CN108471717B - Wheat for improving and preventing gluten intolerance and wheat-dependent exercise-induced allergic reactions - Google Patents

Abstract

The present invention relates to office, a novel wheat variety resulting from a cross between Keumkang wheat and Olgeuru wheat, in which the low molecular weight glutenin subunit (LMW-GS) allele (the main cause of gluten intolerance) and the omega-5 prolamin gene (the main cause of wheat-dependent-movement induced anaphylaxis (WDEIA)) at the Glu-B3 locus have been deleted; a method for developing a novel wheat variety using Ofree; wheat flour produced by office; a method for producing processed food from wheat flour; and a processed food produced by the above production method. It is expected that the office provided by the present invention can be widely used for producing processed foods capable of preventing celiac disease and WDEIA attacks caused by gluten intolerance.

Description

Wheat for improving and preventing gluten intolerance and wheat-dependent exercise-induced allergic reactions

Technical Field

The present invention relates to a novel wheat variety for improving or preventing gluten intolerance and wheat dependent-exercise induced hypersensitivity (WDEIA) and uses thereof.

Background

Celiac disease is a disease caused by gluten intolerance, which is a digestive disorder characterized by difficulty in digesting gluten components contained in, for example, wheat, barley, rye, and oats, and affecting the intestines between digestive organs. Unlike most people who have no problems in digesting and absorbing gluten, people who have gluten digestion problems experience immune reactions induced in the gastrointestinal tract that cause inflammation of mucosal cells of the digestive organs and destroy villi. In this case, nutrients are not easily absorbed, and they cause various symptoms such as developmental delay, loss of appetite, chronic diarrhea, abdominal distension, dermatitis herpetiformis, and anemia due to the appearance of iron deficiency, thereby causing malabsorption of nutrients and ultimately causing serious diseases. That is, when ingested by a person who is naturally deficient in gluten-degrading enzymes, gluten is absorbed into the intestine without being decomposed, and in this case, the function of the intestine is impaired, and the villi in the mucosa is weakened and damaged, resulting in malabsorption, which may cause allergic reactions or endocrine disorders.

As described above, gluten is considered to be a protein composed of prolamin (monomer molecule) and glutenin (complex polymer) as a cause of celiac disease. Glutenins consist of high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) linked to each other by disulfide bonds. Generally, gluten is known to exhibit the properties required to produce wheat products by containing prolamines, which are found to be responsible for viscosity in the dough, and glutenins, which impart elasticity and toughness to the dough.

Prolamines are the major component of wheat gluten and represent 40% to 50% of the total stored protein. Prolamines exist primarily as monomers and are classified into four categories based on their mobility in low pH acid polyacrylamide gel electrophoresis (acid-PAGE or a-PAGE) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): omega-5-prolamin, omega-1, 2-prolamin, alpha-/beta-prolamin, and gamma-prolamin, and having a molecular weight of about 30kDa to 75 kDa. Gamma-and omega-prolamins are present in the short arm of chromosome 1 and encoded by genes located at the loci Gli-A1, Gli-B1 and Gli-D1, and alpha-/beta-prolamins are present in the short arm of chromosome 6 and encoded by genes located at the loci Gli-A2, Gli-B2 and Gli-D2. Among all types of prolamines, α -/β -and γ -prolamines are classified as major proteins, accounting for 28% to 33% and 23% to 31%. Omega-prolamins are present in very small amounts, with omega-1, 2 prolamins accounting for 4% to 7% and omega-5 prolamins accounting for 3% to 6%.

Omega-5 prolamins are known as the major antigens of wheat-dependent-exercise-induced anaphylaxis (WDEIA). Wheat-dependent-exercise-induced anaphylaxis is a severe food allergy and in severe cases can lead to death.

With the increasing number of wheat allergic patients and the expansion of the korean gluten-free market, attempts have been made to find hypoallergenic wheat lines without omega-5, omega-1, 2 and alpha-/beta-prolamines, or gamma-prolamines (the main cause of wheat allergy or celiac disease), and our research group has identified and studied some wheat lines without gluten proteins.

Although research on the loci of high and low molecular weight glutenins and their impact on wheat quality has been much advanced to date, the study of prolamins has been conducted in a limited range compared to glutenins, due to the complexity and difficulty of the study, in addition to some allergic reactions and basic properties.

Accordingly, the present inventors continued to study wheat varieties for reducing the risk of gluten allergy and wheat allergy, and developed wheat varieties from which all omega-5 prolamin (the main antigen of WDEIA) genes and all LMW-GS alleles at the Glu-B3 locus (the main cause of gluten intolerance) were deleted, thereby completing the present invention.

[ non-patent document ]

Matsuo H,Morita E,Tatham AS,Morimoto K,Horikawa T,Osuna H,Ikezawa Z,Kaneko S,Kohno K,Dekio S.2004.Identification of the IgE-binding epitope in ω-5 gliadin,a major allergen in wheat-dependent exercise-induced anaphylaxis.J Biol Chem 279:12135-12140.

Disclosure of Invention

The aim of the present invention is to provide a new wheat variety in which the genes responsible for gluten intolerance and/or wheat dependent-exercise induced allergic reactions (WDEIA) have been deleted.

It is another object of the present invention to provide a method for developing an improved wheat variety comprising the step of crossing the above wheat variety used as a paternal or maternal plant with another wheat variety.

It is a further object of the present invention to provide wheat flour produced from the aforementioned wheat varieties.

It is still another object of the present invention to provide a method for producing a processed food, which comprises the step of processing the above wheat flour.

It is a further object of the present invention to provide a processed food produced by the above method.

The inventors of the present invention conducted various studies to develop a method for eliminating the cause of celiac disease and WDEIA by destroying glutenin and prolamin more effectively. A novel wheat variety was developed by crossing Keumkang wheat and Olgeuru wheat in which all low molecular weight glutenin subunit (LMW-GS) alleles at the Glu-B3 locus (major cause of gluten intolerance) and all omega-5 prolamin genes (cause of WDEIA) have been deleted. The novel wheat variety is genetically distinct from Keumkang and Olgeurux in that the LMW-GS allele and/or the omega-5 prolamin gene at the Glu-B3 locus has been deleted, but it has been found that the novel wheat variety has inherent characteristics, agricultural traits, yield characteristics, disease resistance and baking-related quality characteristics similar to or intermediate to Keumkang or Olgeuru.

The present inventors named the novel wheat variety "office" and deposited it at Korea Agricultural Culture Collection (KACC) (korean total northwest completion county, illinam, gmbh 166) of korea national institute of agricultural science at 9/21 of 2015 with a deposit number of KACC88001 BP. To date, no wheat variety has been developed or reported in which all LMW-GS alleles at the Glu-B3 locus and/or all omega-5 prolamin genes have been deleted, except for the office provided by the present invention. The office developed by the inventors of the present invention was the first of such wheat varieties.

To achieve the above object, one aspect of the present invention provides a novel wheat variety wherein the LMW-GS allele and/or the omega-5 prolamin gene at the Glu-B3 locus has been deleted, which wheat variety has been deposited under accession number KACC88001 BP.

The term "low molecular weight glutenin subunit (LMW-GS)" as used herein refers to a component having a relatively low molecular weight of 36kDa to 44kDa in all glutenin constituents. The genes encoding LMW-GSs are the Glu-A3, Glu-B3 and Glu-D3 genes located at the Glu-3 locus on chromosome 1 of wheat, and the genes can be subdivided into several alleles, the combination of which can produce various characteristics of wheat flour.

The term "glutenin" as used herein refers to a protein consisting of 18 amino acids, in the form of globular proteins having a globular shape due to bridge formation caused by interactions between side chains, and is a type of glutenin, which is a relatively simple protein in various grains including wheat. The glutenin is composed of high molecular weight glutenin subunits (HMW-GS) having sizes of 96kDa to 136kDa and one of the above LMW-GSs linked to each other by disulfide bonds, and the combination of three HMW-GS (Glu-A1, Glu-B1, and Glu-D1) and three LMW-GS (Glu-A3, Glu-B3, and Glu-D3) can produce various forms of glutenin.

The term "prolamin" as used herein refers to the major component of wheat gluten, accounting for 40% to 50% of the total storage protein. Prolamines exist primarily as monomers and are classified into four categories based on mobility in low pH acid PAGE (a-PAGE) or SDS-PAGE: omega-5-prolamin, omega-1, 2-prolamin, alpha-/beta-prolamin, and gamma-prolamin, and having a molecular weight of about 30kDa to 75 kDa. Gamma-and omega-prolamins are present in the short arm of chromosome 1 and encoded by genes located at the loci Gli-A1, Gli-B1 and Gli-D1, and alpha-/beta-prolamins are present in the short arm of chromosome 6 and encoded by genes located at the loci Gli-A2, Gli-B2 and Gli-D2. Among all prolamin types, α -/β -and γ -prolamin are classified as major proteins, accounting for 28% to 33% and 23% to 31%, respectively. Omega-prolamins are present in very small amounts, 4% to 7% omega-1, 2 prolamins and 3% to 6% omega-5 prolamins, respectively.

Omega-5 prolamin is known as the main antigen of WDEIA, which is a severe food allergy.

The term "wheat-dependent-exercise-induced anaphylaxis (WDEIA)" refers to an anaphylaxis that occurs when a person ingests a food containing wheat is performing physical exercise and is known to occur primarily in adults. Omega-5 prolamin is considered to be the major epitope for binding to serum immunoglobulin e (ige) of patients with WDEIA.

The novel wheat variety Ofree provided by the invention does not contain omega-5 prolamin, and the omega-5 prolamin is a main antigen of WDEIA, so that WDEIA can be improved and/or prevented.

The term "prevention" as used herein refers to any inhibition or delay of gluten intolerance and WDEIA activity due to the ingestion of the above-mentioned wheat variety designated as office. The office provided in the present invention does not contain the gene at the Glu-B3 locus (responsible for gluten intolerance) and omega-5 prolamin (WDEIA inducing antigen), which means that office does not have any causative substance of allergic reaction. Therefore, office can prevent WDEIA and celiac disease caused by gluten intolerance.

The term "improving" as used herein encompasses activities aimed at alleviating the severity of gluten intolerance and WDEIA. In particular, it may encompass alleviating the severity of celiac disease and WDEIA that have already occurred.

The inventors of the present invention developed a novel wheat variety in which the LMW-GS allele (the major cause of gluten intolerance) and/or the omega-5 prolamin gene (the cause of WDEIA) located at the Glu-B3 locus had been deleted by Keumkang and Olgeuru crossing, said wheat variety being named "office" and deposited at KACC on 21 days 9 and 2015 with a accession number KACC88001 BP. In the present invention, expressions such as "office wheat", "office" and "new wheat variety" can be interpreted as representing substantially the same wheat variety.

The novel wheat varieties provided by the present invention exhibit characteristics similar to or intermediate between those of Keumkang or Olgeuru as parent wheat plants. For example, it has been found that the characteristics of Ofree are the same as or similar to those of Olgeuru in terms of leaf color, stem length, disease resistance, and the same as or similar to those of Keumkang in terms of growth habit and spike length. However, it has been found that the baking-related quality properties of office, such as milling rate, ash content, flour color, protein content, gluten content, precipitation value, dough properties, loaf volume and crumb hardness, are between those of Keumkang and Olgeuru. Wherein the yield characteristics of Ofree are lower than those of Keumkang or Olgeuru and the thousand kernel weight is higher than that of Keumkang or Olgeuru.

In one embodiment of the present invention, a novel wheat variety named as Ofree and having high cold tolerance and ideal growth habit was developed by Keumkang and Olgeuru crossing (FIG. 1), and the genetic characteristics of Ofree were analyzed. The results show that in office, the LMW-GS allele (the major cause of gluten intolerance) and the ω -5 prolamin gene (the cause of WDEIA) located at the Glu-B3 locus have been deleted and therefore not expressed (table 1, fig. 2A to 2C and fig. 3 to 7).

Furthermore, the inherent characteristics of Ofree, such as seedling rate, cold tolerance, growth level, leaf color, growth habit, branches and leaves, uniformity, etc., are determined. The results show that the intrinsic properties of Ofree are substantially similar to those of Keumkang and Olgeuru, except that the leaf color is similar to that of Olgeuru, and the growth habit is similar to that of Keumkang (Table 2).

Next, agricultural traits of Ofree, such as heading, maturity, stem length, and ear length, were determined, and the results showed that heading and maturity of Ofree were substantially similar to those of Keumkang and Olgeuru, stem length of Ofree was the same as that of Olgeuru, and ear length of Ofree was similar to that of Keumkang (table 3).

Next, yield characteristics of office, such as ears per area, grains per ear, thousand grain weight, grain weight per liter, and yield per area, were determined, and the results indicated that office had more desirable product quality than Keumkang or Olgeuru in some respects: has a thousand kernel weight greater than Keumkang or Olgeuru, and a median kernel weight per liter between Keumkang and Olgeuru, although the yield per area is significantly reduced compared to Keumkang or Olgeuru (table 4).

In addition, resistance of office to scab, viral infection, wheat flour mildew and sharp eyespot (sheath eyespot) was also evaluated, and the results showed that office has the same level of disease resistance as Olgeuru (table 5).

Finally, the roast related quality characteristics of Ofree are determined. The flouring rate, ash content and flour color of the wheat flour made from Ofree (i.e., the Ofree flour) were roughly between those of Keumkang flour and Olgeuru flour, but were more similar to those of Keumkang flour (Table 6) than those of Olgeuru flour (Table 6). Furthermore, the protein content, gluten content, precipitation value and dough properties of the office flour were roughly between those of Keumkang flour and Olgeuru flour, but were more similar to those of Keumkang flour (table 7) than to those of Olgeuru flour (table 7). Bread produced using the office flour (i.e., office bread) was rated as having greater volume and greater softness compared to the Olgeuru bread due to having loaf volume and core firmness between Keumkang bread and Olgeuru bread (table 8).

Another aspect of the present invention provides a method for developing an improved wheat variety capable of improving or preventing gluten intolerance and/or WDEIA, and comprising the step of crossing with another wheat variety using office as a paternal or maternal plant.

The Ofree provided by the present invention is a wheat variety in which the LMW-GS allele (the major cause of gluten intolerance) and/or the omega-5 prolamin gene (the cause of WDEIA) at the Glu-B3 locus has been deleted. Thus, when using office as a paternal or maternal plant in a cross with another wheat variety, it is possible to develop a wheat variety in which genes other than the LMW-GS allele and the ω -5 prolamin gene at the Glu-B3 locus are also deleted, and this wheat variety may be more effective in preventing gluten intolerance and WDEIA than office. In this case, the other wheat variety to be crossed with the office is not limited to a specific wheat variety, and is, for example, Jokyung, jopooom, baekjong, or Goso.

Therefore, the Ofree provided by the present invention can be used to develop a new wheat variety capable of more effectively preventing gluten intolerance and WDEIA.

Other aspects of the present invention provide wheat flour produced from the above wheat variety, a method for producing a processed food comprising a step of processing the wheat flour, and a processed food produced by the method.

The novel wheat variety Ofree provided by the present invention can prevent gluten intolerance and/or WDEIA because the LMW-GS allele and the omega-5 prolamin gene located at the Glu-B3 locus have been deleted therein. Therefore, wheat flour produced by office may be used as a raw material for processed foods capable of preventing gluten intolerance and WDEIA risk, and processed foods produced using the wheat flour may be used as functional foods for preventing gluten intolerance and WDEIA risk.

In particular, the method for producing a processed food product capable of preventing gluten intolerance and/or WDEIA risk according to the present invention comprises the steps of: (a) grinding the new wheat variety to produce wheat flour; (b) preparing a dough comprising said wheat flour; and (c) processing said dough.

In the method for producing processed foods, the wheat flour described in said step (a) may be produced alone from a novel wheat variety or from a mixture of a novel wheat variety and another wheat variety. Since the wheat flour produced from the novel wheat variety according to the present invention cannot exhibit material properties sufficient to produce any type of processed food when used alone, it can be used in combination with wheat flour produced from another wheat variety to provide properties suitable for producing processed food.

The amount of wheat flour produced from the new wheat variety to be mixed with wheat flour produced from another wheat variety is not particularly limited as long as the amount is sufficient to prevent gluten intolerance, celiac disease, and the onset of WDEIA. For example, wheat flour produced from the novel wheat variety is included at 50% v/v to 99% v/v relative to the total wheat flour mixture. In other alternative embodiments, the wheat flour produced from the novel wheat variety is included at 60% v/v to 80% v/v or 65% v/v to 70% v/v relative to the total wheat flour mixture. In addition, other wheat varieties to be included in the wheat flour mixture are not limited to a specific wheat variety, and are, for example, Keumkang, Olgeuru, Jokyung, jopooom, baekjong, or Goso.

In the method for producing a processed food, the dough described in the step (b) may be prepared according to a method known in the art, and in this case, factors such as water content, the type and amount of additives used, aging properties, and aging time may vary depending on the characteristics of various processed foods to be produced using wheat flour.

In the method for producing a processed food, the processing of the dough described in said step (c) may be carried out by methods known in the art. For example, the dough is processed by: a method of heat-treating the dough for a short period of time to produce a processed food product (such as a cookie or biscuit); a method of preparing a sheet by stretching the dough and then drying the sheet to produce a processed food such as noodles; or by heat treating the dough first at a mild temperature and then at an elevated temperature to produce a processed food product such as bread.

The processed food produced by the above method may be bread, noodles, cookies, etc., but is not limited to the listed processed foods as long as it can prevent gluten intolerance and risk of WDEIA by reflecting the characteristics of the novel wheat variety of the present invention in which the LMW-GS allele and/or the ω -5 prolamin gene located at the Glu-B3 locus have been deleted.

When bread is ingested by a person who is naturally deficient in gluten-degrading enzymes, the person often suffers from gluten intolerance caused by the absorption of undecomposed gluten into the intestine without decomposition, and may cause celiac disease, which impairs intestinal function, destroys and weakens the villi in the mucous membrane, thereby causing malabsorption, eventually leading to allergic reactions, endocrine disorders, and the like. However, since the LMW-GS allele (the main cause of gluten intolerance) located at the Glu-B3 locus has been deleted in the novel wheat variety provided by the present invention, processed foods produced using wheat flour of the wheat variety as a main component do not induce gluten intolerance even when ingested by humans naturally lacking gluten-degrading enzymes, and thus the onset of celiac disease can be prevented.

The present invention provides an office that is a wheat variety in which the LMW-GS allele (the major cause of gluten intolerance) and/or the omega-5 prolamin gene (the cause of WDEIA) at the Glu-B3 locus have been deleted; it can be widely used for the production of processed foods which can prevent the onset of celiac disease and/or WDEIA caused by gluten intolerance.

Drawings

FIG. 1 is a family chart for explaining the procedure of developing a novel wheat variety Ofree.

FIG. 2A is an electrophoretic image showing the results of Polymerase Chain Reaction (PCR) amplification of the gene located at the Glu-B3 locus in Keumkang, Olgeuru and Ofree.

FIG. 2B is an electrophoretic image showing HMW-GS-related proteins and LMW-GS-related proteins in Keumkang, Olgeuru and Ofree.

FIG. 2C is an electrophoretic image showing LMW-GS related proteins in various amounts of the Ofree extract.

Fig. 3 is an electrophoretic image for showing the detection result of omega-5 prolamin by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the prolamin fraction in Keumkang, Olgeuru, and office, wherein the first, second, and third lanes represent Keumkang, Olgeuru, and office, respectively.

Fig. 4 is an electrophoretic image for showing the detection result of omega-5 prolamin protein analyzed by acid polyacrylamide gel electrophoresis (a-PAGE) of glutenin fraction in Keumkang, Olgeuru and office, wherein the first, second and third lanes represent office, Keumkang and Olgeuru, respectively.

FIG. 5 shows the results (top to bottom) of reverse phase high performance liquid chromatography (RP-HPLC) analysis of the prolamin portion in Keumkang, Olgeuru and Ofree.

FIG. 6 shows immunoblot results of omega-5 prolamin-specific antibodies in Keumkang, Olgeuru and Ofree.

FIG. 7 shows the results of two-dimensional electrophoresis of all seed storage protein fractions in Keumkang, Olgeuru, Ofree (DH20) and button 86 followed by immunoblotting using the sera of wheat-dependent exercise-induced hypersensitivity (WDEIA) patients; and a protein analysis result superimposed on the immunoblot result by tandem mass spectrometry (MS/MS).

FIG. 8 is a set of images showing Ofree (DH20), Keumkang and Olgeuru at their heading date (A), and grain (B) and slice of bread (C) from Ofree (DH20), Keumkang and Olgeuru.

Detailed Description

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: development of new wheat varieties

A novel wheat variety with high cold tolerance and ideal growth habit was developed by artificial crossing Keumkang and Olgeuru to obtain progeny wheat plant F1. F1 haploids were bred at CIMMYT in mexico and tested for production of F1 in the alpine city for four years. The new wheat variety was named "office" (FIG. 1).

FIG. 1 is a family chart for explaining the procedure of developing a new wheat variety Ofree.

Example 2: genotyping of Ofree

Example 2-1: confirmation of deletion of Gene at Glu-B3 Gene

The genetic profile of Ofree developed by the procedure described in example 1 was determined based on the expression levels of the gene located at the Glu-1 locus as the high molecular weight glutenin subunit (HMW-GSs) component in gluten and the gene located at the Glu-3 locus as the low molecular weight glutenin subunit (LMW-GSs) component in gluten.

Briefly, genomic DNA was extracted from young leaves of Keumkang, Olgeuru and office using a genomic DNA extraction kit for plants (SolGent co., ltd., korea). Subsequently, Polymerase Chain Reaction (PCR) was performed using the genomic DNA extracted earlier as a template and primers capable of detecting genes located at the Glu-A1, Glu-B1, Glu-D1, Glu-A3, Glu-B3 or Glu-D3 loci (listed below) to determine which genes located at the Glu-1 and Glu-3 loci are contained in the genetic composition of Ofree (Table 1).

Glu-A1ab F:5'-AAGACAAGGGGAGCAAGGT-3'(SEQ ID NO:1)

Glu-A1ab R:5'-GTGCTCCGCGCTAACATG-3'(SEQ ID NO:2)

Glu-A1c F:5'-ACGTTCCCCTACAGGTACTA-3'(SEQ ID NO:3)

Glu-A1c R:5'-TATCACTGGCTAGCCGACAA-3'(SEQ ID NO:4)

Glu-B1bcf F:5'-TTCTCTGCATCAGTCAGGA-3'(SEQ ID NO:5)

Glu-B1bcf R:5'-AGAGAAGCTGTGTAATGCC-3'(SEQ ID NO:6)

Glu-D1d F:5'-GCCTAGCAACCTTCACAATC-3'(SEQ ID NO:7)

Glu-D1d R:5'-GAAACCTGCTGCGGACAAG-3'(SEQ ID NO:8)

Glu-D1adf F:5'-TTTGGGGAATACCTGCACTACTAAAAAGGT-3'(SEQ ID NO:9)

Glu-D1adf F:5'-AAAAGGTATTACCCAAGTGTAACTTGTCCG-3'(SEQ ID NO:10)

Glu-D1adf R:5'-AATTGTCCTGGCTGCAGCTGCGA-3'(SEQ ID NO:11)

Glu-A3a F:5'-AAACAGAATTATTAAAGCCGG-3'(SEQ ID NO:12)

Glu-A3a R:5'-GGTTGTTGTTGTTGCAGCA-3'(SEQ ID NO:13)

Glu-A3b F:5'-TTCAGATGCAGCCAAACAA-3'(SEQ ID NO:14)

Glu-A3b R:5'-GCTGTGCTTGGATGATACTCTA-3'(SEQ ID NO:15)

Glu-A3ac F:5'-AACAGAATTATTAAAGCCGG-3'(SEQ ID NO:16)

Glu-A3ac R:5'-CTGTGCTTGGATGATACTCTA-3'(SEQ ID NO:17)

Glu-A3d F:5'-TTCAGATGCAGCCAAACAA-3'(SEQ ID NO:18)

Glu-A3d R:5'-TGGGGTTGGGAGACACATA-3'(SEQ ID NO:19)

Glu-A3e F:5'-AAACAGAATTATTAAAGCCGG-3'(SEQ ID NO:20)

Glu-A3e R:5'-GGCACAGACGAGGAAGGTT-3'(SEQ ID NO:21)

Glu-B3d F:5'-CACCATGAAGACCTTCCTCA-3'(SEQ ID NO:22)

Glu-B3d R:5'-GTTGTTGCAGTAGAACTGGA-3'(SEQ ID NO:23)

Glu-B3fg F:5'-TATAGCTAGTGCAACCTACCAT-3'(SEQ ID NO:24)

Glu-B3fg R:5'-CAACTACTCTGCCACAACG-3'(SEQ ID NO:25)

Glu-B3g F:5'-CAAGAAATACTAGTTAACACTAGTC-3'(SEQ ID NO:26)

Glu-B3g R:5'-GTTGGGGTTGGGAAACA-3'(SEQ ID NO:27)

Glu-B3h F:5'-CCACCACAACAAACATTAA-3'(SEQ ID NO:28)

Glu-B3h R:5'-GTGGTGGTTCTATACAACGA-3'(SEQ ID NO:29)

Glu-B3i F:5'-TATAGCTAGTGCAACCTACCAT-3'(SEQ ID NO:30)

Glu-B3i R:5'-TGGTTGTTGCGGTATAATTT-3'(SEQ ID NO:31)

Glu-D3ab F:5'-TTGGGCCTAATCGCTCGC-3'(SEQ ID NO:32)

Glu-D3ab R:5'-TAGTCTCCATCTGCGCAATT-3'(SEQ ID NO:33)

Glu-D3c F:5'-CAGCTAAACCCATGCAAGC-3'(SEQ ID NO:34)

Glu-D3c R:5'-CAATGGAAGTCATCACCTCAA-3'(SEQ ID NO:35)

[ Table 1]

Genetic constitution of Glu-1 and Glu-3

As shown in Table 1, most of the genes expressed in one of Keumkang and Olgeuru were also expressed in Ofree, in addition to Glu-B3 (FIG. 2A).

FIG. 2A is an electrophoretic image showing the results of Polymerase Chain Reaction (PCR) amplification of genes located at the Glu-B3 locus of Keumkang, Olgeuru and Ofree. As shown in FIG. 2A, the gene at the Glu-B3 locus was not detected from Ofree.

Therefore, in order to detect the gene at the Glu-B3 locus at the protein level, Keumkang, Olgeuru and Ofree extracts obtained from young leaves were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 2B).

FIG. 2B is an electrophoretic image showing HMW-GS-related proteins and LMW-GS-related proteins in Keumkang, Olgeuru and Ofree. As shown in FIG. 2B, no gene was detected at the Glu-B3 locus of Ofree, even at the protein level.

In addition, in order to find out whether the results shown in FIG. 2B were obtained due to the insufficient amount of the Ofree extract used for SDS-PAGE, SDS-PAGE was performed with different amounts of the Ofree extract (FIG. 2C).

FIG. 2C is an electrophoretic image for displaying LMW-GS related proteins in various amounts of the Ofree extract. As shown in fig. 2C, as the amount of the office extract increased, the amount of each protein detected increased accordingly, except for the gene at the Glu-B3 locus, which was not detected even when a large amount of the office extract was examined.

Summarizing the results shown in fig. 2A to 2C, since the office provided by the present invention is a wheat variety in which the LMW-GS allele located at the Glu-B3 locus (the main cause of gluten intolerance) has been deleted, the office can be used to produce baked products that can be ingested by gluten intolerant people without causing side effects.

Example 2-2 confirmation of deletion of omega-5 prolamin Gene in Ofree

By consulting the prior art literature (Dziuba M,

d, Szerszunowicz I, Waga J.2014.proteomic analysis of heat α/A-and β -gliadins. Czech J Food Sci 32:437 442) for prolamin extraction. Mixing 1ml of0.15M aqueous NaCl was mixed with 100mg of each of micro-pulverized Keumkang, Olgeuru, and Ofree. Each mixture was reacted at 25 ℃ for 2 hours, and centrifuged at 15,000rpm at 20 ℃ for 5 minutes, and then the supernatant was removed. The precipitate was mixed with 1ml of 70% aqueous ethanol solution, the mixture was reacted at a constant temperature for 12 hours, and centrifuged at 15,000rpm at 20 ℃ for 5 minutes, and then the supernatant of the mixture was stored at 20 ℃.

Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) analysis and results

Prolamin stored at-20 ℃ was mixed with sample buffer (50mM Tris-HCl, pH6.8, 8% beta-mercaptoethanol, 2% SDS, 20% glycerol) at a 1:2 ratio to make a mixture, and 5. mu.l of the mixture was loaded onto 12.5% SDS-PAGE gels, electrophoresed at 70V for 13 hours, and electrophoresed at 110V for 4 hours. After electrophoresis, the gel was stained with Coomassie Brilliant blue R-250 staining solution (Bio-Rad Laboratories, Inc.) for 4 hours, followed by decolorizing with decolorizing solution (10% glacial acetic acid, 10% methanol, 80% distilled water) for 5 hours, and the obtained protein band pattern was analyzed.

From the results shown in FIG. 3, unlike the cases of Keumkang and Olgeuru, no omega-5 prolamin was detected from Ofree.

Acid Polyacrylamide gel electrophoresis (A-PAGE) analysis and results

1ml of 70% ethanol was mixed with 100mg of each of micro-pulverized Keumkang, Olgeuru and Ofree. Each mixture was stirred at 25 ℃ for 1 hour, centrifuged at 14,000rpm for 10 minutes at constant temperature, and 500. mu.l of the supernatant was mixed with 250. mu.l of 5: 1 and mixing the solutions.

10 μ l of prolamin was added to a 12% gel solution (12% acrylamide, 0.6% bisacrylamide, 0.1% ascorbic acid, 0.004% ferrous sulfate heptahydrate, 0.25% aluminum lactate, pH adjusted to 3.1 with lactic acid), the mixture was loaded and electrophoresed at 400V for 5 hours. Then, the gel was stained with Coomassie Brilliant blue R-250 staining solution for 3 hours, followed by decolorization with 10% acetic acid and 10% methanol for 4 hours, and the obtained protein band pattern was analyzed.

From the results shown in FIG. 4, unlike the cases of Keumkang and Olgeuru, no omega-5 prolamin was detected from Ofree.

Reverse phase high performance liquid chromatography (RP-HPLC) analysis and results

1ml of 70% ethanol was mixed with 100mg of each of micronized Keumkang, Olgeuru and Ofree. Each mixture was reacted at 25 ℃ for 1 day, centrifuged at 15,000rpm at 20 ℃ for 5 minutes, and the supernatant was dissolved with 300ml of 0.1% trifluoroacetic acid (TFA) in 20% Acetonitrile (ACN) and filtered with Whatman polyvinylidene fluoride (PVDF) syringe filter (0.45 μm, Sigma-Aldrich Co.LLC). An Agilent Zorbax 300SB-C18 column (pore size) was used

Particle size 5 μm, inner diameter 4.6mm, length 150mm) and a Waters Alliance e2695HPLC system. Solvents used for HPLC include solvent a (0.1% TFA in water), solvent B (0.1% TFA in Cerium Ammonium Nitrate (CAN)), solvent c (meoh), and solvent d (meoh). For analysis, 10. mu.l of each sample was injected and separation was accomplished with a linear gradient (solvent B) of 25% to 50%, a flow rate of 1 ml/min for 70 minutes, maintaining a column temperature of 65 ℃ and a sample temperature of 15 ℃. The analytical sample at 210nm was observed.

FIG. 5 shows the results of RP-HPLC analysis of prolamin moieties in Keumkang, Olgeuru and Ofree. As shown in fig. 5, unlike the cases of Keumkang and Olgeuru, no omega-5 prolamin was detected from the office.

Results of immunoblotting Using omega-5 prolamin-specific antibodies

Total protein extraction, dimensional electrophoresis and immunoblotting were performed by reference to the literature (Susan B. Altenbach et al.2015. Association of the analytical potential of analytical wastewater (Triticum aestivum) with reduced levels of ω 5-glucans, the major sensing allergen in wastewater-dependent assay-induced and analytical chemistry. journal of Agricultural and Food chemistry. DOI: 10.1021). 2% SDS sample buffer (2% SD)S, 10% Glycerol, 0.04M Tris-HCl, pH8.5, 50mM DTT) 100mg of each of micro pulverized Keumkang, Olgeuru, and Ofree was subjected to total protein extraction. Isoelectric focusing (IEF) was performed manually using ampholytes (pI 3-10) and capillaries, and on NuPAGE^TMSecond electrophoresis was performed on 4-12% Bis-Tris protein gel. Proteins in the two-dimensional gel electrophoresis (2-DE) spots were identified by tandem mass spectrometry (MS/MS). MS/MS analysis was performed by the method described in the literature (Dupont, FM, Vensel WH, Tanaka CK, Hurkman II WJ, Altenbach SB.2011. characterization of the present floor flow protocol using quantitative two-dimensional electrophoresis, present protocols and tandem mass spectrometry. protocol science.9(10), available in http:// www.proteomesci.com/content/9/1/10). For each spot, three classes of proteases (trypsin, chymotrypsin, thermolysin) were used to obtain maximum amino acid sequence coverage. The NCBI protein accession number obtained as a result of protein identification within each spot is provided in fig. 6. Omega-5 prolamins (BAE20328), i-type LMW-GS (AAS10189) were identified from Keumkang, and omega-5 prolamins (BAE20328, AII26682), i-type LMW-GSs (AGM38903, ACY08811) and s-type LMW-GS (ACY08813) were identified from Olgeuru. After 2-DE, the total protein was transferred to nitrocellulose membrane. Sera from the same patient were immunoblotted.

As shown in fig. 6, office did not produce omega-5 prolamin.

Immunoblot analysis results using WDEIA patient sera

Total protein extraction, dimensional electrophoresis and immunoblotting were performed by reference to the literature (Susan B. Altenbach et al.2015. Association of the analytical potential of analytical wastewater (Triticum aestivum) with reduced levels of ω 5-glucans, the major sensing allergen in wastewater-dependent assay-induced and analytical chemistry. journal of Agricultural and Food chemistry. DOI: 10.1021). Total protein extraction was performed on 100mg of each of micro-pulverized Keumkang, Olgeuru and Ofree using 2% SDS sample buffer (2% SDS, 10% glycerol, 0.04M Tris-HCl, pH8.5, 50mM DTT). Using amphoteric electricityThe electrolytes (pI 3-10) and capillaries were isoelectric focused (IEF) manually and on NuPAGE^TMSecond electrophoresis was performed on 4-12% Bis-Tris protein gel. Proteins were identified in the 2-DE spots by MS/MS. MS/MS analysis was performed by the method described in the literature (Dupont FM, Vensel WH, Tanaka CK, Hurkman IIWJ, Altenbach SB.2011. degradation of the complex of the floor flow protocol using quantitative two-dimensional electrophoresis, and the same and same mass spectrometry. protocol science.9(10), available in http:// www.proteomesci.com/content/9/1/10). For each spot, three classes of proteases (trypsin, chymotrypsin, thermolysin) were used to obtain maximum amino acid sequence coverage. The NCBI protein accession number obtained as a result of protein identification within each spot is provided in fig. 7. After 2-DE, the total protein was transferred to nitrocellulose membrane. Antibodies were prepared using the omega-5 prolamin-specific sequence RLLSPRGKELG and used for immunoblotting.

All seed storage protein fractions from Keumkang, Olgeuru, Ofree (DH20) and button 86 were subjected to dielectrophoresis and immunoblotting using WDEIA patient sera. MS/MS protein analysis results were provided in an overlay manner by combining the western blot results of WDEIA patients (fig. 7). Omega-5 prolamins (BAE20328), i-type LMW-GS (AAS10189), s-type LMW-GSs (BAD12055, AEI00677) and m-type LMW-GS (AEI00671) were identified from Keumkang, and omega-5 prolamins (BAE20328, AII26682), i-type LMW-GSs (AGM38903, ACY08811), s-type LMW-GSs (ACY08813, ACA63868, AEI00677) and m-type LMW-GSs (AGK83389, ACP27643) were identified from Olgeuuru. On the other hand, as clearly seen in fig. 7, no antigen-antibody response was observed in WDEIA patients in relation to omega-5 prolamin. Thus, it was confirmed that office (DH20) can reduce allergic immune responses leading to WDEIA.

Accordingly, the present inventors deposited Ofree at 21.9.2015 in the Korean Agricultural Culture Collection (KACC) (166, Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabbuk-do, Republic of Korea, Ito, Korea, national institute of agricultural sciences, Japan) under the number KACC 01 88001 BP.

Example 3: characteristics of Ofree

Various characteristics of Office developed through the steps described in example 1, such as inherent characteristics, Agricultural traits, yield characteristics, disease resistance, quality characteristics and baking-related characteristics, were determined according to crop characteristics guidelines provided by the Agricultural Science and Technology Research Analysis Standards (Analysis Standards for Research in Agricultural Science and Technology, Rural Development Administration, 2012) and the National farm and Forestry department Seed Management Office (National Seed Management of Agriculture of Agricultural and Forestry) for testing new varieties, and the results obtained were compared with the characteristics of the parent wheat plants Keumkang and Olgeuru.

Examples 3-1. intrinsic Properties

The intrinsic characteristics of Ofree in terms of seedling rate, cold tolerance, growth level, leaf color, growth habit, branches and leaves, and uniformity were determined (Table 2).

[ Table 2]

Inherent characteristics of Ofree

As shown in table 2, office has inherent characteristics that are generally similar to Keumkang and Olgeuru, except that the leaf color is similar to that of Olgeuru and the growth habit is similar to that of Keumkang.

Example 3-2 agricultural traits

Agricultural traits of office in heading, maturity, stem length, and ear length were determined (table 3).

[ Table 3]

Agricultural traits of Ofree

As shown in Table 3, the heading and maturity periods of Ofree were substantially similar to those of Keumkang and Olgeuru, the stem length of Ofree was the same as that of Olgeuru, and the ear length of Ofree was similar to that of Keumkang.

Examples 3-3 yield Properties

Yield characteristics of office in terms of ears per area, grains per ear, thousand grain weight, grain weight per liter, and yield per area were determined (table 4).

[ Table 4]

Yield characteristics of Ofree

As shown in table 4, Ofree has a similar number of grains per ear compared to Keumkang or Olgeuru, but a significantly reduced number of ears per area, and thus a significantly reduced yield per area compared to Keumkang or Olgeuru. However, since there is a greater thousand kernel weight and a kernel weight per liter between Keumkang and Olgeuru compared to Keumkang or Olgeuru, office has a more desirable yield quality in some respects compared to Keumkang or Olgeuru.

Examples 3-4 disease resistance

To evaluate disease resistance of office, disease resistance of office to head blight, viral infection, wheat flour mildew and tip eye spot was examined (table 5).

[ Table 5]

Disease resistance of Ofree

As shown in table 5, office has the same level of disease resistance as Olgeuru.

Examples 3-5 baking-related Properties

The baking related characteristics of Ofree in terms of breading characteristics, dough characteristics and baking characteristics were determined.

Example 3-5-1 powdering characteristics

The harvested office was ground to flour in a Buhler mill and the flour milling rate, ash content and flour color of the wheat flour were determined after 1 month of flour aging (table 6). In this case, the ash content of the flour was measured according to AACC method 08-01(AACC International 2000) and the flour color was measured in terms of whiteness, redness and yellowness with Minolta JS-555(Konica Minolta, Inc., Japan), and the flour color results were expressed in terms of CIELAB L (whiteness), a (redness) and b (yellowness) axes.

[ Table 6]

Pulverizing characteristic of Ofree

As shown in table 6, the flouring rate, ash content, and flour color of the flour made from officee (i.e., officee flour) are roughly between those of Keumkang flour and Olgeuru flour, but are more similar to those of Keumkang flour than to those of Olgeuru flour. The L value of Ofree is lower than that of Keumkang or Olgeuru.

Examples 3-5-2 dough Properties

The total protein content, gluten content and precipitation value of the office flour produced according to example 3-5-1 were measured, and the characteristics of the dough made from office flour were measured in terms of the amount of water added, dough time and dough stability (table 7). In this case, the total protein content and the gluten content were measured according to AACC method 46-30(AACC International 2000); determining the sedimentation value by: placing 3g of flour (based on 14% moisture content) into a 100ml cylinder, adding 50ml of a 0.0004% bromophenol blue solution to the cylinder, shaking the cylinder twice for 15 seconds at two minute intervals, adding 50ml of a 2% SDS solution containing 12.5% lactic acid to the cylinder, and shaking the cylinder three times for 15 seconds at two minute intervals, allowing the cylinder to stand for 20 minutes, and then measuring the precipitate level; and dough properties were determined according to AACC method 54-40A (AACC International 2000) using a 10g mixer (National mfg.co., usa).

[ Table 7]

Dough Properties of Ofree

As shown in table 7, the protein content, gluten content, precipitation value, and dough properties of the office flour were approximately between those of Keumkang flour and Olgeuru flour, but were more similar to those of Keumkang flour than to the Olgeuru flour. The Ofree flour had the characteristics of strong wheat flour by requiring the addition of the same amount of water as Keumkang flour and, at the same time, had similar characteristics to those of medium wheat flour in terms of dough mixing time.

Examples 3-5-3 baking characteristics

The bread was baked using the office flour produced according to example 3-5-1, and the loaf volume and the crumb hardness of the obtained bread, which represent the bread characteristics, were determined (table 8). In this case, the bread volume was measured using a bread volume meter (National mfg.co., usa) immediately after baking in an oven, and the crumb hardness was determined by the following steps: the bread was cooled at room temperature for 2 hours, and bread having a thickness of 2.0cm was cut from the central portion of the bread loaf and a texture analyzer having a plastic plunger of 2.5cm diameter at a speed of 1.0mm/sec at 25% strain relative to the thickness of the crumb.

[ Table 8]

Torrefaction Properties of Ofree

As shown in table 8, the breads produced using the office flour (i.e., the office bread) were rated as having greater volume and greater softness than the Olgeuru bread because the loaf volume and the crumb firmness were between those of the Keumkang bread and the Olgeuru bread.

FIG. 8 is a set of images showing the grains (B) and slices of bread (C) of Ofree (DH20), Keumkang and Olgeuru at their heading date (A) with Ofree (DH20), Keumkang and Olgeuru.

Production examples: production of cookies Using flour obtained from Ofree

The office cookie was produced by the following method.

150g of butter was gently beaten in a mixer to loosen the butter, 100g of sugar was added to the mixer, and the contents were homogenized with a whisk to effect creaming. Then, 120g of egg was added to the mixer and stirred to form a mixture, and the mixed wheat flour (100g of office flour and 300g of typical soft wheat flour), 30g of almond flour, 24g of cocoa were gently stirred with the mixture to prepare a base dough.

50g of milk was gradually added to the base dough to adjust the consistency of the dough, and 20g of whipped cream was added to the dough, which was then uniformly rolled to a thickness of 1cm and stored in a freezer. In a freezer, egg white and sugar were applied to the surface of the frozen dough, which was then cut into pieces having dimensions of 1cm (width) by 10cm (length). These pieces were placed on a baking pan and then baked in a preheated oven for 15 minutes at a top heating temperature of 190 ℃ and a bottom heating temperature of 165 ℃ to produce the office cookie. The baked cookies were cooled at room temperature for about 4 hours.

It will be understood by those skilled in the art that various changes or modifications may be made to the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

[ deposit No. ]

The name of the depository institution: korean Agricultural Culture Collection (KACC)

The preservation number is as follows: KACC88001BP

Preservation day: 9/21/2015

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Claims (10)

1. A method for developing an improved wheat variety, said method comprising the step of using one wheat variety as a paternal plant or the maternal plant in a cross with another wheat variety, wherein in said one wheat variety, it is located thereinGlu-B3The low molecular weight glutenin subunit (LMW-GS) allele of the locus has been deleted, the omega-5 prolamin gene has also been deleted, and said one wheat variety has been deposited under accession number KACC88001 BP.

2. The method according to claim 1, wherein the other wheat variety is Jokyung wheat, Jopoom wheat, baekjong wheat or Goso wheat.

3. Wheat flour obtained by grinding a wheat variety wherein among the wheat variety, wheat flour is locatedGlu-B3The low molecular weight glutenin subunit (LMW-GS) allele of the locus has been deleted, the omega-5 prolamin gene has also been deleted, and said one wheat variety has been deposited under accession number KACC88001 BP.

4. A food composition capable of improving or preventing gluten intolerance and wheat-dependent-exercise induced hypersensitivity (WDEIA), said food composition comprising the wheat flour according to claim 3.

5.A processed food product capable of improving or preventing gluten intolerance and WDEIA, said processed food product comprising the food composition according to claim 4.

6. The processed food according to claim 5, wherein the processed food is noodles, cookies or bread.

7. A method of producing a processed food product capable of improving or preventing gluten intolerance and WDEIA, the method comprising:

(a) obtaining wheat flour by grinding a wheat variety wherein in said one wheat variety, among othersGlu-B3The low molecular weight glutenin subunit (LMW-GS) allele of the locus has been deleted, the omega-5 prolamin gene has also been deleted, said one wheat variety being deposited under accession number KACC88001 BP;

(b) preparing a dough comprising said wheat flour; and

(c) processing said dough.

8. The method of claim 7, further comprising:

mixing the wheat flour obtained from (a) with wheat flours obtained from other wheat varieties to form a mixed flour.

9. The method according to claim 8, wherein the wheat flour obtained from (a) is contained in the mixed flour in an amount of 50 to 99% v/v.

10. The method of claim 8, wherein the other wheat variety is selected from the group consisting of Keumkang wheat, Olgeuru wheat, Jokyung wheat, Jopoom wheat, Baekjoong wheat, Goso wheat, and combinations thereof.

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