JP2012019742A - Rice mutant, method for producing starch, starch and method for producing rice mutant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new rice mutant in which a rice starch synthase and a rice branching enzyme are mutated.SOLUTION: There is provided a genetically immobilized rice mutant in which the gene loci of rice starch synthase IIIa type (SSIIIa) and rice branching enzyme IIb type (BEIIb) are recessive homozygous. The rice mutant has SSIIIa activity reduced compared with that of wild type rice. The rice mutant keeps ≥80% in seed weight and maintains an agricultural character in comparison with wild type and parent line rice. The starch formed by the rice mutant is a high-amylose starch rarely found in rice and has an amylose ratio of ≥40%.

Description

  The present invention relates to a rice mutant, a method for producing starch, a starch, and a method for producing a rice variant, and in particular, a rice variant having a mutation in rice starch synthase and rice branching enzyme, a method for producing starch, starch, And a method for producing rice mutants.

Starch is insoluble and is a storage polysaccharide unique to plants. Most organisms on the earth use starch as a carbohydrate source.
Starch as a chemical substance is a glucose polymer containing a linear structure with glucose α1,4 and a branched structure with α1,6 glucoside bonds.
Starch is also an aggregate of amylose mainly composed of straight chain and amylopectin polymer having a branched structure.

It has been found that at least four different enzymes are involved in starch biosynthesis. These four types of enzymes include ADP glucose pyrophosphorylase (AGPase) which is a substrate supply enzyme, starch synthase (SS) which extends α1,4 glucoside bond, and branching enzyme which forms a branched structure consisting of α1,6 glucoside bond ( BE), a debranching enzyme (DBE) that trims an extra branching structure added by BE to maintain the cluster structure that is characteristic of amylopectin.
However, it is considered that there are other enzymes involved in starch biosynthesis (see Non-Patent Document 1).

Thus, at least four types of enzymes are involved in plant starch biosynthesis.
In addition, in higher plants, these enzymes have a number of isozymes and are involved in starch biosynthesis. Isozymes are a group of enzymes with different amino acid sequences that catalyze similar enzyme reactions. For example, there are 10 types of SS, 3 types of BE, and 4 types of DBE in rice.
In recent years, it has been found that these isozymes share roles depending on tissue specificity and subtle substrate specificity.
Elucidating the functions and roles of such isozymes leads to elucidation of the functions of isozymes. And elucidation of the function of isozymes is indispensable for elucidating the whole picture of the plant starch biosynthesis mechanism.
For this reason, mutants of each isozyme have been conventionally developed. By examining the phenotype of a mutant lacking a specific isozyme, it is possible to know the function and role of that isozyme.

For example, in order to clarify the function of each isozyme, mutants that have already been isolated and analyzed in rice include SSI (Non-patent Document 2, Patent Document 1), SSIIa (Non-patent Document 3, Patent Document). 2), SSIIIa (Non-patent document 4, Patent document 3), GBSSI (Non-patent document 5), BEI (Non-patent document 6), BEIIb (Non-patent document 7, Patent document 2), ISA1 (Non-patent document 8) , PUL (Non-Patent Document 9, Patent Document 4), PHO (Non-Patent Document 10), and the like.
These mutants may differ from the wild type in the structure of starch accumulated in the endosperm. Along with the difference in structure, physical properties such as different starch granule sizes may be exhibited.

JP 2003-79260 A Japanese Patent No. 4358006 JP 2006-51023 A JP 2007-20475 A

Y. Nakamura, Towers a letter understand of the metabolomic system for amylectin biosynthesis in plants: rice endosperma asa model tiss. Plant Cell Physiol. , Japan, Society of Plant Physiology, 2002, 43, p. 718-725 N. Fujita et al., Function and charac- terization of start synthase I using mutants in rice. , Plant Physiol. United States, American Society of Plant Biology, 2006, 140, p. 1070-1084 Y. Nakamura et al., Essential amino acids of start synthase IIa differentiated amylectin structure and stark quality between japonica and indicaratica. Plant Mol Biol. 2005, 58, p. 213-227 N. Fujita et al., Characteristic of SSIIIa-defective mutants of rice (Oryza sativa L.); the function of SSIIIa and pleiotropic effects by SS. , Plant Physiol. , United States, American Society of Plant Biology, April 2007, 144, 4, p. 2009-2023 Y. Sano, Differential regulation of waxy gene expression in rice endosperm. Theor Appl Genet, 1984, 68, p. 467-473 H. Satoh et al., Star-branching enzyme I-defective mutation specific effects the structure and properties of starfish endosperm. , Plant Physiol. American Society of Plant Biology, United States, 2003, 133, p. 1111-1121 A. Nishi et al., Biochemical and genetic analysis of the effects of amylase-extender mutation in rice endosperm. , Plant Physiol. American Society of Plant Biology, 2001, 127, p. 459-472 Y. Nakamura et al., Correlation between activities of starting de- veloping enzyme and α-polyglucan structure in endosperms of suger-1 mutants. Plant J .; Blackwell, 1997, 12, p. 143-153 N. Fujita et al., Characteristic of PUL-defective mutuals of rice (Oryza sativa L.) and the function of PUL on the search biosynthesis. , J Exp Bot. Oxford University Press, UK, March 2009, 60, 3, p. 1009-1023 H. Satoh et al., Plastic α-glucan phosphorous mutational dramatic effects the synthesis of structure in rice endosperm. Plant Cell, United States, American Society of Plant Biology, 2003, 20, p. 1833-1849 T.A. Hirose et al., A complete expression analysis of the start syngene gene family in rice (Oryza sativa L.). , Planta, 2004, 220, p. 9-16 T.A. Ohdan et al., Expression profiling of genes involved in star synthesis in sink and source organs of rice. , J Exp Bot. , Oxford University Press, UK, December 2005, 56, 422, p. 3229-3244

Thus, in the rice mutant having a mutation in an enzyme involved in conventional starch biosynthesis, the effects of each single mutant have been clarified.
However, although it was difficult to make recessive homozygous by combining a plurality of mutants, it was not considered to show a special phenotype.
Among these, the combination of rice starch synthase type IIIa (SSIIIa) and rice branching enzyme type IIb (BEIIb) was not known.

  This invention is made | formed in view of such a condition, and makes it a subject to eliminate the above-mentioned subject.

The rice mutant of the present invention is characterized in that the locus of rice starch synthase type IIIa (SSIIIa) and rice branching enzyme type IIb (BEIIb) are recessive homozygous and genetically fixed.
The rice mutant of the present invention is further characterized in that SSIIIa activity and BEIIb activity are reduced compared to the wild type.
In the rice mutant of the present invention, the seed weight is maintained at 80% or more, and the agricultural traits are maintained as compared with the parent line of the wild type, the recessive parent line of SSIIIa, or the parental line of BEIIb locus. It is characterized by being.
The method for producing starch according to the present invention is characterized by using the rice mutant.
The starch production method of the present invention is characterized in that the rice mutant endosperm is used.
The starch of the present invention is produced by the above-mentioned starch production method.
The starch of the present invention is characterized in that it has a shape different from that of wild-type rice, parental rice resulting from decreased activity of SSIIIa, or starch produced using parental rice derived from decreased activity of BEIIb. .
The starch of the present invention is different in size compared to the starch produced using the wild-type rice, the parent line rice resulting from the decreased activity of SSIIIa, or the parent line rice resulting from the decreased activity of BEIIb. It is characterized in that the surface of starch granules having a different size is smooth.
The starch of the present invention has a chain length of amylopectin compared to the starch produced using the wild-type rice, the parent line rice resulting from the decreased activity of SSIIIa, or the parent line rice resulting from the decreased activity of BEIIb. The distribution is different.
The starch of the present invention has gelatinized viscosity characteristics as compared with the starch produced using the wild type rice, the parent line rice resulting from the decreased activity of SSIIIa, or the parent line rice resulting from the decreased activity of BEIIb. Are different.
The starch of the present invention is characterized in that the starch has a amylose ratio of 40% or more.
The starch of the present invention is characterized in that the amylopectin of the starch is a B-type crystal.
The starch of the present invention is characterized in that the starch gelatinization viscosity pattern has a low viscosity.
The starch of the present invention is characterized in that the starch has a high aging property compared to the starch produced using the parent line rice resulting from the decreased activity of SSIIIa or the parent line rice resulting from the decreased activity of BEIIb. Features.
The method for producing a rice mutant of the present invention is characterized in that it is a double recessive homology of a rice starch synthase type IIIa (SSIIIa) mutant and a rice branching enzyme type IIb (BEIIb) mutant.
The rice mutant of the present invention is produced by the method for producing a rice mutant.

  According to the present invention, both SSIIIa and BEIIb loci are recessive homozygous so that SSIIIa-only mutants, BEIIb-only mutants, and rice mutants that can produce starches that differ in shape and properties from the wild type Can be provided.

It is a photograph which shows the form of the fully-ripened brown rice and section | slice of (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concern on embodiment of this invention, those parent variants (e1, EM10), and a wild type (Nippon Hare, Kinnan style). Chain length distribution (A, Molar%) of amylopectin in mature endosperm with ΔSSIIIa / ΔBEIIb (# 4019) and their parent mutants (e1, EM10) according to an embodiment of the present invention, and wild type (Nipponbare) ) Is a graph showing the difference (B, ΔMolar%) obtained by subtracting the pattern. It is a graph which shows the gel filtration pattern of (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concerns on embodiment of this invention, those parent variant (e1, EM10), a wild type (Nipponbare) starch, and refined amylopectin. It is the photograph which observed the starch grain of (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concerns on embodiment of this invention, those parent variant (e1, EM10), and a wild type (Nipponbare) with the scanning electron microscope. It is a figure which shows (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concerns on embodiment of this invention, those parent variant (e1, EM10), and the X-ray-diffraction pattern of the starch grain of a wild type (Nipponbare). FIG. 6 shows gelatinization characteristic patterns (dynamic viscoelasticity) of starch suspensions of ΔSSIIIa / ΔBEIIb (# 4019) and their parent mutants (e1, EM10) and wild type (Nihonbare) according to an embodiment of the present invention. FIG. It is a figure which shows the viscosity characteristic pattern of (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concerns on embodiment of this invention, those parent variant (e1, EM10), and a wild type (Nipponbare) starch paste. It is a figure which shows the storage elastic modulus at the time of cooling of (DELTA) SSIIIa / (DELTA) BEIIb (# 4019) which concerns on embodiment of this invention, those parent variant (e1, EM10), and a wild type (Nipponbare) starch paste. ΔSSIIIa / ΔBEIIb (# 4019) and their parent variants (e1, EM10) and wild-type (Nipponbare) Native-PAGE / SS activity staining method and Western blotting of enzyme activity and protein according to the embodiment of the present invention It is a figure which shows the result of deletion.

<Embodiment>
Embodiments of the present invention will be described below with reference to the drawings.
Conventionally, research is progressing on some isozyme mutants related to starch biosynthesis. For example, in the SSIIIa mutant (ΔSSIIIa) and the BEIIb mutant (ΔBEIIb), the structure and physical properties of starch have already been known.
However, there was no double mutant (ΔSSIIIa / ΔBEIIb) combining the SSIIIa mutant and the BEIIb mutant. The reason for this is that the double mutant is usually only a combination of the phenotypes of the two mutants, and is difficult to create, so the skilled person is motivated to create a double mutant. It was because there was not.
In contrast, the inventor of the present invention comprehensively studied the metabolic system of rice starch, searched for double mutants with beneficial phenotypes, and double mutants of SSIIIa and BEIIb (ΔSSIIIa / We have found that ΔBEIIb) has an unexpected effect.
The SSIIIa and BEIIb double mutant (ΔSSIIIa / ΔBEIIb) produced by the inventors of the present invention had the following characteristics compared to the parent mutant and the wild type.

First, referring to FIG. 1, SSIIIa and BEIIb double mutant (ΔSSIIIa / ΔBEIIb) clone # 4019, its parent mutant, SSIIIa single mutant (ΔSSIIIa) clone e1, and another parent The morphology of the fully-brown brown rice and slices of clone EM10 of a single variant of BEIIb (ΔBEIIb), which is a mutant, and a wild type clone of wildtype (Wild type), which is a control, will be described. In each clone, the left figure is a photograph of the whole shape of fully-ripened brown rice, and the right figure is a photograph of the shape of the slice. Hereinafter, clones with the same reference numbers indicate the same thing.
EM10 was selected using MNU (methane nitrosourea) treatment, which is an embryo mutation source. The MNU process is described in the literature (Yano, M., Okuno, K., Kawakami, J., Satoh, H. and Omura, T. (1985) High amylose mutants of rice et al. 257.).

In FIG. 1, the seed of e1 which is a clone of SSIIIa single mutant (ΔSSIIIa) showed a “heart-white” form in which the center of brown rice was clouded white. On the other hand, the seed of EM10 which is a clone of BEIIb single mutant (ΔBEIIb) was cloudy and smaller than the wild type.
On the other hand, the seed of # 4019 which is a clone of SSIIIa and BEIIb double mutant (ΔSSIIIa / ΔBEIIb) shows a form of “white turbidity” in which the whole brown rice becomes cloudy like EM10 of BEIIb single mutant. However, the seed size was larger than that of EM10. In addition, the seed # 4019 had a feature that the seed fragment was cloudy at the center but transparent on the outside near the seed coat.

  Further, when the seed weight of ΔSSIIIa / ΔBEIIb was measured, it was 80% or more of the wild type. The measurement results are shown in Table 1 below.

Table 1 shows the SSIIIa and BEIIb double mutant clone # 4019 (ΔSSIIIa / ΔBEIIb), their parent mutant clones e1 (ΔSSIIIa) and EM10 (ΔBEIIb), wild type (Wild type). The data measured about the brown rice weight (mg) of Nihonbare and Kimnan style are shown.
As shown in Table 1, e1 which is a clone of SSIIIa single mutant was almost the same size as the wild type of Nipponbare or Kinnan style.
On the other hand, EM10, which is a clone of BEIIb single mutant, had a seed weight of about 60% of the wild type.
In addition, the brown rice weight of # 4019, a clone of SSIIIa and BEIIb double mutant, was slightly smaller, 83%, more than 80% of the wild type, but larger than EM10.

Next, the chain length distribution of the SSIIIa mutant amylopectin will be described with reference to FIG.
FIG. 2A is a graph showing the chain length distribution of amylopectin in mature endosperm of ΔSSIIIa / ΔBEIIb (# 4019), their parental variants ΔSSIIIa (e1) and ΔBEIIb (EM10), and control Nipponbare (wild type) .
FIG. 2B is a graph showing a difference in molar ratio (ΔMolar%) obtained by subtracting the molar percentage of the wild-type Nipponbare from each. DP (degree of polymerization) is a value indicating the degree of polymerization of glucose.
First, e1, which is a clone of SSIIIa single mutant, had a long chain with a chain length DP ≧ 37 as a typical feature, which was reduced to about 70% compared to the wild type. On the other hand, in EM10, which is a clone of BEIIb single mutant, the short chain of DP ≦ 13 was drastically decreased compared to the wild type, and the long chain of DP ≧ 14 was increased instead.
In addition, ΔSSIIIa / ΔBEIIb clone # 4019 was characterized by a large decrease in the short chain of DP6-12 similar to the pattern of EM10, but increased from EM10 at DP10-15, and DP ≧ 17 The long chain of was reduced from EM10. As for the amount of long chain, it inherited the properties of the parents, and in the double mutant, it was an intermediate amount of the parental line, and as a result, it was slightly increased from the wild type.

Next, a gel filtration pattern will be described with reference to FIG. FIG. 3 shows gel filtration patterns of clones of ΔSSIIIa / ΔBEIIb (# 4019), parent mutants ΔSSIIIa (e1) and ΔBEIIb (EM10), and wild-type Nipponbare, respectively. In each clone, the black solid line is a graph of the gel filtration pattern of the starch cut. The black wavy line “AP” is a graph of the gel filtration pattern of amylopectin purified from the starch by the butanol method, and the same was cut in the same manner as starch. The gray dotted line “λ max” is a graph showing the wavelength (λ max (nm)) of the maximum absorbance of the starch-iodine complex when the starch is subjected to gel filtration.
The gel filtration pattern of the starch debranched is divided into three peaks, the fastest detected peak (Fraction I) is the apparent amylose, the second peak (Fraction II) is the B2 chain that connects the clusters The longer amylopectin long chain, the third detected peak (Fraction III) is the short chain in the amylopectin cluster.
These are quantified in Table 2 below. In Table 2, the ratio of each fraction and the ratio of the short chain to the long chain of amylopectin (III / II) are shown.

Referring to Non-Patent Document 4, SSIIIa single mutant (ΔSSIIIa) has an apparent amylose content of about 1.5 times higher than that of Nipponbare as a result of gel filtration of debranched starch. Also in the experimental results of this embodiment, the apparent amylose content of Nipponbare was 23.5%, while the clone e1 of ΔSSIIIa showed 31.4%, which is about 1.4 times the value.
In contrast, the amylose content of ΔSSIIIa / ΔBEIIb was 46.6%, 1.4 times higher than the clone e1 of ΔSSIIIa. In ΔBEIIb, the ratio of the long chain of amylopectin was very large because the III / II value, which is the ratio of the short chain, was as small as 1.1. ΔSSIIIa / ΔBEIIb was found to have a higher proportion of amylopectin long chains than ΔBEIIb, but higher than Nipponbare and ΔSSIIIa. This result is consistent with the fact that the long chain of DP ≧ 37 in the chain length distribution was increased from the clone e1 of ΔSSIIIa.
Fraction I obtained from gel filtration of amylopectin purified from starch shows the amount of extra long chain (ELC). ΔSSIIIa and ΔSSIIIa / ΔBEIIb were slightly higher than those of Nipponbare and EM10.
Further, a value obtained by subtracting the ELC amount from the apparent amylose content indicates the true amylose content. In ΔSSIIIa / ΔBEIIb, the true amylose content was 46.6% −2.4% = 44.2%, indicating a high value.
From the above, it was clarified that the amylose content of ΔSSIIIa / ΔBEIIb is extremely high in conventional rice.
Thereby, it is considered that the starch of ΔSSIIIa / ΔBEIIb has a unique texture and can produce products characteristic of the food processing field, additives, and industrial materials.

Next, with reference to FIG. 4, the shape and particle size of starch granules of ΔSSIIIa / ΔSSIVb will be described.
FIG. 4 is a photograph of ΔSSIIIa / ΔBEIIb clone # 4019, their parent mutant clones e1 and EM10, and wild-type Nipponbare starch granules observed with a scanning electron microscope.
Referring to FIG. 4, the Nipponbare starch particles of wild type were polygons of about 3 to 5 μm. On the other hand, the starch granules of clone e1 which is ΔSSIIIa were slightly smaller than those of Nipponbare, and rounded starch granules were mixed. On the other hand, the starch particles of ΔBEIIb were large and small and had a characteristic that the surface was rough. The ΔSSIIIa / ΔBEIIb clone # 4019 had starch granules of various sizes similar to ΔBEIIb, but had a smooth surface and peanut-like starch granules.
This peanut-like starch granule has a shape characteristic of high amylose starch. For example, the literature (Jiang H, Campbell M, Blanco M, Jane J-L (2010) Characterization of maize amylase-extender (ae) mutant starches:. Part II Strucuture and properties of starch residues remaining after enzymatic hydrolysis at boiling-water temperature Carbohydrate Polymers 80: 1-12), corn high amylose starch was also provided with peanut-like starch granules.

Next, with reference to FIG. 5, the crystallinity of ΔSSIIIa / ΔBEIIb endosperm starch will be described.
According to the X-ray diffraction pattern of the starch granules in FIG. 5, the wild-type Nipponbare shows typical A-type crystal characteristics, and the ΔSSIIIa clone e1 also shows the A-type crystal characteristics. Since it is higher amylose, the crystallinity is low. Non-Patent Document 4 describes that high amylose starch has low crystallinity.
On the other hand, the crystallinity of EM10, which is ΔBEIIb, shows the characteristics of typical B-type crystals found in potato starch and the like. Note that the definition of the B-type crystal is described in the literature (Tanaka N, Fujita N, Nishi A, Satoh H, Hosaka Y, Ugaki M, Kasawaki S, Nakamura Y. (2004) The stricture structure. level of starting branching enzyme IIb in rice endosperm. Plant Biotechnology Journal 2: 507-516.).
The crystallinity of the endosperm starch of ΔSSIIIa / ΔBEIIb showed the characteristics of B-form crystals similar to ΔBEIIb, but the sharpness of the peak was lower than that of ΔBEIIb. It is done.

Next, with reference to FIG. 6, the gelatinization characteristic patterns of ΔSSIIIa / ΔBEIIb (# 4019) and their parent mutants (e1, EM10) and wild-type (Nipponbare) starch suspension will be described. This pasting characteristic pattern is measured by graphing dynamic viscoelasticity, that is, a change in viscosity characteristic value, according to a predetermined temperature program.
The viscosity characteristic pattern of Nipponbare, the wild type, showed a clear peak during heating and a sudden drop immediately after heating. On the other hand, ΔSSIIIa / ΔBEIIb (# 4019) had the highest viscosity characteristic value rising temperature, no peak, and the viscosity characteristic value remained low. Parental mutants ΔSSIIIa (e1) and ΔBEIIb (EM10) also had lower peak viscosities than wild type, but were larger than ΔSSIIIa / ΔBEIIb (# 4019).
Decrease in peak viscosity indicates that swelling and gelatinization of starch granules are suppressed (Tetsu Takahashi, Kazuo Shinoda, Satoshi Miura, Kintetsu, Shoichi Kobayashi, Effects of heat treatment on physicochemical properties of rice flour (2002) (See Japanese Society for Food Science and Technology, 49, 757-764.)

Next, the viscosity characteristic pattern of ΔSSIIIa / ΔBEIIb (# 4019) and their parent mutants (e1, EM10) and wild type (Nipponbare) starch paste will be described with reference to FIG.
When stirring at a constant speed for 10 minutes at a constant temperature of 35 ° C., the viscosity characteristics patterns of wild-type Nipponbare and ΔBEIIb (EM10) were flat.
On the other hand, the viscosity characteristic patterns of ΔSSIIIa / ΔBEIIb (# 4019) and ΔSSIIIa (e1) increased with the passage of time, and the viscosity characteristic value was larger for ΔSSIIIa / ΔBEIIb (# 4019). This suggests that the viscosity characteristic pattern of ΔSSIIIa / ΔBEIIb (# 4019) under this temperature condition is derived from the character of ΔSSIIIa (e1).

Next, the storage elastic modulus by dynamic viscoelasticity measurement during cooling will be described with reference to FIG.
Specifically, ΔSSIIIa / ΔBEIIb (# 4019), these parent mutants ΔSSIIIa (e1) and ΔBEIIb (EM10), wild-type Nipponbare starch paste samples from 30 ° C. to a constant rate (1 ° C. per minute) And the storage elastic modulus was measured.
The wild-type Nipponbare had a flat storage modulus and did not harden even when the temperature was lowered. On the other hand, the storage elastic modulus of ΔSSIIIa / ΔBEIIb (# 4019), ΔSSIIIa (e1), and ΔBEIIb (EM10) increases rapidly as the temperature decreases, and among them, ΔSSIIIa / ΔBEIIb (# 4019) is three times the base value. It turned out above and it turned out that it hardens | cures rapidly. This is because ΔSSIIIa / ΔBEIIb (# 4019) has an extremely high amylose content.

  In addition, ΔSSIIIa / ΔBEIIb was markedly aged as compared to ΔBEIIb. In fact, referring to FIG. 8, the fact that the double mutant of ΔSSIIIa / ΔBEIIb has a higher storage elastic modulus than the other strains is data that shows that aging is remarkable.

The following properties and effects can be obtained from ΔSSIIIa / ΔBEIIb according to the embodiment of the present invention as described above.
In rice mutants having a mutation in an enzyme involved in conventional starch biosynthesis, the structure and physical properties of starch are clarified in ΔSSIIIa and ΔBEIIb, and the functions of each enzyme are also clarified to some extent.
However, there was no double mutant combining ΔSSIIIa and ΔBEIIb, and the effect of deleting both enzymes was unknown.
Here, in the usual case, even if a double mutant is created, each trait of each mutant only appears as it is. For example, according to Mendel's law of classical genetics, when a “wrinkle” bean mutant is multiplied by a “green” bean mutant, there is a “wrinkle” and a “green” mutant. . In addition, it takes considerable effort to create mutants. For these reasons, one skilled in the art usually has no incentive to create double mutants.
However, the inventors of the present invention elucidated the influence on starch properties when a double mutant of ΔSSIIIa / ΔBEIIb was made and both enzymes were deleted, and as described above, an unexpected effect was obtained. .

Specifically, starch having characteristics and shape different from ordinary rice starch can be obtained from the mutant of ΔSSIIIa / ΔBEIIb according to the embodiment of the present invention.
Among these properties, the uniqueness is that the amylose content is much higher than that of the parental mutant, which is unparalleled in the rice reported so far. Moreover, it can be used suitably for foodstuffs, such as a pilaf and a risotto, from the high aging property which will be attributed to high amylose.
Moreover, since it becomes indigestible when the content of amylose becomes high, it can be used for various health foods and diet foods.
Moreover, the rice flour obtained by pulverizing the seed of the mutant of ΔSSIIIa / ΔBEIIb can be used by mixing it with bread, noodles or the like. Thereby, the bread | pan, noodles, etc. of the texture and taste which are different in taste from a normal rice flour bread | pan can be manufactured.
Furthermore, the pulverized product and starch of the mutant of ΔSSIIIa / ΔBEIIb can be used in industrial applications due to the high content of amylose. This is because when aging is necessary, such as biodegradable plastic, it is advantageous that the aging property is higher. For example, since amylose is soluble in warm water, it can be formed into a film and used for applications such as biodegradable films, medical materials, and sutures. Moreover, it can be used as a “scaffolding” member for regenerative medicine by using the property of being absorbed and decomposed in the body.
Furthermore, it can be used for various industrial uses such as materials for producing glass fibers, industrial glue, building material compounding agents, and plant cultivation assets.
Thus, the double mutant which is ΔSSIIIa / ΔBEIIb can be used industrially.

In addition, the starch produced from the rice mutant of ΔSSIIIa / ΔBEIIb according to the embodiment of the present invention has a unique amylopectin structure.
Unlike the amylose content, the structure of amylopectin had characteristics that inherited the properties of the parental mutants, but had remarkable characteristics beyond simply combining the characteristics of each parental mutant.
For example, the starch of ΔSSIIIa / ΔBEIIb has the characteristic that the short chain is greatly reduced similarly to the starch of ΔBEIIb, and therefore the starch crystallinity was a B-form crystal similar to ΔBEIIb. This B-type crystal is not seen in ordinary rice starch, but is a type of crystal form exhibited by potato starch and the like.
However, ΔSSIIIa / ΔBEIIb starch was different in starch granule morphology from ΔBEIIb. Specifically, it was similar to ΔBEIIb with respect to the mixing of large and small grains, but ΔSSIIIa / ΔBEIIb starch was different in that the surface was smooth.
In addition, the starch of ΔSSIIIa / ΔBEIIb was completely different in the gelatinization viscosity pattern from that of ordinary rice starch, had a very low viscosity, and had a very high aging property.
These properties are thought to derive from their structure and starch granule characteristics and are very different from the single mutants ΔSSIIIa and ΔBEIIb.
Thereby, it is possible to use the double mutant which is (DELTA) SSIIIa / (DELTA) BEIIb for the foodstuffs, processed products, and industrial products from which texture is different from usual.
In addition, it is considered that the ΔSSIIIa / ΔBEIIb starch of the present embodiment can shorten the time for saccharification and fermentation as compared with the conventional method by pulverizing and using it for brewing.

Furthermore, the rice mutant of ΔSSIIIa / ΔBEIIb according to the embodiment of the present invention is a genetically fixed line, and all seeds planted by this seed and sowed by self-propagation are all isomorphous.
In addition, the seed of ΔBEIIb, which is the parent, is about 60% of the wild type, whereas the rice mutant of ΔSSIIIa / ΔBEIIb has a seed weight of 8 although it lacks two enzymes. Over 10% are maintained, and fertility and growth are comparable to the wild type. That is, there is no decrease in agricultural traits, and the agricultural traits are maintained. Furthermore, since it is not a gene recombinant, it can be planted in a general paddy field and can be cultivated on a large scale.

  As described above, in the method for producing a rice mutant according to an embodiment of the present invention, a rice (Oryza sativa) mutant of a japonica rice cultivar having a rare amylose starch, a method for producing the same, and the rice A starch derived from the mutant and a method for producing the starch can be obtained. Furthermore, this invention can be used for the food-drinks and industrial products using the said starch.

  Examples of the present invention will be described below with reference to the drawings, but the present invention is not limited to these examples.

(Isolation of double mutant lacking SSIIIa and BEIIb)
A double mutant was created by crossing the SSIIIa mutant and the BEIIb mutant, and their properties were examined.

  Hereinafter, the mating of the double mutant will be described with reference to FIG. The inventors of the present invention crossed the BEIIb mutant EM10 (white cloudy seed) with the SSIIIa mutant (heart white seed) e1 and sown the F1 seed of the compensation dividend in the next year to obtain F2 seed. It was. In this, about 25% of cloudy seeds were contained. In the following fiscal year, cloudy seeds were sown, and then seeds having a recessive homology with the SSIIIa gene were selected by the PCR method (see Non-Patent Document 4). The SSIIIa activity of soluble fractions obtained from these ripened seeds was examined using the Native-PAGE / SS activity staining method (Activity staining) and the presence or absence of BEIIb protein (see FIG. 9). As a result, a clone in which deletion of both SSIIIa activity and BEIIb protein was observed was obtained and named clone # 4019. In addition, the defect | deletion of BEIIb protein means the defect | deletion of BEIIb activity.

Specifically, SSIIIa activity and BEIIb protein were detected according to the method described in Non-Patent Document 2 as follows.
About 10 to 15 days after flowering, the grain, embryo and pericarp of the ripened seed are removed, and 3 times volume extraction buffer [50 mM imidazolol (pH 7.4), 12.5% glycerol, 8 mM magnesium chloride] , 500 mM 2-mercaptoethanol], homogenized in a microtube using a plastic homogenizer (manufactured by Greiner), and centrifuged at 15,000 rpm for 10 minutes at 4 ° C. to obtain a native- PAGE sample buffer [0.3 M Tris-HCl (pH 7.0), 0.1% bromophenol blue, 50% glycerol] was added in 1/2 volume and used for electrophoresis.
For electrophoresis of the Native-PAGE / SS activity staining method, 7.5% acrylamide gel in which oyster glycogen was dissolved at 60 ° C. was used as a primer necessary for the glucosyltransferase reaction. Electrophoresis was performed at 4 ° C. with a steady current of 7.5 mA until the front passed through the concentrated gel and then 15 mA after passing, and the current was stopped 60 minutes after the front came out.
Thereafter, a reaction solution excluding the substrate ADP glucose [sodium citrate buffer (pH 7.5), 0.5 M Citrate-Na, 100 mM Bicine-NaOH (pH 7.5), 0.5 mM EDTA, 10 % Glycerol, 2 mM dithiothreitol, 1 mM ADP glucose] (15 minutes each), ADPG was added, and the mixture was allowed to react at 30 ° C. for 20 hours. After the reaction, it was stained with iodoiodoli solution (1% KI / 0.1% I2).
For SDS-PAGE electrophoresis for Western blotting, a 7.5% acrylamide gel containing 0.1% SDS was used. After SDS-PAGE, one membrane (PVDF membrane) for transferring the protein separated on the gel and six filter papers are cut into approximately the same size as the gel, and the membrane is immersed in 100% methanol once. Then, the filter paper was directly immersed in transfer buffer (25 mM tris, 192 mM glycine, 20% methanol, 0.1% SDS) for 30 minutes or more. The gel subjected to SDS-PAGE was transferred to a plastic case containing a transfer buffer and soaked for 15 minutes. Three filter papers soaked in transfer buffer on the anode side of the blotting electrode are stacked so that bubbles do not enter, the membrane is stacked so that bubbles do not enter, and then the gel is stacked so that bubbles do not enter. Three filter papers were stacked so as not to contain bubbles. The upper electrode plate (cathode) was set and energized at 10 V for 1 hour. After 1 hour, the energization was stopped, the membrane was transferred to a plastic container, and the membrane was washed for 15 minutes while being immersed in TBST2 (50 mM NaCl, 1 mM tris-HCl (pH 7.5), 1% Tween 20). Thereafter, washing was performed twice for 5 minutes while being immersed in TBS (10 mM tris-HCl (pH 7.5), 500 mM NaCl). Excluding TBS, 3% geratin / TBS was added and soaked for 1 hour. After 1 hour, 3% geratin / TBS was removed, and 1% geratin / TBS was added together with a primary antibody (anti-BEIIb serum (non-patent document 7) diluted 1000-fold with 1% geratin / TBS) overnight. I let it soak slowly. The next day, the membrane was washed 3 times for 15 minutes each with TBST2 and 3 times for 5 minutes each with TBS. A secondary antibody (antirabbit serum (manufactured by BioRad) diluted 2000 times with 1% geratin / TBS) was added together with 1% geratin / TBS except for TBS, and the mixture was slowly immersed for about 6 hours. The membrane was then washed 3 times for 15 minutes each with TBST2 and 3 times for 5 minutes each with TBS. Remove TBS, add coloring solution (dissolve 15 mg of 4-chloro-1-naphthol in 5 ml of methanol, add 20 ml of TBS, mix well, add 25 μl of H ₂ O ₂ immediately before use) and soak for a while. After thoroughly washing with distilled water, water was thoroughly wiped off and dried.
As described above, a line of ΔSSIIIa / ΔBEIIb was established.

[Measurement of RVA and dynamic viscoelasticity]
(Measurement of thermal gelatinization viscosity characteristics by Rapid Visco Analyzer (RVA))
The moisture content of the purified endosperm starch was measured with an infrared moisture content measuring device (Basic moisture meter MA150, manufactured by Sartorius Mechatronics Japan Co., Ltd.) and calculated so that the actual dry weight was 2.5 g. Measure in a cup. Distilled water was added thereto so that the total amount was 25.0 g.
Plastic wings were inserted, and the viscosity characteristic value was measured with RVA (manufactured by Foss Japan Co., Ltd., RVA3D). The temperature program was performed at a temperature (° C.) as shown in FIG. This is the same as the method described in Non-Patent Document 4.
After completion of the measurement, the weight of the RVA aluminum cup containing the starch paste was weighed, and distilled water was added to correct the weight of the sample. Again, an aluminum cup was attached to RVA (manufactured by Foss Japan, RVA3D), and the viscosity characteristic value was measured by stirring at 35 ° C. for only 10 minutes.

(Measurement of storage modulus and loss modulus of starch paste with dynamic viscoelasticity measuring device)
The starch paste prepared by RVA measurement is placed on the lower disk controlled at 30 ° C. of a dynamic viscoelasticity measuring apparatus (manufactured by Thermo Scientific Haake, MARS), and the gap is 1 with the upper parallel disk made of titanium having a diameter of 35 mm. It was sandwiched so as to be 0.00 mm. The linear range of each glue sample is obtained in advance by strain dependence measurement, and the strain (amplitude) corresponding to 80% of the elastic limit is applied at an angular velocity (frequency) of 1.0 rad / sec. The storage modulus at a temperature drop was measured at 1 ° C. per minute in the range.

(Observation of starch granules by scanning electron microscope (SEM))
The purified endosperm starch was pasted on a brass stage with a silver double-sided tape and gold deposited.
The images were observed with a SEM (IET Co., Jeol 5600) magnified 1000 times and 4000 times. Specifically, observation was performed according to the method described in Non-Patent Document 4.

(Observation of crystallinity of endosperm starch by X-ray diffractometer)
0.5 g of purified endosperm starch was exposed to 100% humidity for 24 hours in a sealed container containing distilled water.
Thereafter, starch was pasted on a slit having a depth of 0.5 mm, and a diffraction pattern of 2θ = 4 to 40 ° was detected with an X-ray diffractometer (Rigaku RINT2000).
Specifically, observation was similarly performed according to the method described in Non-Patent Document 4.

(Starch purification method)
For the purification of starch, a cold alkali immersion method (Yamamoto et al., Denpun Kagaku 28: 241-244 (1981)) was used. 10 g of brown rice was polished to 80%, 200 ml of 0.1% NaOH was added and left at 4 ° C. overnight.
The next day, the supernatant was discarded, ground in a mortar, passed through a 150 μm mesh, and centrifuged at 3,000 g, 4 ° C. for 10 minutes.
600 ml of 0.1% NaOH was added to the precipitate, shaken in ice for 3 hours, and left overnight at 4 ° C. The next day, the supernatant was discarded, suspended in distilled water, and neutralized with 1N acetic acid. Further, it was washed 5 times with distilled water, dried, and powdered with a mortar.

(Method for purifying amylopectin from starch)
As the amylopectin purification method, a butanol precipitation method (see, for example, “Schoch TJ (1954) Purification of start and the start fractions. Methods Enzymol 3: 5-6”) was used. 1 g of purified starch was weighed into a flask, 100 ml of dimethyl sulfoxide (DMSO) was added and stirred well. Was replaced with air and N ₂ in the flask was filled with N ₂ to flask. The mixture was slowly stirred at 80 to 85 ° C. for 2 hours while being charged with N ₂ . The temperature was gradually lowered to 60 ° C., 500 ml of ethanol was added, the mouth of the flask was closed with a wrap and placed at 4 ° C. overnight. The precipitate was collected by centrifugation at 2,000 G, 5 ° C., 15 min, and transferred to a flask. To the precipitate, 100 ml of DMSO was added and stirred, N ₂ was charged in the same manner, and the mixture was slowly stirred at 80 to 85 ° C. for 2 hours. After the reaction, it was left overnight at 4 ° C. This was transferred to a 50 ml tube and centrifuged at 2,000 G, 5 ° C., 15 min. The precipitate was dissolved in 170 ml of distilled water at 60 ° C. and transferred to a flask. The mixture was slowly stirred at 70 to 80 ° C. while charging N ₂ to dissolve the precipitate. 10 ml each of n-butanol (n-Butyl Alcohol) and isoamyl alcohol (3-Methyl-1-Butanol) was added to the flask, and the mixture was slowly stirred at 80 ° C. for 3 hours. The temperature was gradually lowered to 60 ° C., the mouth of the flask was closed with a wrap, placed in a styrofoam container, and left overnight at room temperature. The next day, the polystyrene foam container was transferred to a 4 ° C. refrigerator and left for 2 days. The solution was stirred well, transferred to a 50 ml tube and centrifuged at 8,500 G, 5 ° C., 20 min. 20 ml ethanol was added to the precipitate and ground finely in a mortar. The solution was transferred to a 50 ml tube and centrifuged at 2,000 G, 5 ° C., 10 min. The supernatant was discarded, 20 ml of ethanol was added and stirred, and centrifuged under the same conditions. The supernatant was discarded, 20 ml of acetone was added and stirred, and centrifuged under the same conditions. I repeated this again. The supernatant was discarded, 20 ml of diethyl ether was added and stirred, and centrifuged under the same conditions. I repeated this again. The supernatant was discarded, Kimwipe was placed on the lid of the tube and fixed with a rubber band, and the precipitate was naturally dried in a draft chamber. The dried sample was stored at −30 ° C. until use.

(Starch debranching and gel filtration)
0.25 ml of distilled water was added to 22.5 mg of the purified starch and amylopectin and mixed, and 0.25 ml of 2N NaOH was added and gelatinized at 37 ° C. for 2 hours.
To this was added 3.26 ml of distilled water, and 90 μl of 5N HCl was added for neutralization. Next, 5 ml of 100 mM acetate buffer (pH 3.5) was added. 12.5 μl (about 875 units) of amyloderamosa isoamylase (EC 3.2.1.68, manufactured by Hayashibara Biochemical Laboratories) was added, and the reaction was carried out while shaking at 40 ° C. for 24 hours.
And 5 ml of ethanol was added, and it was made to dry with a rotary evaporator. 0.4 ml of distilled water and 0.4 ml of 2N NaOH were added thereto, gelatinized at 5 ° C. for 30 minutes, filtered through a 5 μm filter, and the filtrate was applied to a gel filtration column.

  The column used was one TSKgel toyopearl HW55S (300 × 20 mm) and three TSKgel toyopearl HW50S (300 × 20 mm) (both columns manufactured by TOSOH) connected in series, and the eluent was 0.2%. NaCl / 0.05N NaOH was used. 3 ml was collected per test tube and fractionated into 68, and λ max of the starch iodine complex of each fraction was determined. The amount of sugar was detected with an RI detector (TOSOH RI8020) connected to the column.

(Chain length distribution analysis)
The chain length distribution analysis will be described with reference to FIG.
The sample used was a powder obtained by removing outer inner cocoons and embryos from one ripe seed, pulverizing the endosperm with pliers, and further grinding with a plastic homogenizer (made by Greiner) in an Eppendorf tube.
5 ml of methanol was added to each and boiled for 10 minutes. Next, the mixture was centrifuged at 2,500 × g for 10 minutes, the supernatant was removed, and 5 ml of 90% methanol was added and washed twice.
Further, 15 μl of 5N sodium hydroxide was added to the precipitate and boiled for 5 minutes to gelatinize the starch.
The gelatinized solution was neutralized with 9.6 μl of glacial acetic acid, 1089 μl of distilled water, 100 μl of 0.6 M acetate buffer (pH 4.4), 15 μl of 2% sodium azide, P.I. 2 μl (about 210 units) of amyloderamosa isoamylase (EC 3.2.1.68, Hayashibara Biochemical Laboratories) was added, and the mixture was reacted at 37 ° C. for 8 hours or more while stirring with a stir bar.
Next, 2 μl of isoamylase was added and reacted for 8 hours or longer, followed by centrifugation at 10,000 × g at room temperature, and the supernatant was filtered through a deionization column (AG501-X8 (D), Bio-Rad).
Next, in order to fluorescently label the non-reducing end of the α-glucan chain, the sugar content in the sample was quantified by the method of Hizukuri et al. (See Carbohydrate Research, 94, 205-213 (1981)), and the reducing end corresponding to 5 nmol was removed. The α-glucan chain having the chain is dried by a centrifugal concentrator, and 2 μl of 1-aminopyrene-3,6,8-trisulfate (APTS) solution [2.5% APTS, 15% acetic acid], sodium borohydride 2 μl of the solution [1M sodium cyanoborohydride, 100% tetrahydrofuran] was added and reacted at 55 ° C. for 90 minutes.
At the time of analysis, it was diluted 12.5 times with distilled water. Chain length distribution analysis was performed using a capillary electrophoresis apparatus (P / ACE MDQ, Beckman Coulters).
Each peak area having a degree of glucose polymerization (DP) of 3 or more was quantified, and the ratio (Mole%) of each DP when the total peak area up to DP60 was 100% was calculated. Moreover, the graph of the difference ((DELTA) Mole%) which pulled the pattern of the wild type rice from each mutant rice was created.

  Note that the configuration and operation of the above-described embodiment are examples, and it is needless to say that the configuration and operation can be appropriately changed and executed without departing from the gist of the present invention.

  INDUSTRIAL APPLICABILITY The present invention can provide a rice mutant having reduced rice starch synthase type IIIa (SSIIIa) activity and rice branching enzyme type IIb type (BEIIb), and the starch of this rice mutant is rare in rice. It is a high amylose starch and can be used industrially.

Claims (16)

A rice mutant characterized in that the locus of rice starch synthase type IIIa (SSIIIa) and rice branching enzyme type IIb (BEIIb) are recessive homozygous and genetically fixed. Furthermore, the SSIIIa activity and BEIIb activity fell compared with the wild type, The rice variant of Claim 1 characterized by the above-mentioned. The seed weight is maintained at 80% or more and the agricultural traits are maintained as compared with a parent strain having a recessive locus of wild type, SSIIIa locus, or a recessive parent strain of BEIIb. The rice mutant of any one of 1-3. A method for producing starch, wherein the rice mutant according to any one of claims 1 to 3 is used. A method for producing starch, comprising using the rice mutant endosperm according to any one of claims 1 to 3. A starch produced by the method for producing starch according to claim 4 or 5. The shape is different from that of starch produced using wild-type rice, parental rice resulting from decreased activity of SSIIIa, or parental rice derived from decreased activity of BEIIb. starch. Contains starch grains that are different in size compared to starch produced using the wild-type rice, the parental rice resulting from the decreased activity of SSIIIa, or the parental rice derived from the decreased activity of BEIIb The starch according to claim 6 or 7, wherein the surface of the starch granules having different sizes is smooth. The amylopectin has a different chain length distribution compared to starch produced using the wild-type rice, the parental rice resulting from the decreased activity of SSIIIa, or the parental rice derived from the decreased activity of BEIIb. The starch according to any one of claims 6 to 8. Compared to starch produced using the wild-type rice, the parental rice resulting from the decreased activity of SSIIIa, or the parental rice derived from the decreased activity of BEIIb, the gelatinization viscosity characteristics are different. The starch according to any one of claims 6 to 9. The starch according to any one of claims 6 to 10, wherein the starch has an amylose ratio of 40% or more. The starch according to any one of claims 6 to 11, wherein the amylopectin of the starch is a B-type crystal. The starch according to any one of claims 6 to 12, wherein the starch has a low gelatinization viscosity pattern. The said starch is high in aging property compared with the starch manufactured using the parental line rice resulting from the activity fall of the said SSIIIa, or the parental line rice resulting from the activity fall of the said BEIIb. The starch of any one of thru | or 13. A method for producing a rice mutant, wherein the rice starch synthase type IIIa (SSIIIa) mutant and the rice branching enzyme type IIb (BEIIb) mutant are double recessive homozygote. A rice mutant produced by the method for producing a rice mutant according to claim 15.

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