Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
  • A21D13/00—Finished or partly finished bakery products
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
  • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
  • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
  • A21D2/36—Vegetable material
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
  • A23L7/10—Cereal-derived products
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention relates to a novel wheat that can be used as a raw material for wheat flour compositions used in food manufacturing, more specifically, wheat that lacks the enzyme activity of two granule-bound starch synthase I (GBSSI) and two starch synthase type IIa (SSIIa) and has a d-type allele of the high molecular weight glutenin gene Glu-D1.
• GBSSI granule-bound starch synthase I
• SSIIa starch synthase type IIa
• Wheat flour is widely used as an ingredient in various processed foods that are eaten daily as meals or snacks. Many of these processed foods are produced through a heating process, and changes in quality occur immediately after the heating process is completed. For example, bread has a very soft and moist texture immediately after baking. However, after a few hours or days, it becomes hard and dry. This phenomenon is generally known as food aging, but in this invention it is referred to as deterioration. The progression of deterioration is directly linked to the taste of the food, so reducing this progression is an important issue for the food industry.
• wheat flour (GA-SX wheat flour) was developed by milling wheat that does not lack granule bound starch synthase I (GBSSI)-A1, which is responsible for amylose synthesis, but lacks the enzyme activity of GBSSI-B1 and D1, and lacks the enzyme activity of any two of starch synthase IIa (SSIIa)-A1, B1, and D1, which are involved in the side chain elongation of amylopectin (Patent Document 1: Patent No. 6226165).
• This wheat starch has a low amylose content and a structure in which the amylopectin side chains are shortened, resulting in a slow rate of retrogradation of the starch itself.
• the texture is soft, deterioration after production is slow, and the deliciousness can be maintained for a long time; these effects are particularly excellent in GA-SA wheat flour (wheat flour obtained by milling wheat that is not deficient in GBSSI-A1, is deficient in the enzyme activity of GBSSI-B1 and D1, is not deficient in SSIIa-A1, and is deficient in SSIIa-B1 and D1).
• GA-SA wheat flour wheat flour obtained by milling wheat that is not deficient in GBSSI-A1, is deficient in the enzyme activity of GBSSI-B1 and D1, is not deficient in SSIIa-A1, and is deficient in SSIIa-B1 and D1).
• glutenin and gliadin are the main proteins in wheat, and when they coexist and come into contact with water, they form viscoelastic gluten.
• Glutenin forms huge polymers by forming disulfide bonds between its molecules, and is involved in the elasticity (strength) of wheat flour dough.
• Gliadin exists in the form of monomers weakly bonded by hydrogen bonds, etc., and is involved in the extensibility of wheat flour dough.
• Glutenin is broadly divided into high molecular weight glutenin and low molecular weight glutenin.
• High molecular weight glutenin is encoded by the Glu-A1, B1, and D1 loci located on the long arms of wheat chromosomes 1A, 1B, and 1D
• low molecular weight glutenin is encoded by the Glu-A3, B3, and D3 loci located on the short arms of chromosomes 1A, 1B, and 1D.
• Numerous alleles (alleles) are known at each of these six loci, and it is known that the molecular weight and expression level of the encoded subunits differ depending on the type of allele, which affects the secondary processability of wheat flour.
• Glu-D1 alleles include a (having high molecular weight glutenin subunit pairs "2+12"), c (4+12), d (5+10), and f (2.2+12) types (Non-Patent Document 3), of which Glu-D1d is known to be more effective in strengthening dough during bread making and increasing the volume of bread than other Glu-D1 alleles (Non-Patent Documents 4, 5).
• Glu-B3 alleles such as Glu-B3b, B3h, and B3i have a relatively large effect of strengthening dough during bread making compared to other Glu-B3 alleles (c, j, ae types) (Non-Patent Documents 4, 6).
• the present invention aims to provide foods that have improved chewiness and melt-in-the-mouth properties compared to foods made from GA-SX wheat flour.
• the inventors discovered that the defects of GA-SX wheat flour can be eliminated by converting the high molecular weight glutenin gene Glu-D1 of GA-SX wheat (wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1, and SSIIa-D1) to a d-type allele, which led to the completion of the present invention.
• the inventors discovered that the defects of GA-SX wheat flour can be further eliminated by converting the low molecular weight glutenin gene Glu-B3 to a b-type allele or h-type allele, which led to the completion of the present invention.
• the present invention includes the following aspects.
• Wheat flour (GA-SX/GD1d wheat flour) obtained by milling a harvested wheat which does not lack the enzyme activity of GBSSI-A1, lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1 and SSIIa-D1, and in which the high molecular weight glutenin gene Glu-D1 is a d-type allele.
• the wheat flour according to [1] further comprising a b-type allele in the low molecular weight glutenin gene Glu-B3 (GA-SX/GD1d/GB3b wheat flour).
• a flour composition comprising the wheat flour described in [1] to [3].
• the wheat used in the present invention is wheat that does not lack the enzyme activity of GBSSI-A1, lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1 and SSIIa-D1, and has a d-type allele in the high molecular weight glutenin gene Glu-D1 (GA-SX/GD1d wheat).
• the wheat further has a b-type allele in the low molecular weight glutenin gene Glu-B3 (GA-SX/GD1d/GB3b wheat), or the wheat further has an h-type allele in the low molecular weight glutenin gene Glu-B3 (GA-SX/GD1d/GB3h wheat).
• Common wheat is an allohexaploid with three homoeologous genomes, A, B, and D, numbered 1 to 7 (1A to 7A, 1B to 7B, and 1D to 7D).
• "GBSSI” is a granule-bound starch synthase involved in the synthesis of amylose contained in wheat endosperm starch, and is also called Waxy (Wx).
• Wx Waxy
• GBSSI(Wx)-A1, GBSSI(Wx)-B1, and GBSSI(Wx)-D1 are encoded by genes located on chromosomes 7A, 4A, and 7D, respectively. Mutants lacking enzyme function are known for each, and the amylose content differs depending on the combination of mutants.
• SSIIa is an enzyme involved in the elongation of the side chain (branch chain) of amylopectin in wheat endosperm starch. Like GBSSI, SSIIa-A1, B1, and D1 function in normal wheat, and defective mutants of each are also known. SSIIa-A1, SSIIa-B1, and SSIIa-D1 are encoded by genes located on chromosomes 7A, 7B, and 7D, respectively. When one of the three enzymes is defective, the amylopectin side chain is slightly shortened.
• the degree of shortening increases, and when all three enzymes are defective, the side chain is shortened to the greatest extent, and as a secondary effect, the wheat has an amylose content of over 30%, resulting in a high amylose content.
• “Lack of enzyme activity” means that a protein with normal enzyme activity is not functioning in the wheat plant, and preferably that a protein with normal enzyme activity is not expressed. Specific examples include mutations in gene sequence (such as substitutions, deletions, insertions, inversions, translocations, and other mutations of one or more bases, including deletion of the entire gene region), lack of mRNA transcription, lack of protein translation, and inhibition of enzyme activity in the wheat plant. Any of these may be acceptable as long as the enzyme activity is reduced or lost to less than 10%, preferably less than 5%, and more preferably less than 1% of the wild-type enzyme activity.
• GA-SX wheat includes the following wheat varieties, which are derived from the combination of two types of SSIIa that lack enzyme activity: GA-SA wheat: wheat not deficient in the enzyme activity of GBSSI-A1, deficient in the enzyme activities of GBSSI-B1 and GBSSI-D1, not deficient in the enzyme activity of SSIIa-A1, and deficient in the enzyme activities of SSIIa-B1 and SSIIa-D1; GA-SB wheat: wheat not deficient in the enzyme activity of GBSSI-A1, deficient in the enzyme activities of GBSSI-B1 and GBSSI-D1, not deficient in the enzyme activity of SSIIa-B1, and deficient in the enzyme activities of SSIIa-A1 and SSIIa-D1; GA-SC wheat: Wheat that is not deficient in GBSSI-A1 enzyme activity, is deficient in GBSSI-B1 and GBSSI-D1 enzyme activities, is not deficient in SS
• sequences (genomic DNA, protein) of GBSSI-A1, B1, D1, SSIIa-A1, B1, D1 are known as examples of so-called wild-type genes that do not lack enzyme activity, and are registered in GenBank under the following accession numbers. Each of these sequences is shown in the sequence table as shown in Table 1 below.
• sequences are examples of wild-type sequences, and naturally occurring wheat (including improved wheat varieties) may have enzyme proteins with the same activity but with partially different nucleotide or amino acid sequences.
• the terms "GBSSI-A1 gene” and "GBSSI-A1 protein” include not only those having a completely identical nucleotide or amino acid sequence to the sequence listing, but also those having a sequence containing a natural mutation that does not impair the enzyme activity. The same applies to other enzymes.
• Such naturally occurring mutant sequences usually have an identity of 90% or more, for example 95% or more, or 98% or more, to each nucleotide or amino acid sequence shown in the sequence listing.
• these GBSSI-A1, B1, D1, SSIIa-A1, B1, and D1 genotypes are referred to as GBSSI-A1a, B1a, D1a, SSIIa-A1a, B1a, and D1 alleles.
• GBSSI-A1a, B1a, D1a, SSIIa-A1a, B1a, and D1 alleles As an example of a mutant lacking enzyme activity compared to these wild types, a mutant of GBSSI-A1 is known in which the expression of the encoded protein GBSSI-A1 is lost due to a genetic mutation in which 23 base pairs are deleted at the junction site of the first exon and the following intron in the gene sequence of wild-type GBSSI-A1 (Wx-A1) and a different sequence of 4 bases is inserted (Non-Patent Document 7).
• this mutant is referred to as the GBSSI-A1b allele.
• a mutant of GBSSI-B1 is known in which the entire gene region from the initiation codon to the termination codon of wild-type GBSSI-B1 (Wx-B1) is deleted (Non-Patent Document 8), and in this specification, this mutant is referred to as the GBSSI-B1b allele.
• a mutant of GBSSI-D1 is known in which expression of GBSSI-D1 is lost due to a genetic mutation in which 588 bases around the stop codon are deleted and a different sequence of 12 bases is inserted in the gene sequence of wild-type GBSSI-D1 (Wx-D1) (Non-Patent Document 7).
• this mutant is referred to as the GBSSI-D1b allele.
• a mutant of SSIIa-A1 is known in which a 289-base region including the initiation codon is deleted from the wild-type SSIIa-A1 gene sequence and a different sequence of 8 bases is inserted, resulting in loss of expression of SSIIa-A1 (Non-Patent Document 9).
• this mutant is referred to as the SSIIa-A1b allele.
• a mutant of SSIIa-B1 is known in which a 175-base insertion occurs in exon 8 of the wild-type SSIIa-B1 gene sequence, and this insertion creates a stop codon, resulting in a loss of normal expression of the SSIIa-B enzyme protein (Non-Patent Document 9). In this specification, this mutant is referred to as the SSIIa-B1b allele.
• a mutant of SSIIa-D1 is known in which the expression of normal SSIIa-D enzyme protein is lost due to a genetic mutation in which 63 bases are deleted around the junction region of exon 5 and the following intron in the wild-type SSIIa-D1 gene sequence (Non-Patent Document 9). In the present invention, this mutant is referred to as the SSIIa-D1b allele.
• Glutenin is the main protein in wheat, and when it comes into contact with water in the presence of gliadin, it forms viscoelastic gluten.
• Glutenin forms disulfide bonds between its molecules to become giant polymers, and is involved in the elasticity (strength) of wheat flour dough.
• Gliadin exists in the form of monomers weakly bonded by hydrogen bonds, etc., and is involved in the extensibility of wheat flour dough.
• Glutenin is broadly divided into high molecular weight glutenin and low molecular weight glutenin.
• “High molecular weight glutenin” is encoded by the Glu-A1, B1, and D1 loci located on the long arms of wheat chromosomes 1A, 1B, and 1D
• “low molecular weight glutenin” is encoded by the Glu-A3, B3, and D3 loci located on the short arms of wheat chromosomes 1A, 1B, and 1D. Numerous alleles (alleles) are known at each of these six loci, and the molecular weight and expression level of the encoded subunits vary depending on the type of allele, which is known to affect the secondary processability of wheat flour.
• the wheat used in the present invention is GA-SX wheat, and the high molecular weight glutenin gene Glu-D1 is a d-type allele (GA-SX/GD1d).
• the low molecular weight glutenin gene Glu-B3 is a b-type allele (GA-SX/GD1d/GB3b wheat), or the low molecular weight glutenin gene Glu-B3 is an h-type allele (GA-SX/GD1d/GB3h wheat).
• the wheat is GA-SX wheat
• the high molecular weight glutenin gene Glu-D1 is a d-type allele
• the low molecular weight glutenin gene Glu-B3 is an i-type allele (GA-SX/GD1d/GB3i wheat).
• the wheat used in the present invention can be produced by crossbreeding known wheat varieties lacking six enzymes in any combination, with respect to the genetic traits related to the lack of enzyme activity of GBSSI and SSIIa to be GA-SX wheat.
• the desired enzyme-deficient mutants may be selected for crossbreeding by radiation treatment (gamma rays, beta rays, X-rays, neutron rays, etc.), chemical treatment (ethyl methanesulfonate, etc.), or other mutagenesis treatment.
• radiation treatment gamma rays, beta rays, X-rays, neutron rays, etc.
• chemical treatment ethyl methanesulfonate, etc.
• various methods for producing monocotyledonous plant transformants are known, and genetic engineering techniques for causing the function of a target gene to be lost are also known.
• Non-Patent Document 10 there are methods for inhibiting the expression of a target gene by RNAi or antisense methods, and a gene disruption method for destroying only a target gene in a plant is also known (Non-Patent Document 10), so it can also be produced by genetic engineering techniques.
• the high molecular weight glutenin gene Glu-D1 allele type is made d type.
• the allele type of the low molecular weight glutenin gene Glu-B3 is type b or type h.
• Wheat having two GBSSI and two SSIIa enzyme activity defects may be produced, and then the desired allele type may be introduced for the high molecular weight glutenin gene Glu-D1, and preferably for the glutenin gene Glu-B3, or a wheat variety may be selected in the process of producing wheat having two GBSSI and two SSIIa enzyme activity defects so that the high molecular weight glutenin gene Glu-D1, and preferably for the glutenin gene Glu-B3, has the desired allele type.
• Glu-D1d alleles are based on the classification and nomenclature proposed in Non-Patent Document 3.
• the presence or absence of Glu-D1d alleles can be identified by detecting a specific base sequence in the Dx5 gene (GenBank accession number X12928: SEQ ID NO: 13) that codes for subunit 5 of the Glu-D1d allele.
• the method of identification can be performed by PCR using the Dx_F, Dx5_F, and Dx_R primers (Dx_F: SEQ ID NO: 20, Dx5_F: SEQ ID NO: 21, Dx_R: SEQ ID NO: 22) described in Non-Patent Document 11.
• the region containing this specific mutation may be amplified using appropriately designed primers, and the gene sequence may be analyzed to confirm that it matches this sequence.
• a fraction containing high molecular weight glutenin protein can be extracted from wheat, separated by SDS-PAGE, and compared with known wheat to identify a band specific to the Glu-D1d allele (Non-Patent Document 3).
• Glu-B3b, B3h, and B3i alleles are based on the classification and nomenclature proposed in Non-Patent Document 12. The following method can be used to distinguish these alleles. Whether or not a gene has the Glu-B3b allele can be identified by examining whether or not it has a sequence specific to Glu-B3b among the gene sequences (GenBank accession number EU369719: SEQ ID NO: 14) described in Non-Patent Document 14. This can be achieved by performing PCR using the SB2F and SB2R primers (SB2F: SEQ ID NO: 23, SB2R: SEQ ID NO: 24) described in Non-Patent Document 14, and examining whether an amplified fragment of the desired length is obtained.
• PCR can be performed using LB1F and LB1R or LB4F and LB4R primers (LB1F: SEQ ID NO: 25, LB1R: SEQ ID NO: 26, LB4F: SEQ ID NO: 27, LB4R: SEQ ID NO: 28) described in Non-Patent Document 14, and the gene sequence of the amplified fragment obtained is analyzed to check whether it matches the Glu-B3b gene sequence (GenBank accession number EU369700: SEQ ID NO: 15 or EU369719: SEQ ID NO: 14).
• a fraction containing low molecular weight glutenin protein can be extracted from wheat, separated by SDS-PAGE, and compared with known wheat to identify a band specific to the Glu-D1d allele (Non-Patent Document 12).
• Wheat varieties known to have the Glu-B3b allele include "Takunekomugi” and “Nanbukomugi” (Non-Patent Document 13).
• Whether or not a gene has the Glu-B3h allele can be identified by examining whether or not it has a sequence specific to Glu-B3h among the gene sequences described in Non-Patent Document 14 (GenBank Accession No. EU369717: SEQ ID NO: 16). This can be done by performing PCR using the SB8F and SB8R primers (SB8F: SEQ ID NO: 29, SB8R: SEQ ID NO: 30) described in Non-Patent Document 14 and examining whether an amplified fragment of the desired length is obtained.
• PCR can be performed using the LB3F and LB3R primers (LB3F: SEQ ID NO: 31, LB3R: SEQ ID NO: 32) described in Non-Patent Document 14, and analyzing the gene sequence of the amplified fragment obtained to determine whether it matches the Glu-B3h gene sequence (GenBank Accession No. 369717: SEQ ID NO: 18). Alternatively, it can be confirmed by SDS-PAGE, as with the Glu-B3b allele. Wheat varieties known to have the Glu-B3h allele include "Horoshiri Komugi" and "Haruyutaka.” (Non-Patent Document 13)
• Whether or not a gene has the Glu-B3i allele can be identified by examining whether or not it has a sequence specific to Glu-B3i among the gene sequences (GenBank accession number EU369720: SEQ ID NO: 19) described in Non-Patent Document 14. This can be done by performing PCR using the SB9F and SB9R primers (SB9F: SEQ ID NO: 33, SB9R: SEQ ID NO: 34) described in Non-Patent Document 14, and examining whether an amplified fragment of the desired length is obtained.
• PCR can be performed using the LB3F and LB3R or LB4F and LB4R primers (LB3F: SEQ ID NO: 35, LB3R: SEQ ID NO: 36, LB4F: SEQ ID NO: 37, LB4R: SEQ ID NO: 38) described in Non-Patent Document 14, and the gene sequence of the amplified fragment obtained can be analyzed to determine whether it matches the Glu-B3i gene sequence (GenBank accession numbers EU369718: SEQ ID NO: 18 or EU369720: SEQ ID NO: 17 or EU369714: SEQ ID NO: 19). Alternatively, like the Glu-B3b allele, it can be confirmed by SDS-PAGE. Wheat varieties known to have the Glu-B3i allele include "Norin 61", “Minaminokaori", and "Iwainodaichi”. (Non-Patent Document 15)

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