JP2024106134A - Rice mutant with high resistant starch content, rice flour production method, resistant starch production method, rice flour gel production method, food production method, and method for producing rice mutant with high resistant starch content - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rice mutant with high resistant starch content and large seed weight, produced without using genetic engineering techniques.

SOLUTION: The present invention relates to a non-genetically engineered rice, the rice branching enzyme type IIb (BEIIb) being defective, the gene for granule-bound starch synthase GBSSI being a high-expression type derived from indica rice and the gene for starch synthase SSIIa being a low-activity type derived from japonica rice, which are genetically fixed, as well as the starch synthase SSIIIa being normally expressed. In these mutants, the average weight of a single grain of brown rice of the rice mutant is 25 mg or more. Furthermore, the raw rice flour of the mutant rice has a resistant starch content of 20% or more.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2024,JPO&INPIT

Description

Application for application of Article 30, Paragraph 2 of the Patent Act has been filed. Date of publication: June 4, 2022. Address of publication: https://doi.org/10.1186/s12284-022-00573-5

The present invention relates to a rice mutant with a high resistant starch content, a rice flour production method, a method for producing resistant starch, a rice flour gel production method, a food production method, and a method for creating a rice mutant with a high resistant starch content, and in particular to a rice mutant with a high resistant starch content that has a high seed weight and is high in resistant starch due to a specific combination of a branching enzyme and a starch synthase isoenzyme, a rice flour production method, a method for producing resistant starch, a rice flour gel production method, a food production method, and a method for creating a rice mutant with a high resistant starch content.

Starch is an insoluble storage polysaccharide unique to plants, and is used as a carbohydrate source by most organisms on Earth.
Starch as a chemical substance is a glucose polymer containing linear chains of glucose linked by α1,4 and branched structures linked by α1,6 glycosidic bonds.
Starch is also an aggregate of polymers consisting mainly of straight-chain amylose and amylopectin, which has a branched structure.

At least four types of enzymes are known to be involved in starch biosynthesis. These four types of enzymes are ADP glucose pyrophosphorylase (AGPase), which is a substrate supplying enzyme; starch synthase (SS), which extends α1,4 glucosidic bonds; branching enzyme (BE), which forms branched structures consisting of α1,6 glucosidic bonds; and debranching enzyme (DBE), which trims the extra branched structures added by BE to maintain the cluster structure that is characteristic of amylopectin. Each enzyme has many isozymes encoded by different genes. It is also believed that there are other enzymes involved in starch biosynthesis (see Non-Patent Document 1).

On the other hand, there are two types of starch: starch that is easily digested by alpha amylase, etc., and resistant starch that is difficult to digest. Resistant starch (hereinafter also referred to as "RS") is starch that is difficult to break down with human digestive juices, and passes through the small intestine as a polymer before reaching the large intestine. Resistant starch has a calorie-reducing effect because it is difficult to break down into glucose in the small intestine. Resistant starch can be found in large amounts in starches with a high amylose content. For this reason, high-amylose corn is used as a diet ingredient.

Patent Document 1 describes a rice mutant with a high resistant starch content that was produced without using genetic engineering techniques. In this mutant, the rice branching enzyme type IIb (BEIIb) gene locus is recessive homozygous, and a non-genetically modified rice mutant is obtained by introducing the SSIIa gene and GBSSI gene derived from indica rice by crossbreeding. Compared to the wild type, this rice mutant maintains 80% or more of its seed weight and agricultural traits. Furthermore, the rice seeds of this rice mutant contain resistant starch, and the resistant starch content of cooked rice is 10% to 30%, and the amylose ratio is 35% or more.

JP 2017-038588 A

Y. Nakamura, Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue. , Plant Cell Physiol. , Japan, Japanese Society of Plant Physiology, 2002, 43, p. 718-725 N. Fujita et al., Function and characterization of starch synthase I using mutants in rice., Plant Physiol., American Society of Plant Biologist, USA, 2006, 140, pp. 1070-1084 Y. Nakamura et al., Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between japonica and indica rice cultivars. , Plant Mol Biol. , 2005, 58, p. 213-227 N. Crofts et al., Lack of starch synthesis IIIa and high expression of granule-bound starch synthesis I synergistically increase the a Parent amylose content in rice endosperm. , Plant Sci, 2012, 193, 194, p. 62-69 A. Nishi et al., Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm., Plant Physiol., American Society of Plant Biologist, USA, 2001, 127, pp. 459-472

Here, there is a trade-off between the resistant starch content and the weight of rice seeds; in other words, lines with a high resistant starch content have smaller seeds.
Therefore, there has been a demand for the development of a rice mutant having a resistant starch content equal to or greater than that of the rice mutant having a high resistant starch content described in Patent Document 1, while also having a high seed weight.

The present invention was made in light of these circumstances, and aims to resolve the above-mentioned issues.

The rice mutant with high resistant starch content of the present invention is characterized in that it is deficient in rice branching enzyme type IIb (BEIIb), the gene for granule-bound starch synthase GBSSI is of a high-expression type derived from Indica rice, the gene for starch synthase SSIIa is of a low-activity type derived from Japonica rice, and starch synthase SSIIIa is normally expressed, is genetically fixed, and is a non-genetically modified organism.
The resistant starch-rich rice mutant of the present invention is characterized in that it is homozygous dominant for starch synthase SSIIIa.
The rice mutant with high resistant starch content of the present invention is characterized in that the average weight of one grain of brown rice of the rice mutant is 25 mg or more.
The rice mutant with high resistant starch content of the present invention is characterized in that the content of resistant starch in raw rice flour of the rice mutant is 20% or more.
The method for producing rice flour of the present invention is characterized in that rice flour is produced by powdering the endosperm of rice seeds of the above-mentioned rice mutant with high resistant starch content.
The method for producing resistant starch of the present invention is characterized in that resistant starch is extracted from cooked rice made from rice seeds of the rice mutant having high resistant starch content as described above and/or from rice flour made by powdering the rice seeds.
The method for producing rice flour gel of the present invention is characterized in that rice flour from the rice mutant with high resistant starch content is hydrated and heated to produce rice flour gel.
The method for producing a food product of the present invention is characterized in that the food product is produced using any one or any combination of rice seeds of the rice mutant with high resistant starch content, rice flour obtained by powdering the rice seeds, resistant starch extracted from the rice flour, and rice flour gel produced by adding water to and heating the rice flour.
The method for producing a rice mutant with high resistant starch content of the present invention is characterized in that, by a non-genetically modified method, the rice branching enzyme type IIb (BEIIb) is deficient, the gene for granule-bound starch synthase GBSSI is of a high-expression type derived from Indica rice, the starch synthase SSIIa is of a low-activity type derived from Japonica rice, and starch synthase SSIIIa is normally expressed and genetically fixed.

According to the present invention, a rice mutant can be provided that is deficient in rice branching enzyme type IIb (BEIIb), has a high expression type gene for granule-bound starch synthase GBSSI derived from indica rice, has a low activity type starch synthase SSIIa derived from japonica rice, and has normal expression of starch synthase SSIIIa, thereby providing a rice seed resistant starch content equal to or higher than that of conventional rice and a high seed weight.

1 is a photograph showing the results of Western blotting of proteins extracted from ripe endosperm according to an example of the present invention. 1 is a photograph showing the morphology of brown rice of each line according to an embodiment of the present invention. 1 is a graph showing (a) apparent amylose content and (b) ratio of short chains to long chains of amylopectin by gel filtration chromatography using debranched starch according to an embodiment of the present invention. 1 is a graph showing a comparison of the chain length distribution patterns of endosperm amylopectin of #1203 (BC ₃ ), Akita 63, Kasalath, and EM10 (BC ₃ ) according to an embodiment of the present invention. 1 is a graph showing a difference curve of the chain length distribution of #1203 (BC ₃ ) according to an embodiment of the present invention. 1 is a graph showing difference curves of chain length distributions of (a) #1203 (BC ₃ ) and (b) #1206 (BC ₃ ) according to an embodiment of the present invention. 1 is a graph showing a comparison of the chain length distribution patterns of endosperm amylopectin of #1206 (BC ₃ ), Akita 63, Kasalath, and #4019 (BC ₃ ) according to an embodiment of the present invention. 1 is a graph showing a difference curve between #1206 (BC ₃ ) and Akita No. 63 according to an embodiment of the present invention. 13 is a graph showing difference curves between (a) #1203 (BC ₃ ) and (b) #1206 (BC ₃ ) according to an embodiment of the present invention. 1 is a graph showing the RS values of rice treated in different ways according to an embodiment of the present invention.

<Embodiment>
Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
The present inventors aimed to develop and popularize functional rice that contributes to health, and created practical rice mutants with a high content of resistant starch (hereinafter referred to as "RS value") that is expected to suppress postprandial blood glucose rise and regulate the intestines. In order to achieve higher functionality with a small mixing ratio when producing processed foods, it was necessary to develop varieties with an even higher RS value.
Specifically, in a line of a rice mutant with a high RS value as described in Patent Document 1, the RS value and the rice seed weight were in a trade-off relationship. In other words, a line with a high RS value had smaller seeds and lower seed weight. The lower seed weight led to a decrease in yield and a decrease in rice milling efficiency. Therefore, there was a technical demand for the development of a line with both a high RS value and a high seed weight.

In response to this, the inventors have been conducting extensive research and testing on starch biosynthesis. In the process, they discovered that among the enzymes involved in starch biosynthesis, the RS value of seeds increases significantly when branching enzyme (BE) IIb is deficient. Furthermore, they have discovered that the starch structure and properties can be controlled by combining the deficiency and strong expression of enzymes involved in starch biosynthesis other than BEIIb. In the course of trial and error, they discovered that the combination of a deficient BEIIb, a highly expressed granule-bound starch synthase (GBSSI) derived from Indica rice, weak activity of starch synthase (SS) IIa, and normal activity of SSIIIa results in a high RS value and high seed weight, which led to the completion of the present invention.

That is, the rice mutant according to the embodiment of the present invention is characterized in that it is deficient in rice branching enzyme type IIb (BEIIb) ("be2b"; hereinafter, defective genotypes will be indicated by gene names in lowercase letters), the gene for starch granule-bound starch synthase GBSSI is a high-expression type derived from Indica rice ("GBSS1"; hereinafter, high-activity or high-expression genotypes will be indicated by gene names in uppercase letters), starch synthase SSIIa is a low-activity type derived from Japonica rice ("ss2a ^L "; hereinafter, low-activity genotypes will be indicated by a superscript " ^L "), SSIIIa is expressed normally, it is genetically fixed, and it is a non-genetically modified organism.

Here, when considering indica rice as the wild type (SS2a), the SSIIa gene of japonica rice (ss2a ^L ) has amino acid substitutions at four positions, which causes the expression of an enzyme with reduced activity. Therefore, in this embodiment, SS2a derived from indica rice is called "high activity type" and ss2a ^L derived from japonica rice is called "low activity type".
On the other hand, the GBSSI gene of japonica rice (GBSS1 ^{L ) has a mutation in the last base of intron 1, whereas the GBSSI gene of indica rice (GBSS1 L} ) has a mutation in the last base of intron 1, which results in low protein expression. Therefore, in this embodiment, GBSS1 derived from indica rice is called the "high expression type" and gbss1 ^L derived from japonica rice is called the "low expression type."
In addition, the SSIIIa gene is designated as ss3a if it is recessive and as SS3a if it is active (normally expressed, dominant homo).

Specifically, the rice mutant according to the present embodiment was obtained by crossing the SS3a be2b mutant with indica rice having GBSS1 as the mutant rice of the above combination (ss2a ^L SS3a GBSS1 be2b) in a non-recombinant (non-genetically modified) manner using techniques such as antibodies against specific enzymes, molecular markers, and starch trait identification, and was selected by molecular markers. After this, the super high-yielding rice variety "Akita 63", which is suitable for cultivation in the Akita region, was backcrossed three times. Finally, a lineage lacking BEIIb and having GBSSI derived from indica rice was established as a lineage with a high RS value and high seed weight. Specifically, as shown in the Examples described later, two lines, "#1203B-BC ₃ " (in the Examples, these are indicated in parentheses as "#1203B (BC ₃ )"), whose agronomic characteristics are similar to those of "Akita No. 63", were isolated. The #1203B-BC ₃ line had a high RS value and was about 1.35 times heavier in seed weight than the conventional rice mutant seeds with a high RS value described in Patent Document 1.
In this way, when using high-RS rice mutants with high RS values as processed foods, the rice mutants of this embodiment have the advantage of containing more RS and having better agricultural traits such as seed size and yield.

Here, Akita 63 is an elite rice variety with high yield, large grains, and early maturity. The enlargement of the seed size of Akita 63 is due to loss-of-function mutations in the GS3 and qSW5 genes that determine seed size. Furthermore, Akita 63 also exhibits excellent nitrogen absorption ability, and its yield is 150% of that of Akitakomachi, a commonly consumed Japonica rice variety under the same fertilization conditions. Therefore, in the rice mutant according to the present embodiment, sequential backcrossing using Akita 63 suppresses the release of fertilizer-derived nitrogen into the environment, leading to the development of environmentally friendly rice varieties.
Since the amylopectin structure and amylose content may be affected by the ambient temperature during seed ripening, the elite rice variety to be backcrossed when producing the rice mutant of this embodiment may be selected based on the growth environment, and may be selected to be a variety other than Akita 63. In this case, too, by using the same genotype (ss2a ^L SS3a GBSS1 be2b), it is possible to increase the RS value while retaining the characteristics of the elite rice variety.

In any case, if the activity of SSIIa is low and the expression of GBSSI is high in the seeds, the same effect is exhibited. Therefore, even if it is a mutant other than the above-mentioned #1203B-BC ₃ line, a person skilled in the art can create a line with a similar phenotype.
Furthermore, it goes without saying that a mutant in which BEIIb activity is zero due to a deletion will have a similar phenotype. There are no particular limitations on how the deletion is introduced into the BEIIb gene (e.g., base substitution), and the same effect can be obtained as long as the activity is zero.
Similarly, for other genes, the base substitutions and the like are not particularly limited, and it is sufficient that the activity is dominant or recessive to the extent that a similar effect is obtained with respect to the high or low activity.

In addition, the rice mutant according to the embodiment of the present invention is characterized in that SSIIIa is normally expressed, specifically, that SSIIIa is wild-type, dominant homozygous (SS3a).
Here, the above-mentioned #1203B-BC ₃ line is a wild-type dominant homozygous (SS3a) starch synthase SSIIIa, whereas the #1206-BC ₃ line, which is shown in the examples and lacks the SSIIIa gene, lacks SSIIIa (ss3a). When these lines were compared, the #1203B-BC ₃ line had a higher RS value and larger seeds than the #1206-BC ₃ line. In other words, it can be expected that the line in which the starch synthase SSIIIa gene is dominant homozygous will have a higher RS value and larger seed weight.

Furthermore, the rice mutant according to the embodiment of the present invention is characterized in that the average weight of one grain of brown rice of the rice mutant is 25 mg or more.
Specifically, the rice mutant according to the present embodiment showed a significant increase in seed weight for all lines after backcrossing with Akita 63. As shown in the Examples below, the seeds of the #1203B-BC ₃ lines were 150% heavier (28-29 mg per grain) than typical wild-type Japonica rice seeds (19-20 mg per grain).
Thus, the rice mutant according to the present embodiment has a larger seed weight than conventional rice mutants with a high RS value, which is a great advantage in terms of rice milling. In addition, the rice mutant according to the embodiment of the present invention can be backcrossed with a super high-yielding rice variety to impart the traits of the super high-yielding rice variety, resulting in a high yield, and the high productivity contributes to economic efficiency.
This leads to lower costs when acquiring RS and producing food, and therefore improves the practicality when actually producing food or providing it as cooked rice.

Furthermore, the rice mutant according to the embodiment of the present invention is characterized in that the RS value of the rice mutant in raw rice flour is 20% or more.
Thus, the rice mutant according to the present embodiment can increase the RS value even if the seed weight is large, such as the #1203B-BC ₃ line, compared to the rice mutant of Patent Document 1. Furthermore, the rice mutant according to the present embodiment also increases the RS value of rice flour and cooked rice. In fact, as shown in the examples described below, the RS value of wild-type Akita 63 was very small (0.4%), but in the #1203B-BC ₃ line according to the present embodiment, the RS value of raw rice flour was 20 to 29%, and the RS value of unmashed cooked rice was 30 to 35%, as shown in FIG. 6. In this embodiment, unless otherwise specified, the example of "rice flour" is "raw rice flour".
Furthermore, in the rice mutant according to the present embodiment, flowering occurs earlier than in the rice mutant of Patent Document 1 due to backcrossing. This is expected to increase the temperature during seed ripening, thereby ensuring an optimal temperature for RS biosynthesis. This is expected to change the properties of RS to more suitable ones and increase the RS value.

Note that the RS value varies greatly depending on the cooking method. In contrast, the mutant rice of this embodiment has RS values equal to or greater than those of the rice mutant described in Patent Document 1 in raw rice flour, rice flour gel (gelatinized rice flour), cooked rice, and mashed cooked rice (rice gel). Therefore, although the RS value in foods also varies depending on the cooking method, when the mutant rice of this embodiment is added as rice flour or refined starch to produce foods, it is possible to obtain the effect that a predetermined RS value can be achieved with a smaller amount of addition than conventionally. That is, even in this case, since the rice mutant of this embodiment has a large seed weight, the number of seeds added itself can be reduced, which means costs can be reduced.

Furthermore, the ss2a ^L SS3a GBSS1 be2b rice mutant according to the embodiment of the present invention is a genetically fixed line, and all seeds ripened by self-pollination from the seeds are homozygous, which allows for stable production.
Furthermore, since the rice mutant of the present embodiment is a non-genetically modified organism (non-recombinant organism), it can be cultivated in ordinary paddy fields and on a large scale, and as a result, it is possible to provide a rice mutant having an RS value that is highly practical as a cultivated crop.
Furthermore, by determining the agronomic traits of the ss2a ^L SS3a GBSS1 be2b rice mutant, such as the #1203B-BC _3- line rice mutant according to this embodiment, assessing its suitability for cultivar development, and cultivar development, it will be possible to establish a more agricultural production system.

In addition, starch produced from the endosperm of rice seeds according to an embodiment of the present invention is characterized in that, similar to the rice mutant seeds of Patent Document 1, the chain length distribution of amylopectin has a decrease in DP13 or less and an increase in DP14 or more.
Furthermore, the starch produced from the endosperm of the rice seeds according to the embodiment of the present invention is characterized by a gelatinization temperature that is at least 1°C higher, typically about 7°C higher, than starch produced using EM10, similar to the rice mutant seeds of Patent Document 1.

Furthermore, in the method for producing rice flour according to the embodiment of the present invention, it is possible to produce rice flour by powdering the endosperm of the rice seeds of the rice mutant according to the embodiment of the present invention. That is, the rice seeds of the rice mutant according to the embodiment of the present invention are characterized in that they can be used as rice flour by powdering them.
Specifically, the seeds of the rice mutant according to this embodiment (mutant rice) can be pulverized with a grinder or the like and used as rice flour. When this rice flour is used as raw rice flour, the RS value can be increased. That is, the method for producing rice flour according to this embodiment may be a method for producing raw rice flour.

Furthermore, the starch extracted from the mutant rice of this embodiment can itself be used as starch containing RS.
Furthermore, as a method for producing RS according to an embodiment of the present invention, it is also possible to extract RS itself from cooked rice made from rice seeds of the rice mutant according to this embodiment and/or from rice flour made by powdering the rice seeds.

Furthermore, as a method for producing rice flour gel according to an embodiment of the present invention, it is also possible to produce rice flour gel by adding water to rice flour from the rice mutant according to this embodiment and heating it. This rice flour gel can be produced and provided in a similar manner to the "gelatinized rice flour" in the examples described below. Furthermore, this rice flour gel also includes processed products such as mochi and dumplings, as well as the ingredients used for these.
In addition, instead of using rice flour itself, it is also possible to process rice gel by boiling water-added rice itself and processing it into a gel-like form by high-speed shear stirring. In this case, it can be produced in the same manner as the "mashed cooked rice" in the examples described later.

Furthermore, in the food production method according to this embodiment, it is possible to produce food containing any one or any combination of the following: rice seeds of the rice mutant according to this embodiment, raw rice flour powdered from the rice seeds, starch containing RS extracted from the raw rice flour, rice flour gel (gelatinized rice flour) produced by adding water to rice flour and heating it, cooked rice, and rice gel (mashed cooked rice).

Specifically, the mutant rice according to the present embodiment can be provided as functional rice either as brown rice or after polishing. Furthermore, rice flour of the mutant rice, as well as starch and rice flour gel containing RS, can be used by being mixed into foods.
More specifically, the rice seeds, rice flour produced from the rice seeds, and starch and rice flour gel containing RS of this embodiment can be used for polished rice, retort cooked rice, various other foods (bread, noodles, confectionery, etc.), gluten-free foods, supplements containing refined RS, etc. More specifically, the rice seeds, rice flour, starch and rice flour gel containing RS of this embodiment can be suitably used for foods such as pilaf or risotto, rice confectionery, and granola, and can produce foods that are characterized by taste, mouth feel, texture, chewing feeling, flavor, etc. That is, they can be used as a processing agent to add this characteristic to foods. For this reason, the rice seeds, rice flour, and starch and rice flour gel containing RS according to this embodiment can also be used as texture improvers.

Furthermore, the rice seeds, rice flour, starch containing RS, and rice flour gel according to the present embodiment can be used in various health foods, diet foods, and the like. In particular, as described below, brown rice with red skin, such as some strains of the #1203B-BC ₃ strain, contains a lot of polyphenols and is expected to have antioxidant activity, etc. For this reason, it can be used in food processing, health food manufacturers, hospitals, meals at elderly care facilities, bento boxes, beef bowl manufacturers, convenience stores, drug stores, and the like.

Furthermore, the mutant rice according to this embodiment and foods using the powder thereof can be used as rice-derived diet ingredients such as noodles, bread, pilaf, porridge, and rice crackers. Furthermore, by habitually consuming these foods, they are low in calories and are expected to be effective in preventing diabetes, colon cancer, and constipation by improving the large intestine environment.

In other words, the rice seeds, rice flour, starch containing RS, and rice flour gel according to the embodiment of the present invention have a higher amylose content than conventional EM10, and therefore exhibit different physical properties from EM10. Here, the rice seeds, rice flour produced from rice seeds, and starch containing RS and rice flour gel according to the present embodiment have high amylose content and high retrogradation properties due to the large number of long chains of amylopectin, and therefore can improve cooking properties in cooking methods where retrogradation properties are advantageous.

Here, RS is simply defined as starch that is not easily degraded by digestive enzymes, but increasing evidence indicates that RS structure differs among different mutant rice lines. Structural analysis of residual starch after digestive enzyme digestion suggests that fewer but longer amylopectin branches (DP>15) and higher amylose content correlate with higher resistance to digestion by digestive enzymes. This is because adjacent glucan chains form double helices, which, although incomplete, make the starch less digestible. The content of long glucan chains in residual starch is higher in gelatinized rice flour than in raw rice flour. This is because retrograded amylopectin is more likely to form double helices with nearby branches and amylose, making it less digestible by digestive enzymes.

One consideration in improving starch for health is that starch structure influences the rate of glucose release into the bloodstream. Thus, increasing the proportion of slow-digesting starch in addition to RS, as in the rice flour from seeds of the rice mutant according to the present embodiment, may be beneficial in preventing post-prandial blood sugar spikes. RS, which consists of residual glucan chains after digestion, acts like fiber in a prebiotic manner, promoting the production of short-chain fatty acids by the large intestine flora, and may have the effect of preventing colon cancer and allergies.

In addition, there are currently two strains of #1203B- _BC3 according to this embodiment, one of which has a red skin and is rich in polyphenols, so it is expected to have strong antioxidant activity when processed with brown rice. Therefore, when used with brown rice, it is possible to impart new functionality in addition to the functionality provided by RS.

Furthermore, the rice flour and starch of the rice mutant of this embodiment can be used for industrial purposes due to their high amylose content. This is because when molding is required, such as for biodegradable plastics, a high aging property is advantageous. For example, since amylose dissolves in warm water, it can be molded into a film and used for applications such as biodegradable films, medical materials, and sutures. Furthermore, its property of being absorbed and decomposed in the body can be used as a component for "scaffolding" in regenerative medicine.
Furthermore, it can be used for various industrial purposes, such as a material for making glass fiber, industrial glue, a compounding agent for building materials, and a material for plant cultivation.
In this way, the rice mutant according to this embodiment can be used industrially.

As described above, the method for producing rice mutants according to the embodiment of the present invention makes it possible to obtain rice mutants with high RS values and their production methods, as well as starch and rice flour gel with high RS values derived from the rice mutants and their production methods. Furthermore, the present invention can be used in food and beverage products and industrial products that utilize the starch.

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

[Experimental Methods and Materials]
(Plant material)
The rice (Oryza sativa L.) be2b single mutant, EM10 (ss2a ^L SS3a gbss1 ^L be2b), was isolated from an N-methyl-N-nitrosourea (MNU) mutagenized population of Kinmaze (ss2a ^L SS3a gbss1 ^L BE2b), a wild-type (WT) variety of Japonica rice, as described in Patent Document 1. The lower case letters indicating the genes of each of the above-mentioned lines indicate recessive, and the upper case letters indicate dominant. The " ^L " of each gene indicates a low activity type with low activity or expression level, and those without " ^L " indicate a normal or high activity or expression level (high expression type). For example, EM10 (ss2a ^L SS3a gbss1 ^L be2b) indicates that the SSIIa gene is of the low activity type derived from Japonica rice, SS3a indicates that the SSIIIa gene is of the dominant type, gbss1 ^L indicates that the GBSSI gene is recessive and of low expression type, and the be2b gene is recessive and has no expression. EM10 has a mutation in the last nucleotide of intron 9 of BEIIb, which inhibits splicing and results in no protein production.

The ss3a be2b mutant line #4019 (ss2a ^L ss3a gbss1 ^L be2b) was generated by crossing the ss3a mutant (e1, ss2a ^L ss3a gbss1 ^L BE2b), a Tos17 insertion mutant of the japonica rice cultivar Nipponbare, with the be2b mutant EM10.
Furthermore, the e1 strain has a Tos17 insertion in the first exon 1 of SSIIIa and does not produce the SSIIIa protein.

To introduce the SS2a and/or GBSS1 alleles, the be2b or ss3a be2b lines were crossed with an indica rice cultivar, Kasalath (SS2a SS3a GBSS1 BE2b), and named #1203 or #1206, respectively.
Next, the #1206 and #1206C (SS2a ss3a GBSS1 be2b) lines, which have high activity SSIIa and high expression levels of GBSSI derived from indica rice, were crossed with Akita 63 (ss2a ^L SS3a gbss1 ^L BE2b), an elite japonica rice variety with high yield. The resulting F ₁ seeds were grown and self-pollinated to obtain F ₂ seeds. DNA was isolated from the F ₂ seeds, and individuals showing patterns of SSIIa and GBSSI genes derived from indica rice varieties were selected using molecular markers, and those with the desired genotypes in the SSIIIa and BEIIb genotypes (i.e., the #1203 line has a dominant SSIIIa gene and a recessive BEIIb gene, and the #1206 line has recessive genes for both genes) were selected and continued to be cultivated.

In this manner, homozygous #1203A (BC ₃ ), #1203B (BC ₃ ), and #1203C (BC ₃ ) lines were isolated. For #1203B (BC ₃ ), two lines, #1203B (BC ₃ )-1 and #1203B (BC ₃ )-2, were used. For #1203C (BC ₃ ), two lines, #1203C (BC ₃ )-1 and #1203C (BC ₃ )-2, were used. For #1203C (BC ₃ ), two lines, #1206A (BC ₃ ), #1206B (BC 3 ), and #1206C (BC ₃ ), were also isolated. The genotypes of the lines used in this example are shown in Table 1 below. Furthermore, F ₃ seeds and F ₄ seeds obtained by repeated selfing were used for subsequent analysis. All rice lines were grown under natural light conditions during summer in the experimental paddy fields of Akita Prefectural University.

(DNA Extraction Method)
For the selection by the molecular markers described above, DNA was extracted from the seedlings. Specifically, about 2 cm of the leaf blade of the seedlings transplanted from the medium to the cell tray was transferred to a tube for a multi-beads shocker, placed in liquid nitrogen, frozen, and crushed with a multi-beads shocker (SPEED METER: 1600 rpm, ON TIME: 20 seconds, CYCLE: 1 time). 400 μL of DNA extraction buffer (200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5% SDS) was added, vortexed, and centrifuged at 15000 rpm and 20 ° C. for 5 minutes. 300 μL of the supernatant was transferred to a new 1.5 mL tube, 300 μL of isopropanol was added, mixed well, and left to stand for 15 minutes. Then, the mixture was centrifuged under the same conditions as above, and after confirming whether there was a semi-transparent precipitate at the bottom of the tube, 1 mL of 70% ethanol was added and stirred, and after centrifugation, the supernatant was removed. The remaining precipitate was dried under reduced pressure and suspended in 25 μL of TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA-2Na) on ice. The mixture was then frozen and stored at -30°C until use in an experiment.

(Western Blotting)
For Western blotting, each protein was fractionated according to the method described in US Pat. No. 5,399,633, with slight modifications.
Specifically, mature rice grains were ground into a fine powder, and soluble proteins and proteins loosely bound to the starch granule (hereafter referred to as "SP+LBP") were extracted using 20 volumes (w/v) of buffer containing 55 mM Tris-HCl (pH 6.8), 8 M urea, 2.3% (w/v) sodium dodecyl sulfate (SDS), 5% (v/v) β-mercaptoethanol, and 10% (w/v) glycerol. After centrifugation, half of the supernatant was added with 3xSDS-sample buffer containing 0.1M Tris-HCl (pH 6.8), 10% SDS, 12% β-mercaptoethanol, 20% glycerol, and 0.2% bromophenol blue, and boiled for 5 minutes. The remaining starch pellet obtained after extraction of SP+LBP was
The mixture was washed with 1 mL of a buffer containing 55 mM Tris-HCl (pH 6.8), 2.3% (w/v) sodium dodecyl sulfate (SDS), 5% (v/v) β-mercaptoethanol, and 10% (w/v) glycerol. Proteins tightly bound to starch (hereinafter referred to as "TBP") were extracted overnight at room temperature with 30 volumes (w/v) of a buffer containing 125 mM Tris-HCl (pH 6.8), 8 M urea, 4% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 0.05% (w/v) bromophenol blue. After centrifugation, the supernatant was used.

All protein samples were separated by SDS-polyacrylamide gel electrophoresis in a 7.5% acrylamide gel and blotted onto a membrane (PVDF membrane, MILLIPORE, IPVH0010). The membrane was shaken in 10 mL of 4% skim milk/TBS (10 mM Tris-HCl (pH 7.5), 500 mM NaCl) and blocked for 10 minutes. After blocking, anti-GBSSI (1:5000 dilution, Non-Patent Document 2), anti-SSIIa (1:1000 dilution, Non-Patent Document 3), anti-SSIIIa (1:1000 dilution, Non-Patent Document 4), and anti-BEIIb (1:5000, Non-Patent Document 5) were added and incubated overnight at 4°C.

The next day, the membrane was rinsed three times with approximately 50 mL of distilled water and washed once with approximately 50 mL of TBST2 (10 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.02% Tween 20). It was then immersed in 10 mL of a secondary antibody solution in which peroxidase-labeled secondary antibody (Bio Rad) was diluted 5,000-fold with 4% skim milk/TBS (10 mM Tris-HCl (pH 7.5), 500 mM NaCl) and allowed to slowly immerse for approximately 2 hours. The membrane was then rinsed three times with approximately 50 mL of distilled water, washed once with approximately 50 mL of TBST2, washed three times with TBS, and then immersed in fresh TBS for use in detection.

1 mL of ECL solution (Pierce West Pico Chromiluminescent substrate) was applied to the washed membrane to cause it to emit light, and the emitting bands were photographed using a Fuji LAS4000 (manufactured by Fujifilm Corporation). The image data was then edited using software: MultiGauge to determine the brightness and contrast of the detected bands.

(Observation of seed morphology)
The morphology of brown rice seeds was observed using a stereomicroscope (SZX7-ILST-0 (SP), manufactured by OLYMPUS) with light sources arranged above and below. Photographs were taken using a digital camera.

(Starch Refining Method)
Starch was purified from polished rice grains using the cold alkali steeping method (Yamamoto et al., Denpun Kagaku 28:241-244 (1981)).
10 g of brown rice was polished to 80%, and 100 mL of 0.1% NaOH was added thereto and allowed to stand overnight at 4°C.
The next day, the supernatant was discarded, and the mixture was ground in a mortar with 50 mL of 0.1% NaOH on ice, passed through a 100 μm mesh, and centrifuged at 3,000 g at 4° C. for 10 minutes.
150 mL of 0.1% NaOH was added to the precipitate, which was then shaken in ice for 3 hours and centrifuged at 3000 g and 4° C. for 10 minutes. The precipitate was suspended in 200 mL of 0.1% NaOH and allowed to stand overnight at 4° C. The next day, the supernatant was discarded, the precipitate was suspended in distilled water, and neutralized with 1N hydrochloric acid. The precipitate was then washed five times with distilled water, dried, and powdered in a mortar.

Starch debranching and gel filtration
0.25 mL of distilled water was added to 5 mg of purified starch and mixed, and 0.25 mL of 2N NaOH was added thereto, followed by gelatinization at 37° C. for 2 hours.
3.65 mL of distilled water was added to the mixture, and 95 μL of 5N HCl was added to neutralize the mixture. Next, 1.5 mL of 100 mM acetate buffer (pH 3.5) was added, and 12.5 μL (approximately 875 units) of Pseudomonas isoamylase (EC 3.2.1.68, manufactured by Hayashibara Biochemical Laboratories) was added, and the mixture was reacted at 37° C. for 24 hours with shaking.
Then, 5 mL of ethanol was added and the mixture was dried using a rotary evaporator, 0.4 mL of distilled water and 0.4 mL of 2N NaOH were added thereto, and the mixture was gelatinized at 4° C. for 30 minutes. The mixture was then filtered through a 5 μm filter, and the filtrate was applied to a gel filtration column.

The columns used were one TSKgel Toyopearl HW55S (300 x 20 mm) and three TSKgel Toyopearl HW50S (300 x 20 mm) (both columns manufactured by Tosoh Corporation) connected in series, and the eluent used was 0.2% NaCl/0.05N NaOH. The amount of sugar was detected with an RI detector (RI8020, manufactured by Tosoh Corporation) connected to the columns.

(Amylose content)
Starch was debranched using Pseudomonas isoamylase (Hayashibara Biochemical Laboratories) and analyzed by gel filtration chromatography (Toyopearl HW-55S and HW-50S x 3, Tosoh Corporation). The peaks of amylose (Fraction I), long amylopectin chains (Fraction II), and short amylopectin chains (Fraction III) were quantified in the same manner as described in Patent Document 1.

(Analysis of chain length distribution of amylopectin)
Starch was extracted from mature rice seeds and debranched using Pseudomonas isoamylase (Hayashibara Biochemical Laboratories, Inc.), and the debranched starch was then fluorescently labeled and analyzed by capillary electrophoresis (P/ACE MDQ Plus Carbohydrate System, AB Sciex, Inc.).

Specifically, the sample was prepared by removing the lemma and embryo from one fully ripe seed, crushing the endosperm with pliers, and then further grinding the powder in an Eppendorf tube using a plastic homogenizer (Greiner).
Each was added with 5 mL of methanol, boiled for 10 minutes, centrifuged at 2500 g for 10 minutes, the supernatant was removed, and each was washed twice with 5 mL of 90% methanol.
Furthermore, 300 μL of 0.3 N NaOH was added to the precipitate, and the mixture was boiled for 5 minutes to gelatinize the starch.
The gelatinized liquid was neutralized with 19 μL of glacial acetic acid, and then 1100 μL of distilled water, 100 μL of 0.6 M acetate buffer (pH 4.4), 15 μL of 2% sodium azide, and 2 μL (approximately 210 units) of Pseudomonas isoamylase (EC 3.2.1.68, Hayashibara Biochemical Laboratories) were added, and the mixture was allowed to react at 37° C. for more than 8 hours while stirring with a stirrer bar.

Next, 2 μL of isoamylase was added and reacted for more than 8 hours, after which the mixture was boiled for 15 minutes and centrifuged at 10,000 g at room temperature. Deionizing resin (AG501-X8(D), Bio-Rad) was added to the supernatant, which was then swirled for 30 minutes and deionized.
Next, in order to fluorescently label the non-reducing ends of the α-glucan chains, the sugar content in the sample was quantified, and the α-glucan chains having reducing ends equivalent to 5 to 10 nmol were dried using a centrifugal concentrator, and 2 μL of 1-aminopyrene-3,6,8-trisulfate (APTS) solution (2.5% APTS, 15% acetic acid) and 2 μL of sodium borocyanide solution (1 M sodium borocyanide) were added, followed by reaction for 90 minutes at 55° C. The reaction was stopped by adding 46 μL of distilled water.
For analysis, the solution was diluted 20-fold with distilled water. Chain length distribution analysis was performed using a capillary electrophoresis apparatus (P/ACE MDQ, manufactured by AB Sciex).
The area of each peak with a degree of glucose polymerization (DP) of 5 or more was quantified, and the percentage (Molar%) of each DP was calculated when the total area of the peaks up to DP 70 was taken as 100%. In addition, a graph was created showing the difference (ΔMolar%) between the pattern of each mutant rice and that of wild-type rice.

(Measurement of RS value)
The RS content of the samples in various forms (raw rice flour, gelatinized rice flour, unmashed cooked rice, and mashed cooked rice) was determined similarly to US Pat. No. 5,399,633.
First, the rice cooking method will be described. Approximately 100 mg of polished rice (5-8 grains) was weighed into a 15 mL centrifuge tube with a lid, and the sample was washed twice in the centrifuge tube using a Pasteur pipette. Distilled water was added so that the water content in the centrifuge tube was 1.5 times the weight of the polished rice ± 0.3 mg. In other words, the total weight in the centrifuge tube was 2.5 times the weight of the polished rice. The water on the side of the centrifuge tube was removed using a centrifuge. Approximately 250 mL of tap water was placed in the rice cooker of a 5-go rice cooker, a microtube stand and a glass petri dish (diameter 12 cm) were placed on top of them, and the 15 mL centrifuge tube containing the sample was placed diagonally on top of them, and the rice was cooked in the 5-go rice cooker.
Next, the RS content was measured using an RS Assay Kit (hereinafter referred to as "RS measurement kit") sold by Megazyme.

The method for preparing pancreatin α-amylase (10 mg/mL) + amyloglucosidase (3 U/mL) for measurement with the RS measurement kit is as follows.
50 mL of 0.1 M sodium malate buffer (pH 6.0) and 0.5 g of porcine pancreatic α-amylase included in the RS measurement kit were dissolved in a 50 mL centrifuge tube and stirred on ice for 5 minutes with a shaker. 0.5 mL of amyloglucosidase (AMG) solution diluted to 300 U/mL was added and stirred, then centrifuged at 3000 rpm, 25°C, for 10 minutes, and the supernatant was transferred to a new 50 mL centrifuge tube. This reagent was changed to the same amount as needed and prepared each time.

4 mL of the prepared pancreatin α-amylase (10 mg/mL) + AMG (3 U/mL) was added to each cooked rice and vigorously shaken at 164 rpm and 37°C for 16 hours using a thermostatic shaker (MH-10, manufactured by TAITEC). 4 mL of 100% ethanol was added and stirred, then centrifuged at 3000 rpm, 25°C, and 10 minutes, and the supernatant was quickly collected in a 50 mL centrifuge tube. 2 mL of 50% ethanol was added to the precipitate and stirred, and then 8 mL of 50% ethanol was added again to make the total volume 10 mL and stirred. Centrifuged at 3000 rpm, 25°C, and 10 minutes, and the supernatant was quickly transferred to the above 50 mL centrifuge tube. This process was repeated to separate the precipitate (the resistant starch contained therein is referred to as rs) and the supernatant (the amount of solubilized starch contained therein is referred to as non-rs). The centrifuge used in this series of operations was an Allegra X-30R (manufactured by BECKMAN), and the buckets of a swing rotor (SX4400) were used.

The supernatant remaining in the precipitate was removed as much as possible with a pipette, and 2 mL of 2M KOH was added while crushing the rs in the 15mL centrifuge tube with a plastic pestle. A stir bar was added and the mixture was stirred in ice for 20 minutes. 8 mL of 1.2M acetate buffer (pH 3.8) and 0.1 mL of AMG (3300 U/mL) were added to decompose the rs into glucose. The mixture was heated at 50°C for 30 minutes while stirring with a vortex once every 5 minutes, and centrifuged at 3000 rpm, 25°C, and 10 minutes.
The centrifuge tube was filled up to 11 mL with distilled water for rs decomposed to glucose, and 30 mL for non-rs. The rs was a sample with a high content of resistant starch (more than 15% resistant starch), and the non-rs was diluted 10-fold with 0.1 M sodium acetate buffer (pH 4.5). 10 μL of each dissolution solution and dilution solution and 150 μL of glucose measurement reagent (GOPOD solution) were applied to each well of a 96-well microplate, two holes each. In addition, a glucose solution standard solution (mg/mL) for creating a calibration curve was applied to another well, followed by 0.1 M sodium acetate buffer (pH 4.5) and glucose measurement reagent (GOPOD solution). The microplate was covered with a sheet and reacted in a hybrid oven (TAITEC HB-80) at 50° C. for 20 minutes. After the reaction, the absorbance at 510 nm was measured using a microplate reader.
A calibration curve was drawn from the measured values of the glucose solution, the glucose concentration (mg/mL) of each sample was determined, and the amounts of rs and non-rs in 100 mg of polished rice were calculated. The RS value (%) was calculated using the following formula (1).

RS value (%) = rs (mg) / [rs (mg) + non-rs (mg)] ... Formula (1)

(Measurement of gelatinization temperature)
Approximately 3 mg of starch dried at 105° C. for 2 hours was mixed with 9 μL of distilled water, and the temperature was raised from 5° C. to 200° C. at a heating rate of 3° C./min. The differential scanning calorimetry (DSC) was measured using a DSC6100 (manufactured by Seiko Instruments Inc.).
Then, the gelatinization start temperature, gelatinization peak temperature, gelatinization end temperature, and gelatinization heat were calculated using the application software of the same model.

(Data Analysis)
Seed weight, amylose content, thermal pasting properties, and RS value were statistically analyzed by the Tukey-Kramer method (P<0.05).

〔result〕
(With or without SS and BE)
In order to develop rice lines with high RS values and large seed weights, we produced all combinations of SSIIa genotypes, whether they were high activity (SS2a) or low activity (ss2a ^L ), GBSSI genotypes, whether they were high expression (GBSS1) or low expression (gbss1 ^L ), and SSIIIa and BEIIb genotypes, whether they were dominant (SS3a and BE2b, respectively) or recessive (ss3a and be2b, respectively), and isolated lines by backcrossing these three times with Akita 63, a super high-yielding rice variety.
The genotypes of all the lines used in this example and their grain weight-related data are shown in Table 1 below.

Specifically, Table 1 shows the line, genotype, grain weight, mean ± standard error % (Mean ± SE), and rate of increase after backcrossing (%).
In the table, the grain weight is the weight (mg) of one brown rice grain for each rice line, and is shown as the mean ± standard error (SE, n = 20) and as a percentage of the seed weight of the wild-type (WT) variety Kinmaze. Different lowercase letters indicate significant differences between different lines (P < 0.05, Tukey-Kramer method).
The increase rate indicates the increase rate of seed weight after backcrossing relative to the seed weight before backcrossing. When multiple lines with the same genotype were available, the average seed weight was used.

Of these backcross lines, #1203A (BC ₃ ) was SS2a SS3a gbss1 ^L be2b. #1203B (BC ₃ ) was ss2a ^L SS3a GBSS1 be2b. #1203C (BC ₃ ) was SS2a SS3a GBSS1 be2b. In addition, the two lines of #1203B (BC ₃ ) were named #1203B (BC ₃ )-1 and #1203B (BC ₃ )-2, and the two lines of #1203C (BC ₃ ) were named #1203C (BC ₃ )-1 and #1203C (BC ₃ )-2.
Backcross line #1206A ( _BC3 ) was SS2a ss3a gbss1 ^L be2b. #1206B ( _BC3 ) was ss2a ^L ss3a GBSS1 be2b. #1206C ( _BC3 ) was SS2a ss3a GBSS1 be2b.

FIG. 1 shows the results of Western blotting analysis of the starch biosynthetic enzymes SSIIa, SSIIIa, GBSSI, and BEIIb using an extract from this ripe endosperm.
FIG. 1(a) shows the results for soluble proteins and proteins loosely bound to starch (SP+LBP), and FIG. 1(b) shows the results for proteins tightly bound to starch (TBP).

SSIIIa was shown to be absent in e1, #4019, and all three #1206( _BC3 ) lines. Similarly, Western blotting using anti-BEIIb antibody confirmed the absence of BEIIb in EM10, #4019, and all #1203( _BC3 ) and #1206( _BC3 ) lines.

GBSSI and active SSIIa are known to be tightly bound to starch. Western blotting of TBP using anti-SSIIa antibody revealed that SSIIa was more abundant in rice lines with SS2a genotype (i.e., active SSIIa) such as wild-type Kasalath, #1203A (BC ₃ ), #1203C (BC ₃ ), #1206A (BC ₃ ), and #1206C (BC ₃ ) than in lines with ss2a ^L genotype such as Akita 63, e1, #1203B (BC ₃ ), and #1206B (BC ₃ ). The amount of SSIIa protein in the TBP fraction of EM10 and #4019 was also increased in the absence of BEIIb.

GBSSI was enriched in the TBP fraction. Lines carrying the GBSS1 genotype, such as Kasalath, #1203B (BC ₃ ), #1203C (BC ₃ ), #1206B (BC ₃ ), and #1206C (BC ₃ ), contained higher levels of GBSSI protein than lines carrying the gbss1 ^L genotype, such as Akita 63, EM10, e1, #4019, #1203A (BC ₃ ), and #1206A (BC ₃ ).
These results confirmed that all strains used in this example had the correct genotype and showed the expected patterns of SSIIa, SSIIIa, GBSSI, and BEIIb protein abundance.

(Seed morphology and seed weight)
Figure 2 shows the morphology of fully ripe brown rice (molted mature seeds) of each rice line. For each line, the upper image is a photograph taken with light shining on the fully ripe brown rice from above, and the lower image is a photograph taken with light shining on the fully ripe brown rice from below. The scale bar is 5 mm. If the brown rice is cloudy, it will look black when light is shone on it from below.

Seeds of all rice lines lacking BEIIb were opaque, whereas seeds of WT, Akita 63, and Kasalath were translucent. Seeds of #1203B(BC ₃ )-2 and #1203C(BC ₃ )-2 showed red pericarp, a trait inherited from Kasalath. All BC 3 lines formed large short grains, except for Kasalath, which produced long grains, and #1203A(BC ₃ ), whose seeds were smaller and sometimes flatter than those of the _other lines.

As shown in Table 1 above, the seed weight of Akita 63 was large (28 mg per kernel), 149% of that of Kinnamifu, a typical Japonica rice variety (19 mg per kernel). The seed weights of all the RS-rich lines after backcrossing were significantly improved compared to the RS-rich lines before backcrossing, increasing by about 132-190% (EM10: 181%, #4019: 134%, #1203A: 135%, #1203B: up to 190%, #1203C: 153%, #1206A: 145%, #1206B: 132%, and #1206C: 145%). That is, the seed weight of the #1203B line was almost 25 mg or more.

The seed weights of almost all lines after backcrossing, except #1203A (BC ₃ ), were equal to or heavier than that of Kin-nampu, a typical japonica rice. #1203B (BC ₃ ) line produced the largest seeds among all backcross lines, with an average weight per seed of 28-29 mg, almost the same as that of Akita 63. #1203A's seed weight averaged 12.7 mg per seed. Compared to before backcrossing, the seed weight increased by 135% after backcrossing, which was the lowest among all backcross lines. These results indicate that backcrossing successfully increased the seed weight of the lines with high RS content. This was probably caused by the inheritance of loci involved not only in large seed size but also in early flowering, which ensures optimal temperature during starch synthesis. The backcross lines flowered in early August, about 10-30 days earlier than the flowering time of the lines before backcrossing. Early flowering was accompanied by an increase in the cumulative temperature from 5 to 30 days after flowering (DAF), when endosperm starch biosynthesis peaks. The cumulative temperature after backcrossing was 680-693°C, whereas the cumulative temperature before backcrossing was 452-640°C.

(Amylose content and ratio of short to long chains of amylopectin)
The expression level of GBSSI is affected by temperature during seed development. High temperatures during seed development impair GBSSI RNA splicing, leading to reduced transcription of mature GBSSI mRNA, resulting in reduced GBSSI protein levels and reduced amylose content. Since high amylose content is known to contribute to increased RS values, the apparent amylose content of the backcrossed high RS lines was analyzed by gel filtration chromatography.
Specifically, debranched starch was used and compared with that of rice lines containing different combinations of starch biosynthetic enzymes before and after backcrossing.

FIG. 3 shows the results of the apparent amylose content measured by gel filtration chromatography using debranched starch and the ratio of short chains to long chains of amylopectin.
The gel filtration chromatography pattern of debranched starch is divided into three peaks, the first peak detected (Fr. I, fraction I) is apparent amylose, the second peak detected (Fr. II, fraction II) is a long chain of amylopectin longer than the B2 chain that connects the clusters, and the third peak detected (Fr. III, fraction III) is a short chain within the amylopectin cluster. That is, in this gel filtration chromatography, fraction I contains amylose and very long chains of amylopectin, but mainly contains amylose. Fraction II contains long chains of amylopectin. Fraction III contains short chains of amylopectin. Figure 3 shows the numerical values of these.

FIG. 3(a) shows the apparent amylose content (fraction I).
Figure 3(b) shows the ratio of short to long amylopectin chains (fraction III/fraction II), i.e. the ratio of short to long amylopectin chains calculated by dividing the value of fraction III by the value of fraction II.
In all cases, different lowercase letters indicate significant differences (P<0.05; Tukey-Kramer method).

As a result, the apparent amylose content of ss3a be2b lines, such as #4019 and #1206, was higher than that of be2b lines, such as EM10 and #1203, both before and after backcrossing. The ratio of short to long amylopectin chains in be2b lines, such as EM10 and #1203, was lower (0.8-1.2) than that of ss3a be2b lines, such as #4019 and #1206 (1.1-1.5), regardless of backcrossing. These results indicate that #1203 contains longer amylopectin chains than #1206.
Among the be2b lines, the amylose content (35-39%) of lines with the GBSS1 genotype, such as #1203B and #1203C, was higher than that of gbss1 ^L lines, such as EM10 (20-27%) and #1203A (15-16%). The apparent amylose content of #1203A was lower than that of EM10, regardless of backcrossing.

The apparent amylose content of all ss3a be2b lines was high, and no significant differences were observed between lines carrying the GBSS1 or gbss1 ^L alleles. This is likely because the absence of SSIIIa increases AGPase activity, and the increased levels of ADP-glucose, a substrate for starch synthase (SS) including GBSSI, promote amylose biosynthesis even at low expression levels of GBSSI. Furthermore, high expression levels of GBSSI may not be able to further increase the apparent amylose content due to the limiting amount of ADP-glucose.
The apparent amylose content of almost all lines decreased slightly after backcrossing, probably due to high temperatures during seed development.

(Amylopectin branch structure)
Next, to reveal the detailed amylopectin branching structure, the starch was debranched and analyzed by capillary electrophoresis.

Figure 4A shows the comparison of the chain length distribution patterns of endosperm amylopectin of #1203 (BC ₃ ), Akita 63, Kasalath, and EM10 (BC ₃ ). Figure 4A (a) shows the chain length distribution patterns of #1203A (BC ₃ ), (b) shows the chain length distribution patterns of #1203B (BC ₃ ), and (c) shows the chain length distribution patterns of #1203C (BC ₃ ). Each graph shows a typical pattern obtained from at least three trials.

As a result, the peak amylopectin chain lengths of EM10 (BC ₃ ) and #1203 (BC ₃ ) lines lacking BEIIb increased by 3 glucose chains from DP11 to DP14 compared to the chain length distribution pattern of Akita 63. Also, the heights of these peaks were significantly lower than those of Akita 63. This indicates that the loss of BEIIb reduced the number of short chains and increased the number of long chains in EM10 (BC ₃ ) and #1203 (BC ₃ ) lines compared to the distribution of Akita 63. The chain length distribution pattern of #1203 (BC ₃ ) line was essentially the same as before backcrossing, with only a slight increase (DP<20).

Here, the differences in the chain length distribution patterns between #1203 (BC ₃ ) and Akita No. 63 were examined in more detail.
To assess the impact of BEIIb loss, difference curves were created by subtracting the values for Akita 63 from the values for the #1203 (BC ₃ ) system, as shown in Figure 4B. Figure 4B (a) shows the results for "#1203A (BC ₃ ) - Akita 63", (b) for "#1203B (BC ₃ ) - Akita 63", and (c) for "#1203C (BC ₃ ) - Akita 63".
As a result, the number of short chains (DP<13) was significantly decreased, whereas the number of long chains (DP>15) was increased in the #1203 (BC ₃ ) lineage compared with that in Akita 63.

Figure 4C(a) shows the difference curves generated to investigate the effect of active SSIIa on amylopectin structure in the be2b genotype, comparing #1203A(BC ₃ )-EM10(BC ₃ ) and #1203C(BC ₃ )-#1203B(BC ₃ ) with Kasalath-Akita 63.

The chain length distribution pattern of Kasalath was shifted to the larger side by two glucose chains compared to Akita 63, and the difference curve obtained by subtracting the values of Akita 63 from those of Kasalath showed that Akita 63 had more short chains than Kasalath, which was consistent with previous results. This is because active SSIIa functions to extend amylopectin branches from DP6-12 to DP12-24.

Thus, the reduction rate of short chains for Akita No. 63 was greater for #1203A (BC ₃ ) and #1203C (BC ₃ ) than for #1203B (BC ₃ ), indicating that active SSIIa utilizes short chains and produces longer chains.

As a result, the lines with active SSIIa contained fewer short amylopectin branches (DP ≤ 12) and longer amylopectin branches (13 < DP < 30), indicating that active SSIIa synthesizes longer amylopectin branches (13 < DP < 30) from shorter amylopectin chains (DP ≤ 12). However, the absolute value of the mole percentage was much smaller in the absence of BEIIb compared to Kasalath-Akita 63. This was probably due to the lack of short branches due to the loss of BEIIb, which acted as a primer for SSIIa.

Next, a comparison of the chain length distribution patterns of endosperm amylopectin between #1206 line (BC ₃ ) and Akita 63 line, Kasalath line, and #4019 line (BC ₃ ) will be described with reference to Figure 5A. Figure 5(a) shows the chain length distribution patterns of #1206A (BC ₃ ), (b) #1206B (BC ₃ ), and (c) #1206C (BC ₃ ). Each graph shows a typical pattern obtained from the results of at least three trials.

As a result, the chain length distribution patterns of #4019 (BC ₃ ) and #1206 (BC ₃ ) strains lacking SSIIIa and BEIIb were also shifted from DP11 to DP12 or 14, approximately two glucose chains larger, compared with Akita 63.
Lines #4019 (BC ₃ ) and #1206 (BC ₃ ) showed a higher peak at DP 14 and, in contrast to #1203 (BC ₃ ), a characteristic decline at DP 19. The chain length distribution pattern of line #1206 (BC ₃ ) was almost the same as before backcrossing, with only a slight increase (DP < 20) detected.

Figure 5B shows difference curves generated by subtracting the values for Akita 63 from the values for the #1206 (BC ₃ ) line to determine the effect of deletion of SSIIIa and BEIIb. Figure 5B (a) shows the difference curves for #1206A (BC ₃ ), (b) for #1206B (BC ₃ ), and (c) for #1206 (BC ₃ ).
As a result, the number of short chains (DP ≤ 12) was significantly decreased, and the number of long chains (DP > 15) was increased in the #1206 (BC ₃ ) lineage compared with that in Akita 63. The rate of decrease in short chains was greater in the #1206A (BC ₃ ) and #1206C (BC ₃ ) lines than in the #1206B (BC 3 ) lineage compared with _that in Akita 63, indicating that active SSIIa utilizes short chains and produces longer chains.

Next, the effect of active SSIIa on the amylopectin structure of the ss3a be2b genotype was determined.
Referring again to FIG. 4C, FIG. 4C(b) shows the difference curves of "#1206A (BC ₃ ) - #4019BC ₃ " and "#1206C (BC ₃ ) - #1206B (BC ₃ )" created and compared with "Kasarath - Akita No. 63".
As a result, the lines with active SSIIa contained fewer shorter amylopectin branches (DP ≤ 12) and more longer amylopectin branches (15 < DP < 30), and the effect of active SSIIa in these lines was milder than that in Kasalath but stronger than that in #1203 (BC ₃ ).

Figure 5C shows difference curves were generated to compare the effect of the GBSS1 or gbss1 ^L allele in the absence of BEIIb alone or both BEIIb and SSIIIa, and shows that in strains carrying GBSS1 but lacking BEIIb, #1203B(BC ₃ )-EM10(BC ₃ ) and #1203C(BC ₃ )-#1203A(BC ₃ ) had more amylopectin chains with 8<DP<20 and less amylopectin chains with DP>20. In contrast, the absolute values for "#1206B(BC ₃ )-#4019(BC ₃ )" and "#1206C(BC ₃ )-#1206A(BC ₃ )" were small, suggesting that GBSSI protein levels did not affect amylopectin structure even in the absence of SSIIIa and BEIIb.

(Thermal gelatinization properties of starch)
Differences in amylopectin branch length are strongly correlated with starch gelatinization temperature and susceptibility to retrogradation, both of which are affected by temperature during seed development. Because retrograded starch also functions as RS, the thermal gelatinization properties of purified starches were analyzed by differential scanning calorimetry and compared between lines containing different combinations of starch biosynthetic genes before and after backcrossing.
The thermal properties of the starches analyzed by differential scanning calorimetry for each series are shown in Table 2 below:

For each line and genotype in Table 2, T _O (°C) is the gelatinization onset temperature, T _P (°C) is the peak gelatinization temperature, T _C (°C) is the final gelatinization temperature, and ΔH (J/g) is the heat of gelatinization of starch. Data are shown as mean ± SE (n=3). Different lowercase letters indicate significant differences between rice genotypes (P<0.05, Tukey-Kramer method).

As a result, the lack of BEIIb in EM10 (BC ₃ ) and the presence of the SS2a allele of Kasalath resulted in a higher gelatinization temperature than Akita 63. The peak gelatinization temperatures of EM10 (BC ₃ ) and #1203 (BC ₃ ) were approximately 7°C higher than those of #4019 (BC ₃ ) and #1206 (BC ₃ ). This is probably because EM10 (BC ₃ ) and #1203 (BC ₃ ) lines had fewer short amylopectin chains (DP≦12) and more long amylopectin chains (DP>13) than those of #4019 (BC ₃ ) and #1206 (BC ₃ ). The gelatinization peak temperature of #1203A (BC ₃ ) was about 3°C higher than that of EM10 (BC ₃ ) and other #1203 (BC ₃ ) lines. On the other hand, the gelatinization peak temperature of #1206B (BC ₃ ) was about 3°C lower than that of other #1206 (BC ₃ ) lines. These results are considered to be due to the fact that the amount of amylopectin chains with DP>13 was higher in #1203A (BC ₃ ) than in #1203B (BC ₃ ) or #1203C (BC ₃ ). In addition, the amount of amylopectin chains with DP>13 was lower in #1206B (BC ₃ ) than in #1206A (BC ₃ ) or #1206C (BC ₃ ). These results suggest that the increase in the gelatinization temperature was due to the increased abundance of amylopectin chains with a DP of 13 < 25. The peak gelatinization temperatures of starches in all lines increased by 2–4°C after backcrossing, which is likely due to the higher ripening temperature resulting from the earlier flowering time.

(RS value)
The RS value of rice grains varies significantly depending on the cooking method, which alters the accessibility of starch to digestive enzymes, so we measured the RS value in total starch using raw rice flour, gelatinized rice flour, unmilled cooked rice, and mashed cooked rice.

Figure 6 shows the results of the RS values of rice processed in different ways. Figure 6 (a) shows the RS values of raw rice flour, (b) gelatinized rice flour, (c) unmashed cooked rice, and (d) mashed cooked rice. Data are shown as mean ± standard error (SE, n = 3). Different lowercase letters indicate significant differences between the lines (P < 0.05, Tukey-Kramer method).

Overall, the RS values of high RS lines, such as EM10 (BC ₃ ), #4019 (BC ₃ ), #1203 (BC ₃ ), and #1206 (BC ₃ ), were significantly higher (up to 290-fold) than Akita 63, regardless of the treatment method. The RS values decreased when rice was gelatinized and mashed. The RS values of raw rice flour (5-29%) and unmashed cooked rice (15-35%) were higher than those of gelatinized rice flour (1-11%) and mashed cooked rice (3-15%), respectively.
More specifically, the RS value of raw rice flour #1203B (BC ₃ ) was 20% or more.
On the other hand, the RS value of Akita 63 was negligible (<0.7%) and Kasalath was also low (<2.7%), regardless of the processing method.

The RS values of raw rice flour samples #1203A (BC ₃ ) and #1206A (BC ₃ ), which had active SSIIa, were higher than those of samples EM10 (BC ₃ ) and #4019 (BC ₃ ), which had low active SSIIa, respectively. The RS value of raw rice flour #1206C (BC ₃ ), which had high GBSSI activity, was also higher than that of #1206A (BC ₃ ), which had low GBSSI activity. These results suggest that the RS value of raw rice flour increases with increasing long-chain amylopectin and amylose content. When rice flour was gelatinized or cooked rice was mashed, the RS values of EM10 (BC ₃ ) and #1203A (BC ₃ ) were significantly lower than those of #1203B (BC ₃ ) and #1203C (BC ₃ ). Also, the RS values of gelatinized rice flour and mashed cooked rice of #4019 (BC ₃ ) were lower than those of the other #1206 (BC ₃ ) lines. In the absence of BEIIb, it can be speculated that amylose and long amylopectin branches interact to form incomplete helices during retrogradation, preventing degradation by digestive enzymes and functioning as RS types "RS2" and "RS3". In contrast, the number of long amylopectin chains is less in the line lacking both SSIIIa and BEIIb compared to the line lacking only BEIIb, making it easier to digest. Among all the treatments tested, the RS of unmashed cooked rice showed not only RS1 but also RS2 and RS3 aspects, so that unmashed cooked rice showed the highest RS value regardless of genotype. The RS values of the unground cooked rice samples such as EM10 (BC ₃ ) and all #1203 (BC ₃ ) were high and similar.

The RS values of brown rice of the EM10, #1203B and #1203C lines before backcrossing were 20 to 26%, and the RS values increased slightly after backcrossing.
The results of this example suggest that the selection of rice varieties for specific food uses should be based not only on RS value but also on starch and RS structure.

(Effect of amylopectin structure, thermal properties, and amylose content on RS value)
The chain length distribution patterns of the high RS rice lines before and after backcrossing were essentially the same, with a slight increase in DP after backcrossing. After backcrossing, the gelatinization temperature of all lines increased slightly (2-4°C), likely due to higher ripening temperatures associated with earlier flowering. Specifically, the expression levels and/or activities of other starch biosynthetic enzymes, such as SSI, which increased the proportion of amylopectin chains with 8<DP<20, may have been increased.

In EM10 (BC ₃ ) and #1203 (BC ₃ ), the number of short amylopectin chains (DP≦12) was significantly decreased and the number of long amylopectin chains (12<DP<30) was increased compared to Akita 63. However, #4019 (BC ₃ ) and #1206 (BC ₃ ) had more short chains and fewer long chains than EM10 (BC ₃ ) and #1203 (BC ₃ ). The increased SSI expression due to the deficiency of SSIIIa may explain the higher amount of short chains (DP≦12) in #4019 (BC ₃ ) and #1206 (BC ₃ ) compared to EM10 (BC ₃ ) and #1203 (BC ₃ ). As shown in Table 2, the reduction in the proportion of short chains in EM10 (BC ₃ ) and #1203 (BC ₃ ) lines affected their gelatinization temperatures, which were about 7°C higher than those of #4019 (BC ₃ ) and #1206 (BC ₃ ). The difference in chain length distribution pattern also affected the RS values. The RS values of rice flour and gelatinized rice flour of the BEIIb-deficient line (#1203) were higher than those of the line lacking both SSIIIa and BEIIb (#1206).

In addition, the high expression level of GBSSI also contributed to the increase in RS value. Comparison of EM10 (BC ₃ ) with #1203B (BC ₃ ), and #1203A (BC ₃ ) with #1203C (BC ₃ ), showed that the lines with the high expression of GBSS1 allele contained higher amylose and RS value than the lines with the low expression of gbss1 ^L allele. In the lines with ss3a be2b genotype, such as #4019 (BC ₃ ) and #1206 (BC ₃ ), the amylose content was high (37-42%) regardless of the GBSSI allele. This is because the level of GBSSI protein increases in the absence of SSIIIa, regardless of the genotype (GBSS1 or gbss1 ^L ). The lack of SSIIIa also increases AGPase activity. That is, as shown in FIG. 1, it suggests that amylose was efficiently synthesized in the #4019 (BC ₃ ) and #1206A (BC ₃ ) lines, even though the GBSSI protein level was lower than that in the #1206B (BC ₃ ) and #1206C (BC ₃ ) lines. Nevertheless, as shown in FIG. 6, among the ss3a be2b lines, those with the high expression GBSS1 allele showed higher RS values than those with the low expression gbss1 ^L allele. The valley between fraction I and fraction II in the elution pattern of gel filtration chromatography was higher in the line carrying the GBSS1 allele than in the line carrying the gbss1 ^L allele. Long amylopectin chains or short glucan chains such as amylose are synthesized by GBSSI and therefore contribute to the formation of RS. Therefore, it is considered possible that the high expression level of GBSSI contributed to the elongation of very long amylopectin chains in the #1203B and #1203C lines before backcrossing.

As shown in Patent Document 1, the RS values of polished rice from EM10, #1203B, and #1203C lines before backcrossing were 20-26%, and the RS values increased slightly after backcrossing. Although the amylose content decreased slightly after backcrossing, the proportion of long amylopectin chains increased slightly, so the level of RS value increased slightly after backcrossing. The RS value of unmashed cooked rice may also be affected by the grain size, degree of polishing, and the amount of water added to the rice for cooking. However, it is believed that backcrossing caused earlier flowering, which resulted in higher temperatures during seed ripening, ensuring the optimal temperature for RS biosynthesis.

It goes without saying that the configurations and operations of the above-described embodiments are merely examples and can be modified as appropriate without departing from the spirit and scope of the present invention.

The present invention provides a rice mutant with a high RS value and high seed weight, and can be used to produce rice flour, starch containing RS, rice flour gel, and food products based on the mutant, making it industrially applicable.

Claims (9)

A rice mutant with a high resistant starch content, characterized in that it is deficient in rice branching enzyme type IIb (BEIIb), the gene for granule-bound starch synthase GBSSI is of a high expression type derived from indica rice, the gene for starch synthase SSIIa is of a low activity type derived from japonica rice, and starch synthase SSIIIa is normally expressed, is genetically fixed, and is a non-genetically modified organism. The rice mutant with high resistant starch content according to claim 1, characterized in that starch synthase SSIIIa is dominant homozygous. 2. The rice mutant with high resistant starch content according to claim 1, wherein the average weight of one grain of brown rice of the rice mutant is 25 mg or more. 4. The rice mutant with high resistant starch content according to claim 3, wherein the resistant starch content in raw rice flour of the rice mutant is 20% or more. A method for producing rice flour, comprising powdering endosperm of rice seeds of the rice mutant with high resistant starch content according to any one of claims 1 to 4 to produce rice flour. 5. A method for producing resistant starch, comprising extracting resistant starch from cooked rice produced from rice seeds of a rice mutant having a high resistant starch content according to any one of claims 1 to 4, and/or from rice flour produced by powdering said rice seeds. A method for producing rice flour gel, comprising adding water to rice flour of the rice mutant with high resistant starch content according to any one of claims 1 to 4 and heating the rice flour to produce a rice flour gel. 5. A method for producing a food product comprising any one or any combination of rice seeds of a rice mutant with high resistant starch content according to claim 1, rice flour obtained by powdering the rice seeds, resistant starch extracted from the rice flour, and rice flour gel produced by adding water and heating the rice flour. A method for producing a rice mutant with high resistant starch content, characterized in that, by a non-genetically modified method, the rice branching enzyme type IIb (BEIIb) is deficient, the gene for granule-bound starch synthase GBSSI is a high-expression type derived from Indica rice, starch synthase SSIIa is a low-activity type derived from Japonica rice, and starch synthase SSIIIa is normally expressed and genetically fixed.

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