JPH10215884A - Sourness represant, plasmid for plant, transformed cell and plant, and production of curculin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce curculin B repressing sourness and having activity of reducing sourness or making itself tasteless as an effective component in sourness repressive agent suitable for foods by producing curculin B through transformed cells originated from plants. SOLUTION: Curculin B which is a polypeptide including an amino acid sequence of the formula is produced through transformed cells or transformed plants containing them. Deoxyribonucleic acids coding the curculin B are prepared by means of a known genetic technology method and/or a chemical synthetic method. The base sequence wholly coding the complementary deoxyribonucleic acid(cDNA) of the curculin B is disclosed in the Japan Patent official gazette H06-189771. The curculin B is obtained e.g. by transforming cells originated from plants with a plasmid including the cDNA between a sequence containing a promoter able to functionate in plants and a sequence containing a terminator able to functionate in plants and then by extracting the curculin B from the resultant transformant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an acidity inhibitor, a plant plasmid, a transformed cell and a transformed plant obtained by transforming with the plasmid, and a curculin using the transformed cell or the transformed plant. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Curcugo Latifolia (Curcu)
Ligo latifolia is a plant belonging to the family Aphidaceae (or Amaryllidaceae depending on the classification method) which grows naturally in Malaysia, Thailand, and the like. Curculin, which is a protein contained in the fruit of Curculigo latifolia, has a sweetness in itself, and has an activity of making tasteless food and drink (for example, water) feel sweet,
It is known that it also has an activity to make sour taste sweet. Malaysia has a history of using Curculigo latifolia fruit as a sugar substitute for drinking coffee and tea, sometimes as a tenderizer for cooking meat, or as an appetite enhancer. Regarding the amino acid sequence of curculin, the entire amino acid sequence of curculin A, which is one of the curculin homologues, is disclosed in JP-A-3-19089.
Japanese Patent Application Laid-Open No. 6-189771 discloses a nucleotide sequence and an amino acid sequence of a mature form and a precursor of curculin B which is one of curcurin homologs.

[0003] The production of curculin is disclosed in
No. 63, JP-A-2-84157, JP-A-2-8416
No. 0 and Japanese Patent Application Laid-Open No. 2-84161. In these methods, curculin A is extracted from the fruit of Curculigo latifolia. Therefore, for example, the yield of the fruit or the content of curculin A is susceptible to fluctuations, and the treatment of the curculigo-latifolia fruit is complicated, so that mass production of curculin has been difficult. In recent years, many attempts have been made to introduce useful genes into microorganisms using genetic engineering techniques, and it has become possible to mass-produce useful proteins, which have been difficult to mass produce. For example, Japanese Patent Application Laid-Open No. 6-189771 discloses the entire nucleotide sequence encoding the curculin B cDNA,
Recombinant curculin B is produced by a microorganism transformed with a plasmid in which the cDNA encoding curculin B has been cloned. However, since this recombinant curculin B is produced by using Escherichia coli as a host and is derived from Escherichia coli, it tends to be avoided by some consumers as a food. Development was awaited.

[0004]

DISCLOSURE OF THE INVENTION The present inventors diligently searched for a method for producing curculin, which is more suitable for food use and can be mass-produced, and found that plant-derived transformed cells or transformed plants containing the same. It has been found that these problems can be solved by using. In addition, the present inventor has found that the curculin B produced by the production method has an activity of unexpectedly suppressing acidity and reducing or eliminating acidity. This sourness suppressing activity is completely different from curcurin activity known in the art (that is, an activity having a sweetness itself or an activity of causing sourness to feel sweet). The present invention is based on these findings.

[0005]

The present invention relates to (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or (2) one or more amino acids added to the amino acid sequence, It has a deleted or substituted amino acid sequence, and further comprises a polypeptide having an acidity suppressing activity as an active ingredient,
It relates to an acidity inhibitor.

The present invention also relates to (1) a DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or (2) one or more amino acids added to the amino acid sequence, A DNA having a deleted or substituted amino acid sequence and encoding a polypeptide having an acidity-suppressing activity, comprising a sequence containing a promoter capable of functioning in a plant, and a terminator capable of functioning in a plant And a plasmid, characterized in that the plasmid comprises In addition, the present invention relates to a method for producing a plant (however,
(Excluding Latifolia), which is obtained by transforming cells derived therefrom. The present invention also relates to a transformed plant containing the transformed cell. Furthermore, the present invention relates to (1) a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or (2) one or more amino acids in the amino acid sequence from the transformed cell or the transformed plant. Amino acids added,
The present invention relates to a method for producing the polypeptide, comprising extracting a polypeptide having a deleted or substituted amino acid sequence and having an acidity suppressing activity.

[0007] As used herein, the term "plant" refers to a plant in which the whole organism is classified into microorganisms, animals, and plants, and is a multicellular differentiated organism capable of photosynthesis, or a part thereof. I do. The “plant” includes moss, fern, gymnosperm, angiosperm, and the like. In addition, a part of the organism capable of photosynthesis and a part that does not itself perform photosynthesis (eg, a fruit or a root) are included in the plant of the present invention. The “plant cell” means any cell derived from a plant, such as protoplasts, undifferentiated tissues (eg, callus), seeds, embryos, pollen, plant embryos, adventitious embryos, and artificial seeds. Including.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The sour suppressant of the present invention comprises, as an active ingredient, (1) a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or (2) one polypeptide in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Or, an amino acid sequence in which a plurality of amino acids are added, deleted, or substituted,
Moreover, it contains a polypeptide having sourness suppressing activity.
Examples of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing include, for example, SEQ ID NO: 1 in the sequence listing.
Curcurin B consisting of the amino acid sequence represented by One or more amino acids are added, deleted, or substituted in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing,
Moreover, the polypeptide having sourness-suppressing activity is hereinafter referred to as curculin B analog.

[0009] As used herein, the term "curcurin B analog" refers to a polypeptide comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence of curculin B. ,
In addition, it means a polypeptide having an acidity suppressing effect.
The number and types of amino acids that can be added, deleted, or substituted are not particularly limited, and those skilled in the art can appropriately prepare analogs based on the acidity-suppressing action.

[0010] In the present specification, "acidity suppressing activity" means
It means an activity of suppressing sourness of foods and beverages exhibiting sourness, that is, an activity of reducing or eliminating sourness. Whether or not a certain compound has the acidity-suppressing activity can be determined, for example, by a sensory test shown below. That is,
First, an aqueous solution of a sour level reference substance (for example, about 0.
(1M citric acid aqueous solution) in the mouth and memorize the reference level of acidity. The mouth is then rinsed thoroughly with water (eg, several times) until no sourness is felt. A test compound aqueous solution of a predetermined concentration (for example, an aqueous solution of curculin B analog)
A predetermined amount (for example, about 2 to 5 ml) is contained in the mouth for a predetermined time (for example, about 2 to 5 minutes), and then, it is exhaled. If necessary, rinse mouth lightly with water. Subsequently, the aqueous solution of the sour level reference substance is filled in the mouth in the same manner as described above, and a difference between the sour level felt at that time and the sour level reference level is determined. Depending on whether the acidity level is significantly suppressed from the acidity reference level,
It can be determined whether or not it has sourness suppressing activity.

[0011] The plasmid of the present invention comprises at least
(1) a sequence containing a promoter capable of functioning in a plant, (2) a DNA encoding curculin B or a curculin B analog, and (3) a sequence containing a terminator capable of functioning in a plant, on the plasmid. Included. The sequence containing the promoter is located 5 ′ upstream of the DNA encoding curculin B or an analog thereof,
The sequence containing the terminator is located 3 ′ downstream of the DNA encoding curculin or an analog thereof.

Curcurin B has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Curculin B has a signal peptide at the N-terminus (from the -22nd amino acid to the-amino acid in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing).
An amino acid sequence comprising an amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 2 SEQ ID NO: 2 in the Sequence Listing
The precursor consisting of the amino acid sequence represented by
It is presumed that it is produced in plant cells in the form of an immature form, and the signal peptide and the extended peptide are eliminated by processing to form a mature form consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. .

In the plasmid of the present invention, a DNA containing a nucleotide sequence encoding curculin B can be used in place of the DNA encoding curculin B. Examples of such DNA include "mature" curculin B (For example, SEQ ID NO: 3 in the sequence listing)
The 77th base to the fourth base in the base sequence represented by
A base sequence encoding a “precursor type” curculin B containing both a DNA consisting of a base sequence consisting of the 18th base, a signal peptide and an extension peptide (for example, the 11th base in the base sequence represented by SEQ ID NO: 3 in the sequence listing) A base sequence encoding a "extended peptide deletion-precursor type" curculin B containing a signal peptide but lacking an extended peptide (e.g., SEQ ID NO: 3 in Sequence Listing
11th to 4th bases in the base sequence represented by
DNA consisting of a base sequence consisting of the 18th base, or a base sequence encoding "signal peptide deletion-precursor type" curculin B containing an extended peptide but lacking a signal peptide (for example, a sequence in the sequence listing) The 77th base to the 484rd base in the base sequence represented by No. 3
(A base sequence consisting of the second base) can be used.

In the plasmid of the present invention, a DNA encoding a curculin B analog or a DNA containing a base sequence encoding a curculin B analog can be used in place of the DNA encoding curculin B. As the DNA containing the nucleotide sequence encoding curculin B analog, as in the case of the DNA containing the nucleotide sequence encoding curculin B, for example, mature, precursor, extended peptide deletion-precursor type, or signal peptide deletion DNA consisting of a nucleotide sequence encoding various curculin B analogs such as apo-precursors can be used.

[0015] DNA encoding curculin B or a curculin B analog can be prepared, for example, by known genetic engineering techniques or chemical synthesis methods, or a combination thereof. When a plasmid containing curculin B cDNA or curculin B analog cDNA has been obtained, the polymerase chain reaction method (hereinafter referred to as P
The desired DNA can be easily prepared by the CR method). For example, a plasmid pQ9 carrying a cDNA containing the nucleotide sequence encoding the precursor curculin B (the nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing) (for the structure and preparation method of plasmid pQ9, see
No. 189771 and the outline of the preparation method is shown in Example 1 described later). The nucleotide sequence encoding the signal peptide (the 11th nucleotide in the nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing) is used. A base sequence consisting of the 76th base to the 76th base) and a base sequence encoding mature curculin B (the 77th base to the 418th base in the base sequence represented by SEQ ID NO: 3 in the sequence listing) Base sequence) and a base sequence encoding the extended peptide (base sequence consisting of the 419th base to the 484th base in the base sequence represented by SEQ ID NO: 3 in the sequence listing). Then, by performing a PCR method using the plasmid pQ9 as a template, the mature form, precursor, extended peptide deletion-precursor type Or signal peptide deletion - can be prepared nucleotide sequences encoding a variety of curculin B, such as precursors.

As the primer used in the PCR method, for example, oligodeoxyribonucleotide or oligoribonucleotide can be used. As the sense primer, for example, an arbitrary length (usually,
At the 5 'end of the base sequence encoding the amino acid sequence (6 to 10), a protein translation initiation codon (ATG)
Synthetic nucleotides with linked sequences can be designed. When the amino terminus of the target protein is ATG, the ATG sequence is overlapped when the ATG sequences are linked. Therefore, the nucleotide sequence encoding the amino acid sequence of any length at the amino terminus of the target protein can be used as it is. It can be used as a primer. In addition, the sense primer has a so-called Kozak's law [Ko]
Zak et al., Microbiol. Reviews, 47
Vol., Pp. 1-45 (1983)] is also preferable for increasing protein production.

Examples of the antisense primer include, for example, any length (usually 6 to 10) at the carboxy terminal portion (including the carboxy terminal amino acid) of the target protein.
At the 3 ′ end of the base sequence encoding the amino acid sequence of the present invention, one or more (preferably 1 or 2) stop codon (TAA, TAG, or TGA) sequences for protein translation.
, And more preferably two) can be designed.

A restriction enzyme cleavage site corresponding to a restriction enzyme capable of cleaving a site into which the fragment is to be inserted at the 5 'end of the sense primer and / or antisense primer of the fragment amplified by the PCR method. Is preferable, since the fragment can be inserted into the restriction enzyme cleavage site of a sequence comprising a promoter sequence or a terminator sequence and, if desired, an intron sequence.

The promoter that can be used in the plasmid of the present invention is not particularly limited as long as it can function in plants, and is preferably a promoter other than the curculin promoter of Curculigo latifolia. Here, the term "promoter capable of functioning in a plant" means a promoter capable of specifically binding RNA polymerase and starting transcription in the downstream direction when the promoter is introduced into a plant. Examples of the promoter include a cauliflower mosaic virus 35S promoter [The Embojournal, Vol. 6, No. 390].
1 to 3907 (1987)], an actin promoter (particularly a rice actin promoter) [R. Wu
Et al., The Plant Cell, Volume 2, pages 163 to 171.
(1990)], a ubiquitin promoter (particularly, a corn ubiquitin promoter) [Christ
Ensen, Plant Molecular Biology,
18, pp. 675-689 (1992)],
Alternatively, a TR1 ′ promoter or TR2 ′ promoter can be used. When rice is used as a host, a ubiquitin promoter (particularly, a corn ubiquitin promoter) is particularly preferred. When a plant belonging to the family Rutaceae (for example, lemon or orange) is used as a host, the TR1 ′ promoter or TR2 ′ promoter [A. Vardi et al.
Science, Vol. 69, pp. 199-206 (19
1990)] or cauliflower mosaic virus 35S
Promoter [T. Hidaka et al., Japan. J.
Breed. 40, 199-207 (1
990)] is particularly preferred.

The terminator that can be used in the plasmid of the present invention is not particularly limited as long as it can function in a plant.
The terminator is preferably a terminator other than the curculin lacrifolia curculin terminator. Here, "terminator that can function in plants"
When the terminator is introduced into a plant, it means a terminator capable of terminating transcription from the upstream direction and adding polyA. Examples of the terminator include a nopaline synthase terminator, a cauliflower mosaic virus 35S terminator, and an octopine synthase terminator. When rice is used as a host, a nopaline synthase terminator is preferred. When a plant belonging to the family Rutaceae (for example, lemon or orange) is used as a host, an octopine synthase terminator [A. Vardi et al., Plant Science, No. 69
Pp. 199-206 (1990)], or the nopaline synthase terminator [T. Hidaka
Et al., Japan. J. Breed. , Vol. 40, 19
9 to 207 (1990)]. Plasmids that do not contain a promoter and / or terminator that can function in plants (eg, plasmids for microorganisms) do not normally express when introduced into plants.

In the plasmid of the present invention, the 3 'end of the sequence containing the promoter control sequence in the sequence containing the promoter and the 5' end of the sequence containing the control sequence of the terminator in the sequence containing the terminator are included. In between, by further providing an intron that can function in plants, it is possible to increase the gene expression efficiency,
Alternatively, it is preferable because the stability of mRNA can be increased. Here, “an intron that can function in a plant” means that when the intron is introduced into a plant,
It means an intron that can be removed during mRNA export or splicing. Examples of the intron include Hima catalase intron [Tanaka et al., Nucleic Acid Research, 18th Edition]
Vol. 6767-6770 (1990)] or a corn ubiquitin intron [Christe
nsen, Plant Molecular Biology, Vol. 18, pp. 675-689 (1992)]. When rice is used as a host, corn ubiquitin intron is particularly preferred.

In the plasmid of the present invention, when an intron is provided, the place where the intron is inserted is not particularly limited as long as the intron is within the above-mentioned range, but the promoter-containing sequence, curculin B or curculin B analog Is preferably inserted between the DNA and the DNA encoding When the intron is inserted into DNA encoding curculin B or a curculin B analog, the position of the intron is not particularly limited.

The procedure for constructing the plasmid of the present invention is not particularly limited. For example, a sequence containing a promoter capable of functioning in a plant, a DNA encoding curculin B or a curculin B analog, and a function functioning in a plant After all the sequences containing the terminator that can be ligated, they can be inserted into an appropriate plasmid at one time, or they can be inserted separately and sequentially into an appropriate plasmid. In general, it is preferable to prepare beforehand a plasmid containing a promoter and a terminator that can function in a plant, and optionally further contain an intron, and insert a DNA encoding curculin B or a curculin B analog into the plasmid.

[0024] A plasmid containing a promoter and a terminator that can function in a plant, and optionally a further intron, wherein the desired DNA can be inserted between the promoter and the terminator includes, for example, pUBA [ Toki and other plants
Physiology, Vol. 100, pp. 1503-150
7 (1992)] or pBI121 (Clontech). Plasmid pUB
A can be used for a direct introduction method such as a particle gun method, and a desired DNA can be inserted between an intron following a promoter and a terminator. Further, the plasmid pBI121 can be used for an Agrobacterium-mediated introduction method, and a desired DNA can be inserted between a promoter and a terminator.

The plant in the plant-derived cells which can be used as a host of the transformed cell according to the present invention may be any plant except curculigo-latifolia, preferably higher plants, and preferably edible plants. It is more preferable to use As used herein, the term "edible plant" means a plant that can directly or partially consume itself or a plant that can partially or wholly be a raw material of food. . Examples of the edible plants include solanaceae (tomato, potato, and the like), cruciferous (rape,
Radish, cabbage, broccoli, cauliflower, etc.), Apiaceae (carrot, etc.), Cucurbitaceae (melon, cucumber, etc.), Poaceae (rice, corn, etc.), Rosaceae (apple, peach, etc.), Rutaceae Examples include plants such as (citrus, orange, or lemon), legumes (soybean, etc.), asteraceae (lettuce, sunflower, safflower, etc.), or convolvulaceae (sweet potato, etc.).

As a method for introducing the constructed plasmid into a plant, any method established as a method for transforming a plant can be used. Examples of such a method include a direct introduction method and an Agrobacterium-mediated introduction method. As a direct introduction method, for example, an electroporation method or a polyethylene glycol method can be used to directly introduce into a plant protoplast. Alternatively, a particle gun method in which microprojectiles are directly introduced into plant cells can be used. When rice is used as a host, a particle gun method or an Agrobacterium-mediated introduction method is preferred. Further, as the cells to be transformed, cells suitable for the introduction method can be used depending on the introduction method to be used. When rice is used as a host, suitable cells include, for example, embryos extracted from immature seeds or ripe seeds, or induced calli.

When transforming plant cells with the plasmid of the present invention, a cassette capable of expressing curculin B or a curculin B analog in a plant (hereinafter sometimes referred to as a curculin B cassette), A sequence comprising a promoter capable of functioning in plants, DNA encoding curculin B or a curculin B analog,
And a cassette containing a sequence containing a terminator capable of functioning in a plant and a cassette capable of expressing a selectable marker gene in a plant (hereinafter sometimes referred to as a selectable marker gene cassette) simultaneously in a plant cell. Can be introduced.

Examples of the selection marker gene include a hygromycin resistance gene, a kanamycin resistance gene,
Alternatively, a bialaphos resistance gene or the like can be used.
When the plant to be introduced is a monocotyledonous plant such as a grass plant, the bialaphos-resistant (bar) gene is very effective [Toki et al., Plant Physiology, No. 10
0, pages 1503 to 1507 (1992)],
Particularly preferred. When a plant belonging to the family Rutaceae (for example, lemon or orange) is used as a host, the kanamycin resistance gene [A. Vardi et al., Plant Science, Vol. 69, pp. 199-206 (1990).
Year)], or a kanamycin resistance gene or a hygromycin resistance gene [T. Hidaka et al., Japan
n. J. Breed. , Vol. 40, pp. 199-20
7 (1990)].

When a plant cell is transformed with the plasmid of the present invention using a direct introduction method such as a particle gun method, for example, a curculin B cassette and a selectable marker gene cassette can be used in a variety of E. coli commonly used. Cloned into a copy plasmid (eg, pUC19),
It can be introduced into plant cells. In that case, the curculin B cassette and the selectable marker gene cassette are
(1) It can be introduced while being retained on the same plasmid, or (2) It can be retained on different plasmids, and these plasmids can be introduced as a mixture (so-called cotransformation). When transforming plant cells with the plasmid of the present invention by co-transformation, for example, curculin B
Among the cassette and the selection marker gene cassette, as a plasmid holding only the selection marker gene cassette, plasmid pDM302 [R.R.] containing the bar gene between the rice actin promoter and the nopaline synthase terminator. Wu et al., Plant Cell Reports, Vol. 11, pp. 586-591 (1992).
Year)], two types of plasmid DNAs, a plasmid having a curculin B cassette prepared separately and the plasmid pDM302, can be mixed and cotransformed.

In the present invention, when a plant cell is transformed with the plasmid of the present invention using the Agrobacterium-mediated transfer method, for example, the curculin B cassette and the selectable marker gene cassette are placed on the same plasmid. A binary plasmid for Agrobacterium to be retained can be prepared and introduced into plant cells. For example, DNA encoding curculin B or a curculin B analog
(Eg, curculin B cDNA) is cloned between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator of the binary vector pBI121 having a kanamycin resistance gene,
Transformed cells can be obtained by infecting plant cells with Agrobacterium having this binary plasmid and selecting for kanamycin resistance.

From the cells into which the gene thus obtained is introduced, a plant can be regenerated by any method after screening. In the case of rice, for example, the cell into which the gene has been introduced is replaced with an N6 medium containing a selection drug corresponding to the selection marker (eg, 10 ppm of bialaphos when the selection marker is a bialaphos resistance gene) and a plant hormone. When cultured, cells (callus) transformed by gene transfer can be obtained in 4 to 8 weeks. The obtained callus is transferred to a suitable growth medium (for example, N6 medium containing 10 ppm of bialaphos and plant hormone), and further cultured in a suitable regeneration medium (for example, MS medium containing 10 ppm of bialaphos and plant hormone). Thus, a transformed rice plant can be obtained.
The expression of curculin B or a curculin B analog in cells can be confirmed by immunologically analyzing (for example, Western analysis).

Curcurin B or curculin B analog can be extracted from the thus obtained transformed cells or transformed plants by a known method. For example,
The transformed cells or transformed plants (whole or part thereof) are disrupted, and an appropriate washing solution [eg, 50 mM-Tr
is buffer (pH 7.0) or 0.015N sulfuric acid] to remove water-soluble proteins. An extract suitable for the precipitate [e.g. 50 containing 0.5 M NaCl
mM-Tris buffer (pH 7.0) or 0.0
5N sulfuric acid etc.], shake and extract overnight, and add curculin B
Alternatively, a curculin B analog extract can be obtained. 70% saturated ammonium sulfate is added to this extract, and the precipitate is collected by centrifugation. The precipitate is washed with 0.2 M acetic acid 2.5
Then, the solution is dissolved in water and desalted using a desalting column PD10 (Pharmacia). Curcurin B thus obtained
Alternatively, curculin B or a curculin B analog can be obtained by freeze-drying the curculin B analog protein solution. The transformed plant that can be used for curculin B or curculin B analog extraction is not particularly limited as long as it contains curculin B or curculin B analog, and the whole plant or a part thereof, for example, leaves, stems, etc. ,
Rhizomes, roots, tubers, pulp, or seeds can be used. In addition, if it is sufficient that only extraction of curculin B or a curculin B analog can be achieved, it is not necessary to regenerate the plant, and the transformed cells such as protoplasts or callus can be crushed and extracted as they are. it can.

The sour suppressant according to the present invention is not limited to, but includes, for example, foods and drinks to which a compound exhibiting a sour taste (eg, ascorbic acid or acetic acid) is added.
Alternatively, it is useful for reducing or eliminating only the sour taste of foods and drinks that themselves show sour taste. For example, foods and drinks to which ascorbic acid is added as an oxidizing agent and / or a vitamin C reinforcing agent (for example, cans or bottled beverages such as tea)
By adding the acidity inhibitor according to the present invention, only the acidity of ascorbic acid can be reduced. Foods and drinks to which acetic acid is added as a bacteriostatic or bacteriostatic agent (for example, lunches, pickles, salads, or packed rice)
By adding the acidity inhibitor according to the present invention, only the acidity of ascorbic acid can be reduced. Examples of foods and drinks that themselves exhibit sour taste include fruit juice beverages, and the acidity can be adjusted by adding the acidity inhibitor of the present invention to foods and drinks that themselves exhibit sour taste.

The transformed plant of the present invention can be used as it is as such. For example, crops whose added value is improved when only the acidity is suppressed [e.g., citrus fruits (for example, Citrus unshiu, Yuzu, Kumquat, grapefruit, Valencia, Pomelo, Hassac, Lemon, Iyokan, Bampeiyu, Sampoucan, Lime, or Summer orange) Etc.), kiwi-fruit, pineapple, acerola,
Or tomato etc.], curculin B or curculin B
When the analog is expressed, it is possible to obtain a transformed plant in which only the acidity inherent in the plant is suppressed. In addition, curculin B or a curcurin B analog is expressed in a crop which does not have a sour taste itself but may be eaten simultaneously with food and drink to which ascorbic acid or acetic acid is added as described above. By doing so, the same effect as that of the above-mentioned sour suppressant can be obtained. The above-mentioned various transformed plants can be produced using the above-mentioned known method.

[0035]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 Construction of Plasmid for Expression of Curculin B or Curcurin B Analog Gene First, a cDNA encoding precursor curculin B containing a signal sequence and an extension sequence, and an extension sequence containing a signal sequence but containing a signal sequence were prepared by the following procedure. CDNA encoding the deleted extended peptide deletion-curcurin B precursor was converted to PC
Created by the R method. A primer CURN1 consisting of the base sequence represented by SEQ ID NO: 4 and a primer CURC4 consisting of the base sequence represented by SEQ ID NO: 5 were used as primers, and the plasmid pQ9 described in JP-A-6-189771 was used as a template for amplification. Pf as enzyme
PCR was performed using u-DNA polymerase (Takara Shuzo). The plasmid pQ9 is disclosed in Japanese Patent Laid-Open No. 6-1897.
No. 71 was prepared by the method described in Examples 1 to 10. That is, a cDNA library was prepared using mRNA extracted from the fruit of Curculigo latifolia, and the amino acid sequence of curculin A (Japanese Unexamined Patent Publication No.
No. 99), a plasmid pQ9 was obtained by selection with a probe designed.

The amplified DNA fragment is transformed into an E. coli vector p.
After cloning into the SmaI site of UC19 (Toyobo), BamHI (Toyobo) and SacI (Toyobo)
Was cut twice to purify a DNA fragment containing curculin B cDNA. This fragment was transformed into a plasmid p containing a corn ubiquitin promoter, a corn ubiquitin intron, and a nopaline synthase terminator.
UBA [Toki et al., Plant Physiology, 100th
Volume, pages 1503 to 1507 (1992)]
Subcloning was performed so as to replace the bialaphos resistance (bar) gene existing between the amHI site and the SacI site, thereby obtaining a plasmid pMCU42. pMCU42
Had a precursor curculin B gene containing a signal sequence and an extension sequence. FIG. 1 shows the plasmid pMCU.
42 schematically shows the structure. In FIG. 1, "nos"
Means nopaline synthase.

Similarly, the primer CURN1 and the primer CU comprising the base sequence represented by SEQ ID NO: 6
As described above, using two kinds of primers of RC5,
After amplifying the DNA fragment and cloning it at the SmaI site of pUC19, it was double-cut with BamHI and SacI,
Subcloning was performed so as to replace the bialaphos resistance (bar) gene existing between the BamHI site and the SacI site of pUBA, to obtain a plasmid pMCU43. p
MCU43 had an extended peptide-deleted-precursor curculin B gene containing a signal sequence but lacking an extended sequence. FIG. 2 schematically shows the structure of the plasmid pMCU43. In FIG. 2, “nos” means nopaline synthase.

[0038]

Example 2 Transformation of rice 2-1. Extraction of embryo tissue from ripe seeds After dehulling the ripe seeds of rice cultivar Nipponbare, sterilization was carried out in a 3.5% calcium hypochlorite solution while degassing and stirring.
Minutes. Then, after sufficient washing with sterile water, water was removed on sterile filter paper, and the plate was placed on a callus induction medium A shown in Table 1 and cultured in a dark place at 25 ° C. Six days after the culture, only the enlarged embryonic tissue was removed. The excised tissues were arranged on a 6 cm dish containing callus induction medium A with the scutellum side up. 10 to 20 pieces were arranged along a circumference having a radius of 1 cm to form a shot dish.

[0039]

[Table 1] (“N6” shown in the column of basic salt composition in the table is 463 m
g / l- (NH ₄ ) ₂ SO ₄ , 2830 mg / l-KN
O ₃ , 400 mg / l-KH ₂ PO ₄ , 1.6 mg / l
_{-H 3 BO 3, 4.4mg / l} -MnSO 4 · 4H
_{2 O, 1.5mg / l-ZnSO} 4 · 7H 2 O, 0.8
mg / l-KI, 166mg / l-CaCl 2 · 2H 2
_{O, 185mg / l-MgSO 4} · 7H 2 O, 37.3
mg / l-Na ₂ -EDTA, and 27.8 mg / l-
Means FeSO ₄ · 7H ₂ O, "MS" shown in the column of the basic salt _{composition, 1650mg / l-NH 4 NO} 3, 19
00 mg / l-KNO ₃ , 170 mg / l-KH ₂ PO
_{4, 6.2mg / l-H 3} BO 3, 22.3mg / l-
MnSO ₄ .4H ₂ O, 8.6 mg / l-ZnSO _{4.
7H 2 O, 0.83mg / l-} KI, 0.25mg / l
_{-Na 2 MoO 4 · 2H 2 O} , 0.025mg / l-C
_{uSO 4 · 5H 2 O, 0.025mg} / l-CoCl 2
6H ₂ O, 440 mg / l-CaCl ₂ .2H ₂ O,
_{370mg / l-MgSO 4 · 7H} 2 O, 37.3mg
/ L-Na ₂ -EDTA, and 27.8 mg / l-Fe
Means SO ₄ · 7H ₂ O, "vitamin" is, 0.5m
g / l nicotinic acid, 0.5 mg / l pyridoxine hydrochloride,
1 mg / l thiamine hydrochloride and 2 mg / l glycine, "2,4-D" means 2,4-dichlorophenoxyacetic acid, "NAA" means naphthaleneacetic acid)

2-2. Gene Transfer Using a Particle Gun A pneumatic Reebok Chamber Model 260 was used as the particle gun device. Plasmid p constructed in Example 1
MCU42 or pMCU43, and pDM302 [R. Wu et al., Plant Cell Reports, Vol. 11, pp. 586-591 (1992).
)) Into 1 micron gold particles according to the method of Morikawa et al. [Plant Cell Engineering, Vol. 4, pp. 43-48 (1992)]. It was coated, placed on a special projectile (bullet), and air-dried. 0.1mg gold particles and 0.4μgD per bullet
Two shots of a shot containing NA are shot on a shot dish (firing pressure = 8 times of pumping, distance between samples = 6c)
m) Two types of genes were introduced.

2-3. Selection culture of transformed callus and regeneration into plants Replacing embryo tissue from the dish shot in Example 2-2 with callus induction medium B shown in Table 1 further containing 10 ppm of selection drug bialaphos, 25 After culturing in a dark place at 7 ° C. for 7 days, culturing was continued in a light place (3000 lux). In 4-8 weeks of culture, bialaphos-resistant callus due to gene transfer was confirmed on the scutellum, the callus portion was cut out, grown and cultured in callus induction medium B, and transformed with pMCU42 to obtain a rice-derived callus MCU42-15, pMCU
43-derived callus MCU43-4 transformed with rice
6 was obtained. This callus was transplanted to a regeneration medium shown in Table 1 and cultured at 25 ° C. in a light place (3000 Lux) for 4 to 8 weeks to obtain a redifferentiated individual.

[0042]

Example 3 Detection of Curculin B Protein by Western Analysis Each of the transformed callus lines was subjected to Western analysis to examine curculin B expression. For extraction of the protein sample from the callus, 200 μl of 50 mM Tris-hydrochloric acid (pH 7.0) and sea sand were added to 0.1 g of the callus and extracted with a hand homogenizer. The obtained extract was 12,
After centrifugation at 000 rpm for 10 minutes, the supernatant was used as a sample by measuring the protein concentration. A sample in an amount of 50 μg of protein per well is subjected to a 15-25% gradient SDS.
After developing by polyacrylamide gel (Daiichi Kagaku, multi-gel) electrophoresis and transferring to PVDF membrane, rabbit anti-curculin B serum was used as the primary antibody, and HRP-labeled anti-rabbit IgG was used.
Using the antibody as a secondary antibody, a reacting band was immunochemically detected using a Konica color development kit (Konica).

FIG. 3 shows the results. Lane 1 shows transformed callus MCU42- transformed with plasmid pMCU42.
15 shows the results, lane 2 shows the results of MCU43-46 of the callus transformed with plasmid pMCU43, lane 3 shows the results of non-transformed callus (rice cultivar = Nipponbare) as a control, and lane 4 shows the results of natural curculin. The results are shown respectively. In the transformed callus sample, a band was detected at almost the same position as that of native curculin, confirming the expression of curculin B protein in rice callus. No reactive band was observed in the non-transformed callus.

[0044]

Example 4 Purification of Recombinant Curculin B and Determination of N-Terminal Amino Acid Sequence 40 of rice callus MCU43-46 obtained in Example 2
g was crushed using a mortar and pestle, and 50 mM-Tris-
Extraction was performed with 80 ml of a hydrochloric acid buffer (pH 7.0), and the supernatant after centrifugation was discarded to remove water-soluble proteins. 50 mM Tris containing 0.5 M NaCl in callus precipitate
-80 ml of a hydrochloric acid buffer (pH 7.0) was added, the mixture was shaken and extracted overnight, and a curculin extract was obtained by centrifugation. 70% saturated ammonium sulfate was added to this extract, and the precipitate was collected by centrifugation. The precipitate is washed with 0.2 M acetic acid 2.5
The solution was dissolved in the same solution, and desalted using a desalting column PD10 (Pharmacia). Curcurin B thus obtained
The crude fraction containing the protein is lyophilized to give 0.5M-
It was dissolved in 3 ml of 50 mM Tris-HCl buffer (pH 7.0) containing NaCl. A portion of this solution
5% gel (manufactured by Daiichi Kagaku Co., Multigel) was subjected to SDS-polyacrylamide gel electrophoresis, separated, and
The membrane was electroblotted. The membrane was stained with Coomassie Brilliant Blue and then destained with 50% methanol. Cut out the desired protein band,
The N-terminal amino acid sequence was determined using a protein sequencer (Shimadzu PPSQ10).
The amino acid sequence up to the 0th position was found to be the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing. This amino acid sequence completely matched the amino acid sequence predicted from the nucleotide sequence of the introduced cDNA.

[0045]

Example 5 Sensory Test of Crude Purified Recombinant Curculin B Curculin B protein was contained from 40 g of each of the rice callus MCU43-46 or rice callus MCU42-15 obtained in Example 2 according to the method described in Example 4 above. A crude fraction was obtained. As a control, a crude fraction was similarly obtained from 40 g of non-transformed callus (rice cultivar = Nipponbare). These crude fractions were lyophilized, and each 25 mg of the obtained lyophilized product was dissolved in 12 ml of water, and two types of curculin B protein-containing samples were obtained.
One type of control sample was obtained. The obtained sample was used for the following sensory tests.

The sensory test was performed according to the following procedure. That is, first, 3 ml of a 0.1 M aqueous citric acid solution was contained in the mouth, and the reference level of acidity was stored. Next, the mouth was strongly rinsed three times with water so that no acidity was felt. Subsequently, 3 ml of the above-mentioned curculin B protein-containing sample or control sample was contained in the mouth for 3 minutes, after which it was exhaled. After rinsing the mouth lightly once with water, 3 ml of 0.1 M aqueous citric acid solution was again contained in the mouth, and the difference between the acidity felt at that time and the acidity at the above-mentioned reference level was compared. As a result, no difference was observed in the level of sourness in the case of the control sample, whereas the acidity of citric acid was not felt in the case of the curculin B protein-containing sample.

[0047]

According to the present invention, curculin B or a curculin B analog suitable for food can be produced in large quantities. According to the acidity inhibitor of the present invention,
It is useful for reducing sourness only in foods and drinks to which a compound showing sourness (for example, ascorbic acid or acetic acid) is added, or in foods and drinks showing sourness itself.

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence length: 114 Sequence type: amino acid Sequence type: protein Origin: Organism name: Curculigo
latifolia) Sequence Asp Asn Val Leu Leu Ser Gly Gln Thr Leu His Ala Asp His Ser Leu 1 5 10 15 Gln Ala Gly Ala Tyr Thr Leu Thr Ile Gln Asn Lys Cys Asn Leu Val 20 25 30 Lys Tyr Gln Asn Gly Arg Gln Ile Trp Ala Ser Asn Thr Asp Arg Arg 35 40 45 Gly Ser Gly Cys Arg Leu Thr Leu Leu Ser Asp Gly Asn Leu Val Ile 50 55 60 Tyr Asp His Asn Asn Asn Asn Asp Val Trp Gly Ser Ala Cys Trp Gly Asp 65 70 75 80 Asn Gly Lys Tyr Ala Leu Val Leu Gln Lys Asp Gly Arg Phe Val Ile 85 90 95 Tyr Gly Pro Val Leu Trp Ser Leu Gly Pro Asn Gly Cys Arg Arg Val 100 105 110 Asn Gly

SEQ ID NO: 2 Sequence length: 158 Sequence type: amino acid Sequence type: protein Origin: Biological name: Curculigo
latifolia) Sequence Met Ala Ala Lys Phe Leu Leu Thr Ile Leu Val Thr Phe Ala Ala Val -20 -15 -10 Ala Ser Leu Gly Met Ala Asp Asn Val Leu Leu Ser Gly Gln Thr Leu -5 1 5 10 His Ala Asp His Ser Leu Gln Ala Gly Ala Tyr Thr Leu Thr Ile Gln 15 20 25 Asn Lys Cys Asn Leu Val Lys Tyr Gln Asn Gly Arg Gln Ile Trp Ala 30 35 40 Ser Asn Thr Asp Arg Arg Gly Ser Gly Cys Arg Leu Thr Leu Leu Ser 45 50 55 Asp Gly Asn Leu Val Ile Tyr Asp His Asn Asn Asn Asn Asp Val Trp Gly 60 65 70 Ser Ala Cys Trp Gly Asp Asn Gly Lys Tyr Ala Leu Val Leu Gln Lys 75 80 85 90 Asp Gly Arg Phe Val Ile Tyr Gly Pro Val Leu Trp Ser Leu Gly Pro 95 100 105 Asn Gly Cys Arg Arg Val Asn Gly Gly Ile Thr Val Ala Lys Asp Ser 110 115 120 Thr Glu Pro Gln His Glu Asp Ile Lys Met Val Ile Asn Asn 125 130 135

SEQ ID NO: 3 Sequence length: 1166 Sequence type: Nucleic acid Sequence type: cDNA to mRNA sequence CGCAAAGACA ATG GCG GCC AAG TTT CTT CTC ACC ATT CTT GTC ACC TTT 49 Met Ala Ala Lys Phe Leu Leu Thr Ile Leu Val Thr Phe -20 -15 -10 GCG GCC GTC GCT AGC CTT GGC ATG GCC GAC AAT GTC CTG CTC TCC GGG 97 Ala Ala Val Ala Ser Leu Gly Met Ala Asp Asn Val Leu Leu Ser Gly -5 1 5 CAA ACT CTG CAT GCC GAC CAC TCT CTC CAG GCG GGC GCC TAT ACC TTA 145 Gln Thr Leu His Ala Asp His Ser Leu Gln Ala Gly Ala Tyr Thr Leu 10 15 20 ACC ATA CAA AAC AAG TGC AAC CTG GTG AAA TAC CAG AAC GGG AGG CAG 193 Thr Ile Gln Asn Lys Cys Asn Leu Val Lys Tyr Gln Asn Gly Arg Gln 25 30 35 ATC TGG GCT AGC AAC ACT GAC AGG CGG GGC TCC GGC TGC CGC CTC ACA 241 Ile Trp Ala Ser Asn Thr Asp Arg Arg Gly Ser Gly Cys Arg Leu Thr 40 45 50 55 TTG CTG AGT GAC GGG AAC CTC GTT ATC TAC GAC CAC AAC AAC AAC GAC 289 Leu Leu Ser Asp Gly Asn Leu Val Ile Tyr Asp His Asn Asn Asn Asp 60 65 70 GTG TGG GGG AGC GCC TGC TGG GGG GAC A AC GGC AAG TAT GCT CTT GTT 337 Val Trp Gly Ser Ala Cys Trp Gly Asp Asn Gly Lys Tyr Ala Leu Val 75 80 85 CTT CAG AAG GAT GGC AGA TTT GTC ATC TAT GGC CCG GTT TTG TGG TCC 385 Leu Gln Lys Asp Gly Arg Phe Val Ile Tyr Gly Pro Val Leu Trp Ser 90 95 100 CTT GGC CCT AAT GGG TGC CGC CGT GTT AAT GGT GGA ATC ACA GTT GCT 433 Leu Gly Pro Asn Gly Cys Arg Arg Val Asn Gly Gly Ile Thr Val Ala 105 110 115 AAG GAT TCT ACT GAA CCA CAA CAT GAG GAT ATT AAG ATG GTG ATT AAT 481 Lys Asp Ser Thr Glu Pro Gln His Glu Asp Ile Lys Met Val Ile Asn 120 125 130 135 AAT Asn 136 TAATCAAGTG AGAGGATTGT TATGAGAATA ATGAGTGGAA TGGAAGTCTGCT CATGACTAGCTCATGCATG AATAACACAT TGGGGAATAA 604 TAAAGTGAAA CTATATAGAT TGGTTCAGCA AATTTTCTGT TCAGTTTTCC TCTCACATGT 664 CAATGTCGAT TTTTTGCCGC GGATCATACA TGTGCTTGGT ATTCTAATCG ATAGAATTAT 724 GGCTCAAATG GAGGCAGGGA TTATGAGAGT TTATTCGCAT CTCCGGGTCT TCCAACTTAC 784 GAATTATAAC AAGATTCAAG GATGCATCTG AGAGCCAACT TAACGTCTTA CATCAAAGGA 844 GCTAGCCGAA GTTTATTCCC AGAGCTAG AG GAAGTTCGCT GCCATGGTTG ATAGTACAAG 904 TAGAACGACG CATGTATTGC TTCCAGGAAT CACTTCCAGC TTCTCGACAC CTCCAGTGGC 964 CTTTTCACCA CCGAAAGCAC CACCAATTTC AGCACCATTG GTAGGTATAT TTACATTAAC 1024 AATACCACAG TCACTGCCAT GGGGTCCAAT CCACTTGAAA ATAACTTCAG GTCTACGAGT 1084 GAAAATAGAA CTGCTTAAAC TTGCGGTACA GAGTTATTTA TTTCAATTGC TTCTTTCAGA 1144 GTCTGGAATT TCATTACGTA AA 1166

SEQ ID NO: 4 Sequence length: 29 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Antisense: No Sequence type: Other nucleic acid Synthetic oligonucleotide sequence GGATCCACCA TGGCGGCCAA GTTTCTTCT 29

SEQ ID NO: 5 Sequence length: 29 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Antisense: Yes Sequence type: Other nucleic acid Synthetic oligonucleotide sequence GAGCTCCTAT TAATTATTAA TCACCATCT 29

SEQ ID NO: 6 Sequence length: 29 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Antisense: Yes Sequence type: Other nucleic acid Synthetic oligonucleotide sequence GAGCTCCTAT TAACCATTAA CACGGCGGC 29

SEQ ID NO: 7 Sequence length: 10 Sequence type: amino acid Sequence type: peptide

[Brief description of the drawings]

FIG. 1. Signal sequence, mature curculin B, downstream of the corn ubiquitin promoter and intron.
FIG. 4 is an explanatory view schematically showing the structure of a plasmid pMCU42 in which a precursor curculin B cDNA comprising an extension sequence and a nocurin synthase terminator are further connected downstream thereof.

FIG. 2: Signal sequence and mature curculin B downstream of the corn ubiquitin promoter and intron
Elongated peptide deletion-precursor curculin BcD
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram schematically showing the structure of a plasmid pMCU43 in which NA is connected and a nopaline synthase terminator is further connected downstream thereof.

FIG. 3 is a photograph instead of a drawing, showing the results of electrophoresis of recombinant curculin B expressed in rice calli, detected by Western analysis.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/415 C07K 14/415 C12N 5/10 C12P 21/02 C C12P 21/02 C12N 5/00 C // (C12N 15 / 09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Yoshie Kurihara 7-4-7 Okusawa, Setagaya-ku, Tokyo (72) Inventor Arai Soichi 38, Shichishima-cho, Kanagawa-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Hiroyuki Anzai 760, Shioka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture Meiji Seika Co., Ltd.Pharmaceutical Research Laboratory (72) Inventor Kazuko Katsumata, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture 760 Norooka-cho Meiji Seika Co., Ltd.Pharmaceutical Research Laboratory (72) Inventor Haruyuki Yamashita 7-35 Higashiogu, Arakawa-ku, Tokyo Asahi Denka Kogyo Co., Ltd. (72) Inventor Asahi Denka Kogyo Co., Ltd. Hiroshi Sugiyama 7-35 Higashiogu, Arakawa-ku, Tokyo

Claims (11)

[Claims] 1. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or (2) an amino acid in which one or more amino acids have been added, deleted or substituted in the amino acid sequence An acidity inhibitor comprising a polypeptide having a sequence and an acidity-suppressing activity as an active ingredient. 2. (1) DN encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing
A, or (2) a DNA encoding a polypeptide having an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence and having an acidity suppressing activity, A plasmid comprising a sequence containing a promoter capable of being activated and a sequence containing a terminator capable of functioning in a plant. 3. The corn ubiquitin promoter, cauliflower mosaic virus 3
The plasmid according to claim 2, which is a promoter selected from the group consisting of a 5S promoter and a rice actin promoter. 4. The plasmid according to claim 2, wherein the terminator is a nopaline synthase terminator or a cauliflower mosaic virus 35S terminator. 5. A sequence between the 3 ′ end of the sequence containing the control sequence of the promoter and the 5 ′ end of the sequence containing the control sequence of the terminator in the sequence containing the terminator, in the sequence containing the promoter. The plasmid according to any one of claims 2 to 4, further comprising an intron capable of functioning in a plant. 6. The plasmid according to claim 5, wherein the intron is a corn ubiquitin intron. 7. A trait obtained by transforming a cell derived from a plant (excluding Curculigo latifolia) with the plasmid according to any one of claims 2 to 6. Converted cells. 8. The transformed cell according to claim 7, wherein the plant is an edible plant. 9. It comprises the transformed cell according to claim 7.
Transformed plant. 10. The plant according to claim 9, wherein said plant is an edible plant.
A transformed plant according to item 1. 11. The transformed cell according to claim 7 or 11 or the transformed plant according to claim 9 or 13, comprising (1) an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. A polypeptide, or (2) having an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence,
In addition, a method for producing the polypeptide, comprising extracting a polypeptide having an acidity suppressing activity.