild-type. Food or drink may be used to provide nutrients or increased amount of nutrients, persons suffering from celiac disease, reducing the risk of provoking symptoms.

In another aspect the present invention provides for the use of food or beverage on the basis of the malt of the invention, or grains of the invention, for the manufacture of a medicinal product intended for oral administration to the person of nutrients, preventing or reducing at the same time the frequency or severity of celiac disease.

Obviously, the preferred features and characteristics of one aspect of the invention is applicable to any other aspect of the invention.

Throughout the text of the present description, the word "includes", or such variations as "comprising"should be understood as the inclusion of the specified element, integer or stage, or group of elements, integers or steps, but not the exclusion of any other element, integer or stage, or group of elements, integers or steps.

Hereinafter the invention is described by pogranicze the operating Examples with reference to the accompanying illustrations.

A BRIEF description of the ACCOMPANYING ILLUSTRATIONS

Figure 1: Reversed-phase FPLC total extract of prolamins, which shows the chromatogram A280 nm of wheat (a)barley (b), oats (c)maize (d) or pure gradient (e). Put the prolamins, equivalent to 0.2 g of flour. Shown oxidized DTT (DTTox); chromatogram were reduced for clarity.

Figure 2: Reversed-phase FPLC Gordeyev. A representative chromatogram is shown A280 nm (solid line) and solvent mixture (dashed line) during the selection gardenboy faction 1 (#1), 2 (#2), 3 (#3), 4 (#4), 5 (#5), or 6 (#6) from the extract of barley. These fractions were combined, as shown (bold line), of the successive introductions of samples.

Figure 3: Analysis of 20 µg gordiyovych fractions #1-6 by electrophoresis in LTO-SDS page, gel painted 0,06% Coomassie Blue G250. On the left of the track indicates the position of molecular weight standards (in kDa, BenchMark, Invitrogen).

Figure 4: Stimulation produce IFN-γ in T-cells isolated from patients with celiac disease six days after diet provocation, the total drugs prolamins, made from barley, wheat, oats or corn in the presence (•, n=21)or absence (o, n=13) pre-treatment tTG. IFN-γ-positive colonies were counted and presented as mean SFU ± SE. Error bars not shown, when SE was smaller than the symbols.

Figure 5: the item is while accumulation to produce IFN-γ in T-cells, selected patients with celiac disease six days after diet provocation Gordeyeva factions#1, 2, 3, 4, 5 and 6, in the presence (•, n=21)or absence (o, n=13) pre-treatment tTG. IFN-γ-positive colonies were counted and presented as mean SFU ± SE. Error bars not shown, when SE was smaller than the symbols.

6: Chromatogram of analytical reversed-phase HPLC allocated gordiyovych fractions. On a representative chromatograms are shown A280 nm during HPLC gordiyovych fractions#1, 2, 3, 4, 5, 6, isolated from barley. For comparison, shows the chromatogram for barley wild-type (Himalaya), which shows the elution (solid line) hordein D, C and B, as well as mutant R56, which accumulate mainly C hordein.

7: Characteristics of prolamins in Riso56 and Riso1508 by electrophoresis in LTO-page and Western blotting. Twenty micrograms of prolamine, purified as in Example 1 from the specified lines of barley were incubated for 30 minutes at room temperature in buffer containing 6.6 M urea, 2% (wt./about.) LTOs, 1% (wt./about.) DTT, 62.5 mm Tris-HCl (pH 6,8) and 0.01% (wt./about.) bromophenol blue, and then put on two tracks 12% acrylamide gel and electrophoresis was performed at 200 V for 40 min, the Gel was washed in buffer for transferring containing 192 mm glycine, 25 mm Tris-base and 20% (vol./about.) methanol, in accordance with the ie 10 minutes after which he completed a transfer to nitrocellulose (Amersham Hybond C+) at 100 V for 1 hour. The membrane on the left were stained with 0.2% Ponceau S (wt./about.) in 3% (wt./about.) trichloroacetic acid, 3% 5-sulfosalicylic acid and quickly remove excess dye in the water; the membrane on the right was blocked in 5% fat-free milk in PBST for 1 hour, then incubated with mouse monoclonal antibody 12224 (Skerrit, 1988) in PBST, washed 3x in PBST for 10 minutes, incubated with the antibody sheep against mouse antibody conjugated with HRP (Selenius)in PBST, washed 3x in PBST for 5 minutes, incubated in ECL-reagent Amersham according to the manufacturer's instructions, and kept on Amersham Hyperfilm. MAb 12224 induced against the total glutening extract, therefore, were detected all Gordini and prolamins (Skerrit, 1988).

Fig: Reversed-phase FPLC gordiyovych extracts in Riso56 and Riso1508 in comparison with the wild type Bomi and Carlsberg II. Gordini was isolated from these lines, as in Example 1, with FPLC column was applied, the number, equivalent to 0.2 g of flour, using the first method FPLC in Example 1. Indicates the time of elution of C-hordein (C-Hor) and B-hordein (B-Hor).

Figure 9: Representative electrophoresis LTO-PAG soluble protein applied per seed. Prolamine extracts (10 µl) of individual seeds of barley F2 from crosses Riso1508 and Riso56 received, as described above. The provisions of the protein standard the mouths 30, 50, 70 and 100 kDa are indicated on the track on the left. Also shows the protein profiles of the parental lines Riso1508 and Riso56, and wild-type (Bomi). Six tracks of the alleged double zero samples contain very little protein (zero), six other tracks contain low levels of protein (reduced).

Figure 10: Representative electrophoresis LTO-PAG soluble proteins caused on the basis of equal amounts of protein. Samples containing 20 μg soluble protein extracted from single seeds of barley F2, separated by electrophoresis, and the gel was stained with Coomassie blue. Put samples from the parental lines (Riso 1508 and Riso 56), and wild-type (Bomi). External and Central track (10 kDa) contained protein standards of known molecular weight, are the provisions of the strips 30, 50, 70 and 100 kDa.

11: Chromatogram OF FPLC soluble extracts from seeds of barley F3. Soluble proteins were extracted from single seeds F3, as described; supernatant two seeds from each line were pooled, 50 µl was injected into the column OF FPLC and suirable as described in Example 1.

Fig: the Content of water-soluble (A), solarstorm (B)alcohol (C) and soluble in urea solution (D) protein in duplicated samples of flour from barley wild-type (Large, Carlsberg II, Bomi), one-zero parent (Riso 56 And Riso 1508) and F4 seeds plants of lines J4, J1, BB5, G, 5RB was determined as in Example 4. Determined the content of total extractable protein (E), summarizing the content of the individual fractions. The content of total protein was also evaluated by means of elemental analysis according to the method Dumas (F). The protein is shown as mean ± SE.

Fig: Toxicity in celiac disease patients Gordeyev isolated from different samples of flour were determined using T-cells isolated from patients with celiac disease, 6 days after the provocation, as in Example 5, and has led the average number petrobrazi units (SFU) + SE depending on the WW flour. For clarity, the mean SFU shown only for Gordeyev isolated from barley wild-type (Sloop) or double zero line (G1) in the presence (+tTG) or absence (-tTG) enzyme, tissue transglutaminase (A). In all cases the processing tTG increased toxicity Gordeyev expected celiac disease. SFU also provided for processed tTG Gordeyev (B)selected from the flour of barley wild-type (Sloop), one-zero parent (R, R1508) and seeds F4 (4BH).

Fig: the sequences of genes that are specific either to the control gene (gamma3-Hor)or to gene B-hordein (B-Hor), amplified from DNA extracts of individual shoots F4 or lines 9RE, 4BH, or parental line R as in Example 9.

General methodology and definitions

Unless specifically defined otherwise, all technical the definition and scientific terms, used in this application have a standard value, which generally understood by the average person skilled in the art (for example, in agriculture, technology, food, cell cultivation, molecular genetics, immunology, protein chemistry, and biochemistry).

Unless otherwise stated, the methods of genetic engineering, cell culture, immunological techniques used in the present invention, are standard techniques known to experts, skilled in the art. Such methods are widely described and explained in the literature, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions at the moment), Ed Harlow and David Lane (EDS) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all revisions at the moment).

Used in this application, the term "barley" refers to any species of the genusHordeumincluding predecessors of this kind, as well as the offspring resulting from krasiva the Oia with other species. The preferred form of barley are species ofHordeum vulgare.

Celiac disease or gluten enteropathy is an autoimmune disease of the small intestine that occurs in genetically predisposed people of all age groups after the first year of life. It affects approximately 1% of the Indo-European populations, although it is diagnosed largely insufficient. Celiac disease is caused by a reaction to gliadin, a gluten protein present in wheat (and similar proteins psenicnik (Triticeae), which includes other crops such as barley and rye). Upon contact with gliadin, the enzyme tissue transglutaminase modifies the protein, and the immune system cross-reacts with the tissue of the intestine, causing an inflammatory reaction. This leads to the alignment of the membranes of the small intestine and, as a consequence, the malabsorption of nutrients. The only effective treatment is a lifelong gluten-free diet. This condition has several other names, including: celiac disease (cœliac disease, with a ligature), disease- (c(o)eliac) sprue, non-tropical sprue, endemic sprue, gluten enteropathy or patentability enteropathy, and gluten intolerance. Symptoms of celiac disease vary greatly from person to person. Symptoms of celiac disease may include one or the more of the following: gas, periodic abdominal bloating and pain, chronic diarrhea, constipation, pale, foul-smelling or fatty stool, weight loss/weight gain, fatigue, unexplained anemia (low number of red blood cells causing fatigue), pain in the bones or joints, osteoporosis, osteopenia, behavioral changes, tingling with numbness from nerve damage), muscle spasms, cramps, menstrual disorder (often because of excessive weight loss), infertility, habitual miscarriage, growth retardation, delayed development in children, grayish ulcers in the mouth, called aphthous ulcers, the teeth discoloration or loss of enamel, as well as itchy skin rash called dermatitis herpetiformis. Some of the most common symptoms include: fatigue, alternating diarrhoea, abdominal pain or cramps, indigestion, flatulence, bloating, and weight loss. Celiac disease can be diagnosed, for example as described in WO 01/025793.

Used in this application, the term "non-toxic to people with celiac disease" refers to the consumption of food or drink, not leading to the development of a symptom of celiac disease in persons suffering from specified diseases. As described in the present application, food or drink is prepared from the barley plants of the wild type, when the result in development of symptoms.

The terms "seed" and "seed" used in this application interchangeably. "Grain" usually refers to Mature, harvested grain, but can also refer to the grain after processing, for example, after grinding or polishing, in which the composition of the greater part of the grain is left intact, or after soaking or germination, according to the context. Mature grain usually has a moisture content of less than approximately 18-20%. Grain barley wild-type (whole grains) usually contains 9-12% protein, of which approximately 30-50% prolamin, usually 35%, so, grain of barley wild-type and contains approximately 3-4% of prolamine by weight. Prolamins are present almost exclusively in the endosperm, which makes up approximately 70% of the weight of whole grains.

Used in this application, the term "corresponding wild-type barley plants refers to plants that includes at least 50%, more preferably at least 75%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99%, and most preferably 99.5% of the genotype of the invention, but gives the grain with unmodified content Gordeyev. In one embodiment, the "corresponding wild-type barley plants is a plant variety that is used in EC the experiments in plant breeding with the aim of introducing genetic variants, which lead to decreased production Gordeyev in the grain. In another embodiment, the corresponding wild-type barley plants is the parent variety, which was introduced transgene, which reduces production Gordeyev in the grain. In the following embodiment, a corresponding wild-type barley plants is the variety that applies at the date of registration of commercial production of barley grain, such as, among others, Bomi, Sloop, Carlsberg II, K8, L1, Vlamingh, Stirling, Hamelin, Schooner, Baudin, Gairdner, Buloke, WI3586-1747, WI3416, Flagship, Cowabbie, Franklin, SloopSA, Sloop Vic, Quasar, VB9104, Grimmett, Cameo*Arupo 31-04, Prior, Schooner, Unicom, Harrington, Torrens, Galleon, Morex, Dhow, Capstan, Fleet, Keel, Maritime, Yarra, Dash, Doolup, Fitzgerald, Molloy, Mundah, Onslow, Aka Skiff, Unicorn, Yagan, Chebec, Hindmarsh, Chariot, Diamant, Koral, Rubin, Bonus, Zenit, Akcent, Forum, Amulet, Tolar, Heris, Maresi, Landora, Caruso, Miralix, Wikingett Brise, Caruso, Potter, Pasadena, Annabell, Maud, Extract, Saloon, Prestige, Astoria, EIo, Cork, Extract, Laura. In the embodiment, "the corresponding wild-type barley plants gives the grain with unmodified content Gordeyev because it includes a full set of functional gordiyovych genes encoding functional proteins Gordeyev, including B, C, D, and γ-hordein encoded by lociHor2,Hor1,Hor3andHor5.

Used in this application, the term "one or more proteins of barley grain" refers to natural proteins produced by barley grain. Examples of such proteins are known in the art, kVA is inficirovannym in the art. Specific examples include, among others, albumin barley, for example, a 9 kDa protein transfer of lipids 1 (LTP1) (see review Douliez et al. (2000) and Swiss-prot P07597, as an example)and protein Z (see Brandt et al. (1990) and Genbank P06293), including appropriate versions of (Mature) forms and their denatured forms and/or fragments resulting from the production of malt, flour, wholemeal flour, food product or beverage on the basis of malt according to the invention.

Used in this application, the term "malt" refers to barley malt, "flour" refers to barley flour, whole-wheat flour" refers to barley flour from whole grains, and "beer" refers to barley beer. More specifically, the source of malt, flour, beer, flour from whole grains, food, etc. according to the invention is produced by means of processing (for example, grinding and/or fermentation of barley grain. These terms include malt, flour, beer, flour from whole grains, food, etc. obtained from a mixture of grains. In a preferred embodiment, at least 50% of the grain used for the production of malt, flour, beer, flour from whole grains, food, etc. is a grain of barley.

The term "plant"also used in this application refers to the whole plant, such as, for example, a plant growing in a field with the spruce commercial production of barley. "Plant part" refers to vegetative structures of plants (e.g. leaves, stems, roots, Tsvetkov organs/structures, seed (including embryo, endosperm and the seed's shell), plant tissues (e.g. vascular tissue, epithelial tissue, and the like), cells, starch grains, or the offspring of the above.

"Transgenic plant", "genetically modified plant" or variants of the above apply to plant that contains a gene construct, "transgene"), not present in the wild type plant of the same species, races or varieties. "Transgene"as described in this application has a normal value of biotechnology and includes a genetic sequence that was obtained or modified using genetic methods DNA or RNA, and which was introduced into the plant cell. The transgene may include genetic sequences derived from the plant cells. Typically, the transgene is introduced into the plant artificially, for example, using the transformation, however, can be used by any method known to a person skilled in the technical field.

"Nucleic acid molecule" refers to polynucleotide, such as, for example, DNA, RNA, or oligonucleotides. It can be a DNA or RNA of genomic or synthetic origin, Dunaeva eletronical, and also associated with carbohydrate, lipids, protein or other materials to perform specific functions defined in this application.

"Functionally linked", as used in this application refers to the functional relationship between two or more segments of nucleic acids (e.g. DNA). Typically, this refers to the functional element of the regulation of transcription (promoter) with the transcribed sequence. For example, a promoter functionally linked to the coding sequence, such as polynucleotide defined in this application, if it stimulates or modulates the transcription of the coding sequence in the appropriate box. In most cases, promoter elements, transcription regulation, which is functionally linked to the transcribed sequence, physically adjacent to the transcribed sequence, i.e., they are incis-position. However, some elements of the regulation of Transkei, such as enhancers, not necessarily physically adjacent or located in close proximity to the coding sequences, the transcription of which they reinforce.

Used in this application, the term "gene" should be understood in its broadest sense, it includes desoxyribonucleic Tinnie sequence, including the region of the structural gene encoding the protein, and includes the sequence adjacent to the coding region at the 5' and 3' ends for at least about 2 TPN at either end, and are involved in gene expression. Sequences located at the 5'end of the coding region and which are present on the mRNA are referred to as 5'-untranslated sequences. Sequence located on the 3'-end or the right of the coding region and which are present on the mRNA are referred to as 3'-untranslated sequences. The term "gene" includes cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region, which may be interrupted by non-coding sequences, called introns" or "intermediate areas" or "intermediate sequences". Introns are segments of a gene that are transcribed into nuclear RNA (Garc), introns may contain regulatory elements such as enhancers. Introns are removed or "cut out" from the nuclear or primary transcript, therefore, introns are not present in the transcript of the messenger RNA (mRNA). Functions of mRNA during translation, determining the sequence or order of amino acids in the polypeptide synthesized. The term "gene" includes manufactured the definition or fused molecule, encoding all or part of the proteins of the invention described in this application, as well as the nucleotide sequence complementary to any of the above. Used in this application, the term "another component of food or drink" refers to any substance suitable for use in animals, preferably to any substance suitable for human consumption. Examples include, without limitation, the water, the grain of other species, sugar, etc.

Used in this application, the term "genetic variation, which leads to a decrease in the level of at least one gardein"refers to any polymorphism in barley plants, which leads to decreased production hordein. Genetic variation can be, for example, a deletion of Gordeyeva gene (genes) or part thereof, or a mutation that results in decreased transcription of the gene in barley. Examples of such genetic variations present in Riso 56 And Riso 527 and Riso 1508. Therefore, such plants can be used in the methods of the invention. In addition, the plant of the invention may be a hybrid of any of these mutants of barley. In a preferred embodiment, the plant of the invention is a hybrid Riso 56 and Riso 1508 or their offspring, including mutationhor2andLys3present in these lines. In variationbetween plant is not a hybrid Riso 527 and Riso 1508.

Used in this application, the phrase "approximately", unless otherwise specified, refers to any allowable range with regard to values. In a preferred embodiment, the term "approximately" is +/- 10%, more preferably +/- 5% from the specified value.

Prolamins and Gordini

The prolamins of cereal crops (known as the gliadins in wheat, hordein barley, scalini in the rye, Avenida in oats and Seine in corn) are the main spare proteins of the endosperm in all grain cereals except oats and rice (Shewry and Halford, 2002). Gordini are 35-50% of the total protein in the seeds of barley (Jaradat, 1991). They are divided into four groups (also known as γ-hordein), B, C, and D, in order of decreasing mobility (Field et al., 1982). B-hordein are a major fraction of the protein and differ from C-Gordeyev in sulfur content (Kreis and Shewry, 1989). B-hordein constitute 70-80% of the total number, and C is Gordini - 10-20% (Davies et al., 1993). A-hordein in General is not considered as the fraction of the stock, whereas D-hordein homologous glutenins with high molecular weight. Gordini, along with the rest related prolamins cereal crops, is not expressed in the zygote embryo directly, unlike other spare proteins, such as napisy, they believed, the former is reservada exclusively in the starchy endosperm during the middle/late stages of seed development.

Examples of amino acid sequences Gordeyev barley (given in the form of non NCBI with the description, ID, gi), include, among others:

Examples of genes and/or cDNA encoding Gordini barley (given in the form of non NCBI with the description, ID, gi), include, among others:

One of the embodiments of the present invention relates to transgenic barley plants, comprising prolamin, which is not toxic to people with celiac disease. As shown in the present application, examples of such prolamine are Avenyn oats and corn Zein.

Examples of amino acid sequences evennow oats (given in the form of non NCBI with the description, ID, gi), include, among others:


<> The malting

Based drink malt provided by the present invention, includes alcoholic beverages (beverage obtained by distillation) and soft drinks, which are produced by using malt as part or all of the source material. Examples include beer, happoshu (drink like beer, low-malt)whisky, alcoholic drinks on the basis of malt (for example, based beverages malt, containing less than 1% alcohol), and non-alcoholic drinks.

The malting is a process controlled soaking and germination, followed by drying of barley grain. This sequence of events is important for the synthesis of many enzymes, which cause modification of grain, a process in which mainly depolymerized walls of dead cells of the endosperm and mobilise nutrients of the grain. In the subsequent drying process as a result of chemical reactions darkening develops flavor and color. Although the primary use of malt is a beverage, it can also be used in other production processes, for example as a source of enzymes in the baking industry or as a flavouring and colouring agent in food industry, for example, in the form of malt or malt flour, or indirectly, in VI is e malt syrup, etc.

In one of the embodiments the present invention relates to a method for producing a malt composition. The method preferably includes the following stages:

(i) obtaining grains of barley plants inventions

(ii) soaking the specified grain,

(iii) germination soaked the grain in the given conditions, and

(iv) drying specified, sprouted grains.

For example, the malt may be obtained by any of the methods described in Hoseney (Principles of Cereal Science and Technology, Second Edition, 1994: American Association of Cereal Chemists, St. Paul, Minn.). However, in the present invention can also be used any other suitable method to produce malt, such as methods of obtaining special malts, including, among others, methods of roasting malt. One of the non-limiting examples are described in Example 6.

Malt can be made using grain obtained only from barley plants of the invention or mixtures comprising other grains.

Malt is mainly used in brewing, but it is also used in the production of spirits. Warka beer involves the production of jam, the primary and secondary fermentation, and postprocessing. First malt propalyvayut, stir in water and heated. During this "mash" the enzymes activated during malting, break down starch grain is with the formation of fermentable sugars. Cooked jam lighten, add the yeast mixture is fermented and perform postprocessing.

In another embodiment of the malt can be cooked mash songs. The specified congestion may be the first and/or second and/or as jam. Usually mash the composition has a high content of amide nitrogen and fermentable carbohydrates, mainly maltose. Typically, congestion prepare, soaking the malt with water, that is, when mashing. In the mashing process in the composition of malt/water can be added carbohydrate-rich compositions, such as barley, corn or rice supplements. Neocolonies grain supplements usually do not contain any active enzymes and, therefore, to ensure that the enzymes required for hydrolysis of polysaccharides necessary malt or externally administered enzymes.

Usually the first stage in the production process of the congestion is the grinding of malt required to ensure that water could get access to the grain particles in the phase mashing, which is essentially a continuation of the process of malting with enzymatic depolymerization of substrates. During mashing milled malt withstand liquid fraction, such as water. Temperature or maintained at a constant level (isothermal mashing)or gradually stand is the culprit. In any case, soluble substances formed during malting and mashing, moving in a specified liquid fraction prior to its separation by filtering on congestion and the residual solid particles called grains. The specified congestion may also be referred to as the first jam. After filtering, receive a second congestion. These blockages can be obtained by repeating this procedure. Non-limiting examples of suitable methods of preparation of congestion described in Hoseney (above).

Mash the composition may also be prepared by treatment of barley plants of the invention or parts thereof with one or more suitable enzymes, such as enzyme compositions or mixed enzyme compositions, for example Ultraflo or Cereflo (Novozymes). Mash the composition may also be prepared using a mixture of malt and neocolony barley plants or parts thereof, optionally with the addition of one or more suitable enzymes during the specified cooking. In addition, the enzymes prolyl-endopeptidase that destroy specific toxic aminokwasy involved in celiac disease, can be added in the process of fermenting mash to reduce the toxicity of residual Gordeyev (De Angelis et al., 2007; Marti et al., 2005; Stepniak et al, 2006).

Processing grain

The barley grain of the invention can be obrabotano in order to obtain food or non-food product, using any methodology known in the art.

In one embodiment, the implementation of the product is flour from whole grains (whole-wheat flour ultrafine grinding, for example, whole-wheat flour ultrafine grinding; whole-wheat flour, or flour obtained from approximately 100% of the grain). Flour from whole grains include purified component of flour (refined flour)and coarse fraction (coarse fraction of ultrafine grinding).

Refined flour may be flour, which is obtained, for example, when the grinding and sieving of peeled barley. Management of quality control of food products and medicines (FDA) requires that the meal meets certain standards of particle sizes, so it can be included in the category of purified barley flour. The particle size of the treated flour is described as flour, in which not less than 98% of the particles pass through a sieve with holes the size of which does not exceed the size of the holes woven wire mesh, defined as "212 micrometers (U.S. Wire 70)".

The coarse fraction includes at least one of: bran and germ. For example, the sprout is an embryo of plants found in barley grain. Seedlings include lipids, fiber, vitamins, protein, minerals and nutrients flora the CSOs origin, such as flavonoids. Bran include several cell layers and contain substantial amounts of lipids, fiber, vitamins, protein, minerals, and nutrients of plant origin, such as flavonoids. In addition, the coarse fraction may include the aleurone layer, which also includes lipids, fiber, vitamins, protein, minerals and nutrients of plant origin, such as flavonoids. The aleurone layer, which is actually considered part of the endosperm, exhibits many of the same traits as bran, and therefore usually removed with the bran and alfalfa sprouts during the grinding process. The aleurone layer contains proteins, vitamins and nutrients of plant origin, such as Frolovo acid.

Next, the coarse fraction may be mixed with purified component of the flour. Preferably, the coarse fraction is homogeneous mixed with purified element of flour. Homogenous mixing of coarse fraction and a purified component of flour can help to reduce stratification of particle size in the shipping process. The coarse fraction can be mixed with purified component of the flour, the flour from whole grains, providing, thus, the whole-wheat flour with a high nutritional value, fiber content and antioxidant capable of the TEW compared with refined flour. For example, the coarse fraction or whole-wheat flour can be used in varying amounts to the replacement of treated flour or flour from whole grains in baked goods, snack foods and food products. Flour from whole grains of the present invention (e.g. flour from whole grains ultrafine grinding) may also be supplied directly to consumers for use in the manufacture of their homemade cakes. In the embodiment, the particle size of the flour from whole grains is such that 98% by weight of the particles of flour from whole grains less than 212 micrometers.

In other embodiments, implementation of the enzymes present in the bran and the germ flour from whole grains and/or coarse fraction inaktivirovannye to stabilize the flour from whole grains and/or coarse fraction. In the present invention is also seen that the vaccine may indicate inhibited, denatured or other Stabilization is a process that uses steam, heat, radiation or other treatments designed to inactivate enzymes present in the bran layer and germ. The natural enzymes in the bran and seedlings catalyze change connections in the flour, negatively affecting the culinary properties of flour and shelf life. Inactivated the enzymes do not catalyse of the change of connections, present in the flour, so the flour that was stable, retains its culinary properties, and has a longer shelf life. For example, the present invention can provide a technology of grinding with two threads for grinding the coarse fraction. After separation and stabilization of the coarse fraction, the coarse fraction passes through the mill, preferably a roller mill, with the formation of coarse fraction having a grain size distribution less than or equal to about 500 micrometers. In the example implementation roller mill usually works with the speed of the roller 115 - 144 m/s, high speed roll leads to heating. The heat generated during the process, and the air flow, reducing the content of microorganisms in the coarse fraction. In other embodiments, the implementation before grinding in a roller mill, the coarse fraction may have an average content of aerobic microorganisms 95000 colony forming units/gram (CFU/g) and the average content of a group of bacteria Escherichia coli 1200 CFU/g After passing through the roller mill, the coarse fraction may have an average content of aerobic microorganisms 10000 CFU/g and the average content of a group of bacteria Escherichia coli 900 CFU/g Thus, the microbial content in the coarse fraction of the present invention may be substantially red eye reduction is but. After sieving the crushed coarse fraction with a particle size greater than 500 microns can be returned to the process for additional grinding.

In additional embodiments, the implementation of the whole-wheat flour or coarse fraction may be a component of the food product. For example, the food product can be a croissant, biscuit, bread, muffin, croissant, gnocchi, English butter bun, sweet muffin, pita bread, unleavened bread, refrigerated/frozen dough, the dough is ready beans, burritos, chili, Taco, Tamal, tortilla pie, cooked Breakfast, box lunch, stuffing, dinner to cook in the microwave, brownie, pie, cheesecake, pastry and coffee, biscuits, dessert, pastries, sweet rolls, sweet candy bar, crisp basis for pie fillings pie, product for baby food, a mixture of baking, batter, breading mixture for the sauce, filler for meat, meat substitute, mixture of spices, soup mix, sauce, salad sauce, dressing to the salad, soup, sour cream, noodles, pasta, macaroni, noodles Chow main noodles lo Maine, filler for ice cream, cake ice cream, ice cream cone, ice cream, waffles, crackers, toast, doughnut, egg roll, extruded snacks, fresh muesli with fruit, snack foods for cooking in the microwave the furnace is nutritional the second bar, damn, bakery product, cake mix, pretzel, pudding, product-based granola, chips, snacks, a mixture of snacks, waffles, pizza crust, animal feed or pet food.

In alternative embodiments, the implementation of the whole-wheat flour or coarse fraction may be a component of the dietary Supplement. For example, a dietary Supplement may be a product, which is added to food, and which contains one or more components, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber. Flour from whole grains or coarse fraction of the present invention include vitamins, minerals, amino acids, enzymes and fiber. For example, the coarse fraction contains a concentrated amount of dietary fiber and other essential nutrients, such as B vitamins, selenium, chromium, manganese, magnesium and antioxidants needed for a healthy diet. For example, 22 grams of coarse fraction of the present invention provide 33% of the recommended daily intake of fiber for humans. Further, to ensure the person 20% of the daily recommended intake of fiber, you need 14 grams. Thus, the coarse fraction is an excellent additional light source is whom to meet human needs in the fiber. Thus, in the present embodiment, the whole-wheat flour or coarse fraction may be a component of the dietary Supplement. A food additive may include any known food components that contribute to the improvement of the General condition of man, the examples include, without limitation, vitamins, minerals, and other fibrous components, fatty acids, antioxidants, amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids and/or other nutritional components.

In additional embodiments, the implementation of the whole-wheat flour or coarse fraction may be fibrous additive or component. Many existing fiber supplements, such as plantain seed husk, derivatives of cellulose and hydrolyzed guar gum in addition to the fiber content have no significant nutritional value. In addition, many fiber supplements have unwanted structure and bad taste. Fiber supplements made from flour from whole grains or coarse fraction, can help provide fiber and protein and antioxidants. Fiber supplements can be made, inter alia, in the following forms: instant mixes drinks, ready-to-drink, food, cakes, waffles, biscuits, crackers, gel, cap the uly, zawacki, chewable tablets and pills. One of the embodiments provides a fibrous additives in the form of flavored cocktail or malt beverage, this option may not be particularly attractive as a fiber Supplement for children.

In an additional embodiment, the milling process can be used to obtain flour of several kinds of grain, flour made from several types of barley or coarse fractions of several kinds of grain. For example, bran and seedlings from one type of barley can be crushed and mixed with crushed endosperm or flour from whole grain barley of other types. In alternative bran and seedlings of the same type of grain can be crushed and mixed with crushed endosperm or flour from whole grains of another type. In an additional embodiment, the bran and the seedlings from the first type of barley or grain can be mixed with bran and seedlings of the second type of barley or grain, to obtain the coarse fraction of the several kinds of grain. Is seen that the present invention comprises mixing any combination of one or more of the bran, germ, endosperm and flour from whole grains, obtained from grains of one or more species. This multigrain, mnogochleny approach can be used for the Holocene flour special varieties and best use of properties and nutritional value of the grain or barley several kinds, in the manufacture of flour for one type.

Flour from whole grains of the present invention can be obtained by using different milling processes. The example implementation includes the grinding of grain in one thread, without separation of the endosperm, bran and germ of the grain in separate threads. Clean and air-conditioned grain is fed to the first mill, such as hammer crusher, roller crusher, rod mill, impact crusher, disk mill, rotary jet mill, roller mill, etc. In one embodiment, the mill may be a roller mill. After grinding the grain is unloaded and served on a sieve. Can be used any sieve, known from the prior art and used for sifting the crushed particles. The material passing through the sieve mesh is flour made from whole grains of the present invention and requires no additional processing. The material that remains on the grid, called the second fraction. The second group requires additional particle size reduction. Therefore, the above mentioned second fraction can be fed to the second crusher. After grinding the second fraction can be fed into the second sieve. The material passing through the mesh of the second sieve is flour made from whole grains of the present invention. The material that remains on the grid, called Chet what ertai faction and requires additional processing to reduce the size of the particles. The fourth fraction on the grid of the second sieve comes back, either in the first crusher or the second crusher for further processing through the feedback circuit. In an alternative embodiment of the invention, the process may include a number of first crusher to provide higher system capacity.

Provided that the whole-wheat flour, coarse fraction and/or cereal products of the present invention can be manufactured using any milling process known in the prior art. In addition, provided that the flour from whole grains, coarse fraction and/or cereal products of the present invention can be modified or improved through a number of other processes, such as fermentation, instantiate, extrusion, encapsulation, roasting, baking, etc.

Polynucleotide that suppress production hordein

In one embodiment, the grain of the invention and/or used in the methods of the invention derived from transgenic barley plants, which includes a transgene encoding polynucleotide, which suppresses production of at least one hordein in the grain. Examples of such polynucleotides include, among others, " antisense polynucleotide, semantic polynucleotide, catalytic polynucleotide, artificial microRNA or RNA duplex. In the learn the presence in the grain of each of these polynucleotides leads to a reduction in the number of mRNA hordein, available for broadcast.

The antisense polynucleotide

The term " antisense polynucleotide" should be understood as meaning a molecule of DNA or RNA, or a combination of both, which is complementary to at least part of a specific mRNA molecule that encodes hordein and can hinder post transcriptional event such as mRNA translation. Application of methods using antisense polynucleotides known in the prior art (see, for example, Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)). Application of methods using antisense polynucleotides in plants has been considered in reviews Bourque (1995) and Senior (1998). Senior (1998) stated that in the present methods using antisense polynucleotides are proven methods to control gene expression.

"Antisense polynucleotide in the plant barley invention hybridizes with the target polynucleotide under physiological conditions. Used in this application, the term " antisense polynucleotide that hybridizes under physiological conditions" means that polynucleotide (which is fully or partially single-stranded) capable of, at least, to the formation of dantewara of polynucleotide with mRNA that encodes a protein, such as gardein barley, under normal the conditions in cells of barley.

Antisense molecules can include sequences that correspond to structural genes or sequences that regulate gene expression or splicing. For example, the antisense sequence may correspond to a target coding region of the genes of the invention or the 5'-untranslated region (UTR)or the 3'-UTR, or combinations thereof. It may be partially complementary to the sequences of the introns that can be cut during or after transcription, preferably only the sequences of the exons of the target gene. Whereas in General the higher the divergence UTR, targeting these areas provides a higher specificity of inhibition of the gene.

The length of the antisense sequence must be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500, or 1000 nucleotides. Can be used full-size sequence, complementary to the entire gene transcript. Length most preferably is 100-2000 nucleotides. The degree of identity antisense sequence of the target transcript should be at least 90%, and more preferably 95-100%. Antisense RNA molecule can certainly include cuger the derivative sequence, which can stabilize the molecule.

Catalytic polynucleotide

The term catalytic polynucleotide/nucleic acid refers to a DNA molecule or a molecule containing DNA (also known in the prior art as "deoxyribozyme"), or RNA molecule, or a molecule containing RNA (also known as a "ribozyme"), which is specific recognizes a specific substrate and catalyzes the chemical modification of this substrate. The bases of nucleic acids in the catalytic nucleic acid can be the basis of A, C, G, T and U for RNA).

Typically, the catalytic nucleic acid contains the antisense sequence for specific recognition of the target nucleic acid, and nucleic acid exhibiting hydralicious enzymatic activity (also referred to in this application as "catalytic domain"). Types of ribozymes, which are the most preferred in the present invention include ribozyme type hammerhead (Haseloff and Gerlach, 1988, perriman Monday et al., 1992) and the hairpin ribozyme (Shippy et al., 1999).

Ribozymes in plants of barley invention and DNA encoding the ribozymes can be chemically synthesized using methods known in the art. Ribozymes can also be obtained from DNA molecules (which, upon transcription yields a RNA molecule), NGF is associated with national promoter RNA polymerase, for example, the promoter of RNA polymerase T7 or RNA polymerase SP6. In the case when the vector also contains the promoter of RNA polymerase, functionally associated with the DNA molecule, a ribozyme can be obtainedin vitrowhen incubation with RNA polymerase and nucleotides. In a separate embodiment, DNA can be integrated into the expression cassette or the cassette transcription. After synthesis, the RNA molecule can be modified by ligation with a DNA molecule which is able to stabilize the ribozyme and make it resistant to hydrolysis by RNase.

As in the case of antimuslim polynucleotide described in this application, the catalytic polynucleotide must also be capable of hybridization with the target nucleic acid molecule (e.g., mRNA, coding hordein barley) under "physiological conditions", namely, when the conditions inside the cells of barley.

RNA interference

RNA interference (Rnci) are particularly useful for specific inhibition of the production of a specific protein. Not wanting to be limited in accordance with any theory, Waterhouse al. (1998) proposed a model of the mechanism by which dnrc (Dunaeva RNA) can be used to decrease the production of the protein. This technology is based on the presence of molecules dnrc that contain the sequence, to the which is essentially identical to the mRNA sequence of a target gene or part thereof, in this case, the mRNA that encodes the polypeptide according to the invention. Conveniently, dnrc can be synthesized from a single promoter in a recombinant vector or host cell, in which sense and antisense sequences are flanked alien sequence that provides the hybridization sense and antisense sequences with the formation of molecules dnrc in which the alien sequence forms a loop structure. The creation and reception of suitable molecules dnrc to the present invention lies within the competence of a specialist skilled in the art, in particular, see Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029 and WO 01/34815.

In one example, the injected DNA that directs the synthesis of at least partially donateware (duplex) RNA product (s), with homology to the target gene to be inactivated. Thus, the DNA includes sense and antisense sequence that, when transcribed into RNA can gibridizatsiya with the formation of the field of double-RNA. In a preferred embodiment, the sense and antisense sequences are separated by spacer elements area, which includes intron exposed splicing during transcription into RNA. This scheme, as was Asano, leads to a higher efficiency of gene silencing. Dunaeva region may include one or two RNA molecules transcribed either from one region of DNA or two. The presence of double molecules, it is believed, induces endogenous response system of plants, which destroys as Dunaeva RNA and homologous RNA transcript of a target gene in plants, effectively reducing or eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize shall, for each, to be of at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500, or 1000 nucleotides. Can be used full-size sequence corresponding to the full transcript of the gene. Length, most preferably consists of 100-2000 nucleotides. The degree of identity of sense and antisense sequences of the target transcript should be at least 85%, preferably at least 90%, and more preferably 95-100%. The RNA molecule can certainly include alien sequence that can stabilize the molecule. The RNA molecule may expressivity under the control of promoters for RNA polymerase II or RNA polymerase III. Examples include the last paragraph is amatory tRNA or mark.

A preferred molecule is a small interfering RNA ("siRNA") includes a nucleotide sequence that is identical to the sequence of about 19 to 21 nucleotides of the target mRNA. Preferably, the target mRNA sequence begins with dinucleotide AA, has a GC content of about 30-70% (preferably 30-60%, more preferably 40-60% and most preferably about 45-55%), and has high identity to any of the nucleotide sequence, with the exception of the target sequence in the genome of barley plants, in which it should be introduced, for example, when you define using standard BLAST search.

MicroRNA

MicroRNA regulation is a specialized branch path RNA silencing, which was developed in the way of regulation of genes other than the usual MKI/PTSG. MicroRNA is a special class of small RNAS that are encoded generatorname elements, organized in a characteristic inverted repeats. During the transcription of microRNA genes with synthesized RNA precursor with the structure of the stem-loop, which are processed microRNA. MicroRNAs typically have a length of approximately 21 nucleotides. Formed microRNA included in RISC-like complexes containing a specific family of proteins argonaute proteins, which is haunted cause sequence-specific repression of the gene (Millar and Waterhouse, 2005; Pasquinelli et al, 2005; Almeida and Allshire, 2005).

Compresse

Other molecular biological approach, which can be used is compresse. The mechanism of compressie not been fully investigated, but as suggested, it involves posttranscriptional gene silencing (PTSG), and in this respect may be very similar to many examples of antisense suppression. It includes the introduction of additional copies of a gene or its fragment in a plant, in a sense orientation relative to the promoter for its expression. The size of the semantic fragment, the regions of the target gene, and the degree of sequence identity to the target gene are the same as in the case of antisense sequences described above. In some cases, an additional copy of the gene sequence disrupts the expression of a target gene in plants. For information on methods of implementation of compressie see WO 97/20936 and EP 0465572.

Structure of nucleic acids

Structure of nucleic acids suitable for producing transgenic plants can be easily created using standard methods.

With the introduction of the field, the coding mRNA, the design may include sequences of introns. These sequences introns may contribute to the expression of tra is of Shana in the plant. The term "intron" is used in the default value and means of genetic segment that is transcribed but does not encode a protein and is cut out of the RNA before translation. Introns can be introduced into the 5'-UTR or coding region, if the transgene encodes the transmitted product, or in any another part of the transcribed region, if the transgene does not encode the transmitted product. However, in the preferred embodiment, any region encoding a polypeptide represented as a single open reading frame. As informed by a qualified specialist, such open reading frames can be obtained by reverse transcription of mRNA that encodes the polypeptide.

To ensure proper expression of the gene encoding the target mRNA, the design of the nucleic acid typically comprises one or more regulatory elements such as promoters, enhancers, and the sequence of the transcription termination or polyadenylation. Such elements are known in the prior art.

The region of transcription initiation, which includes a regulatory element(s)may be present to provide regulated or constitutive expression in plants. Preferably, expression of, at least, occurs in the cells of the seed.

Has been described a number of constitutive promoters which the asset is s in plant cells. Suitable promoters for constitutive expression in plants include, among others, the 35S promoter of cauliflower mosaic virus (CaMV), the 35S promoter of mosaic virus norichika (FMV)promoter rod-shaped virus of sugar cane, the promoter of yellow mosaic virus commelina, segmentectomy the promoter of the small subunit of ribulose-1,5-bisphosphatase, the promoter of the cytosolic triosephosphate-isomerase rice, the promoter adrinfo.standortstr Arabidopsis, the promoter of the gene actin 1 rice, promoters Monophysites and octopunctata, promoter, Adh, sucrose synthase promoter, the promoter of the complex R genes and the promoter of the gene chlorophyll α/β binding protein. These promoters have been used to create DNA vectors that expressibility in plants; see, for example, WO 84/02913. All of these promoters have been used to create various types expressed in plants recombinant DNA vectors.

The promoter may be regulated by factors such as temperature, light or stress. Typically, the regulatory elements located at the 5'end of expressed genetic sequence. Design can also contain other elements that enhance transcription, such as nos-3' or ocs-3' region polyadenylation or transcription terminators.

5'-untranslated leader sequence is eljnosti can be obtained from the promoter, selected for expression of the heterologous sequence of the gene, and can be specific modified, if you want to increase translation of the mRNA. For more information about optimizing expression of transgenes, see review Koziel et al., (1996). 5'-noncoding region can also be obtained from RNAS of plant viruses (tobacco mosaic virus, virus engraving tobacco, virus dwarf mosaic corn, alfalfa mosaic virus, etc.), from suitable eukaryotic genes, genes of plants (wheat and signal sequence of the gene chlorophyll a/b binding protein maize), or from the sequence of the synthetic gene. The present invention is not limited to the use of structures in which noncoding region derived from the 5'-noncoding sequence that accompanies the sequence of the promoter. The leader sequence can also be obtained from non-native promoter or coding sequence. Leader sequence, which can be used within the present invention include the leader sequence of the Hsp70 protein maize (US 5362865 and US 5859347), as well as omega-element TMV.

Termination of transcription is provided by the 3'untranslated DNA sequence operatively linked to a chimeric vector with the target Polina is leotides. 3'-noncoding region of the recombinant DNA molecule contains a polyadenylation signal which functions in plants, causing attach adenylate nucleotides to the 3'-end of RNA. 3'-noncoding region can be derived from various genes that are expressed in plant cells. Usually use a 3'-noncoding region napaliensis, 3'-noncoding region of the gene of the small subunit of Rubisco, 3'-noncoding region of the gene of the protein reserve 7S soy. Also suitable are transcribed 3'-untranslated region containing a polyadenylation signal genes Ti-plasmid (from the English. tumor-inducing-opuholerodnyh)Agrobacteriumtumefaciens.

Typically, the design of the nucleic acids comprises a selective marker. Selective markers assist in identifying and screening plants or cells which have been transformed with exogenous nucleic acid molecule. The selective marker gene may give the cells of barley resistance to an antibiotic or herbicide, or allow the utilization of substrates such as mannose. The selective marker is preferably attached to the cells of barley resistance to hygromycin.

Preferably, the design of the nucleic acid stably integrated into the genome of plants. Accordingly, the nucleic acid includes the necessary elements that call the given molecule to integrate into the genome, or structure is placed in a suitable vector, which can be integrated into a chromosome of the plant cell.

One of the embodiments of the present invention includes the use of a recombinant vector, which includes at least the transgene described in this application, built into any vector capable of delivering the nucleic acid molecule in a host cell. Such a vector contains heterologous nucleic acid sequences, which are sequences of nucleic acids that are not present in nature, merged with the nucleic acid molecules of the present invention, and which preferably come from a species different from the species from which the molecule (molecule) of the nucleic acid. The vector may be RNA or DNA, prokaryotic or eukaryotic, and is usually a virus or a plasmid.

The set of vectors suitable for stable transfection of plant cells or for the establishment of stable transgenic plants have been described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. As a rule, plant expression vectors include, for example, one or more plant genes cloned under transcriptional control of the eat 5'- and 3'-regulatory sequences, as well as a dominant selective marker. Such plant expression vectors may also contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, regulated by external conditions or developments, or cell - or tissue-specific expression), the site of transcription initiation, the binding site of the ribosome, the signal processing RNA, the site of transcription termination and/or polyadenylation signal.

Transgenic plants

Transgenic barley plants, as defined in the framework of the present invention include plants (as well as parts and cells of these plants and their progeny which have been genetically altered using genetic engineering techniques, to induce the production of at least one polynucleotide and/or polypeptide in the desired plant or organ of a plant. Transgenic plants can be obtained using methods known from the prior art, such as described in General terms in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

In the preferred embodiment, transgenic plants are homozygous for each gene, which was introduced (the transgene), resulting in their offspring is not cleaved at the desired fenati is at. Transgenic plants can also be heterozygous for any given transgene (transgenes), for example, in the F1 offspring, which was grown from hybrid seed. Such plants can provide advantages, such as hybrid power, known from the prior art.

Were described four General methods of direct gene delivery into cells: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (see, for example, WO 87/06614, US 5472869, 5384253, WO 92/09696 and WO 93/21335)and the gene gun (see, for example, US 4,945,050 and US 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptore-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that can be used include, for example, microparticle bombardment, etc. One example of a method of transforming delivery of nucleic acid molecules into plant cells is the bombing of the microparticles. This method was described in the review of Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microparticles), which can be coated with nucleic acids and introduced into cells by means of the driving force. Examples of the particles include particles of tungsten, gold, platinum, etc. Special advantage of microparticle bombardment, in addition to the fact that this method is effectivenessa reproducible transformation of monocots, is that not require any allocation of protoplasts or susceptibility to infectionAgrobacterium. Illustrative of the embodiment of the method of delivery of DNA into cells of maize (Zea mays)using the acceleration is neoliticheskaya delivery system α-particles, which can be used for the transmission of particles coated with DNA through a screen, for example, stainless steel or Nytex screen, onto a filter surface covered with a suspension of cells of maize. The delivery system of particles suitable for use in the present invention, is a gun PDS-1000/He accelerated helium, supplied by Bio-Rad Laboratories.

For bombardment, cells in suspension can be concentrated on the filters. The filters containing the bombarded cells placed at an appropriate distance below the plate, stopping microparticles. If necessary, between the gun and the bombarded cells also put one or more screens.

Alternatively, immature embryos or other target cells may be deposited on a solid culture medium. Bombarded cells placed at an appropriate distance below the plate, stopping microparticles. If necessary, between the accelerator and bombarded cells also put one or more screens. Using the methods described in this application, you can get up to 1000 Il is more foci of cells, transient expressing the marker gene. The number of cells in the hearth, which Express a product of an exogenous gene after 48 hours after the bombing usually varies from one to ten and average of one to three cells.

In transformation by bombardment you can optimize the cultivation conditions before the bombing and settings bombing to get the maximum number of stable transformants. In this method it is important to both physical and biological parameters of the bombing. Physical factors include manipulation of the deposition of DNA/microparticles or factors that affect the flight and velocity macro - or micro-particles. Biological factors include all the stages included in the manipulation of cells before and immediately after bombardment, the osmotic regulation of target cells, aimed at mitigating the trauma associated with bombardment, as well as the properties of the transforming DNA, such as linearized DNA or intact superstructure plasmids. Assume that the manipulation carried out prior to bombardment, especially important for the successful transformation of immature embryos.

In another alternative embodiment, can be stably transformed plastids. The described method of transformation of plastids of higher plants, including delivery with Oshu gun on the particle DNA, containing a selective marker, and the direction of DNA into the genome of plastids via homologous recombination (US 5451513, US 5545818, US 5877402, US 5932479 and WO 99/05265).

Thus, it is considered that you may need to adjust various aspects of the parameters bombing in small-scale studies to fully optimize the conditions. May in particular be necessary to adjust the physical parameters such as the distance of the gap, the distance of the flight, the length of the fabric, as well as the helium pressure. You can also minimize traumatic factors, changing conditions that affect the physiological state of the recipient cells, and therefore this may affect the efficiency of transformation and integration. For example, the osmotic state of hydration of the tissue and the phase of the subculture or the cell cycle recipient cells can be adjusted for optimal transformation. Perform other normal functions known to experts in the art given the present description.

Transfer withAgrobacteriumis a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, avoiding, thus, the need for regeneration of intact plants from protoplast. The use of Agrobacterium-mediatedvegetable, integrative vectoroids the introduction of DNA into plant cells is well known in the prior art (see, for example, US 5177010, US 5104310, US 5004863, US 5159135). In addition, the integration of T-DNA is relatively exact process that leads to a small number of rearrangements. Region of DNA that migrated marked borderline sequences, while in between DNA is usually integrated into the genome of a plant.

Modern vectors for Agrobacterium-mediated transformation capable of replication inE. Colias inAgrobacteriumproviding convenient manipulations as described in Klee et al., In: Plant DNA Infectious Agents, clear Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203 (1985). In addition, technological advances in vectors for gene transfer usingAgrobacteriumled to improvements in the arrangement of genes and restriction sites in the vectors, which facilitates the construction of vectors capable of expression of genes encoding different polypeptides. Described vectors are convenient multilinker region between the promoter and the polyadenylation site for direct expression of embedded genes encoding the polypeptide, and are suitable within the present invention. In addition, for transformation can be usedAgrobacteriumcontaining active and inactive Ti-genes. In those varieties in which Agrobacterium-mediated transformation is efficient, this method is preferred due to the simplicity and from whom the well being of gene transfer.

Transgenic plant obtained by the use of Agrobacterium mediated transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as geminiguy on introduced gene. More preferred is a transgenic plant that is homozygous for the introduced structural gene, i.e., a transgenic plant that contains two added gene, one gene at the same locus on each chromosome in a pair of chromosomes. A homozygous transgenic plant can be obtained by sexual reproduction (pollination) generation of independent transgenic plants after splitting, which contains a single added gene, germinating part of the obtained seeds and analysis of plants obtained in the presence of the target gene.

It should also be understood that two different transgenic plants can be crossed with producing offspring that contain two independently split exogenous gene. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Backcross with parental plant and the outcross with nereshennymi plant is also considered, as is the vegetative reproduction. Descriptions of other breeding methods that are commonly used is used for different traits and crops, can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis (1987).

Transformation of plant protoplasts can be carried out using methods based on precipitation with calcium phosphate, the processing of polyethylene glycol, electroporation, and combinations of the above methods. The application of these systems to different types of plants depends on the ability to restore specific line of plants from protoplasts. Examples of methods of regeneration of cereals from protoplasts are described in Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986.

They may also use other methods of transformation of cells, which include, inter alia, the introduction of DNA into plants by direct DNA transfer into pollen, direct injection of DNA into reproductive organs of a plant or direct injection of DNA into the cells of immature embryos followed by the rehydration of the dried embryos.

The regeneration, development and cultivation of plants from single transformed protoplast plants or from various transformed explants is known in the prior art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)). The process of regeneration and growth stage typically includes selection of transformed cells, culturing the obtained individual cells in the normal stages of embryo development to the stage of daveg the roots sprout. Transgenic embryos and seeds recovered in a similar way. Then transgenic shoots with roots planted in a suitable environment for plant growth, such as soil.

The development or regeneration of plants containing the foreign, exogenous gene, known from the prior art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. In other cases, the pollen obtained from the regenerated plants, pollinating grown from seeds plants agrotehnicheskij important lines. In turn, pollen from plants of these important lines used to pollinate regenerated plants. The transgenic plant of the present invention containing exogenous target nucleic acid, grown using methods known to the expert skilled in this technical field.

Methods of transformation of dicotyledonous plants, primarily byAgrobacterium tumefaciensand obtaining transgenic plants have been described for cotton (US 5004863, US 5159135, US 5518908), soybeans (US 5569834, US 5416011), cabbage (US 5463174), peanut (Cheng et al., 1996) and pea (Grant et al., 1995).

Methods of transformation of cereal plants, such as barley, the introduction of a genetic modification into the plant through the introduction of exogenous nucleic acid and the regeneration of plants from protoplay the tov or immature embryos of plants known in the prior art, see, for example, CA 2092588, AU 61781/94, AU 667939, US 6100447, PCT/US97/10621, US 5589617, US 6541257 and WO 99/14314. Preferably, transgenic barley plants produced using the methods of transformation mediated byAgrobacterium tumefaciens. Vectors carrying the target design nucleic acid can be introduced into the regenerated cells of barley cultivated plant tissue or Explant, or suitable plant systems, such as protoplasts.

The regenerated cells of barley preferably taken from the scutellum of immature embryos, Mature embryos, calli from them, or meristematic tissue. In order to confirm the presence of transgenes in transgenic cells and plants can be performed by amplification using the polymerase chain reaction (PCR) or southern blot analysis, using methods known to experts skilled in the art. The products of transgene expression, depending on the nature of the product, can be detected in various ways, which include Western blotting and enzyme analysis. One of the most convenient methods of quantitative analysis of protein expression and detection of replication in different tissues of plants is the use of a reporter gene such as GUS. Once transgenic plants were obtained, they can be grown with the receiving plant the tissues or parts of plants, having the desired phenotype. Can be collected plant tissue or plant part, and/or obtained from the seeds. The seed can serve as a source for growing additional plants, tissues or parts which have the desired characteristics.

TILLING-induced local lesions in genomes

Plants of the invention can be obtained by using the method known as TILLING (from the English. Targeting Induced Local Lesions in Genomes - induced local lesions in genomes). At the first stage in a population of plants induce mutations, for example, new replacement of one base pair with seed treatment (or pollen) with a chemical mutagen, and then select plants from those generations in which mutations are stably inherited. DNA is extracted, and the seeds obtained from all members of the population remain to create a resource to which you can periodically go over time.

For analysis TILLING generate PCR primers that are specific to amplify only the target gene target. Specificity is particularly important if the target is a member of a gene family or part of a polyploid genome. Then to amplify PCR products from the combined DNA of some number of individual plants can be used labeled with dye primers. These PCR products are denatured and re from Haut with the formation of mismatched base pairs. Mismatch, or heteroduplexes pairs are as natural single nucleotide polymorphisms (SNP or SNP) (i.e., a few plants from the population may carry the same polymorphism)and induced by SNP (i.e., only the rare individual plants can be mutation). After the formation of heteroduplex use endonuclease, such asCelI, which recognizes and cleaves unpaired DNA, which is key to the discovery of new SNPs within TILLING population.

Using this approach, you can host a screening of many thousands of plants to identify plants with the replacement of one base, and short insertions or deletions (1-30 BP), any gene or a region of the genome. The analyzed genomic fragments can vary in size approximately from 0.3 to 1.6 TPN. When 8-fold Association, fragments of 1,4 TPN (excluding the ends of the fragments, on which detection of SNP problematic due to noise) and 96 tracks in the analysis, this combination allows the screening of up to a million base pairs of genomic DNA in a single test, which makes TILLING high-performance method. TILLING is also described in Slade and Knauf (2005), and Henikoff et al., (2004).

In addition to efficient mutation detection, high throughput TILLING method is ideal for detecting natural polio is fizmo. Thus, a detailed comparison of the unknown homologous DNA through the formation of heteroduplexes with known sequence shows the number and position of polymorphic sites. Identify and nucleotide substitutions and short insertions and deletions, which include at least some number of repeat polymorphisms. This method is called Ecotilling (Comai et al., 2004).

Each SNP are registered according to its approximate position within a few nucleotides. Thus, each haplotype can be entered into the database on the basis of its variability. Sequence data can be obtained with a relatively small incremental effort, using aliquots of the same amplified DNA, which is used in the analysis by splitting mismatches. Left or right Sequeira measures for one reaction Vybrat on the proximity of its location to the polymorphism. Software sequencing machine performs multiple alignment and detects the change of the bases, which in each case confirms the band on the gel.

“Ecotilling can be performed with lower cost compared to the full sequencing, a method that is currently used for the detection of the majority of the SNP. Tablets containing matrix ectopically DNA can be subjected to screening, without the need for the unification of DNA derived from plants subjected to mutagenesis. Since the detection performed on the gels with a resolution of almost a couple of reasons and background samples are homogeneous along the tracks, the lines that have identical size, can be compared, allowing, thus, to detect and to carry out SNP genotyping in a single stage. Thus, the full sequencing of the SNP is simple and effective, and that is more due to the fact that the aliquots of the same PCR products used for the screening, can be subjected to DNA sequencing.

EXAMPLES

Example 1. Materials and Methods

Isolation and purification of prolamins

To highlight the prolamins of the cereal flour of whole grain (10 g) was stirred for 30 minutes at 25°C in 200 ml of buffer containing 20 mm triethanolamine-HCl (TEA), 1% (wt./about.) the sodium ascorbate, 1% (wt./about.) polyethylene glycol (malves 6000; PEG6000) and 200 ál of a mixture of protease inhibitors plants (Sigma #P9599), all with a pH of 8. The suspension was centrifuged at 7000 g for 15 minutes, the precipitate was washed at least two times to remove proteins soluble in aqueous buffer. The prolamins in the washed precipitate was dissolved in 40 ml of 50% (vol./about.) propane-2-ol containing 1% (wt./about.) dithiothreitol (DTT), 1% (wt./about.) PEG6000, 1% (wt./about.) the sodium ascorbate, was stirred for 30 min at 25°C. the Suspension was centrifuged, and the prolamins about what was Adali of supernatant 2 volumes of propan-2-ol and kept at -20°C. If necessary, an aliquot equivalent to 10 g of flour, besieged by centrifugation at 160 g at 4°C for 10 min, the precipitate was re-dissolved in 10 ml of buffer (buffer A), which contained 25 mm TEA, freshly prepared 8 M deionized urea and 1% DTT, all with a pH of 6, or in other buffers, as described.

Total preliminary fraction was isolated from each sample of grain using reversed-phase fast protein liquid chromatography (RP FPLC) as follows: Prolamins (200 µl) was injected in 1 ml column RPC Resource (Pharmacia)connected with the same 3 ml column. The column was washed with 2 ml of 95% solvent A/5% solvent B and suirable prolamins in 30 ml linear gradient from 95% solvent A/5% solvent B to 100% solvent B at a speed of 2 ml/min Solvent A was 0.1 percent (vol./about.) triperoxonane acid (TFU) in water, solvent B was 0.1 percent (vol./about.) TFU 60% (vol./about.) aqueous acetonitrile. Fractions of the eluate corresponding to the peaks of protein were combined. Control fraction of solvent similarly combined from runs without input protein.

Next Gordini barley was fractionally use OF FPLC as follows: Used the methodology as given above, except that the gradient elution was different in that the concentration of solvent B was 50% at 4 ml, 52% in 17 ml, 56% at 34 ml, 58% at 37 ml, 60% with 41 ml, 62% at 44 is l, 64% at 47 ml, 66% at 50 ml, 100% with 53 ml, 100%, with 57 ml were Collected fractions of 1 ml and fractions 11-14 (#1), 19-23 (#2), 31-34 (#3), 43-51 (#4), 53-58 (#5), 63-64 (#6)corresponding to A280 peaks, together.

Analytical methods

Prolamine fraction was dissolved in 6 M urea, 2% (wt./about.) LTOs, 1% (wt./about.) DTT, 0.01 percent (weight/about.) bromophenol blue, 0,0625 M Tris-HCl (pH 6,8) at 25°C, and analyzed by electrophoresis in LTO-page as follows. An aliquot of 5 μl of a solution prolamin-LTOs were applied to the gel LTO-page, ready-made gels 245x110x0,5 mm with a gradient of polyacrylamide 8-18% (ExcelGel Pharmacia), and phoresis was performed at 600V for 90 min at 15°C. the Gels were washed in 40% MeOH/10% acetic acid for 30 min, and then in water for 10 minutes Prolamins were stained by immersing the gel 0.06% (wt./about.) colloidal Coomassie G250 8.5% phosphoric acid for 30 min, after which the gel was washed in water during the night. Each gel was calibrated standard 10 kDa protein marker (BenchMark, Invitrogen).

Gordeyeva faction was dissolved in 50% (vol./about.) water isopropyl alcohol, 1% (wt./about.) DTT treated in excess of vinylpyridine to restore the disulfide bonds and analyzed using reversed-phase HPLC (RP-HPLC, Larroque et al., 2000), calibrated with prolamins extracted from lines of barley Riso56 or Riso1508, in which, as a result of mutations do not accumulate Gordini groups B or C is respectively (Doll, 1983). The levels of protein in the extracts or fractions were determined by the method of Bradford (1976). Generally, the protein content was measured in the format of 96-well plates by adding 10 ál DTT/propan-2-ol supernatant to 200 ál of dilution 1 to 5 concentrate for analysis of protein Coomassie protein assay concentrate (BioRAD) in water, was calibrated against gamma-globulin, and measured the absorbance at 595 nm.

Ex vivo analysis of T-cell toxicity

The prolamins (50 mg/ml in 2M urea) was dissolved in PBS containing 1 mm CaCl2obtaining the concentration of prolamine of 62.5, 250, 625, 2500 or 6250 ug/ml, was dezaminirovanie by adding 25 µl of each solution to 100 ál of tTG from the liver of the Guinea pig (transglutaminase (Sigma, T5398), 25 mcg/ml tTG in PBS containing 1 mm CaCl2), and were incubated for 6 h at 37°C. Nezamechennye solutions prepared similarly, by incubation in the absence of tTG. The solvent control was added as maximum concentrations prolamine. Other controls contained either known toxic peptide ω-gliadin, designated as 626fEE, at a concentration of 50 μg/ml peptide 626fEE in pure form or with tetanus toxoid (50 sitebrowser particles/ml). The peptide ω-gliadin 626fEE, also known as the DQ2-ω-1, had the amino acid sequence QPEQPFPQPEQPFPWQP (SEQ ID NO:1) was synthesized by the firm Mimotopes, Melbourne, Australia. Its identity and purity (91%) were confirmed using the ACC-spectrometry and HPLC. The tetanus toxoid was obtained from Commonwealth Serum Laboratories, Melbourne. Then all the solutions were frozen at -20°C.

Twenty-one patients with HLA-DQ2+celiac disease confirmed by biopsy, who adhered to a strict gluten-free diet for at least three months, daily for 3 days received 150 g of boiled barley, used as part of their diet, which otherwise remained gluten-free. Heparinized venous blood was taken either prior to, or six days after the beginning of dietary provocation, and from each blood sample by centrifugation in a density gradient Ficoll-Hipac (Anderson et al., 2000) were isolated peripheral blood mononuclear cells (PBMC). Cells PBMC resuspendable in complete medium HT-RPMI (Invitrogen)containing 10% thermoinactivation mixed human AB serum. Desametasone or nezamechennye prolamins and control solutions were thawed and 25 μl were made in wells containing 100 μl of PBMC (3-8×105PBMC in the hole). They were cultured at 37°C overnight in 96-well plates (MAIP-S-45; Millipore, Bedford, MA). The control culture was obtained by adding 25 μl of PBS containing 1 mm CaCl2(the control containing only buffer). Final concentration of prolamine was 2.5, 10, 25, 100, or 250 μg/ml and the final concentration of urea was 50 mm. The level of IFN-γ, wirabuana what about in every culture, served as an indicator of the toxicity of each prolamine, were evaluated visually for the formation of spots, using secondary antibodies according to the manufacturer's instructions (Mabtech, Stockholm, Sweden), and counted the number petrobrazi units (SFU) using an automated ELISPOT tablet reader (AID Autoimmun Diagnostika GmbH, Germany). The results were presented as the average number petrobrazi units (SFU) ±SE. As a rule, the percentage coefficient of variation SFU/106 PBMC in the analysis was 14% based on six duplicate analyses of the positive control, inkubiruemykh 0,5x106cells in six patients with CD (all of >20 SFU/well).

Statistical analysis

Analysis of variance (ANOVA) or t-criteria using subregions and districts used to determine the magnitude of differences observed for the average SFU shown T-cells isolated from patients with celiac disease before (n=10) or after (n=21) diet provocation, and encourageme with gardinali, prolamins or control.

Response curves for 21 people after the provocation was very different, and a significant proportion of the variance was due to these differences. To take into account the different responses of patients was applied to model random coefficients. This is a mixed model analysis, which was performed using the method of residual maximum likelihood (EML), which allows you to enter the amendment random indicators related to the subject (patient) and provocation (protein concentration) in the body of the patient. In order to stabilize the significant heterogeneity of variance before the specified analysis data were transformed logarithmically. To solve the problem of zero values to all data to a logarithmic transformation was added to the unit. Constant values in the model were the presence or absence of tTG and used Gordeyeva fraction, together with their interaction.

The hyperbolic model is also applied to the untransformed average SFU for T-cells from 21 patients after provocation, peered with six tTG Gordeyeva factions or four drugs grain prolamine treated with tTG.

Transformation of barley

Transformed barley plants can be obtained by the method of Tingay et al, (1997). Gene construct binary vectors can be introduced into a highly virulent strain ofAgrobacterium(AGL1) through technovillage conjugation, which are then used for the introduction of T-DNA containing the transgene, and the selective marker gene (encoding resistance to hygromycin expressed from the promoter CaMV35S), the regenerated cells of the scutellum of immature embryos of barley, as follows.

Developing seeds of barley is ORT Golden Promise, after 12-15 days after flowering were removed from the growing ear grown in the greenhouse plants and sterilized for ten minutes in 20% (vol./about.) the sodium hypochlorite solution followed by a single rinse with 95% ethanol and seven times with sterile water. Then the embryos (approximately 1.5 - 2.5 mm) was separated from the seeds in sterile conditions, and each embryo was cut hem. The embryos were placed cut down on a Petri dish containing medium to induce callus formation. TransconjugateAgrobacteriumwere grown in a nutrient medium MG/L (containing 5 g mannitol, 1 g L-glutamic acid, 0.2 g KH2PO4, 0.1 g NaCl, 0.1 g MgSO4·7H2O, 5 g of tryptone, 2.5 g yeast extract and 1 mcg of Biotin per liter, pH 7.0)containing spectinomycin (50 mg/l) and rifampicin (20 mg/l) with aeration at 28°C to a concentration of approximately 2-3×108cells/ml and Then approximately 300 µl of cell suspension was added to the embryos in a Petri dish. After 2 minutes, poured the excess liquid from the Cup, and embryos turned so that the side with the cut (axillary side of the scutellum of the embryo) on top. Then the embryos are transferred into the new Cup with medium induction kalpaamruthaa and placed in the dark for 2-3 days at 24°C. the Embryos are transferred into the environment induction kalpaamruthaa selection (50 μg/ml of hygromycin and 15 µg/ml Timentina).

Sarod the necks were left in this medium for 2 weeks in the dark at 24°C. Then healthy callus was divided, and placed in fresh selective medium, then incubated for two weeks at 24°C in the dark. Thereafter, embryos were incubated at 24°C in the light for 2 weeks on the environment regeneration, containing cytokinin, and then transferred to the medium for root formation containing cytokinin and auxin during three 2-week periods. Then the young plants were transplanted into soil mixture and kept on a shelf with atomized irrigation within two weeks, and then finally transferred into the greenhouse.

Methods of mutagenesis, including gamma irradiation

Mutation of genes in the barley, leading to a reduction in the expression of D-, C-, B -, or γ-Gordeyev, you can enter either by using gamma irradiation, or by chemical mutagenesis, for example, using ethylmethanesulfonate (EMS). In the case of mutations caused by gamma radiation, seeds can be irradiated at a dose of 20-50 cu source60Co (Zikiryaeva and Kasimov, 1972). EMS mutagenesis can be accomplished by seed treatment with EMS (0.03%and about./about.) according to Mullins et al., (1999). Double zero background B+C mutant grains can be identified on the basis of reduced protein or hordein, or a change in the morphology of the grain, and confirmed by the methods described above. Mutants of one gene hordein can be crossed with a second mutant for which Yedinaya mutations and receive nedrencheskogo barley essentially not containing Gordeyev in the endosperm.

Example 2. Toxicity Gordeyev barley for celiacs

The composition of the prolamins in barley and other cereals

Prolamins were allocated in the form of proteins, soluble in water-alcohol mixture, cereal, toxic to celiacs, barley and wheat, less toxicangel oats and non-toxic corn, and was purified in a single phase with the help OF FPLC as described in Example 1. The elution profiles of protein prolamins that are defined in ANmin PF FPLC (Figure 1), showed a number of partially resolved peaks of individual proteins, buervenich with a sharp increase in the gradient of the solvent. Fractions containing protein after 10 cleanings for each cereal, United and liofilizirovanny. Normal output prolamine from different grains (2 g) were: corn - 10 mg, oat - 23 mg, barley - 73 mg, and wheat - 114 mg. Total prolamins from each cereal liofilizirovanny and stored for testing in ex vivo analysis of T-cells (below). Controls solvents also received with the help OF FPLC.

The barley prolamins (hordein) was also fractionally use OF FPLC as described in Example 1. The elution profile obtained in the fractionation process in the initial experiment, shown in figure 2. Received six peaks and isolated from each protein. The appropriate combined fractions of twenty consecutive input the s samples were combined and liofilizirovanny. Normal outputs of 4 g of flour from whole grains were: fraction 1-19 mg, fraction 2-26 mg, fraction 3-14 mg, fraction 4-104 mg, fraction 5-24 mg, and the fraction 6-11 mg

Identity Gordeyev in each fraction was determined by electrophoresis in LTO-SDS page as described in Example 1, and was confirmed by analytical RP HPLC. The results are shown in figure 3 and 6. HPLC showed that the fraction #1 contained approximately 39% D-hordein weight reached 90 kDa by electrophoresis in LTO-PAG, and approximately 61% C-hordein weight was reached 47 and 48 kDa by electrophoresis in LTO-page (Figure 3, #1). Fraction #2 contained the C-hordein, as shown by electrophoresis in LTO-page and HPLC. Fraction #3 contained a broad protein band, which was at approximately 45 kDa by electrophoresis in LTO-PAG, but which were divided into 6 peaks in HPLC, which corresponds to the elution and C-, and B-Gordeyev. The composition was estimated using HPLC, determining the content of approximately 43% and 57% Gordeyev C and B, respectively. Fraction #4, 5 and 6 contained the B-hordein; these fractions may also contain a small amount of gamma hordein. Two-dimensional electrophoresis and mass spectrometry trypticase peptides (fingerprint) specified gordiyovych fractions did not give enough unique peptide fragments to uniquely identify individual Gordini. This may occur because the sky is the greater variations between sequences selected Gordeyev and sequences, available in databases. Therefore, the fractionation in this experiment resulted in the enrichment of specific Gordeyev from barley, but not completely clear. Further purification can be achieved by using additional stages OF FPLC or OF FLPC, combined with ion-exchange methods.

Samples of each gardenboy fractions were treated or not treated with tTG, which converts glutamine residues in proteins in glutamate, and then liofilizirovanny for use in the analysis of T-cells.

Analyses toxicity

Tests on T-cells using PBMC isolated from patients with confirmed cases of celiac disease, was performed as described in Example 1 to determine the toxicity of total products of prolamins and gordiyovych fractions. PBMC were isolated before and after diet provocation barley, and samples of prolamins was treated with tTG, or not processed. T-cells isolated in a group of 10 patients with celiac disease to dietary provocation, were immune to the prolamin. Statistical analysis using ANOVA showed that there was no any significant difference (P=0,77) between the average number of IFN-γ positive spots for the highest concentrations of treated tTG prolamin fractions, peptides or Gordeyev (average value in the group SFU±SE - 1,52±0,18), and the control culture (mean SFU±SE - 1,40±0,45) on the Contrary, the analysis showed that T-cells that were not subjected to provocations, actively responded (P<0,001) with tetanus toxoid, which served as a positive control (mean SFU±SE - 22,3±4,72), compared with prolamins. This indicates that the selected T cells were functional and were capable of reaction with a known toxin, and confirms that in populations selected before dietary provocation, was attended by a small number of prolamin-reactive T-cells.

In contrast to the weak response to the prolamins in front of dietary provocation, T-cells isolated after diet provocation, was very active. T cells isolated from 21 patients with celiac disease, 6 days after the provocation, gave a strong response to the prolamins treated with tTG, in contrast to T-cell subgroups (n=13) in this group, reacting with nezamechennymi prolamins. Figure 4 shows the results of cereals, total barley prolamins induced maximum value SFU, followed by, in descending order of prolamins from wheat, oats, and then maize (figure 4 blocks A, B, C, D, respectively). Although the maize prolamin caused a weak dose-dependent T-cell response in this analysis, it usually does not cause a response in diet provocation and is considered safe for celiac cereals. Digestion in the intestine can destroy epitopes, PR is sutstvie in the whole prolamins of maize, which remained intact in this analysis and stimulated T-cellsin vitro.

From gordiyovych fractions fractions #1, #2 and #3 gave higher values SFU compared to Gordeyeva fractions #4, #5 and #6 (Figure 5).

Since the concentration of prolamins in the analysis was increased, the value of IFN-γ spots increased in a hyperbolic manner similar enzyme kinetics Michaelis-Menten often observed between the enzyme and its substrate (Figure 4 and 5), although it was not clear why this occurred in the data cell assays.

Each 96-well plate contained a number of internal positive and negative controls. Observed a small but significant difference (P<0,001) between the average SFU, when compared to the control cultures and the control solvent (control culture SFU 2,75±0.67 and 1,49±0,24; controlling solvents SFU 2,64±0,23 and 2.75±0,23, in the absence and in the presence of tTG, respectively). Although these values were statistically significant, they were very small compared to SFU after provocation in the positive controls or tests, including the prolamin. It was confirmed that the impurities in the solvents did not give false positive signals. The control peptide 626fEE gave correspondingly high response (mean SFU±SE 29,55±of 4.38 and 33,60±2,97 in the absence and in the presence of tTG, respectively). Insufficient strengthening of the than 626fEE when processing tTG expected as indicated peptide was synthesized from glutamate in the 10th position, and did not require treatment tTG to increase toxicity. Adding control solvent does not substantially inhibit a positive response to peptide 626fEE (P=0,13), confirming that the impurities in the solvents did not give a false signal. Reproducibility targets tetanus toxoid (P=0,193) from tablet to tablet confirmed that differences in the response of T-cells on the prolamins were not due to differences from tablet to tablet, but reflect the different sensitivity of populations of T-cells derived from different patients.

The variation of sensitivity of different patients to the same concentration of prolamins were up to 200 times. Therefore, the normalized data SFU applied the model random coefficients REML, and found that the model that corrects for the deviation in the responses of patients due to different concentrations of the protein, showed a significantly better match to the data (P<0,001)than the model that used the response per patient regardless of the concentration, with a deviation within 1982,28 (1616 df) to 1640,91 (1613 df). The main effects caused by tTG (P<0.001) and prelaminates fraction (P<0,001), was significant, and between them was absent any interaction. This confirmed that sensitive to what rollino T cells were induced in patients with celiac disease six days after diet provocation barley. Processed average values, on a logarithmic scale, for normalized data SFU was only 1,613 Torino (without tTG) and 2,026 (tTG) with standard error (SED) 0,0527, confirming that pre-tTG had a substantial impact on the answers. Processed average values for gordiyovych fractions #1 - #6 were 1,903, 1,909, 1,956, 1,693, and 1,724 1,733 respectively with SED 0,0826. The results indicate that Gordeyeva fractions are divided into two groups with different toxicity, while Gordeyeva fractions #1, #2 and #3 form more toxic group than Gordeyeva fraction #4, #5 and #6.

It is interesting to note that the most toxic Gordeyeva fractions were suirvey in reversed-phase FPLC and HPLC in the beginning, and so was more polar than erwerbende later, less toxic fraction.

Conclusions

T cells isolated from 21 patients with celiac disease, 6 days after the provocation, strongly interacted with prolamins treated with tTG, compared with nezamechennymi prolamins, as should be expected for celiac (Hadjivassiliou et al, 2004; Kim et al., 2004). This can be explained by the interaction between detalizirovannymi prolamins and binding site in a key protein, such as a molecule HLA-DQ2, which is presenting stimulating protein receptor on CD4+T-cells involved in the inflammatory response.

Although observed measurable differences in toxicity gordiyovych factions, all Gordini were significantly more toxic compared to the prolamins of maize and oats, which are considered safe for most celiacs. Statistical analyses showed that the barley prolamins and Gordeyeva fractions #1, #2 and #3 (containing D and C hordein) formed the most toxic group. Gordeyeva fraction #4, #5 and #6, containing mainly B-hordein, and the prolamins of wheat, has formed a second, less toxic group. The prolamins of oats and corn formed the least toxic group. This indicates that T cells induced in patients with celiac disease as a result of provocation barley was less sensitive to wheat and oats. This can be explained by the fact that the dominant epitopes in barley prolamins are significantly different from the epitopes in the prolamins of wheat and oats. Although the processed data showed that the oat prolamins was significantly less toxic in comparison with barley prolamins, between different patients seen pyatidesyatiletnie the discrepancy between the results when the same concentration of prolamine oats, while T-cells five of the 21 patients showed >20 SFU with the greatest concentration of prolamine. This is consistent with other reports of significant responses to oats in celiac disease patients (Arentz-Hansen et a., 2004; Lundin et al., 2003).

Based on these data, considered it possible that when dietary provocation all gardenstorage faction cause significant intestinal response in coeliac patients. This suggests that all gardenstorage faction you want to delete or modify to get the barley, which is absolutely non-toxic to celiacs. Also suggested that you first need to delete or modify basic components - hordein B and C.

Example 3. Obtaining grain barley with reduced content Gordeyev B and C

A number of barley mutants with changes in the synthesis or accumulation Gordeyev were previously identified. These mutants of barley were not allocated to reduce the content Gordeyev in grain, and were identified and selected by the indicator of a high content of lysine in the grain, and later found that they had a low content of Gordeyev.

Mutant Riso 7, first described by Doll et al. (1976), was identified after treatment with fast neutrons original cultivar Bomi. It contained a recessive mutation in a gene, which resulted in a 29%WMD lower levels of prolamins and 10%boost in the content of lysine in proteins, compared with Bomi. The decrease in the content of the poor in lysine prolamins was compensated by the increase in the content of other relative activities is but rich in lysine of Saanich proteins, that led to increased levels of lysine. Grain yield and starch content decreased by 6% and 7%, respectively, compared with the original variety (Talberg, 1982; Doll, 1983).

Riso 56, first described by Doll et al. (1973), was created by induced gamma radiation mutation of the parent varieties Carlsberg II. The grain size, grain yield and content of prolamins decreased by 30%, 47% and 25%, respectively, compared with the original variety, whereas the content of lysine in proteins mutant grain increased by 13% compared with the parent strain. Reduced content Gordeyev was associated with the increased content of non-protein nitrogen and water, and solarstorm proteins (Shewry PR, et al., 1980). The high content of lysine in proteins Riso 56 was caused by a recessive mutation on chromosome 5 (Ullrich and Eslick, 1978) genetic locus, denoted byHor2ca(Doll, 1980). The mutation consisted of a deletion 80-90 TPN DNA locus ofHor2, which encodes B-hordein in barley. Expression of B-Gordeyev in the mutant decreased by 75%, whereas expression of C-Gordeyev increased 2 times (Kreis et al., 1983). The deletion was not associated with a translocation between chromosomes 2 and 5, which were also present in Riso 56 (Olsen, 1977).

Riso 527, first described by Doll et al. (1973), was also obtained using the induced gamma radiation mutation, but from the parent cultivar Bomi. The grain size, grain yield and content of prolamins in see have not been reduced by 13%, 25% and 20%, respectively, compared with the original variety, whereas the content of lysine in proteins of the mutant grew by 12%. The mutation was recessive and was localized in the gene on chromosome 6, denoted bylys6i(Jensen, 1979). This mutant had a reduced content of D-Gordeyev and high content B1-Gordeyev (Klemsdal et al., 1987).

Riso 1508 was identified after EMS-induced mutation of the parent cultivar Bomi (Doll et al., 1973; Ingerversen et al., 1973; Doll, 1973). The grain size, grain yield and content of prolamins in grain decreased by 8%, 12% and 70%, respectively, compared with the original variety, whereas the content of lysine in proteins of the mutant grew by 42%. The high content of lysine was caused by a recessive mutation in a gene, located near the centromeric region of chromosome 7 barley (Karlsson, 1977). Specified gene initially defined assexScXenia with shrunken endosperm (Ullrich and Eslick, 1977), but now it is known aslysSa(Tallberg, 1977). Comparative levels types of mutant proteins in the grain has changed, with increased content of water-soluble protein (albumin/globulin) - from 27% to 46% of the total nitrogen protein seed, and decreased the number of prolamins by 70% compared with the parent variety, from 29% to 9% of the total nitrogen protein seed (Ingerversen et al., 1973; Doll, 1973). In Riso 1508 observed a fourfold increase in the content of free amino acids and the sky is the same N, compared with the parental strain when growing plants under conditions of high nitrogen content fertilizer (Koeie and Kreis, 1978). Shewry with TCS. (1978) confirmed that the level solarstorm nonprotein nitrogen has doubled. The percentage nitrogen content of seeds in the form Gordeyev decreased by 70%, and the content solarstorm proteins in Riso 1508 increased in comparison with Bomi 70%. Detailed molecular analysis showed that the levels of B - and C-Gordeyev decreased by 80% and 93% respectively, while the content of D-Gordeyev increased four times. The influence on the accumulation of the protein was determined by the changes in the copy number or mRNA stability (Kreis et al., 1984). It could be caused by increased methylation of the promoters of the genes encoding B - and C-hordein in the mutant Riso 1508 (Sorensen et al., 1996). Smaller seed Riso 1508 was mainly caused by reduced synthesis of starch (Koeie and Breis, 1978; Kreis and Doll, 1980; Doll, 1983). The content of sugar has doubled, whereas the synthesis of starch in Riso 1508 decreased by about 20-30% compared with the original variety. Kreis (1979) reported that in Riso 1508 levels of β-amylase decreased, whereas Hejgaard and Boisen (1980) reported similar levels of β-amylase.

Hiproly was a spontaneous mutant identified from germplasm Ethiopian barley CI 3947 (Munck et al., 1970), and had increased by 20-30%, the content of total protein and lysine in the protein p is compared with barley wild-type (Doll, 1983). When crossed with barley wild-type and high protein content was lost, while increased lysine content in the protein is preserved, showing that these traits were inherited independently. The high content of lysine was due to a single recessive mutation in the genelyson chromosome 7. The mutation caused an increase in water content and solarstorm proteins and, therefore, the content of lysine. Unlike Riso with a high content of lysine levels Gordeyev and the weight of seeds in the mutants Hiproly in the progeny of backcross not decreased. Nonprotein nitrogen was not raised. The content of β-amylase increased 4 times (Hejgaard and Boisen, 1980).

Characteristics of parental lines Riso 56 and Riso 1508

Properties of prolamins, which are accumulated by the parent lines Riso 56 and Riso 1508, was confirmed by electrophoresis in LTO-page and reversed-phase HPLC. Solarstorm proteins extracted from grain, separated by gel electrophoresis and transferred to membranes (Western blotting). Proteins on the membranes were stained for determination of total protein (7, left side) or were treated prolamin-specific monoclonal antibody (mouse monoclonal antibody MAb 12224, induced against the total glutening extract, which were detected all Gordini and prolamins (Skerritt, 1988) (right side), which showed t is, the levels of B-Gordeyev in Riso 56 were very low, while the content of C-Gordeyev, compared with levels in Riso 527 increased. The detection antibody was confirmed that the level of B-Gordeyev extract Riso 56 was extremely low (dashed square). These three proteins observed in Riso 56, which is moved together with B-gardinali, most likely, was γ-gardinali. In Riso 1508 was reduced accumulation of B-Gordeyev, while C-hordein differed with difficulty (dotted square). This is consistent with the published literature. Levels of D-hordein, which was relatively minor prolamin component, did not seem high when applied to the protein in the quantities used in this gel.

On Fig shows the relative levels of different Gordeyev in purified extracts after analysis of reversed-phase FPLC. Extracts Gordeyev, equivalent to 0.2 g of flour were analyzed using FPLC as described in Example 1. Thus, the area under the chromatogram A280 was proportional to the protein content in each sample. In Riso 56 levels of C-Gordeyev increased by 400%, and B-Gordeyev decreased by 86% compared with the parent variety Carlsberg II. In Riso 1508 C - and B-hordein decreased (91% and 86%, respectively) compared to the parent cultivar Bomi. The results obtained were similar opublikovannym.

Identification of seeds with two Gordeyeva mutations

Plant lines Riso 56 and Riso 1508 crossed by removing pistils Riso 1508, and in two days were pollinated with fresh pollen Riso 56. Ten F1 seeds were germinated and cultivated plants F1, which allowed sampledatabase. The seeds of the F2 was collected in a Mature form.

To identify double mutants in the population, half of each of the 288 F2 seeds were separately crushed and used in the powder in the plastic microprobe using a stainless steel ball, with shaking at 30/sec for 3 × 1.5 min in 96-well vibrating mill Vibration Mill (Retsch Gmbh, Rheinische). To each tube was added an aliquot (400 μl) of the aqueous buffer for extraction of soluble proteins. The buffer contained 20 mm triethylamine-HCl (TEA), 1% (wt./about.) the sodium ascorbate, 1% (wt./about.) PEG6000 and inhibitor of proteases of plants at 1/1000 dilution (Sigma P9599), pH 8 at room temperature (RT). The contents of each tube was again shaken and then centrifuged at 160 g for 10 min at RT. Water-insoluble precipitate flour washed at least two times in a similar way, then the corresponding supernatant United, having water-soluble fractions. Then the precipitate was extracted soluble prolamins, adding 400 ál of 50% (vol./about.) water propan-2-ol containing 1% (wt./about.) DTT, and shook the tube, as Casanovas, with subsequent incubation for 30 minutes at RT and the second stage shaking and centrifugation, as described above. Appropriate supernatant containing prolamins extracted, combined and transferred into new tubes. The protein content in DTT/propan-2-ol supernatant was measured by the Coomassie reagent (BioRAD), then the prolamins in the aliquot 200 ál besieged 400 µl propan-2-ol and kept overnight at -20°C.

An aliquot of each prolinnova extract from half of the seed was analyzed for the reduction of B - and C-Gordeyev by electrophoresis in LTO-SDS page as described in Example 1 (Fig.9). To analyze the gels were applied to the samples corresponding to individual seeds, and each track has caused the amount of protein equivalent to 1/20 of the seed. In particular, the extracts were analyzed for the absence or reduction of the characteristic protein bands Gordeyev 40 kDa (specific B-hordein) and 70 kDa (specific C-hordein). The seeds of the parent lines Riso 56 and Riso 1508 had a low content of B - and C-Gordeyev respectively, but still contained low levels of D-Gordeyev corresponding to the band of 100 kDa (Figure 9). Most of the extracts of seeds of F2 contained a profile of the wild type, including the presence of Gordeyev D, C and B (Figure 9), confirming that which was made effective crossing two parental lines. Chestnut the AMB seeds, as it turned out, did not contain any B-or C-Gordeyev, and therefore, they were evaluated as homozygous for both genetic disorders present in the parental lines. They were identified from 288 policeman (frequency 0,055). This corresponded to a frequency of 1 to 16 (0,0625)expected for the combination of two simple recessive mutation.

The content of total soluble protein in the extracts half of the F2 seeds were compared with similar indicators of seeds of wild type and parental seeds. The data are shown in Table 1. The protein levels in extracts of F2 seeds were reduced to less than 20%, in some cases, less than 15% of wild type. These values may have been overestimated non-protein nitrogen-containing compounds such as free amino acids, which are present in the extracts.

Table 1
The protein content in the soluble extracts halves of F2 seeds of barley
SampleSoluble protein
(μg/seed ± SE)
% Bomi
The control line
Bomi512±130/td> 100%
Riso 56364±4471%
Riso 1508147±2628%
Double zeros
RE9of 129.625%
RF889,618%
RH285,617%
BA985,617%

RB1085,617%
RA982,416%
RG12to 75.215%
W72,814%
BD57214%
BD973,6 14%
BE858,411%
BF859,212%
BB557,611%
RB557,611%

The observed differences in levels of prolamins between the lines F2, possibly due to the splitting of other genes or mutations of the parents.

Additional protein gels were driven, taking the volume of supernatant in DTT/propane-2-Ola, containing 20 µg of protein, dried each SpeediVac vacuum, was dissolved protein in 20 μl of a buffer containing 62.5 mm Tris-HCl (pH 6,8), and 12.5% (wt./about.) glycerol, 2% (wt./about.) LTOs, 1% (wt./about.) DTT and 0,112% (wt./about.) bromophenol blue, and then was heated in a bath of boiling water for 90 seconds. Each solution was applied to a pre-filled LTO-polyacrylamide gel electrophoresis was performed, and then stained and analyzed by gel as described above. Standard gel shown in Figure 10. Most of the selected seeds F2, as it turned out, did not contain any B-or C-Gordeyev and, as expected, was a "double zero". Even though each track inflicted the same amount of protein measured in the analysis of binding Bel is and dye, most of the extracts of the double zeros, as it turned out, contained significantly less protein than the control, in particular, they contain a small amount of protein material with a mass of more than 20 kDa. This can be explained by the presence of non-protein nitrogen-containing compounds such as free amino acids contained in the extracts, which may have caused an overestimation of the true levels of protein when evaluated through analysis of the binding protein with the dye. This effect was also observed for extracts Riso 1508, in which the total number of coated material, coming in the form of protein bands was reduced compared with Riso 56 or Bomi. Riso 1508, as shown, accumulated a greater amount of non-protein N in the form of free amino acids (Koie and Kreis, 1978).

Also researched slice of F2 seeds. Compared with the wild type, in some cases, the endosperm alleged double zero seeds were moderately wrinkled, others more wrinkled.

The second half of each F2 seed were germinated on wet filter paper, the F2 seedlings transferred into soil in the greenhouse and were grown to a Mature state, with the receipt of F3 seeds. Measured various growth parameters and yield of plants (table 2).

Table 2
Growth parameters and yield of barley plants F2, estimated according to the average weight of 100 seeds for seeds F3. Red = reduced for a specified hordein, WT - wild type
The age ofThe phenotype in b - and C-hordeinsHeightQuantity harvested-GOVThe index of crop productivitySeeds/
Kolos
Cf. the weight of 100 seeds (% K8)
SloopWT36,34±2,29,2±0,970,60±0,0210,3±0,75,47±0,16
K8WT54,7±1,16340,63±0,02of 21.9±1,44,65±0,11 (100%)
L1WT46,6±0,9440of 0.66±0.0122,0±1,1to 4.41±0,05 (94,8%)
9REbc downgraded56,8±2,14110,56±0,01 19,0±1,244,19±0,13 (90,1%)

R1508With zero,
Red
36,4±,3427,5±3,5of 0.66±0.0120,0±0,7was 4.02±0,02 (86,5%)
5RBbc downgraded62,0±2,24280,46±0,0215,8±1,24,01±0,01 (86.2 per cent)
Glbc downgraded61,4±1,19340,45±0,0216,0±1,03,83±0,09 (82,4%)
5BDRed63,9±1,68190,45±0,0115±0,93,70±0,09 (79,6%)
R56b zero56,24±0,3420,0±2,00,51±0,0116,8±1,23,70±0,08 (79,6%)
B5WT47,3±1,36340,52±0,0214,3±0,63,52±0,12 (75,7%)
Jlbc downgraded50,7±1,71320,57±0,0223,9±0,43,56±0,03 (76,6%)
4BHRed44,9±0,79190,56±0,0119,7±0,63,29±0,17 (70,7%)
D6bc downgraded42,3±1,23240,47±0,029,2±0,82,90 (62,4%)
6RFRed61,4±1,66230,35±0,056,6±1,62,86 (61,5%)
BlWT51,9±2,79120,37±0,03 9,6±1,52,62±0,11 (56,3%)
J4bc downgraded49,8±0,59170,35±0,037,4±1,12,64±0,01 (56,8%)

Measure plant height, weight of the spike and stem, number of shoots, number of seeds per ear and the average weight of 100 seeds. The yield index was calculated from the ratio of the weight of the ear/(weight of the stem + the weight of the ear). Then F3 seeds were grown in field seeds F4 each line.

Seeds F3 showed a significant change in all measured parameters, compared with the parental lines and the control line, Sloop. Many of the alleged double zero lines, such as J4 and 6RF, had reduced the average weight of 100 seeds, approximately 40%, or reduced the number of seeds per ear compared with wild-type sibs K8. This suggests that other genes, fissile in the population, as well as mutations Gordeyev B or C, have an impact on the harvest. However, several lines of F3 had the seed weight is above or equal to the weight of seeds in the parent lines, and presumably other genes could be split from mutations of B-hordein andlys3a.

The appearance of the F3 seeds on the cut varied from wrinkled (like Riso 1508) to slightly wrinkled (like Riso 56), sravnenie is with siblings of wild-type or control Sloop.

Total water-soluble and soluble proteins from eight seeds F3 lines were extracted as described above. The protein content in the soluble and water-soluble fractions was measured as described in Example 1, using the known number of gamma-globulin as the protein standard. However, the levels of total soluble protein in some samples of seeds F3 essentially was the same as the Riso 1508. Subsequently determined that these samples of seeds were split on wild-type alleles of the gene Lys3a and were not uniformly "double zero".

Quantitative determination of the levels hordein seeds F3 by means OF FPLC

Soluble extracts from the two seeds of each line were pooled and 50 ál was analyzed with the help OF FPLC as described above. The chromatogram is shown figure 11. Total area under the chromatogram corresponding to hordeins, was calculated and expressed relative to levels in the wild type line. The data (table 3) showed that the grain F3 levels hordein were less than 30% of the level of wild-type and in some cases less than 20%, even at 5.3%. The absence of significant protein bands on LTO-PAG supports the claim that the levels of total alcohol protein were inflated due to higher levels of non-protein nitrogen in the seeds F3.

Table 3
Relative levels Gordeyev in F3 seeds that are defined with the help OF FPLC
LineContent Gordeyev
Wild type (K8)100%
R5670%
R150850%
4BH26%
5RB21%
9RE16%
J15%

Example 4. Properties grown in a field of barley grain F4

Compared properties grown in a greenhouse and grown in field F4 seeds of selected lines (9RE, J1, G1, 4BH), odnodolnyh parental lines (Riso 56 and Riso 1508) and barley wild-type (Large, Bomi and K8, restored the wild-type sibs from the same crossing that double zero line).

The seed weight

The average weight of 100 seeds F4 grown in the greenhouse varied within 60-76% by weight Sloop (5,47 + 0.16 g per 100 seeds), whereas the weight of 100 seeds F4 grown in the field was lower and varied from 58-65% by weight Sloop (4,75 + 0.04 g).

Sprouting grain

narashivanie seeds of the two selected lines of barley F4 compared with the sort of wild-type Sloop, soaking each sample of 100 seeds on a wet paper within six days. Germination was observed as the appearance of the tip of the root from the shell of the seed. Grain F4, as it turned out, were germinated at the same rate as the grain wild type, with approximately 60-70%germination after 3 days. Grain storage at 37°C for 4 weeks before the swelling was slightly increased percent germination of both lines F4. Treatment at 4°C for 3 days also provided a similar increase in the freshly harvested material.

This demonstrates that the grain lines F4 did not show any major slow germination, and therefore it was considered agrotehnicheskij suitable.

The levels of protein in the grain F4

The levels of water, salt, alcohol, and soluble in urea protein in the grain of the F4 lines were measured using duplicate samples of 20 mg of flour from whole grains grown in the greenhouse seeds selected F4 lines (9RE, J1, G1, 4BH), odnodolnyh parental lines (Riso 56 and Riso 1508), and barley wild-type (Large, Bomi and K8).

Soluble proteins were extracted from each specimen flour using 0.5 ml of water was stirred for 30 minutes, the mixture was centrifuged at 13,000 rpm for 5 minutes, removing the supernatant, after which the extraction repeated twice on the lees. Supernatant combined (water-soluble extract), and the residue on sledovatelno were extracted three times in a similar way, using 0.5 ml of 0.5 M NaCl (solaractivity extract), and then 0.5 ml of 50% (vol./about.) propane-1-ol, containing 1% (wt./about.) DTT (soluble extract (hordein)), with subsequent extraction of 8 M urea containing 1% (wt./about.) DTT (soluble urea extract). The protein content in each fraction was measured using analysis of binding dye (BioRad) according to the manufacturer's instructions, with the calibration of gamma-globulin as the protein standard. The data shown in Fig. The content of total extractable protein (Fige) was calculated as the total amount of protein contained in all soluble fractions.

In addition, total nitrogen (Total N; Fig.12F) was measured using duplicate samples of 2.5 mg of the same flour, using elemental analysis, after combustion at 1800°C and recovery to N2at 600°C, and quantification by mass spectrometry (Dumas method). The complete protein content was calculated using the following formula: protein = 6,63 × the total number N. the Data obtained for levels of total protein using MS were reasonably similar to the intended content of total extractable protein, demonstrating that the protein extraction was effective.

Content Gordeyev (measured as the level of soluble protein) in see is not the F4 was reduced to 17-39% of the parental lines (R1508 and R56) to 7-16% of the level of grade Sloop wild type. This corresponds to approximately 10-fold decrease in the total number Gordeyev, which, as shown above, are toxic to celiacs, data samples of grain, compared with barley wild-type Sloop.

Other types of proteins, in particular water and solarstorm proteins are considered to have a favorable impact on the brewing properties of barley grain. Since the levels of water and solarstorm protein grain F4 were similar to those of wild-type Sloop, suggested that grain F4 contain enough in brewing purposes, the number of these proteins.

The content and composition of fatty acids

Since the main supply of nitrogen during the growth and development of seeds, has been removed due to a reduced level Gordeyev, mutant grain was analyzed to determine, whether compensated development of seed increased accumulation of other components, some of which may be undesirable when applying grain. Fatty acids in the duplicate sample, 50 mg of flour from whole grains F4 were extracted, metilirovanie and analyzed by quantitative gas chromatography (GC)using the method Folich et al. (1957).

Total concentration of fatty acids in grain F4 lines G1, BB5, J1 and J4 varied from about 2.5% to 3% (by weight), and was similar to UB is Nude in barley grain odnopoloi lines and wild type. Found that double zero grain did not contain elevated levels of fatty acids.

Fatty acids in the lipids of grain included mainly linoleic (C18:2), oleic (C18:1) and palmitic acid (C16:0)and other fatty acids with lower levels. The concentrations of individual fatty acids, which have been accumulated in the selected grain F4, compared with odnomodovymi parental lines or barley wild-type, did not observe any significant differences. In particular, the concentration of erucic acid (C22:1n-9), which is toxic to humans in high concentrations in grain F4 was not raised. Mutant grain, thus, had a normal content and fatty acid composition.

The levels of starch

Starch is the main component grains and is usually approximately 55-65% of the dry weight. The levels of starch are particularly important in the barley used for malting. The starch content that is too low may lead to the production of malt sugar, is insufficient to sustain an effective fermentation in the brewing process, and therefore measured the starch content of barley grain.

Starch mutant grains were isolated and analyzed essentially as described in the Megazyme Method (AACC76.13), using samples of 20 mg of flour from whole grains. The total starch content of C is rnu F4 ranged from 57% to 66% (by weight), and was similar to the starch content in odnodolnyh parental lines and barley wild-type, which was within 51-64% (by weight).

Found that grain barley F4 had enough starch content, in order to obtain the malt from grain.

The levels of β-glucans

The content of β-glucans in the mutant grain was evaluated as described in the Megazyme Method (AACC32.23), using samples of 20 mg of flour from whole grains. The levels of β-glucans in the grain lines G1, BB5, J1, J4 ranged from 1.2 to 2.6% (by weight), and were similar to the content of β-glucans in adenolipoma parent grain and grain barley wild-type, which ranged from 2.4 to 3.3% (by weight).

High levels of β-glucans contribute to the formation of turbidity in beer during storage. Found that the content of β-glucans in cereals F4 was not increased compared with grains of wild-type, while it is unlikely that such levels will adversely affect the brewing characteristics of the grain.

Levels of free amino acids

Increased accumulation of free amino acids may be unfavorable when applying grain. For example, the free asparagine in sufficient quantities may form toxic compound acrylamide when heated to high temperatures in the presence of starch.

The content and the composition of free amino acids in the grain was evaluated, using duplicate samples of 20 mg of flour from whole grains, grown in the greenhouse seeds F4. The samples were dissolved in 0.1 N HCl, selected the sample, and dried, then analyzed amino acids using a set of Waters AccQTag chemistry, on the basis of the Australian Centre for Proteomic Analysis (Australian Proteome Analysis Facility, Sydney).

The most predominant amino acids in barley flour was Proline, asparagine, glutamic acid and aspartic acid, in descending order, ranging from approximately 1.5 mg/g of flour to 0.5 mg/g flour. The content of free Proline in the selected grain F4 were in the range of 0.6-1.5 mg/g, and was similar to the content of free Proline in odnodolnyh parental lines and barley wild-type, which was in the range of 0.2-1.2 mg/g Levels of other free amino acids were respectively similar to the levels in the control beans and F4. In particular, the content of free asparagine in the grain F4 was approximately 0.5 mg/g for lines G1, BB5 and J1, and approximately 1.0 mg/g in line J4. In odnodolnyh parent grains, the level of free asparagine was 0.3 or 0.9 mg/g, and in grains barley wild-type and free asparagine was present in the range of 0.3-0.6 mg/year Since the content of free asparagine in the grain F4 was similar to the levels in the corresponding grain wild type, presumably the or, that the formation of acrylamide of free asparagine in the process of malting, or other use of grain will not differ compared with the grain of the wild type.

Free lysine is known to be a critical amino acid in the diet of animals, and so levels of this amino acid are of interest for potential use of grain as feed. The content of free lysine in the grain F4 lines G1, BB5 and J1 was approximately 0.5 mg/g, and 1.0 mg/g in grain line J4. This corresponded to 181% - 1,020%-WMD increase compared with the level in the grain wild-type varieties Sloop. Thus, the line F4 was more valuable food source of free lysine compared to the Sloop.

Example 5. Analysis of grain F4 - test of the toxicity of T-cells

For analysis of toxicity grain F4 celiac disease, hordein were isolated and purified from 10 g samples of flour from whole grains, obtained from grown in the field of seeds of selected lines 9JAE, J1, G1 and 4BH, odnodolnyh parental lines (Riso 56 and Riso 1508), and barley wild-type (Large, Bomi and K8), as described below. Peeled Gordini was added to T cells selected in a population of patients with celiac disease, to test for toxicity in celiac disease patients. The test consisted of measuring the number of T-cells that produced IFN-γ after an overnight incubation with purified protein using an immunoassay level DIN the a-interferon. Thus, the level of gamma-interferon served as a measure of the degree of toxicity of the proteins in the grain. Then the dimension of the toxicity of flour with celiac disease inflicted on a graph as a function of wet weight of flour produced from grain.

Purification of prolamins (Gordeyev)

The whole-wheat flour (10 g) was stirred for 30 minutes at 25°C in 200 ml of buffer containing 20 mm triethanolamine-HCl (TEA), 1% (wt./about.) the sodium ascorbate, 1% (wt./about.) polyethylene glycol (malves 6000; PEG 6000), and 1 μg/ml of protease inhibitors E64 and AEBSF (Sigma); the buffer was brought to pH 8. The suspension was centrifuged at 5000 g for 5 minutes, supernatant was discarded and the sediment was washed at least two times. Proteins in the washed precipitate was dissolved in 80 ml of 50% (vol./about.) propane-2-ol containing 1% (wt./about.) DTT was stirred for 30 minutes at 60°C. the Suspension was cooled at 4°C for 10 minutes and centrifuged at 10,000 g for 10 minutes at 4°C. Proteins, including Gordini, the supernatant was besieged by 2 volumes of propan-2-ol over night at -20°C, then centrifuged at 10,000 g for 10 minutes at 4°C, and the residue was dissolved in 10 ml of buffer containing 8 M freshly prepared deionized solution of urea, 1% DTT, 20 mm TEA, pH 6.

Gordini was purified using FPLC as follows. The solution Gordeyev (1 ml) was administered in 8 ml column for reversed-phase chromatography Source 15 (chemistry, Pharmaci). The column was washed with 4 ml of 5%solvent B, and hordein was suirable 2.5 ml linear gradient from 5% solvent B to 35% solvent B at a speed of 4 ml/min, then linear gradient from 35% solvent B to 83% solvent B, in the amount of 36 ml of Solvent A consisted of 0.1% (vol./about.) triperoxonane acid (TFU) in water, solvent B consisted of 0.1% (vol./about.) TFU 60% (vol./about.) aqueous acetonitrile. Faction, eluruume between 25 and 43 ml, were combined. Control solvent similar by United from runs without input samples. The respective pools of 10 consecutive injections were combined and liofilizirovanny.

Ex vivo analysis of T-cells

Gordini, purified using FPLC (50 mg/ml in 2 M urea), was dissolved in PBS containing 1 mm CaCl2receiving the solutions with a concentration of 25, 62,5, 125, 250, 625, 3750 or 6250 ug hordein/ml, after which Gordini was dezaminirovanie by adding 25 µl of each solution to 100 ál of tTG from the liver of the Guinea pig (Sigma; 25 µg/ml tTG in PBS containing 1 mm CaCl2), followed by incubation for 6 hours at 37°C. Nezamechennye the solutions were obtained similarly, by incubation in the absence of tTG. Control the solvents were added, both at maximum concentrations Gordeyev. Other control samples contained either the control solvent control solvent and known Tox is h, the tetanus toxoid (50 sitebrowser particles/ml, obtained from Commonwealth Serum Laboratories, Melbourne)or tetanus toxoid (50 sitebrowser particles/ml) in pure form. Then all the solutions were frozen at -20°C.

T-cells were obtained as follows. Six patients with HLA-DQ2+celiac disease confirmed by biopsy, which followed a strict gluten-free diet for at least three months, ate 150 grams of boiled barley daily for 3 days. PBMC were isolated by centrifugation in a density gradient Ficoll-Hipac from heparinized venous blood, taken either prior to, or six days after the beginning of dietary provocation, and resuspendable in complete medium HT-RPMI containing 10% thermoinactivation mixed human AB serum. Desametasone or nezamechennye Gordini and control solutions were thawed and added to 25 μl in the wells containing 100 μl of PBMC (3-8×105PBMC in the hole), were cultured at 37°C overnight in 96-well plates (MAIP-S-45; Millipore, Bedford, MA) and compared with control cultures (without additives), to which was added 25 μl of PBS containing only 1 mm CaCl2. Final concentration was Gordeyev 0, 1, 2,5, 5, 10, 25, 150 or 250 µg/ml Maximum final concentration of urea was 10 mm. IFN-γ visualized, IP is by using secondary antibodies according to the manufacturer's instructions (Mabtech, Stockholm, Sweden)as previously described by Anderson et al. (2005), and measured petrobrazi unit (SFU), using an automated ELISPOT tablet reader (AID Autoimmun Diagnostika GmbH; Germany). The results are presented as the mean value petrobrazi units (SFU) ± SE depending on the equivalent weight of the flour, which contains the estimated number hordein. Content Gordeyev each flour sample was calculated in Example 5, taking into account the calculation of the weight of flour.

Data were analyzed using GraphPAD Prism, the expected curves of best fit and showed averages + SE. Values of r2for data exceeded or 0.83, indicating good agreement between the experimental data and the curve of best fit (Fig).

Results

T cells isolated from one patient with celiac disease diet before provocation, were less sensitive to the prolamin added at a concentration of 25 μg/ml, compared with T cells isolated from the same man after dietary provocation barley. Average SFU±SE of 29.5+3.0 and 104+15,9 registered for T-cells, allocated before and after diet provocation. This indicates that specific celiac disease T-cells were induced by dietary provocation.

Used T-cells isolated six days after the specified diet provocation, polozhitelnyh.html, the tetanus toxoid, gave comparable response in the absence and in the presence of tTG (average SFU±SE of 28.1±5.9 and 20,2±7.4 respectively). Adding control solvent does not significantly inhibit the response of the positive control, tetanus toxoid (average SFU±SE 20,5+4,1 and 17.6±6,0, in the absence and in the presence of tTG, respectively), confirming that the impurities in the solvent did not give false-negative and not inhibited positive responses.

T cells isolated from patients with celiac disease 6 days after the provocation, the more actively engaged with all Gordeyeva fractions treated with tTG, compared to T-cells, peered with nezamechennymi gardinali, as expected celiac disease (Hadjivassiliou et al., 2004, Kim et al., 2004) (Fig A; for clarity, shows only two samples Gordeyev, Sloop and G1). It was confirmed that the measured response of T-cells has been associated with toxicity in celiac disease patients.

Since the concentration of hordein was increased, the number of SFU has also increased in a hyperbolic manner expected for a standard enzyme kinetics Michaelis-Menten between the enzyme and its substrate. To describe such curves typically use two parameters: Bmax, the maximum number of SFU, expected at the highest concentrations; and Kd, the protein concentration required to induce half of the maximum is Isla SFU. The more toxic the sample of flour, the lower Kd.

The coefficients Kd and Bmax were calculated for the curve of best fit. The values of Bmax was not significantly different between wild type and mutants, as expected. In contrast, the Kd values for F4 lines were higher in 10 times in comparison with the lines of the wild type (table 4). Thus, approximately 10 times the quantity of flour obtained from the mutant lines, should cause half-maximal response, toxicity, compared with flour wild type (table 4). Thus, it was found that the toxicity of grain F4 when celiac disease was reduced by approximately 10 times compared with the lines of the wild type. This level of reduction is well correlated with a reduced level of hordein discovered during the determination of protein in grain F4.

td align="center"> 0,47±0,09
Table 4
T-cell toxicity barley flour
LineKd (mg flour for induction premaxillae reply)
Wild type: Sloop0,18±0,03
Bomi0,18±0,02
Odnodolnye: Riso56
Riso15083,31±0,47
Line F4: G12,3±0,3
5RB2,6±0,5
4BH1,7±0,2
J11,4±0,2

Toxicity grain F4 was lower than the toxicity Riso 56, as expected. However, the toxicity of grain F4 was similar to the toxicity of the other parental line - Riso 1508. Subsequently, with further genetic study of grain F4 discovered that it was caused by heterozygosity mutations of the gene encoding the protein B-hordein in the selected F4 lines, which led to an increase in the content of Gordeyev higher than expected.

Example 6. The malting of grain F4

To determine the suitability of a grain of barley for malting, conducted research, including tests with small-scale malting (microcolonies).

One of the factors that affects the ability to malting is the size of the seed. Samples of grain F4 analyzed for the distribution of seed size by counting the percentage of seeds 1000 seeds was delayed by a sieve with cells of 2.8, 2.5, or 2,2 mm Grain F4 on average had smaller ropnet compared with the wild type, and similar to the parent grains Riso 1508 and Riso 56, while less than 5% of the seeds was delayed by a sieve with cells of 2.5 mm (table 5). This contrasted with the control lines Galleon and Sloop, in which the particle size 90% seeds more than 2.5 mm, it Was observed that the grain lines K8, which is the wild type line, obtained from the same cross Riso 1508XRiso56, also had a reduced size, so, at least partially, the decrease in seed size was associated with genetic background and, indirectly, reducing Gordeyev. In addition, smaller seeds can be accounted for by modifications in the method of soaking grains.

Table 5
The size of the seed used in microcolony
Line% of the population of seeds, detain sieve with cells 2.5 mm
G11,0
4BH2,6
5RB3,2
J12,0
9RE6,2
Riso15084,0
Riso 566,9
K824,8
Bomi56,6
Carlsberg IIof 57.5
Galleon83,9
Sloopto 91.6

The levels of moisture content of seeds can affect the efficiency of malting. Before microcolonies measured % moisture and % nitrogen by analysis in the near infrared region (NIR). The moisture level of the seeds of all samples of grain F4 was in the range of 11 -11,4% and was similar to the control lines, except grain varieties Galleon (GA1, 8,9%). The nitrogen content in the seed double zero lines ranged from 2.3% to 2.5%, which was higher compared with the control line malting varieties Galleon (1.6 per cent). For malting levels of nitrogen in the seed optimally be 1.5 to 2.0%.

Samples of barley (170 g) grown in a field with grain selected F4 lines 5RB, G1, J1, 9RE, 4BH, odnodolnyh parental lines (Riso 56 and Riso 1508) and barley wild-type K8, varieties Bomi, Carlsberg II, Sloop and Galleon were soaked at 16°C for 6 h, followed by settling for 7 h in air, then soaked for 6 h, and then were germinated at 15°C for 4 days in the system microcolony JWM. Sprouted grains are dried for 21 h Ave the minimum temperature of 50°C and a maximum temperature of 80°C, then, the obtained malt was purified from the roots by grinding and sifting.

The malt was analyzed by relative humidity (%), total nitrogen (% dry weight) using NIR whole grain, as well as output (expressed as the weight of the pure malt in the percentage of the original weight of barley).

In addition, samples of malt was crushed in a hammer mill and sample weight of 50 g was dissolved in water heated to 45°C - 70°C, obtaining a solution with ultimate weight 450 g, which is analyzed to extract content (solubilities % by weight of grain), color, soluble nitrogen (N), the number of Kilbaha (KI: % soluble protein/total protein), β-glucan, viscosity, AAL (limit visible fermentation or terminterest, % drop density in the fermentation beer yeast), all according to the standard technology of European Brewing Convention (European Brewery Convention, http://www.ebc-nl.com/) (table 6).

The protein content in the malt as a whole exceeded the desired norm. This was associated with a total N of malt and soluble N, however, the percentage of soluble protein compared with the total number (KI) was close to the norm. The color and viscosity of the wort F4 were close to normal, and the levels of β-glucans in wort were low. These results are acceptable for malting.

The malting process included three stages: malting, wort preparation and fermentation. Full the power was calculated from three values of the efficiency of each stage: exit the extract content and AAL, respectively. They indicate that in each stage the grain F4 was approximately 10% less efficient than the reference grain varieties Galleon. In total approximately 1.3 times more grain lines F4 will be required for the production of beer, a fortress equivalent commercial standard, compared to the Galleon.

All of these symptoms showed that grain F4 can be prepared malt.

Example 7. The ELISA analysis of untreated samples of malt

Samples with a volume of approximately 40 ml of wort from Example 6 was suspended, liofilizirovanny and was dissolved in 20 ml of 6M urea, 1% (wt./about.) DTT, 20 mm TEA (pH 6) at room temperature. The protein content in each sample was determined using the Bradford method. Serial dilutions containing 20 µg of protein malt in 100 μl of 6M urea, 1% DTT and 20 mm TEA (pH 6) were applied to nitrocellulose membrane (Amersham Hybond C+), which is pre-balanced with PBS buffer, the device for the dot-blot (BioRad), and grooved purified standard (C-hordein, 2 µg). The solution was passed through the membrane under reduced pressure, and then the membrane was washed with PBS buffer containing 0.1% Tween 20 (PBST), the device was disassembled and the membrane was blocked by incubation in 5% (wt./about.) dry fat-free milk in PBS buffer containing 0.1% Tween 20 for 1 hour at room temp is the temperature. Gordini were detected with primary antibody (the antibody rabbit against gliadins of wheat, conjugated with horseradish peroxidase, Sigma), having diluted 1 in 2000 in PBST buffer for 30 minutes at room temperature. The membrane was washed in three changes of PBST buffer and stained, incubare in 10 ml of 1:1 (vol./about.) the mixture Amersham ECL reagents A and B for Western blotting (GE HealthCare), and then were detected signal, surviving with Amersham Hyperfilm for 30 sec. After application of the film was performed quantitative analysis using software Total Lab TL100 (Non-linear dynamics, 2006).

Untreated malt solutions obtained from selected grain F4, had an average content hordein 58+12,7 ppm

This level was well below the limit of 200 ppm, FSANZ established for Nickolaevich food in Australia, and significantly lower than the average content 687±158 ppm for malt of varieties of wild type, Galleon, Sloop, K8, Bomi and Carlsberg II. He also was significantly lower than the content of Gordeyev malt derived from the parental lines Riso 56 and Riso 1508.

Usually gluten free (Gordeyev) in mixtures falling sharply in the processes of malting, cooking the wort and fermentation, resulting in a ready stable beer can contain 1/1000 of the level in the raw malt (Dostalek et al., 2006).

Therefore, hypothesized that the level Gordeyev in the finished beer, ol is prepared from malt F4, will decrease approximately to 0.05 ppm, well below the range 3-40 ppm, present in beer made from grain barley wild-type (Dostalek et al., 2006).

In the literature there are several new recommendations regarding maximum concentration of gluten in the diet of coeliac patients. The most reliable of them are based on a multicenter, placebo control, double blind test, and show that the use of less than 10 mg/day is safe for celiacs, and recommend that consumption remained at the level of less than 50 mg/day (Catassi et al., 2007). Another recent study confirms the above data and (Collin et al., 2004) recommends that consumption of food containing gluten 100 ppm will lead to the consumption of approximately 30 mg/day and cause little damage in coeliac patients. FSANZ sets food standards for New Zealand and Australia. The Codex Alimentarius Commission (from lat. "Food code") was created in 1963 by FAO and who to develop food standards, guidelines and similar documents such as the code of practice under the supervision of the Program standards for food, FAO/who, and is a common regulation for Europe and North America. The Codex Alimentarius Commission currently sets of gluten limit of less than 0.05 g N (gluten) 100 gra the MOU food product. There is a proposal to revise the standard Code, offering a limit of 20 ppm for a food product made from cereal, gluten-free, and 200 ppm for food products made from cereals containing gluten (p32, PROPOSAL P264, REVIEW OF GLUTEN CLAIMS WITH SPECIFIC REFERENCE TO OATS AND MALT, FSANZ web site: http;//www,foodstandards.gov.au/_srcfiles/P264 Gluten Claims FAR-pάfffsearcbr^/o 22gluten%20free%22s).

From the above analysis has established that the consumption of beer produced from barley lines F4, will be significantly below the safety limit for gluten-free food products for celiacs, in the above-mentioned studies, including guidelines established by FSANZ and the Codex Alimentarius.

Example 8. Additional research lines F4

Soluble proteins were purified from the mass of seeds F4 collected for each of the designated lines, as described above. Purified protein samples (20 µg) from grain F4 lines G1, J1, 4BH, 5RB and 9RE was dissolved in 6M urea, 2% (wt./about.) LTOs, 1% (wt./about.) DTT, 0.01 percent (weight/about.) bromophenol blue, 0,0625 M Tris-HCl (pH 6,8) at 25°C, analyzed by electrophoresis in LTO-page, stained 0,006% colloidal Commassie Blue, compared with gardinali isolated from Riso 56 And Riso 1508 and the wild-type (K8). The movement of proteins correlated with molecular weight standards to determine molecular weight (table 7).

Consequently the STI proteins were obtained using mass spectrometry trypsinogen Perevalov, obtained from the protein spots cut from the gels and processed for protein sequencing by fragmentation in tandem mass spectrometry, as described previously (Campbell et al., 2001), finding in the non-redundant NCBI database.

Table 7
Identification of protein-ordinator-PAG
Toe-but noIDAEqual to the peptides
(% protein)
Room NCBIThe total economic
score
MS-MS
The truth-ness
3D-hordein15 (20%)30421167205infallible
4B3-hordeinB9 (27%)82371122infallible
5gamma 3-hordeinc3(11%)170828047reliable
6predecessor gamma hordein-11 (2%)12346414indicates homology
7predecessor gamma hordein-16 (24%)12346494infallible
8gamma 3-hordeinD14 (30%)1708280199infallible

A: All pervari, as expected, also contain peptides from porcine trypsin.

B: Also contains low levels of D-hordein

C: Also contains low levels of B-hordein.

D: Also contains low levels of precursor gamma-hordein-1.

E: Total score search MS-MS indicates the validity of the attribution of identity. From past experiences, to reliably identify the required score above 15, and a score above 50 indicates virtually error-free identification.

Peptides from each sample was bound to the column Agilent Bond SB-C18 5 μm, 150x0,5 mm at a rate of 0.1% (vol./about.) formic acid/5% (vol./about.) acetonitrile, 20 ml/min for one minute, and then ale is listed in the gradients with increasing concentration of acetonitrile in 0.1% (vol./about.) formic acid/20% (vol./about.) acetonitrile per min at 5 μl/min, then with 0.1% (vol./about.) formic acid/50% (vol./about.) acetonitrile for 28 min, followed by 0.1% (vol./about.) formic acid/95% (vol./about.) acetonitrile per min the Column was washed in a gradient from 0.1% (vol./about.) formic acid/95% (vol./about.) acetonitrile, 0.1% (vol./about.) formic acid/100% (vol./about.) acetonitrile for 5 minutes at 20 ál/min and re-balanced to 0.1% (vol./about.) formic acid/5% (vol./about.) acetonitrile for 7 minutes before applying peptides from a sample.

The eluate from the column was introduced into the mass spectrometer ion trap Agilent XCT through micronebulizers ion source of electrocapillary. As the elution of the peptides from the column ion trap were recorded wide-range scans of positive ions (100-2200 m/z) followed by four MS/MS scans of the ions observed in the full spectrum according to the instrument setup 'SmartFrag' and 'Peptide Scan'. As only two spectra fragmentation was obtained for specific values of m/z, they were excluded from selection for the analysis in the next 30 seconds, to avoid collecting redundant data.

Sets of mass spectrometry data were compared with database sequences using the software Agilent''s Spectrum Mill (Rev A.03.02.060). False positive matches avoided when using the software default settings 'autovalidate'. This includes the requirement that coin is placed peptides was much better than the best match on the reverse databases and a variety of assessment contributing more likely profiles of ionization and fragmentation ('assessment of the mobility of the protons'). Oxidized methionine was allowed as a variable modification.

Results protein sequencing showed that the F4 seed from selected lines unexpectedly contained the band B3-hordein, in addition to the expected gamma-1-hordein and D-hordein. The identity of the bands gamma 1 and 3 Gordeyev established in the sequencing of proteins from mutant Riso 56, in which these proteins when the movement did not masked by the overlapping bands (B-hordein. This indicated that the selected line F4 were not completely devoid B3-hordein.

Example 9. Identification of barley grain that does not contain B and C hordein

Separate half of the seeds from one ear grown in field plants F4 lines G1 left overnight to swell in water containing protease inhibitors E64 and AEBSF (1 μg/ml), individually crushed and used in plastic microprobing with stainless steel ball, shook with a frequency of 30/sec for 3×1.5 min in 96-well vibrating mill Vibration Mill (Retsch Gmbh, Rheinische), and then centrifuged at 3000 g for 5 minutes at RT and discard the supernatant. Water-insoluble precipitate flour washed at least two times in a similar way and threw away supernatant. Then the precipitate was extracted with alcohol Gordini, adding 400 ál of 50% (vol./about.) water propan-2-ol containing 1% (wt./about.) DTT, followed by shaking and centrifugation as described above. Supernatant containing extracted Gordini, was transferred to a new tube, then DTT/propan-2-ol supernatant measured the protein using Coomassie reagent (BioRAD).

An aliquot of each Gordeyeva extract corresponding to 20 µg hordein, liofilizovane in vacuum overnight, was dissolved in 15 μl of boiling LTO-buffer, heated for 3 minutes at 90°C, was applied to a pre-filled gel 12-18% Excell gradient gel (Pharmacia) and analyzed by electrophoresis in LTO-SDS page as described in Example 1. Visible band corresponding to approximately 43 kDa, as it was observed, stood out in a separate seeds and was absent in extracts 5 of 16 seeds. The position of the specified band corresponded to the band B3-hordein identified previously.

Data analysis protein was confirmed that the F4 seed from lines G1 was heterozygous and was cut by one or more B-Gordeyeva proteins. This situation was also confirmed for other lines F4.

Genetic research

Genetic analyses were performed to confirm protein data analysis. Separate half of the seeds grown in the field, selected F4 lines were germinated in moist soil, and satellitedual for 2 weeks in the greenhouse at 25°C day and 20°C at night. DNA was isolated from 0.5 cm of leaf blade, using a set of REDExtract-N-Amp Plant PCR Kit (Sigma) according to the instructions. Gene sequence-specific B1-hordein and gamma hordeins, amplified in separate PCR reactions by adding 10 ál RMix, 1 µl of each primer for B1-hordein (5'B1hor and 3'B1hor) or 0.5 μl of each primer for the gamma 3-hordein (5'gamma hor3 and 3'gamma 3-full), 4 µl of plant DNA and MilliQ water up to 20 ál at room temperature, and then subjected to the following temperature program in Eppendorf thermocycler: 95°C for 10 min, then 35 cycles: 95°C for 30 sec, 56°C for 30 sec and 72°C for 1 min. then heating is performed at 72°C for 10 minutes and cooled to 10°C.

Sequences of PCR primers were as follows:

5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2)

3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3)

5'gamma hor3: 5'-CGAGAAGGTACCATTACTCCAG-3' (SEQ ID NO:4)

3'gamma 3-full: 5'-AGTAACAATGAAGGTCCATCG-3' (SEQ ID NO:5).

20 µl of each PCR mixture was applied to a 1 cm, 1% (wt./about.) the agarose gel containing EtBr, electrophoresis was performed at 100V for 1 hour in TBE buffer, and received the image of a fluorescence DNA products using the GelDoc system image system (uvitec) (Fig).

Band of amplified DNA control gene gamma-3-hordein was present in all the tracks, as expected (Fig, lower block, gamma3-Hor). Amplificatory DNA B-hordein was absent in all tracks PCR from Riso 56 as expected, since the gene in Riso 56 has been removed (Fig, upper block, R56). Bands of amplified DNA genes B-hordein stood out in extracts from seeds taken from one ear, F4 lines 9RE and 4BH (Fig, the upper block 9RE, 4BH). It was indicated that one or more genes B-hordein was present in part of the F4 seeds, and that the F3 seed was not homozygous for deletions of the locus B-hordein in Riso 56. It has also been shown for other lines F4. This method was convenient method based on DNA, allowing to identify and select seeds, nesadurai B1-hordein.

The results of a genetic test used for selection of plants that did not contain B-Gordeyeva genes. Twelve individual plants F5 zero according to PCR for B-Gordeyeva genes were selected and cultivated with the purpose of obtaining a population of seeds F5, known as G1*. Individual G1*, F5 half of the seeds taken from one ear, were germinated in moist soil, and grown for 2 weeks in the greenhouse at 25°C day and 20°C at night prior to extraction of DNA/PCR analysis described above. The appropriate half of the seed used for the isolation and analysis Gordeyev, which took an aliquot corresponding to 40 μg hordein, liofilizovane in vacuum overnight, was dissolved in 15 μl of boiling LTO-buffer, heated for 3 minutes at 90°C, was applied to a pre-filled 1 mm 12% gel Longlife (Lnglife Gels) and perform electrophoresis at 150V for 40 minutes, and then dyed as in Example 1.

PCR analysis showed that the DNA isolated from the lines of the positive control, Sloopa and Riso 1508, gave the band the B-hordein, as expected. The stripe size of the Sloop was more than the PCR product from Riso 1508, as genes B1-hordein was somewhat different. The control gene, gamma-3-hordein, amplified in all plants. Strip PCR was not amplified from extracts of six individual samples Gl*, confirming the absence of the gene in these plants. Profile Gordeyev in the respective halves of the seed has confirmed it is in Gl* any stripes B-Gordeyev not observed. Therefore concluded that Gl* did not contain detectable B-Gordeyev and were homozygous null for the locus, codereuse B-hordein.

The remaining 250 F5 G1* seeds were germinated, were analyzed shoots and confirmed as zeros gene B-hordein. The next generation used to get seeds of this line.

The content analysis Gordeyev

Barley varieties Sloop, R56, R1508 and G1* grown on an adjacent parcel in a field of ripe grain was collected and processed to obtain flour. Levels Gordeyev in the flour samples were analyzed as described above. The fraction of proteins that are soluble in water, salt solution, alcohol/DTT and urea solution, obtained as described in Example 4, and then in each fraction was measured by the protein content. The protein content of websites is but in Table 8 and expressed as mg protein/g dry weight of flour. Each content of total protein was determined by summing the protein content in the fractions of the corresponding sample. Gordini contained in the soluble fraction, along with other soluble proteins, such as serpine, protease inhibitors, LTP1 and Z protein.

Table 8
The protein content in the fractions in flour produced from grain G1*
BarleyWater solubleSolaractivityAlcohol/DTT (% Sloop)Soluble ureaOnly
Sloop17,217,623,1 (100%)48,0106
R5616,719,013.2 (58%)58,6108
R150822,215,08,0 (35%)53,599
1* 19,022,24,8 (21%)58,6105

The data showed that the content of soluble protein in the grain G1* and, consequently, the flour was reduced to less than 22% compared with the sort of wild-type Sloop.

The fraction of soluble protein obtained above, was enriched by hordein using FPLC as in Example 5. Proteins in each eluate FPLC liofilizirovanny and determined the output of FPLC-purified protein with 10 grams of flour. It showed that the content of hordein in G1* has been reduced to less than 8 mg/10 g of flour compared to 105 mg/10 g of flour for a Sloop, 38 for R56 and 24 for R1508. This corresponded to a decrease in the content hordein in the grain and flour G1*, at least 92% compared with the Sloop.

Example 10. More large-scale malting and beer production using grain F4

Experiments on malting conducted on a larger scale, in order to obtain a sufficient amount of malt for trial production of beer using grain F4. In these tests used a modified scheme soaking, taking into account, among other factors, a smaller grain size, as follows. Samples of grain at 800 g were placed in containers for malting. Used scheme soaking 17°C for 5 hours, the temperature germination was 15°C in accordance with the s 94 hours. The drying program was the following: 50-78°C for 17 hours, 50-74°C for 17 hours. In the production of malt is not used gibberelline acid, it was not necessary.

The recipe for the jam: 4,65 kg nitroglitserinovogo malt, 10 liters of water, 10 g of calcium chloride, 2 g of calcium sulfate, 64-65°C within 2 hours.

The boiler was added 17 g of hop pellet Target (alpha acid (AA) 10,0%) for 60 minutes, 21 g hop Hallertau pellets (AA - 4,5%) for 10 minutes.

Fermentation was performed in 19-liter capacity periodic cycle, at a temperature of fermentation 12°C, using a 12 g Fermentis dry yeast W34/70, with the primary fermentation for 8 days, followed by cooling to 0°C for 9 days. Then the beer was filtered through a filter with pores of 1 μm under a pressure of carbon dioxide poured into the keg and poured into bottles through the machine for bottling under pressure. The initial density was 1,044, the final density of the fermented product was 1,013 with an approximate bitterness of 30 Units (IBU), and the approximate alcohol content of 4.0%vol. Other parameters measured during the manufacturing process, were as follows: moisture malt: 4,2%, extractive 71,5; color 3,9; WC 1,0; TN 2,63% dry weight; SN 1,11; KI 51; Viscosity of 1.52; AAL 71,8%; beta - glucosidase 130 mg/l; DP 24.

All these parameters showed that the beer which can be made from malt, obtained from grain F4.

More extensive tests malting and brewing also performed with the use of barley grain G1*. Eight hundred grams of grain salariale in automated colourstyle apparatus of Joe White Maltings, according to the specified Protocol. Preferred conditions for malting grain G1* defined as the following: soak 3 hours at 17°C, germination 4 days at 15°C, followed by drying at 50 to 80°C in a drying oven. The optimal time for soaking grains G1* was somewhat different compared to other grains: Sloop: program 8h-9h-5h steeping/aged/steeping at 17°C; R1508: program 7h-8h-3h steeping/aged/steeping step 17°C; R56: program 8h-10h-5h steeping/aged/ steeping at 17°C. the Applied analysis techniques established in accordance with the European Convention Brewing production (EBC) or the Institute of Brewing (IOB). The moisture content of the grain was determined by spectroscopy in the near infrared region (NIR). The full content of nitrogen was determined by the Dumas method. Data for malt are shown in Table 9. One significant difference between grain G1* and other tested varieties was the fact that the indicators diastolicheskoe activity defined for G1* and R1508, were much lower than for grain Sloop or R56. It is bound, therefore, tolys3Muta what s in G1* and R1508.

Malting was repeated and combined for each grade. Approximately 4 kg of malt each line G1*, R56, R1508 and Sloop (wild type) prepared beer and bottled in the following way. In samples of malt was added bitterness, Tettnang hops for 60 minutes at boiling temperature, reaching 21-22 units of bitterness (IBU). Fermentation is conducted with the use of yeast US-05 (Fermentis) at 18-20°C. the Fermented product was poured in kegs without filtering and aziraphale under pressure before bottling. All samples of the beer was cloudy during the filling process, but after 2-4 weeks of storage became less muddy. The beer had a noticeable aroma and flavor of "milk caramel" due to the presence of diacetyl at the keg and bottle, but he also disappeared during storage.

Data prepared beer products are given in Table 10. The alcohol content in beer made from grain G1*, was 4.2%vol. Beer G1* had a somewhat lower, but a satisfactory level of foam after the spill.

Table 10
Data on the parameters of the beer brewed from grain G1*
SLOOPR1508R56 G1*
Batch size (l)15,014,118,618,0
The weight of the Sload (kg)3,603,334,004,55
Extract protein (Temp/Time)57°C / 20 min56°C / 20 min54°C / 20 min55°C / 20 min
Extract amylase (Temp/Time)65°C /1 h63-65°C / 1 h64-65°C / 2 h64-65°C / 2 h
Initial density (SG)1,0511,0521,0511,049
The final density (SG)1,014 registered1,0131,0121,017
The alcohol content
(about%)
4,8%5,1%5,2%4,2%

These experiments while the Ali, what grain G1* can be successfully used in the production of malt and beer.

Levels Gordeyev in beer made from grain G1*measured by immunoassay, as expected, below 1 ppm, and in some cases, below 0.5 ppm That corresponds to the range of content Gordeyev in wheat beer 10-41 ppm, stout 9-15 ppm, lager beer 3-9 ppm

Specialists in this field will understand that there are numerous changes and/or modifications, as shown in some embodiments, implementation, without derogating from the spirits or scope of the invention as generally described. Real options implementation, therefore, should be considered in all respects as illustrative and non-limiting.

This application has priority application U.S. Serial No. 60/964672, the entire contents of which is hereby incorporated into this description by reference.

All publications described and/or cited in this application are fully incorporated in the present description by reference.

Any examination of documents, acts, materials, devices, products, etc. that were included in the present description, are intended solely for the purpose of providing context for the present invention. Should not be construed as an admission that any of these objects are part of the prior art or were overall is known, available knowledge in the field to which the present invention is, as they existed before the priority date of each claim of this application.

1. Method of manufacturing a food product for human consumption comprising mixing barley grain or malt, flour or whole grain flour derived from the specified grain, at least one other component of the product receiving the food product, and grain derived from a plant that is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

2. The method according to claim 1, wherein said food product is yeast or yeast-free breads, mA is a Arona, noodles, cereal Breakfast, a snack product, cake, confectionery product or a food product containing a sauce based on flour.

3. Method of production-based drink malt for human consumption, including the production of malt from barley grain and mixing of malt with at least one other component of the drink to get a drink, and specified barley grain obtained from a plant that is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

4. The method according to claim 3, in which the obtaining of malt from barley grain involves soaking the grain, germination soaked the grain in the given conditions and drying p is rosenovo grain.

5. The method according to claim 3, in which specified based drink malt is to beer or whiskey.

6. The method according to any one of claims 1 to 5, in which electrophoresis on polyacrylamide gel does not detect In - and-Gordeyev.

7. The method according to any one of claims 1 to 5, in which the grain has one or more of the following characteristics:
i) has an average weight of 100 grains of at least about 2.4 g,
ii) has an average weight of 100 grains from about 2.4 g to about 6 g,
iii) contains at least about 50 wt.% starch or
iv) contains from about 50 wt.% to about 70 wt.% the starch.

8. The method according to any one of claims 1 to 5, in which the toxicity in celiac flour derived from the specified grain matches the:
i) less than about 50% of the toxicity of flour produced from grain parent plants of barley,
ii) less than about 25% of the toxicity of flour produced from grain parent plants of barley,
iii) less than about 10% of the toxicity of flour produced from grain parent plants of barley.

9. The method according to any one of claims 1 to 5, in which at least about 50% of the grain germinates within 3 days after soaking.

10. The plant is barley, which gives the grain and is homozygous for at least two loci for the introduced genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allels locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

11. Plant barley of claim 10, in which the substance - and-Gordeyev is undetectable by electrophoresis on polyacrylamide gel.

12. Grain barley plants of claim 10 or 11, used for manufacture of food or drink made of malt for human consumption.

13. Grain at 12, having one or more of the following characteristics:
i) has an average weight of 100 grains of at least about 2.4 g,
ii) has an average weight of 100 grains from about 2.4 g to about 6 g,
iii) contains at least about 50 wt.% starch or
iv) contains from about 50 wt.% to about 70 wt.% the starch.

14. The method of obtaining barley grain, including
the cultivation of barley plants of claim 10 or 11, and
the harvest of grain.

15. The method of producing flour, including
getting grain in item 12 or 13, and
the processing of grain for the production of flour.

16. Method received what I wholegrain flour including
obtaining grain under item 12 or 13, and
grain processing to obtain whole wheat flour.

17. The method of producing starch, including
obtaining grain under item 12 or 13, and
grain processing for obtaining starch.

18. The method of producing malt, including
getting grain in item 12 or 13, and
the processing of grain for malt production.

19. Beer for human consumption, obtained by the method according to any of p-9, in which the seed obtained from the plant, which is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

20. Beer in claim 19, in which the substance - and-Gordeyev is undetectable by electrophoresis on polyacrylamide the gel.

21. Flour for human consumption, obtained by the method according to any one of claims 1,6 to 9, in which the seed obtained from the plant, which is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor - nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

22. Flour on item 21, in which the content of b - and C-Gordeyev is undetectable by electrophoresis on polyacrylamide gel.

23. Malt for human consumption, obtained by the method according to any one of claims 1,6 to 9, in which the seed obtained from the plant, which is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
b) tantou allele in locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

24. Malt according to item 23, in which the substance - and-Gordeyev is undetectable by electrophoresis on polyacrylamide gel.

25. A food product for human consumption, obtained by the method according to any one of claims 1,6 to 9, in which the seed obtained from the plant, which is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is detectable by the absence of amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.

26. Food product A.25, in which the substance - and-Gordeyev is undetectable by electrophoresis on polyacrylamide gel.

27. A method for the identification of barley grain, suitable for the production of food and/or beverage on the basis of malt, suitable for use by a person with celiac disease, including:
a) obtaining one or more of the following materials:
i) a sample of the plant, able to give a specified grain,
ii) grain,
iii) the malt produced from corn, and/or
iv) extract the specified grain,
b) analysis of the material from step a) in the presence of at least one hordein and/or at least one gene encoding hordein,
moreover, choose barley grain, which is homozygous for at least two loci for genetic variations, which are:
a) allele, which removed most or all of the genes encoding the In-hordein in the locus Hor2, and
(b) mutant allele in the locus Lys3 barley,
so that the grain does not contain nor-nor-Gordeyev, and
these genetic variations are present in the barley lines Riso 56 and Riso 1508, respectively,
this lack of In-Gordeyev is findable on no who Yu amplified DNA using primers:
5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3' (SEQ ID NO:2),
3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:3),
and no-Gordeyev is detectable in the absence of a 70 kDa band in the study of alcohol extract of the grain through the LTO-PAG.



 

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2 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method includes sample preparation, DNA isolation, PCR statement. At that in carrying out PCR, the oligonucleotide primers are used, which are complementary to sequences of the chromosomal genes fliC and hom2, having the following sequences: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3', followed by electrophoretic analysis of the amplification products, when the formation of the amplification product is 153 bps in size it is indicative of belonging of the strain under study to the species B.anthracis, formation of the amplification product with size of 550 bps is indicative of belonging of the strain under study to the other species of the genus Bacillus.

EFFECT: invention enables to differentiate effectively the bacillus anthracis from other species of bacilli.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to chronobiology. A method for measuring a circadian cycle on the basis of time-series data by expression levels obtained by measuring the expression levels of two clock-genes in biological samples taken from a subject three times a day. The clock-genes have different phases of the circadian measuring cycle of the expression level.

EFFECT: method provides the high accuracy measurement of the biological rhythm and minimises the quantities of samplings from the subject.

4 cl, 4 dwg, 6 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of quantitative determination of fixed rabies virus strain "Moskva 3253". Method includes decontamination and separation of RNA from virus-containing material, carrying out reaction of reverse transcription and polymerase chain reaction with hybridization-fluorescence account of results in "real time" mode with application of specific primers RV5-5'-GTTGGGCACTGAAACTGCTA-3', RV6-5'-GAATCTCCGGGTTCAAGAGT-3' and probe RV7-5'-ROX-AATCCTCCTTGAACTCCATGCGACAGA-BHQ2. Quantitative assessment of virus is determined on the basis of registration of signal of analysed sample fluorescence and its comparison with signal of fluorescence of PCR-standards, which contain different quantities of DNA-targets. Claimed method makes it possible to determine quantitative content of virus in rabies antigen of organ-tissue and culture origin.

EFFECT: application of invention contributes to standardisation of stage of rabies antigen preparation in production of heterological anti-rabies immunoglobulin.

2 tbl, 3 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering, more specifically to analysing the disorders related to ovarian carcinoma, and may be used in medicine. The method involves determining the methylation status of CpG-dinucleotide in the genome in each sequence of a group of sequences SEQ ID NO:1-10 using a set of probes specific for the above sequences and able to be hybridised with the sequence along the full length. The above sequences are used as a part of a chip for detection, diagnosis and monitoring of the proliferative disorders related to ovarian cell proliferation, as well as for detection of a predisposition to the proliferative disorders, or treatment of the proliferative ovarian disorders.

EFFECT: invention enables identifying the proliferative disorders in ovarian cells and detecting a genetic predisposition to the above disorders.

10 cl, 3 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and pharmacology. What is presented is a method for the prediction of the pharmaceutical effectiveness of adalimumab for treating rheumatoid arthritis, wherein the method involves measuring a level of at least one of ADAMTS4 iRNA and ADAMTS5 iRNA in a sample taken from a subject, and determining if adalimumab is effective for rheumatoid arthritis in a subject on the basis of a level of at least one of ADAMTS4 iRNA and ADAMTS5 iRNA considered as a value.

EFFECT: method may be used in medicine in treating rheumatoid arthritis.

3 cl, 2 dwg, 8 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with set, which includes oligodeoxyribonucleotide primers and fluorescently labelled probe for identification of DNA of adenovirus of serotypes 3, 4, 7, 14, 21 by method of hybridization-fluorescence polymerase chain reaction in real time mode. Claimed primers and probe have the following structure: external primer 5'→3' 5'-AATGTARTTGGGTCTGTTRGGCAT-3' internal primers 5'→3' 5'-CCCWTCGATGMTGCCCC-3' 5'-TCMACGGGYACRAAGCGCA-3' probe 5'→3' ROX-CCTGTCCGGCGATGTGCAT-BHQ2.

EFFECT: invention possesses higher homology to currently circulating adenoviruses and makes it possible to identify DNA of adenoviruses of serotypes 3, 4, 7, 14, 21.

3 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and deals with recombinant strain of E. coli TG1(pRVMoscow3253G-L) for obtaining PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253".Recombinant strain is created on the basis of strain of E. coli TG1 by transformation with plasmid pRVMoscow3253G-L. Plasmid is obtained by ligation of fragment G-L of the region of genome of fixed rabies virus of strain "Moskva 3253", which has sequence SEQ ID NO1, into plasmid pGem-T. Also claimed is set of PCR-standards for quantitative determination of cDNA of rabies virus of strain "Moskva 3253" in rabies antigen. Set contains solutions of plasmid pRVMoscow3253G-L DNA in concentrations 108, 107, 105, 103 GE/ml. Concentration is determined by method of polymerase chain reaction, with hybridisation-fluorescence account of results.

EFFECT: invention contributes to standardisation of stage of preparation of rabies antigen in production of heterological anti-rabies immunoglobulin.

2 cl, 3 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is separated chimeric polynucleotide for amplification of production of heterologous protein of interest, which contains polynucleotide sequence of promoter SigA or SigH, functionally connected with polynucleotide, coding protein YmaH, with chimeric polynucleotide connecting sequence, which by, at least, 90% is identical to SEQ ID NO: 1, 2, 3 or 13. Also described are: expression vector, containing claimed nucleotide structure, and host cell Bacillus for production of heterologous protein of interest, which contains said vector. Claimed is method of obtaining modified Bacillus cell, including transformation of host cell of Bacillus-producent of heterologous protein of interest with said vector; and growing said modified cell in optimal conditions. Described is method of obtaining protein of interest in modified Bacillus cell, where method includes cultivation of said host cell; and growing said modified Bacillus cell in optimal conditions. Also described is method of amplification of expression of heterologous protein from Bacillus of interest includes obtaining said modified Bacillus cell; growing modified Bacillus cell in optimal conditions; and expression of said protein of interest in modified Bacillus cell, where expression of said heterologous protein of interest in modified Bacillus cell is amplified in comparison with expression of said protein of interest in said parent Bacillus host-cell.

EFFECT: invention makes it possible to increase output of target protein due to superexpression of protein YmaH.

30 cl, 4 dwg, 3 ex

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