JP2001204284A - Transgenic rice plant containing non-rice plant amylose synthase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transgenic rice plant or its cell capable of providing amylose-containing rice, namely, rice simultaneously satisfying low allergen and excellent flavor in spite of the absence of rice plant waxy protein being rice allergen. SOLUTION: This transgenic rice plant or its cell has a genome to which a DNA encoding a non-rice plant amylose synthase lowly allergic to a rice allergic subject is transferred and contains no rice plant waxy protein or its fragment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a transgenic rice (transformed rice) or a cell thereof containing a non-rice amylose synthase gene.

[0002]

2. Description of the Related Art The ingestion of food, which is indispensable on a daily basis, sometimes causes an abnormal reaction in a living body. Among them, a pathological condition in which a substance in food is recognized as an antigen and presents symptoms such as asthma and eczema by a series of immunological mechanisms is called food allergy. Food allergic patients are estimated to be less than about 1% of the population, and especially in children, food allergies frequently occur as a cause of bronchial asthma, atopic dermatitis and the like. It is generally said that food allergies are caused by milk, eggs, soybeans, etc.
In recent years, allergies due to grains such as rice and wheat have been increasing (Pediatrics, Vol. 38, p.
1985; Pediatric Internal Medicine, Vol. 22, pp. 415 et seq., 1
990).

[0003] As a method of treating food allergy patients, in addition to symptomatic treatment with drugs, methods of restricting or not ingesting foods that cause allergies have been attempted.
Restricting food can hinder the maintenance and development of life. Therefore, a method of removing only allergic components and ingesting foods that do not impair other nutritional components has been studied.

In recent years, ingredients that cause food allergies,
That is, allergens have been studied, and in rice, it has been reported that globulin, a protein with a molecular weight of 16 kd, and many other protein molecules are allergens (Chemistry and Biology, Vol. 25, pp. 739 et seq., 1987). ).

[0005] For proteins as cereal allergens, especially rice allergens, salt-soluble proteins have been mainly studied. Accordingly, allergen-reduced rice obtained by treating rice to remove or reduce its salt-soluble protein has been given to rice allergic patients. However, the present inventors and their co-workers have found that some patients develop allergies even with treated rice in which the salt-soluble protein, which is a rice allergen, has been removed or reduced. In addition to identifying their allergen proteins, we also confirmed that among these rice allergens, proteins that are highly homologous not only to rice but also widely to cereals (particularly, corn and wheat). And a method for producing a newly prepared cereal preparation in which allergens have been reduced (Japanese Patent Application Laid-Open No. 10-1).
79059).

More specifically, Japanese Patent Application Laid-Open No. Hei 10-1
No. 79059 discloses one of rice amylose synthases as one of rice allergens other than salt-soluble protein.
Waxy protein (UDP glucose starch glucosyltransferase) (Pl
ant Mol. Biol. , 19, 513-516,
1992). And, in the production method described in the above-mentioned publication, allergen reduction treatment (for example,
Aqueous salt solution). As waxy protein content low cereals, waxy varieties cereal, for example, waxy rice, waxy wheat, or waxy corn, can be used, most of the waxy varieties lack waxy protein, the publication Is disclosed. Rice preparations prepared by the production method described in the publication, for example, allergen-reduced rice preparations lacking or reducing waxy proteins and salt-soluble proteins that are rice allergens,
It can be given to more patients with rice allergies than conventional allergen reduced rice preparations.

By the way, it is known that edible rice having good taste usually has an amylose content of about 15 to 18%. On the other hand, since the waxy protein is an essential enzyme for the synthesis of amylose among amylose and amylopectin, which are main components of starch, cereals lacking the waxy protein (for example,
Mochi varieties) do not contain any amylose. Therefore, the allergen-reduced rice preparation prepared by the production method described in JP-A-10-179059 is excellent in that it exhibits low allergenicity, but has a different taste from amylose-containing rice. Therefore, improvement in taste was desired.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a rice containing amylose, which does not contain rice allergen, rice waxy protein, that is, simultaneously has low allergenicity and good taste. It is to provide rice that can obtain satisfactory rice.

[0009]

The object of the present invention is to provide a rice waxy protein comprising a DNA encoding a non-rice amylose synthase having low allergenicity to a rice allergic patient according to the present invention. Alternatively, it can be solved by rice which does not contain a fragment thereof. Further, the present invention provides a DNA encoding a non-rice amylose synthase exhibiting low allergenicity to a rice allergic patient
Is introduced into the genome, and does not contain the rice waxy protein or a fragment thereof. The present invention also relates to rice obtained from the rice. Further, the present invention relates to an allergen-reduced rice preparation obtained by subjecting the rice to an allergen reduction treatment.

As used herein, “rice” includes a plant (ie, rice as a whole) and a part thereof (eg, a flower,
Leaf, stem, root, or seed, or tissue]. In addition, "rice cells" include, for example, undifferentiated rice cells or undifferentiated rice tissue culture, or
Includes protoplasts, calli (cell clumps), adventitious embryos, or adventitious buds. Furthermore, in this specification, “rice” includes rice seed itself and a part thereof, and examples thereof include paddy rice, brown rice, and polished rice (including partially polished rice).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The transgenic rice or cells thereof according to the present invention are (1) free of rice waxy protein or a fragment thereof, and (2) have a hypoallergenic property in a genome against rice allergy patients. (Hereinafter referred to as "hypoallergenic non-rice amylose synthase").

"Rice waxy protein" as used herein is one of the amylose synthases and is a protein having a molecular weight of 60 kd and consisting of 532 amino acids (Plant Mol. Biol., 19, 513-).
516, 1992). Rice waxy protein is an essential enzyme of amylose synthesis among amylose and amylopectin, which are main components of starch, and can be an allergen for rice allergy.

As used herein, the term "rice or cell thereof does not contain rice waxy protein or a fragment thereof" means that rice rice or cell thereof is rice irrespective of the presence or absence of its enzymatic activity. Not included (ie, not expressed) as a protein or fragment thereof that is allergenic to allergic patients
Means the state.

The rice or cells thereof in such a state include, for example, (1) a gene encoding a rice waxy protein is deficient (natural mutant and artificially deficient by genetic engineering). And both variants)
Although rice or its cells in which rice waxy protein is not expressed, or (2) a gene encoding rice waxy protein is present, for example, it has an abnormality in transcription and / or translation stage, and as a result, rice waxy Rice or a cell thereof in which the protein is not expressed can be mentioned. In addition, the inactive rice waxy protein which does not show the enzyme activity as rice waxy protein, or a partial fragment of the rice waxy protein which does not show the enzyme activity is expressed in rice or its cells, When they show allergenicity, as is clear from the above definition, they do not fall under the condition "rice or its cells do not contain rice waxy protein".

In the transgenic rice plant of the present invention or its cells, the "low allergenic non-rice amylose synthase" encoded by the DNA contained therein is:
(1) Amylose synthesis can be performed in rice instead of rice waxy protein, and (2)
As long as it shows low allergenicity to rice allergy patients,
There is no particular limitation. In addition, in this specification, "non-rice" means all organisms other than rice, for example, plants, animals, or microorganisms other than rice.

The enzyme capable of synthesizing amylose in rice in place of rice waxy protein (ie, amylose synthase) is not limited to, but includes, for example, rice waxy in non-rice plants. Protein corresponding to the protein, for example, waxy proteins of grasses other than rice, or
Starch synthase (granule bound) of plants other than grasses (non-grass plants)
start synthase (GBSS). Waxy protein and GBSS
Not only are the biochemical properties similar, but the partial amino acid sequences have high homology to each other.

Means for determining whether or not a certain amylose synthase exhibits hypoallergenicity in a rice allergic patient include, for example, sera of a plurality of rice allergic patients and sera of healthy individuals (control sera). ELIS using
A (enzyme-linked immunosorbent)
ent assay) method, for example,
It can be carried out by the procedure shown in Reference Example 2 described later. ELISA values for rice waxy protein and ELISA values for amylose synthase (enzyme to be determined) for which allergenicity is to be determined were measured for sera of a plurality of rice allergic patients, and the test values (EL
ISA values) in three stages: negative, weakly positive [ie, positive (+)], and strongly positive [ie, positive (+
+)], More than half (preferably 80% or more) of allergic patients with rice showed higher E-values for the enzyme to be determined than those for rice waxy protein.
If the LISA value has decreased by one or more steps, it can be determined that the patient has low allergenicity in a rice allergic patient.

In the transgenic rice plant of the present invention or the cells thereof, the "low allergenic non-rice amylose synthase" encoded by the DNA contained therein includes, for example, armpits of grasses other than rice (for example, maize). Dye proteins, starch-bound starch synthase (GBSS) of convolvulaceous plants (eg, sweet potato), GBSS of solanaceous plants (eg, potato), or GBSS of legumes (eg, kudzu)
Can be mentioned.

The term "non-rice amylose synthase" used herein includes naturally occurring amylose synthase, as well as functionally equivalent variants thereof, for example, mutants, The obtained recombinant protein or a protein having an amino acid sequence in which one or more (preferably one or several) amino acids have been deleted, substituted, and / or added in the amino acid sequence of amylose synthase ( However, it is limited to those having amylose synthase activity).

The transgenic rice of the present invention includes, but is not limited to, rice-derived cells that cannot express rice waxy protein,
It can be obtained by introducing a DNA encoding a low-allergenic non-rice amylose synthase by a method known per se, and then subjecting the resulting transgenic plant cells to growth and individual regeneration.

Further, the transgenic rice cell of the present invention is not limited thereto. For example, the transgenic rice cell can be transformed into a rice-derived cell which cannot express rice waxy protein by a method known per se. It can be obtained by introducing a DNA encoding an allergenic non-rice amylose synthase.

As a rice cell that can be used for preparing the transgenic rice of the present invention or a cell thereof, that is, a rice-derived cell that cannot express rice waxy protein, a known rice cell can be used as it is. It can be used, or rice cells produced by cultivar selection, generation of mutants, or genetic engineering techniques can be used.

As rice that cannot express rice waxy protein, for example, rice varieties rice can be used. Most rice varieties lack rice waxy protein [Hirano Hiroyuki, "Seed Bioscience" (1995) Gakkai Shuppan Center, 117
Pp. 122-]. In addition, many waxy mutants obtained by mutagen treatment have been isolated, and mutants expressing the protein in an inactive form or mutants lacking the protein itself have been obtained (protein / Nucleic Acids and Enzymes, Vol. 37, pp. 1317-1325, 19
1992), the latter mutant, that is, a mutant lacking the protein itself can be used.

Further, in the genetic engineering technique, rice in which the expression of this waxy protein is suppressed can be produced by, for example, antisense technology (for example, JP-A-4-104791, JP-A-5-13981).
And JP-A-5-168482). For the suppression, a rice waxy promoter, a maize Adh1 promoter, a cauliflower mosaic virus 35S promoter, a rice allergen promoter, and the like, which are expressed in rice endosperm, can be used as the promoter. Examples of the terminator include a nopaline synthase gene NOS terminator and a cauliflower mosaic virus 35S RNA terminator.

When a DNA encoding a low-allergenic non-rice amylose synthase is introduced into the genome of a rice cell that cannot express rice waxy protein, the DNA is inserted into an appropriate vector, It is preferable to construct a vector containing the DNA, and to transform rice cells with the vector. In addition, the vector includes the DN, in addition to the DNA.
More preferably, a promoter and a terminator that function in rice seeds are inserted upstream and downstream of A, respectively.

It is known that rice waxy proteins are mostly localized in amyloplasts in rice seeds. In addition, a precursor of waxy protein in various plants including rice has, at its amino terminus, a transit peptide which is thought to play an important role in transport to amyloplast (for example, a transit peptide of rice waxy protein is It is also known that there are 77 amino acid residues, the transit peptide of potato GBSS consists of 77 amino acid residues, and the transit peptide of maize protein has 72 amino acid residues). Therefore, D encoding low allergenic non-rice amylose synthase
When introducing NA into the genome of a rice cell that cannot express rice waxy protein, it is considered that the amino terminus of the low-allergenic non-rice amylose synthase plays an important role in transport to amyloplast. It is preferable to construct the vector so that the added transit peptide is added.

Examples of promoters that function in rice seeds include rice waxy protein promoter and cauliflower mosaic virus 35S promoter (the.
Embo Journal, Vol. 6, pp. 3901-3907
1987), rice actin promoter (The Plant Cell, Volume 2, pages 163 to 171, 199).
0), the rice glutelin promoter (Plant Molecular Biology, Vol. 30, pp. 1207-1221, 1996), or the maize ubiquitin promoter (Plant Molecular Biology, Vol. 26, No. 1007). Pp. 1012, 199
4 years) can be used, and in that rice seed can be highly expressed in a rice seed-specific manner, a rice waxy protein promoter and a rice glutelin promoter are preferable, and a rice waxy protein promoter is particularly preferable.

As a terminator that functions in rice seed, for example, a nopaline synthase terminator, a cauliflower mosaic virus 35S terminator, or the like can be used.

In the above-mentioned vector, between 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. To
When an intron capable of functioning in rice is further provided, gene expression efficiency can be improved, or mRNA stability can be improved.
Examples of the intron include castor catalase intron (Nucleic Acid Research, No. 18).
Vol. 6767-6770 (1990) or corn ubiquitin intron (Plant Molecular Biology, 26, 1007-1st.
012, 1994), but corn ubiquitin intron is particularly preferred.

When a direct gene transfer method such as a particle gun method is used, the gene can be expressed in rice cells.
A cassette containing a DNA encoding a low allergenic non-rice amylose synthase and a cassette for a selectable marker gene that can be expressed in rice cells were transformed into a multicopy plasmid generally used in E. coli [for example, pUC19 (Takara Shuzo) ] And introduced into rice cells. At this time, the selectable marker gene can be (1) introduced while being retained on the same plasmid, or (2) can be introduced after being retained on separate plasmids and mixed (cotransformation). For example, the bialaphos resistance (bar) gene (Molecular
General Genetics, Volume 205, pp. 42-
50, 1986), the plasmid pDM302 contained between the rice actin promoter and the nopaline synthase terminator (Plant Cell Reports, 11, 586-591, 1992).
By mixing and co-transforming a plasmid having a DNA encoding a low-allergenic non-rice amylose synthase, the gene can be introduced into rice cells.

When the Agrobacterium-mediated transfection method is used, a cassette containing a DNA encoding a low allergenic non-rice amylose synthase that can be expressed in rice cells, and a cassette that can be expressed in rice cells. Thus, a binary plasmid for Agrobacterium containing a cassette of a selective marker gene can be prepared and introduced into rice cells. For example, a DNA encoding a hypoallergenic non-rice amylose synthase is converted into a binary vector pBI.
121 (manufactured by Clontech) and cloned between the 35S promoter and the nopaline synthase terminator, infecting Agrobacterium having a binary plasmid containing the DNA into rice cells, selecting for kanamycin resistance, and transforming cells. And a plant body can be obtained from the transformed cell.

The selectable marker gene may be a hygromycin resistance gene, a kanamycin resistance gene, or ba
The r gene and the like can be used, and the bar gene is particularly preferable (Plant Molecular Biology, Vol. 26, pp. 1007-1010, 1994).

As a method for introducing the constructed plasmid into rice, any method established as a method for transforming rice can be used. For example, a method of direct introduction into plant protoplasts by electroporation or polyethylene glycol, a particle gun method of directly introducing into plant cells using microprojectile, or an introduction method via Agrobacterium can be used. A particle gun method or an introduction method via Agrobacterium is preferred. In addition, cells and tissues suitable for the introduction method can be used as cells to be transformed. For example, embryos extracted from immature seeds, ripe seeds, or induced calli can be used as appropriate tissues.

Cells or tissues into which the gene obtained by the above method has been introduced are selected, and the plant can be regenerated by any method. For example, a transformed cell (callus) can be obtained by transplanting a tissue into which a gene has been introduced into a medium containing a selective drug corresponding to a selectable marker and a plant hormone and culturing it for several weeks. After that, the obtained cells (callus) are transferred to an appropriate growth medium, and further cultured in an appropriate regeneration medium, whereby a transformed rice plant can be obtained. The expression of the hypoallergenic non-rice amylose synthase can be confirmed by immunological analysis.

The rice of the present invention obtained from the transgenic rice of the present invention is further subjected to an allergen reduction treatment, whereby the allergen-reduced rice preparation of the present invention can be prepared. The allergen reduction treatment is not particularly limited as long as rice allergens other than rice waxy protein can be removed or reduced, and can be performed by a known allergen reduction method.

Examples of such known allergen reduction methods include, for example, the allergen reduction methods described in JP-A-10-179059, for example, proteolytic enzymes (for example, papain, collagenase, transglutaminase, and carboxypeptidase). ), A salt [eg, a salt of an inorganic acid (eg, hydrochloride, sulfate, carbonate, or phosphate) or an alkali metal salt (eg,
Aqueous solution, lower alcohol (e.g., ethanol), alkaline aqueous solution (preferably an aqueous solution adjusted to pH 7 to 10, more preferably pH 8 to 10), and / or acidic aqueous solution (preferably pH
5 or less, more preferably an aqueous solution adjusted to pH 4 or less)
Or a method of fermenting with a solution in which saccharides and saccharide-decomposing microorganisms (eg, lactic acid bacteria) are dispersed, and a method of appropriately combining them.

[0037]

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.

[Reference Example 1] << Various gramineous plant waxy proteins and potato starch granule-bound starch synthase (GB
Preparation of SS) >> First, starch powder was prepared from commercially available rice grains, wheat grains, and corn grains according to the following procedure. As for potato, commercially available potato starch (indicated by 100% potato starch) was used as starch powder. Hereinafter, the preparation procedure in the case of using rice grains will be described, but the same procedure was applied to wheat grains or corn grains.

That is, 1 g of a powder obtained by crushing rice grains in a coffee mill was mixed with an extraction buffer [2.3% sodium dodecyl sulfate (hereinafter referred to as SDS), 5%-
2-mercaptoethanol, 10% glycerol] 10
and shaken for 30 minutes. Then, 12000
After centrifugation for 5 minutes by rotation, the obtained precipitate was suspended again in 10 ml of the extraction buffer, and shaken for 30 minutes.
Centrifuge at 00 rpm for 5 minutes. The obtained precipitate is washed with 10 ml of water.
After shaking for 20 minutes, the mixture was centrifuged at 12,000 rpm for 5 minutes, and this operation was repeated once for the obtained precipitate. The obtained precipitate was suspended in 10 ml of acetone,
After shaking for 15 minutes, centrifuge at 12,000 rpm for 5 minutes,
This operation was repeated once for the resulting precipitate.
Finally, the precipitate was air-dried to obtain a white starch powder.

Next, waxy proteins or GBSS were extracted from various starch powders according to the following procedure. That is, 20 mg of various starch powders were weighed into a plastic tube (Eppendorf tube), suspended in 200 μl of the extraction buffer, and heated at 100 ° C.
And then cooled on ice. Further, 200 μl of extraction buffer was added, and the gelled portion was pulverized with a spoonful and mixed well. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was transferred to a new plastic tube. To this solution was added 3 volumes of acetone, mixed well, and the freezer (-4
(0 ° C.) overnight. The precipitate obtained by centrifugation at 12,000 rpm for 5 minutes was dissolved in an equal volume of 8 mol / l urea solution, and subjected to the following experiment as a waxy protein sample or GBSS sample.

[0040]

Reference Example 2 << Evaluation of Allergenicity of Various Waxy Proteins or GBSS Using Rice Allergic Patient Serum >> Waxy Protein Sample and G Prepared in Reference Example 2
About BSS sample, I in serum of rice allergy patient
The reaction with the gE antibody was measured by the ELISA method. That is, the various samples were diluted with a 100-fold volume of a phosphate buffer solution (hereinafter, referred to as PBS), and 100 μl of each of the obtained sample diluting solutions was applied to an ELISA plate (ELISA plate maxisorp; manufactured by Nunc).
And coated at 4 ° C. overnight. After washing each well of the plate twice with PBS, 2% bovine serum albumin (hereinafter referred to as BSA) -containing PBS
After 250 μl was added and blocking was performed at 37 ° C. for 1 hour, the plate was washed twice with PBS. Serum collected from 14 patients diagnosed with rice allergy or healthy adult serum (control serum) was prepared using 0.2% BSA and 0.05% surfactant Tween 20 (Tween2).
The mixture was diluted to 10-fold volume with PBS containing 0), 100 μl of each serum dilution was added to the plate, and reacted at room temperature for 24 hours.

PBS containing 0.05% Tween 20
After washing the plate four times with PBS (hereinafter referred to as PBS-T), 100 μl of a 2,500-fold dilution of biotin-conjugated sheep anti-human IgE antibody (manufactured by Vector) as a secondary antibody was added to the plate. In addition, the reaction was carried out at 37 ° C. for 2 hours. In addition, PBS-T containing 0.2% BSA was used for dilution of the antibody. After the reaction with the secondary antibody was completed, the plate was washed 5 times with PBS-T, and
Avidin-conjugated peroxidase (manufactured by Boehringer) diluted by a factor of 1 was added to perform enzyme labeling. Again, PBS-
The plate was washed 5 times with T, and the substrate solution (0.034%
100 μl of a 0.1% orthophenylenediamine solution to which H ₂ O ₂ was added). After accurately reacting at 37 ° C. for 30 minutes, color development was stopped by adding 40 μl of ₂ mol / l-H ₂ SO ₄ , and the absorbance (490 nm) was measured. All measurements were performed in duplicate, and the average was determined.

The results are shown in Table 1. Since the sum of the average ELISA value and twice the SD value when the control serum was used was 0.030, the case where the ELISA value was 0.03 or less was defined as "negative". Also, ELIS
If the A value is more than 0.03 and less than 0.10 (that is, weakly positive), it is regarded as “positive (+)” and EL
The case where the ISA value is 0.10 or more (that is, the case where the ISA value is strongly positive) is shown in Table 1 as “positive (++)”. As a result, it was found that the reactivity of the IgE antibody in the serum of the rice allergic patient with the waxy protein or GBSS of each crop was different, and the reactivity with the corn waxy protein and the potato GBSS was low.

<< Table 1 >> Negative Positive (+) Positive (++) Rice −6 8 Wheat 5 3 6 Corn 7 7 0 Potato 9 3 2

[0044]

Example 1 << Preparation of Transgenic Rice Introducing cDNA of Potato Starch Granule-Binding Starch Synthase (GBSS) >> (1) Preparation of cDNA of Potato GBSS A phenol-SDS method was used from a potato cultivar, Baron. Edited by Masahiro Sugiura, "Cloning and Sequence
Plant Biotechnology Experiment Manual ”(1989
Year) Rural culture company, pp. 30-33]
An NA (total RNA) fraction was prepared. This all RN
Using A as a template, a reverse transcription reaction was performed using a reverse transcriptase derived from mouse leukemia virus (MuLV-RT; Takara Shuzo) and oligo dT (Takara Shuzo) as a primer, and cD
An NA mix was obtained. This cDNA mix and a known potato GBSS cDNA sequence (MGG., 22
8, 240-248, 1991) based on PCR (polymerase chain area).
ction) When PCR was performed using the primers PWAX3 and PWAX4, a DNA fragment of about 2 kb was amplified. The primers PWAX3 and PWAX3
Each nucleotide sequence of WAX4 is as follows: PWAX3: 5'-CTCAACAAGTGCTTTGG
TAGA-3 'PWAX4: 5'-GGTTCTACATAGTTTCG
CTAG-3 '

The amplified DNA fragment was cloned into the E. coli vector pTZ18U (Toyobo) at the SmaI site. Regarding the plasmid DNA of the obtained 5 clones,
Commercially available sequencing kit (BigDye Term
inator CycleSequencing Ki
t; ABI) and an automatic DNA sequencer (ABI)
377; manufactured by ABI), and the base sequence of the DNA fragment contained in one clone was determined to be the known cDNA of the aforementioned potato GBSS.
Completely matched the sequence. This plasmid containing the potato GBSS gene (including the promoter and the structural gene) was named pAFT300 and was used in the following examples.

(2) Preparation of plasmid for rice introduction The promoter for expression of potato GBSS in rice includes:
The rice waxy gene promoter was used. Further, as the transit peptide controlling the transfer of the waxy protein to amyloplast, the transit peptide of the rice waxy protein was used, and the replacement of the potato GBSS with the transit peptide was performed by recombinant PCR according to the following procedure. Done.

Specifically, the test was performed according to the protocol shown in FIG. First, a plasmid pRWA1 containing a rice waxy gene (including a promoter and a structural gene)
11 (Mol. Biol. Evol., 15, 978-
987, 1998) as a template and a primer pair RP1
By carrying out PCR using RP2 and RP2, DN containing rice transit peptide
A fragment A was obtained. Similarly, by using the plasmid pAFT300 prepared in Example 1 (1) as a template and performing PCR using the primer pair RP3 and RP4, D-containing part of the N-terminal side of the mature potato GBSS is obtained.
NA fragment B was obtained. The primers RP1 to RP
The base sequences of No. 4 are as follows: RP1: 5′-TGCAGAGATCTTTCACAG
CAACAGC-3 'RP2: 5'-CATTCCCTTTCCGGTGGC
GTACACGACGAC-3 'RP3: 5'-GTACGCCCACCGGAAAGGGG
AATGAACTTG-3 'RP4: 5'-GATGTCCGCGGGCTGCAA
GGGCTGG-3 '

The obtained DNA fragment A and DNA fragment B
Were separated and purified by agarose gel electrophoresis, respectively. Subsequently, the purified DNA fragment A and DNA
Fragment B was mixed and recombinant PCR was performed using primer pairs RP1 and RP4. The DNA fragment A
A common sequence: 5′-GTACGCCCACCGGAAAGGGAATG at the 3 ′ end of the DNA fragment B and the 5 ′ end of the DNA fragment B
Due to the presence of -3 ', the DNA fragment amplified as a result of this recombinant PCR is a DNA fragment obtained by fusing a portion of the N-terminal side of mature potato GBSS to the transit peptide of rice waxy protein.

The PCR product obtained by the recombinant PCR was cloned into an E. coli vector pTZ18U to obtain a plasmid pAFT301. An approximately 400 bp DNA fragment obtained by double digestion of this plasmid pAFT301 with restriction enzymes BglII and SacII and double digestion of pAFT300 prepared in Example 1 (1) with restriction enzymes BamHI and SacII are obtained. The obtained DNA fragment of about 4.4 kb was ligated to obtain a plasmid pAFT302. This plasmid pAF
T302 contains the transit peptide of rice waxy protein and whole potato mature GBSS.

On the other hand, the plasmid pRWA containing the rice waxy gene (including a promoter and a structural gene)
111 containing the rice waxy gene promoter obtained by double digestion with BglII and BamHI (however, partial digestion of BglII).
The kb DNA fragment was purified and used for the E. coli vector pB.
The plasmid pAFT303 was obtained by cloning into the BamHI site of luscriptKS + (Toyobo). Furthermore, the XbaI / KpnI-DNA fragment of about 2 kb of the plasmid pAFT302 obtained above was ligated with T4DN
After blunt-ending with A polymerase (Takara Shuzo), the plasmid was cloned into the SmaI site of the plasmid pAFT303 to obtain a plasmid pAFT308. This plasmid pAFT308 contains a rice waxy gene promoter, a transit peptide of rice waxy protein,
And whole potato mature GBSS.

On the other hand, as a vector, a plant plasmid pGAH for Agrobacterium infection method (plant cell engineering,
4, 193-203, 1992). That is, a DNA fragment containing the neomycin resistance gene obtained by double-cutting the plasmid pGAH with the restriction enzymes BamHI and HindIII was inserted into the E. coli vector pKF3 (Takara Shuzo) at the BglII / HindIII site to obtain the plasmid pAFT5. Next, plasmid pAHC27 (TransgenicRe
s. 5, 213-218, 1996), a DNA fragment containing the corn ubiquitin promoter, the E. coli β-glucuronidase gene, and the nopaline synthase terminator was converted to HindIII / KpnI-DNA.
The plasmid pAFT5 was excised as a fragment.
ndIII / KpnI site, the plasmid pA
FT14 was obtained.

Further, the plasmid pAFT14 was first digested with the restriction enzyme SacI, and the sticky end was converted to a blunt end with T4 DNA polymerase (Takara Shuzo).
About 12 kb of DN obtained by digestion with restriction enzyme BglII
After ligating the A fragment and the approximately 6 kb BamHI / EcoRV-DNA fragment of the plasmid pAFT308 obtained above (including the whole rice waxy gene promoter, rice waxy protein transit peptide, and potato mature GBSS), DH5α was transformed into a plant plasmid pAF for Agrobacterium infection.
T310 was obtained. The resulting plasmid pAFT31
The structure of 0 is schematically shown in FIG.

(3) Introduction of plasmid pAFT310 into Agrobacterium For introduction of a gene into rice, Agrobacterium tumefaciens was used.
aciens) EHA101 (plant cell engineering, vol. 4,
193-203, 1992) were used as host cells for infection. The introduction of the plasmid pAFT310 prepared in Escherichia coli into Agrobacterium in Example 1 (2) was performed by a three-way conjugation method (EMBO J., 2, 41).
1-418, 1983). That is, the Agrobacterium tumefaciens EHA101, Escherichia coli DH5α having the plasmid pAFT310, and Escherichia coli DH1 having the conjugative helper plasmid pRK2013 were respectively treated overnight in an LB liquid medium, which is a general Escherichia coli culture medium. After culturing, 1 ml of each culture
Was centrifuged, and the precipitate was resuspended in 1 ml of LB liquid medium. 5 μl of each of the three culture suspensions was spotted at the same location on the LB agar medium, and cultured at 28 ° C. overnight. The grown colonies were suspended in LB liquid medium, diluted appropriately, and mixed with 2.5 μg / ml tetracycline and 25 μg.
/ Ml kanamycin and spread on LB agar medium.
C. for 3 days to obtain Agrobacterium having plasmid pAFT310.

(4) Gene introduction into rice by Agrobacterium method and selection and cultivation of transformant The gene introduction into rice was carried out by the method of Toki et al.
"Regular Biology Reporter,
15 (1), 16-21, 1997]. That is, after the mature seeds of Nipponbare were threshed, they were treated with 70% ethanol for 30 seconds, and subsequently sterilized by stirring with a 1.5% final concentration of sodium hypochlorite solution for 30 minutes. After thoroughly washing with sterile water and removing moisture from the seeds with filter paper,
The cells were seeded on an N6D solid medium, cultured at 28 ° C. and under light for 3 weeks to induce calli. On the other hand, the Agrobacterium prepared in Example 1 (3) was cultured in an AB medium in a dark place at 28 ° C. for 2 days, and the cells were transferred to an AAM medium containing 10 mg / l acetosyringone. Agrobacterium suspension was prepared by suspending the Agrobacterium in such an amount that it became slightly cloudy.

The induced calli were placed in a stainless steel mesh basket, immersed in the Agrobacterium suspension for 2 minutes, and then placed on a sterilized water-absorbing paper (Kimwipe) to remove excess bacterial solution. Removed. 100 pieces of calli infected with Agrobacterium were
The plate was placed on a Petri dish of 6AS medium (20 pieces per petri dish) and cultured in a dark place at 28 ° C. for 3 days to infect Agrobacterium.

The callus was placed again in a stainless steel mesh basket, immersed in an N6D liquid medium, and the N6D liquid medium was replaced until the washing liquid did not become cloudy, and Agrobacterium was washed away. A stainless mesh basket containing callus from which Agrobacterium had been removed was placed on sterilized filter paper to remove excess water, and N6 supplemented with 500 mg / l carbenicillin and 50 mg / l hygromycin.
The callus was placed on the D solid medium and cultured under illumination at 28 ° C. for 3 weeks. 50 pieces of callus grown in 3 weeks of drug selection culture were added to 250 mg / l carbenicillin and 50 mg
Transplanted into regeneration medium containing 1 / l hygromycin,
The cells were cultured under illumination at 8 ° C. for 3 weeks. When the shoots and roots are differentiated from the callus by 1 cm or more, 50 mg
The cells were transplanted to a hormone-free medium supplemented with / l hygromycin, and the culture was continued under illumination at 28 ° C. When the plant has grown to a dish, the culture soil (Bonsol;
(Sumitomo Chemical Co., Ltd.), and 25 transgenic plants were grown in a closed greenhouse, and T1 seeds could be collected from 21 of them.

[0057]

[Evaluation Example 1] << Analysis of Transformant by PCR >> Eighty individual leaves 50 randomly selected from grown transgenic plants
The DNA was extracted from the DNA solution using the CTAB method [edited by Tadashi Watanabe, edited by Masahiro Sugiura, “Manual for Cloning and Sequencing and Plant Biotechnology Experiments” (1989) Rural Bunkasha, pages 253 to 255]. 5
0 μl was prepared. 5 μl of this DNA solution and the primer
PCR using Taq DNA polymerase (manufactured by Takara Shuzo) was performed using PWAX1 and PWAX2. The primers PWAX1 and PWAX2 were designed to be able to amplify the inside of the coding region of the potato GBSS gene, and their base sequences are as follows: PWAX1: 5′-GAATGCCAAGGTCGCT
TTCTG-3 ′ PWAX2: 5′-TGCCGATGAAGCCAAT
CAAAG-3 'The plasmid pAFT310 was used as a control. As a result of PCR, plasmid pA
An amplified band having the same size as that of FT310 was detected.

[0058]

[Evaluation Example 2] << Qualitative detection of amylose by iodine starch reaction of rice slices >> T1 seeds (5 seeds per individual) of the 21 transformants obtained in Example 1 (4) were used. The two halves were halved and one half was stained with the staining solution (0.0
2% iodine, 0.2% iodide potassium). As controls, mochi rice (containing no amylose) and urchi rice (containing amylose) were used. After observing the staining after 20 hours at room temperature,
Eleven individuals were as colorless as the waxy rice without amylose. The remaining 10 individuals were stained in the same blue color as wool rice containing amylose.
However, even among the 10 individuals stained blue, the density of staining varied between individuals. More specifically, four individuals exhibited a pale blue color, and it was presumed that the contained amylose was minimal, while six individuals exhibited a relatively dark blue color. Furthermore, six individuals having relatively dark blue color also had shading between the five grains, indicating that the T1 seed was heterozygous.

[0059]

Evaluation Example 3 << Quantification of Amylose by Iodometric Colorimetry >> (1) Preparation of Standard Amylose Sample Nipponbare Mochi Brown Rice Powder and Amylose (Type III, derived from potato; Sigma) The samples were weighed in the amounts shown in Table 2 and thoroughly ground and mixed in a mortar to prepare four standard amylose samples containing a predetermined amount of amylose.

<< Table 2 >> Amylose Content Mochi Brown Rice Flour (mg) Amylose (mg) 0% 20000 1% 1980 20 2.5% 975 255 5% 380 20

(2) Determination of amylose The iodine colorimetric method [Julia] showing the specific procedure will be described below.
no method (Cereal. Sci. Today, 16, 3)
34-340, 1971)], among the T1 seeds of the 21 transformants sown in Example 1 (4), a transformant showing a relatively dark blue color in Evaluation Example 2 described above. About 4 body lines (4 grains per individual)
Amylose quantification was performed. In addition, Nipponbare mochi was used as a control.

One T1 seed of the transformant was weighed as is with brown rice, crushed with a hammer and placed in a test tube. Subsequently, after adding 0.1 ml of 95% ethanol, 1 mol
/ L sodium hydroxide (0.9 ml) at 100 ° C.
Treated for 0 minutes. After further adding 5 ml of distilled water, the mixture is ground with a polytron, the shaft of the polytron is washed with fresh distilled water, the washing solution is combined, the total volume is adjusted to 20 ml, and the transformation is carried out by sufficiently stirring. This was used as a seed sample liquid. For each transformant, four kinds of T1 seeds and four kinds of transformed seed sample solutions (hereinafter referred to as "n-1" to
"N-4") was prepared. On the other hand, the above operation was repeated except that 20 mg of each standard amylose sample prepared in Evaluation Example 3 (1) was used instead of the T1 seed crushed with a hammer, to prepare a standard amylose sample solution.

Each standard amylose sample solution and each transformed seed sample solution (1 ml) were placed in a test tube, and each sample was taken at 1 mol / mol.
After adding 0.5 ml of acetic acid and mixing, the iodine solution (0.1 ml) was added.
After further adding 0.1 ml of 2% iodine and 2% potassium iodide) and mixing well, the total volume was adjusted to 10 ml with distilled water. After reacting at 27 ° C. for 20 minutes, a wavelength of 620
The absorbance at nm was measured. From the standard curve prepared with the standard amylose sample solution, the amylose content of each transformed seed sample solution was calculated and corrected to 20 mg per brown rice grain. As a result, the amount of amylose shown in Table 3 was detected. . In Table 3, the unit of the amylose content is “%” and the symbol “N
"D" indicates that no amylose was detected.

<< Table 3 >> Transformant n-1 n-2 n-3 n-4 W310-23 2.1 ND 1.0 0.8 W310-24 1.5 3.3 1.1 2.6 W310-25 2.1 ND 1.1 1.8 W310-43 1.8 2.0 0.8 0.1 Nipponbare Mochi ND ND ND ND

[0065]

The transgenic rice of the present invention does not contain the rice allergen, rice waxy protein. Further, since the transgenic rice of the present invention can express a low allergenic non-rice amylose synthase, the seed of the transgenic rice does not contain the rice waxy protein, which is an enzyme essential for amylose synthesis. Contains amylose. Therefore, according to the transgenic rice of the present invention, it is possible to obtain rice that can simultaneously satisfy low allergenicity and good taste.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing a procedure for constructing a plasmid pAFT310.

FIG. 2 is an explanatory view schematically showing a structure of a plasmid pAFT310.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Hiroyuki Anzai 8-4-1 Higashiogu, Arakawa-ku, Tokyo Inside the Allergen-Free Technology Laboratory (72) Inventor Kazufumi Tsubaki 8-4-1 Higashiogu, Arakawa-ku, Tokyo No. Co., Ltd. Allergen-Free Technology Research Institute (72) Inventor Sadasuke Shimada 8-4-1 Higashi-Oku, Arakawa-ku, Tokyo Co., Ltd. (72) Inventor Kazuyuki Mogi 8-Chome Higashi-Oku, Arakawa-ku, Tokyo No.4-1 Inside Allergen-Free Technology Research Institute (72) Inventor Hiroshi Sugiyama 8-4-1 Higashio Higashi Arakawa-ku, Tokyo Inside (72) Inventor Hiroyuki Hirano Aoi Adachi-ku, Tokyo 2-16-4- 312 (72) Inventor Yoshiro Ikezawa 24-8 Nomidaidori, Kanazawa-ku, Yokohama, Kanagawa Prefecture F-term (reference) 2B030 AA02 AB03 AD08 AD20 CA17 CA19 CB02 CD03 CD07 CD09 CD10 CD13 CD17 CD21 4B024 AA05 AA08 BA07 BA80 CA04 DA01 EA04 FA02 FA07 FA10 GA17 4B065 AA88X AA88Y AA89X AB01 AC14 BA02 BA25 BB37 CA22 CA27 CA53

Claims (4)

[Claims] 1. A DNA encoding a non-rice amylose synthase exhibiting low allergenicity to a rice allergic patient
Has been introduced into the genome and does not contain the rice waxy protein or a fragment thereof. 2. A DNA encoding a non-rice amylose synthase showing low allergenicity to a rice allergic patient
Is a rice cell which has been introduced into the genome and does not contain the rice waxy protein or a fragment thereof. 3. Rice obtained from the rice according to claim 1. 4. An allergen reduced rice preparation obtained by subjecting the rice according to claim 3 to an allergen reduction treatment.