JP5070544B2 - Transformed rice, rice that lowers blood pressure, and rice vectors - Google Patents

Description

  The present invention mainly relates to the production of an expression vector for accumulating glutamate decarboxylase in rice at a high concentration in a specific tissue, the production of a transformed rice plant using the same, and the use or use thereof. It is.

  In the 21st century, the number of patients suffering from lifestyle-related diseases such as obesity, hyperlipidemia, and hypertension is increasing rapidly worldwide. Therefore, measures for preventing lifestyle-related diseases and improving their symptoms are urgent issues. In Japan, elderly people are taking large amounts of antihypertensive drugs to prevent lifestyle-related diseases. The cost of the drug was estimated at 800 billion yen in 2004, and there are concerns about side effects as well as pressure on medical finance.

  In recent years, γ-aminobutyric acid (GABA) has attracted attention as a health ingredient because of its excellent effects such as lowering blood pressure, preventing obesity, and improving internal functions, and a wide variety of foods and supplements enriched with GABA such as germinated brown rice It has become. That is, from the viewpoint of the same source of medical foods, germinated brown rice enriched with GABA is mixed with cooked rice and eaten every day, or GABA supplements are consumed daily, and health benefits such as prevention of increased blood pressure are expected. .

  GABA is synthesized by glutamate decarboxylase (GAD), which catalyzes irreversible synthesis from glutamate. This GAD is a kind of enzyme protein that exists universally in organisms from E. coli to humans and rice.

JP 2004-290069 A Kazuhito Akama, Takashi Akihiro, MasatoKitagawa, Fumio Takaiwa (2001) Rice (Oryza sativa) contains a novel isoformof glutamate decarbyoxylase that lacks an authenticcalmodulin-binding domain at the C-terminus. Biochemica et Biophysica Acta1522,147-150. Fumio Takaiwa, Kiyoharu Oono, David Wing and Akira Kato (1991) Sequence of three members and expression of a new major subfamily of glutelin genes fromrice.Plant Molecular Biology 17 (4), 875-885. Elizabeth E. Hood, Jenifer M. Murphy and Robert C. Pendleton (1993) Molecular characterization of maize extensin expression. Plant Molecular Biology, 23 (4), 685-695. Yukoh Hiei, Shozo Ohta, Toshihiko Komari and TAkahashi Kumashiro (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agorbacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6 (2), 271-282. Jeff J. Doyle and Jane I. Doyle (1990) Isolation of plant DNA from fresh tissue. Focus, 12, 135-137. L.Q.Qu, Y.Tada and F.Takaiwa (2003) In situ westernhybridization: a new, highly sensitive technique to detect foreign andendogenous protein distribution in rice seeds.Plant Cell Reports, 22,282-285.

  However, in the case of germinated brown rice, there is a problem that the taste is not always excellent and the constant intake is difficult. In addition, in the case of rice, GABA is accumulated in the rice bran portion, and in the case of brown rice, the intake is secured to some extent, but the polished rice is reduced to less than a fraction. In the case of supplements, there are problems of bothersome intake and high cost.

  This invention is made | formed in view of the above, Comprising: It aims at enabling provision of white rice with much GABA content. More specifically, the present invention provides an expression vector capable of efficiently accumulating GAD protein in seeds (endosperm part) in gramineous plants, and providing a technique capable of accumulating GABA in the edible part. .

  Another object of the present invention is to provide brown rice rich in free amino acids, minerals or vitamins, not limited to GABA.

In order to achieve the above object, transformed rice according to claim 1 is a DNA encoding the amino acid sequence represented by SEQ ID NO: 2, or one or several amino acids of the amino acid sequence represented by SEQ ID NO: 2. Is a fertilized endosperm GABA-accumulating transformation rice introduced with DNA encoding an amino acid sequence having glutamic acid decarboxylase activity consisting of an amino acid sequence containing substitution, deletion, insertion, addition or inversion of .

  The transformed rice according to claim 2 is the transformed rice according to claim 1, wherein the DNA encoding the amino acid sequence is linked to the downstream side of the promoter region of the rice glutelin gene.

  The rice glutelin gene includes the A gene family and the B gene family, of which GluB-1 is preferred. A standard 2.3 kb length can be used as the promoter region of GluB-1.

  Further, the white rice according to claim 3 is obtained from the transformed rice according to claim 1 or 2, and is a white rice having a blood pressure lowering action containing at least 30 mg / 100 g of GABA.

Further, the rice vector of claim 4 comprises a rice glutelin gene promoter region and DNA encoding the amino acid sequence of SEQ ID NO: 2 or one or several amino acids of the amino acid sequence of SEQ ID NO: 2. A vector having a gene linked to a DNA encoding an amino acid sequence having glutamic acid decarboxylase activity , comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion, and increasing the GABA content of the endosperm is there.

  According to the present invention, GAD encoded by an expression vector can be expressed at a high level in the white rice portion of rice, and at the same time, the product GABA can be accumulated at a high concentration. Therefore, GABA acts by eating polished rice obtained from this transformed rice, and can be used for prevention or treatment of various lifestyle-related diseases.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments described herein, and is based on the description of the present specification and techniques known in the art. Techniques that can be easily modified or altered by those skilled in the art are intended to be included within the scope of the present invention.

  GAD catalyzes the production of GABA by decarboxylation of glutamic acid. GAD is a kind of enzyme that organisms have universally from E. coli to humans, but only plant GAD has a calmodulin binding site (CaMBD) in its C-terminal region, and the interaction between this site and CaM is the enzyme. It plays an important role in the regulation of activity.

  Presence of at least 5 types of isoforms is presumed in monocotyledonous rice, among which the gene encoding OsGAD2 (accession number: AB056061) does not have CaMBD exceptionally (see Non-Patent Document 1). In the following, OsGAD2 will be simply expressed as GAD2 as appropriate.

  Furthermore, as has been clarified by the present inventor, it has been confirmed that the enzyme activity is increased by a factor of 5 or more by lacking the CAD-terminal peptide portion of GAD2 (see Patent Document 1). The inventor of the present application further studied GAD2 and made the present invention as a result of intensive studies.

<Overview>
The present inventor modified the rice GAD2 gene (Patent Document 1), prepared a vector for expression with a seed-specific gene promoter, incorporated the gene into a plant cell via Agrobacterium, and recombined Rice individuals were regenerated and GABA was highly accumulated in seeds. Furthermore, the traits were fixed by advancing generations. The developed brown rice not only improved the expected GABA content in the endosperm but also unexpectedly improved the whole mineral, free amino acids, and vitamin content of the brown rice. Using this as a material, a mixed feed was prepared and administered to hypertensive rats, and it was confirmed that it had an effect of lowering blood pressure. More details are as follows.

<Construction of expression vector>
The construction of the plant expression vector was as follows. First, a nucleotide sequence corresponding to amino acids from the start codon to the 469th amino acid was amplified by PCR using a cDNA encoding GAD2 as a template and a specific primer set. GAD2 consists of 500 amino acid residues, and CaMBD (31 residues on the C-terminal side) seen in OsGAD1 is not seen, but 31 residues corresponding to CaMBD, which is thought to regulate enzyme activity, are deleted, An attempt was made to construct a vector with DNA encoding 469 residues, which is considered to be a region that controls the enzyme function itself.

  The sense primer used at this time is 5'-CGTGCGTAGGCCATGGTCTG-3 'as shown in SEQ ID NO: 3, and the antisense primer is 5'-AAAGCATGCCTAGGCGGTCCCGGGCGGGGCC-3' as shown in SEQ ID NO: 4. The underlined part indicates the NcoI recognition sequence in the former and the SphI recognition sequence in the latter. The DNA fragment obtained by PCR is a deletion of 31 amino acid residues on the C-terminal side of GAD2, and hereinafter this DNA fragment will be referred to as GAD2ΔC31 as appropriate.

  The obtained GAD2ΔC31 was incorporated into an Escherichia coli plasmid, and its base sequence was confirmed. The plasmid was first cut with SphI, blunt-ended with T4 DNA polymerase and then cut with NcoI. This DNA fragment was integrated at the same position after removing the DNA fragment encoding the endogenous green fluorescent protein by cutting the plant expression vector pCAMBIA1302 (CAMBIA, Australia) with NcoI / PlmI.

  Then, the promoter region 2.3 kb (Non-patent Document 2) of the rice glutelin gene (GluB-1) was excised with XbaI / NcoI, and treated with the same restriction enzyme, thereby causing endogenous cauliflower mosaic virus 35S promoter and Replaced. One feature of the present invention is that the GluB-1 promoter is used instead of the 35S promoter. This is because, as a result of the study by the inventors of the present application, the commonly used transformed rice using the 35S promoter has a defect of sterility. That is, it can be said that one feature of the present invention is an expression vector having this gene cassette.

  The plant expression vector thus prepared is hereinafter referred to as pCAMBIA GluB-1 :: GADΔC31 as appropriate. FIG. 1 shows the flow up to the construction of this vector. In the present embodiment, GAD2ΔC31 is directly linked downstream of the GluB-1 promoter without using an artificial sequence.

<Introduction of expression vector into Agrobacterium strain>
The prepared vector was introduced into Agrobacterium strain EHA105 by electroporation (see Non-Patent Document 3). The bacterial suspension was cultured at 28 ° C. for 2 days using LB agar medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) containing 100 mg / l kanamycin. . The formed colonies were scraped with a sterile loop, inoculated into AB medium (see Non-Patent Document 4) containing kanamycin at the same concentration as above, and cultured at 28 ° C. for 2 to 3 days.

<Callus induction in 2N6 medium of rice brown rice>
The rice cultivar “Kitaake” was threshed, and about 50 grains of brown rice were sterilized for 30 minutes using bleach diluted 2 times (made by Kao). Thereafter, the brown rice was washed 5 times with sterilized water and placed on a 2N6 callus induction medium (see Non-Patent Document 4). This was cultured in a growth chamber (28 ° C., light: dark = 16 hours: 8 hours) for 2 to 3 weeks to form callus derived from scutellum.

<Transformation of rice callus cells via Agrobacterium>
Rice was transformed with Agrobacterium. The various reagents used for the method or transformation are ordinary methods or reagents. For example, the methods or reagents disclosed in Non-Patent Document 4 widely used for rice can be used.

  Agrobacterium carrying the target expression vector was suspended in N6CO liquid medium. In contrast, an appropriate amount of callus grown to a diameter of 2 to 3 mm was added. The callus was taken out, excess water was removed on a sterilized filter paper, and then placed on an N6CO agar medium and cultured at 23 ° C. for 3 days in the dark.

  The callus in which the bacteria had sufficiently grown was taken out and washed while shaking the callus in an N6CO liquid medium containing a disinfectant. This operation was repeated 5 times. The callus was removed from excess water on sterile filter paper, transferred to N6SE medium (selection medium) containing hygromycin, and cultured in a growth chamber (28 ° C., light: dark = 16 hours: 8 hours) for 3 to 4 weeks.

<Selection of resistant callus and solid regeneration>
The callus resistant to hygromycin was transferred to MSRE medium (regeneration medium) and cultured until shoots appeared. The rooted shoots were transferred to a hormone-free MS medium made in a test tube to promote plant growth.

<Boiling>
Subsequently, the plant body is taken out from the test tube, removed from the agar attached to the roots, transferred to a pot containing the soil for raising seedlings, cultivated in a specific net room, and harvested next-generation seeds (rice). It was used for analysis.

<Confirmation of foreign gene integration in transformed rice plant>
After seeding (T1 generation), brown rice was sterilized and sown on MS agar medium containing 50 μg / ml hygromycin (see Non-Patent Document 4). About 10 days later, total DNA was extracted by a CTAB method using a leaf piece of a resistant plant as a material (for the DNA extraction technique, for example, the technique disclosed in Non-Patent Document 5 can be referred to). PCR was performed using a primer set prepared based on the selection marker hygromycin resistance gene sequence. The sense primer used is shown in SEQ ID NO: 5, and the antisense primer is shown in SEQ ID NO: 6.

  Subsequently, denaturation (95 ° C., 30 seconds) → annealing (60 ° C., 30 seconds) → extension (72 ° C., 60 seconds) was repeated 30 cycles. The reaction sample was fractionated by 2% agarose gel electrophoresis.

  As a result, as shown in FIG. 2, no PCR amplification was observed with the wild-type DNA, whereas those using DNA extracted from the recombinant plant as a template all correspond to the expected 800 bp. An amplification product was observed at the position. From this, it was confirmed that the target gene was incorporated in all the investigated plants.

<Expression analysis at the RNA level of GAD2ΔC31 in brown rice using RT-PCR method>
In order to examine the expression of the target gene during the ripening process of the transformed plant, an immature seed (T1 generation) was crushed under liquid nitrogen and then RNA extraction reagent Sepazol RNAI (manufactured by Nacalai Tesque) was used. Total RNA was extracted. Total RNA and complementary primers were mixed, and cDNA synthesis was performed at 42 ° C. for 1 hour using reverse transcriptase SuperScriptII (GIBCO BRL) in the presence of 0.5 mM dNTP. The complementary primer used is shown in SEQ ID NO: 7.

  PCR was performed using a primer set with a 1/10 volume of the cDNA sample as a template. The sense primer used is shown in SEQ ID NO: 8. The same antisense primer as shown in SEQ ID NO: 7 was used. The reaction conditions were 95 ° C. (30 seconds), 55 ° C. (30 seconds), and 72 ° C. (30 seconds), which were repeated 35 cycles. An appropriate amount of the reaction sample was fractionated by 2% agarose gel electrophoresis. As a result, as shown in FIG. 3, GAD2 is not expressed in the wild-type seed used as a control or is at a very low level, whereas the seed derived from the recombinant plant has a high level of GAD2ΔC31. Expression was observed.

<GAD2ΔC31 expression analysis and GABA analysis at protein level by Western method>
A plurality of seeds (T1 generation) of the same transformed plant were collected, and the relationship between the transgene expression at the protein level and GABA accumulation was examined. First, brown rice was put in a microtube, freeze-fractured with liquid nitrogen, and then added with an extraction buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM DTT, 1 mM PMSF) and suspended.

  After centrifugation (20,000 g, 20 minutes, 4 ° C.), the supernatant was transferred to a new tube. The sample was divided into two equal parts, one for protein analysis and the other for amino acid analysis. Western analysis of the protein was performed as follows. A protein sample (about 10 μg) was fractionated by 12% SDS-polyacrylamide gel electrophoresis, and the protein was transferred to a membrane with a semi-driving apparatus.

  The membrane was immersed in a rabbit anti-GAD2 polyclonal antibody solution diluted 5,000 times in TBST (10 mM Tris-HCl, 500 mM NaCl, pH 7.5, 0.1% Tween-20), and shaken for 30 minutes. The membrane was washed three times for 5 minutes each with TBST, immersed in anti-rabbit IgG-alkaline phosphatase diluted 5,000 times with TBST, and shaken for 30 minutes. The membrane was washed 3 times for 5 minutes each with TBST, transferred to a detection buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl 2) and equilibrated. The membrane was immersed in CSPD (Roche) diluted 200-fold with a detection buffer, reacted for 5 minutes, and observed under an ultraviolet lamp.

  On the other hand, in order to extract free amino acids, trichloroacetic acid (TCA) was added to the sample to a final concentration of 8% (v / v) and stirred. After centrifugation, the supernatant was transferred to a new tube, and an equal volume of diethyl ether was added and stirred. Centrifugation was performed to remove the upper layer diethyl ether. This operation was repeated once more. Diethyl ether was completely volatilized in a fume hood and a part was used for analysis.

Amino acid samples were placed in microtubes, against which 100 mM sodium pyrophosphate (pH 9.0), 0.5 mM NADP ⁺ , 0.6 mM α-ketoglutaric acid, 1.2 mM 2-mercaptoethanol, 0.005 units GABase (Manufactured by Sigma) was added and reacted at 37 ° C. for 1 hour.

  After the reaction, the reduced coenzyme NADPH accompanying GABA synthesis was visualized by ultraviolet irradiation or quantitatively analyzed with a spectrophotometer (340 nm). The result is shown in FIG. As is clear from the figure, it was confirmed that the GABA content was also at a high level when the target enzyme protein GAD2ΔC31 was highly accumulated in the brown rice.

  From the above, it was confirmed that as a result of overexpression of the GAD2ΔC31 gene in seeds, an increase in GAD enzyme activity was caused in the cells, resulting in high concentration accumulation of GABA.

<Analysis of free amino acids in brown rice>
Next, in order to quantify the content of various free amino acids in the brown rice, the amino acid sample extracted by the TCA method was subjected to a quantitative analysis using an automatic amino acid analyzer (JLC-300 manufactured by JEOL Ltd.). The result is shown in FIG. As shown in the chart of FIG. 5-1, it has been found that the recombinant brown rice (T1 generation) has surprisingly increased its content not only with GABA but also with many types of amino acids. . FIG. 5-2 quantifies and summarizes the GABA and proteinous amino acid contents of wild-type brown rice and some recombinant brown rice (T1 generation). It was confirmed that the higher the GABA accumulation, the higher the amino acid content overall.

  In addition, although this result was T1 generation and there were many dispersion | variation, content increase of 2 times or 14 times was recognized by the essential amino acid. In particular, ricin is a kind of amino acid whose content in grain is desired to be increased, and is at least 4 times higher in the chart and 14 times higher in many lines. In addition, the total amount of free amino acids has increased by about 30 times, which is considered to have caused a multifaceted effect that cannot be assumed from the manipulation of one gene.

<Immunohistochemical detection of GAD2ΔC31 protein and GABA in recombinant brown rice using specific antibody>
In order to investigate the localization of the transgene product in the seed and the GABA produced thereby, detection using an immunohistochemical technique was performed. The implementation was mainly based on the method disclosed in Non-Patent Document 6.

  First, seeds soaked in water overnight were divided into two vertically with a scalpel and blocked with TBS (10 mM Tris-HCl, 500 mM NaCl, pH 7.5) containing 3% skim milk for 3 hours or more. As a primary antibody, a rabbit anti-GAD2 polyclonal antibody or a rabbit anti-GABA monoclonal antibody was used after being diluted 200-fold with TBS containing 1% skim milk.

  After reacting overnight, it was washed 3 times with TBST for 15 minutes. As a secondary antibody, an anti-rabbit IgG-alkaline phosphatase diluted 2,000 times with TBST was used for reaction for 2 hours. The detection reaction was performed using Western Blue Stabilized Substrate for Alkaline Phosphatase (manufactured by Promega), and the state of staining was observed using a stereomicroscope. FIG. 6 is an image obtained with a stereomicroscope. As shown in the figure, with any specific antibody against GAD2 and GABA, blue staining indicating their localization was observed in the endosperm tissue in the recombinant brown rice.

<Analysis of various components of brown rice>
In order to analyze various components in brown rice other than free amino acids, chemical components of T3 generation brown rice were analyzed. The results are shown in FIG. Except for water and soluble inorganic nitrogen (mainly starch), vitamins and various minerals had higher values for recombinant brown rice than wild-type for all analysis items. The rate of increase was 1.3 to 2.5 times, and it was confirmed that various items that could not be considered to have a direct influence of GAD were significantly increased.

  In particular, in the mineral content, potassium is increased by a factor of 2 or more, so that an antihypertensive action can be expected in addition to GABA. Vitamin B1 helps enzymes that break down carbohydrates, vitamin B6 promotes protein metabolism, and vitamin E prevents the formation of lipid peroxides. Can be expected to affect the human body.

  That is, neither the results of FIG. 7 nor the results of FIG. 5 are completely unexpected, and these values are considered to be up to about 1.5 times for several items even if significantly increased. A vector can be said to be a vector that not only causes an increase in GABA but also causes activation of intracellular metabolic pathways.

<Analysis of GABA content for T2 generation and T3 generation recombinant rice lines>
In order to develop a genetically stable line for GABA accumulation, 12 seeds of T2 generation by self-pollination of seed (T1 generation) obtained by self-pollination of the recombinant rice line (T0 generation) The analysis was performed by a method using GABase.

  As a result, as shown in FIG. 8A, high GABA accumulation was confirmed in many strains compared to wild type and germinated brown rice. From these, a line with little variation was selected, and the generation was further advanced to perform GABA analysis in the T3 generation. As shown in FIG. 8B, even in the T3 generation, a GABA content of 2-9 times that of germinated brown rice was obtained while having a brown rice weight comparable to that of the wild type (without grain reduction). In other words, the recombinant rice strain of the present invention maintains fertility even after generation renewal, and the brown rice weight is comparable to that of conventional varieties, so it is possible to develop varieties with guaranteed yields. And

<A comparative analysis of GABA accumulation in brown rice and polished rice>
In order to further verify that GABA has accumulated in the endosperm tissue of brown rice obtained from recombinant rice, brown rice (T3 generation) is made into polished rice by applying a small rice mill (Ket Kagaku Co., Ltd.). White rice was pulverized, free amino acids were extracted by the TCA method, and the GABA content was measured by an indirect method using GABase. The result is shown in FIG. As shown in the figure, in the wild type, the GABA content decreased to 1/5 or less by the rice milling treatment, whereas in the recombinant rice, there was almost no difference in the GABA content between the brown rice and the milled rice, and It was confirmed that GABA contained in the polished rice itself (endosperm tissue which is an edible portion) in a surprisingly large amount.

  That is, although it changes with lines | system | groups, the GABA content in white rice is at least 30 mg / 100g, and it rose to 60 mg / 100g in many things. Since at most 0.5 mg / 100 g of wild-type white rice, it was confirmed that it increased by 60 times or more. In addition, since it was 2.9 mg / 100g in wild-type brown rice, it has confirmed that it rose 10 times or more compared with this. This result agrees with the result shown in FIG. In addition, since this result is an evaluation in the T3 generation that is stabilized to some extent in terms of characteristics, the same result can be expected even when the variety is fixed.

<Clinical study using hypertensive rats>
Recombinant rice line brown rice (T3 generation), which was experimentally cultivated and harvested in a specific net room, was crushed and then put into a browning machine. Brown rice is pulverized using a blender, based on AIN-93G purified feed for mice and rats (made by Oriental Yeast Co., Ltd.), and 40% corn starch contained in it is blended with brown rice flour at the same ratio to make a special order. A feed was prepared.

  A clinical trial to examine the effect of recombinant brown rice was conducted as follows. 9 weeks old male spontaneously hypertensive spontaneously hypertensive rats (SHR / NCrlCrlj) purchased from Nippon Charles River Co., Ltd. were acclimatized with a normal diet for 1 week and divided into 2 groups of 8 animals. Wild type brown rice flour supplemented feed, Group B with recombinant brown rice flour added sample, Group C with wild type brown rice flour with added purified GABA to the same extent as germinated brown rice, Body weight and blood pressure were measured before admission and every week until the 5th week.

  As a result, in the 5th week, the average blood pressure of the A group was 235 mmHg, and the B and C groups were both 220 mmHg or less, and a significant blood pressure lowering effect was confirmed. In addition, in terms of blood biochemistry, a decrease in total cholesterol, neutral fat, GOT, and GPT was confirmed in group B as compared to group A.

<Creation and evaluation of other transformed rice plants>
Next, transformed rice was produced for the rice cultivar “Nipponbare” in the same manner as “Kitaake”.
The result of having analyzed the free amino acid content of the polished rice in T4 generation with the automatic amino acid analyzer is shown in FIG. Regarding GABA, the content was increased by 3 to 30 times compared to the wild type. In addition, as with the results of Kitaake, it was confirmed that the higher the GABA accumulation, the higher the amino acid content overall.

  Next, FIG. 11 shows the results of analysis of various components in white rice other than free amino acids for the (47-52) and (78-87) strains of the T4 generation having a high GABA content. Vitamins and various minerals showed that the value of recombinant rice exceeded that of wild type for both analytical items. Here again, it was confirmed that various items that were not considered to have a direct influence of high expression of GAD were significantly increased.

  Similar to the result of introduction to Kitaake (FIG. 7), in particular, minerals have a potassium increase of more than twice, so that an antihypertensive effect can be expected in addition to GABA. Vitamin B1 helps enzymes that break down carbohydrates, vitamin B6 promotes protein metabolism, and vitamin E prevents the formation of lipid peroxides. Can be expected to affect the human body.

<Oral administration test to hypertensive rats>
Next, SHR / Izm, which is a hypertensive rat, was fed a food ground with (47-52) strains of white rice once a day for 5 weeks to observe changes in blood pressure. In SHR / Izm, generally, a tendency toward hypertension starts to appear at the age of 5 or 6 weeks, and the transition to a hypertension state is completed around 11 weeks of age. In the evaluation, 6 animals from 9 weeks of age were divided into 3 groups,
・ Standard diet + ground milled rice (control group)
-White rice administration of (47-52) strain ground so that it can be administered to 0.1 mg of GABA in terms of standard bait + 1 kg of individuals-0.5 mg of GABA in terms of GABA amount for standard baits + 1 kg of individuals (47-52) strains of white rice were crushed so as to be administered. The results are shown in FIG. As shown in the figure, there was no significant difference between the control group and the group with a small amount of GABA, but in the group with a large amount of GABA, inhibition of transition to a hypertensive state could be confirmed. In all three groups, there was no difference in body weight.

  Next, changes in blood pressure over time were observed when this (47-52) strain of recombinant white rice and wild-type (Nipponbare) white rice were orally administered to stroke-prone hypertensive rats (SHRSP). The results are shown in FIG. As is apparent from the figure, a drop in blood pressure was observed after administration, and it was confirmed that the blood pressure dropped most in 8 hours and returned to a normal hypertension state in 24 hours. The dose was determined as the amount of white rice that would give 0.5 mg of GABA per 1 kg of living body.

  From the results shown in FIG. 12 and FIG. 13, it was found that recombinant white rice has a blood pressure lowering effect by ingesting white rice.

<Change in GABA content by water immersion treatment>
GABA is produced by decarboxylation of glutamic acid through GAD and the coenzyme pyridoxal phosphate (PLP). Brown rice is distributed as GABA-reinforced rice as germinated brown rice by dipping treatment. Therefore, using the non-recombinant Nipponbare (WildType: WT) and (47-52) line,
・ Group just immersed in water (w / o)
GATA content change was observed in the group of glutamic acid (Glu) addition + water immersion, coenzyme PLP addition + water immersion group, glutamic acid (Glu) addition + coenzyme PLP addition + water immersion group. The results are shown in FIG. As shown in the figure, the addition rate of both Glu + PLP was high for both non-recombined and recombined ones, but the recombinants were confirmed to have a very high rate of increase and absolute amount. Since PLP is a vitamin, it can be said that, for example, the white rice of the present invention is suitable for use in cooked rice, or α-formation and processing into an emergency food or the like.

  INDUSTRIAL APPLICABILITY The present invention can be used as a diet for lifestyle-related disease patients or as a regular food for middle-aged and older people. In addition, functional ingredient-enhanced rice can be developed using the vector of the present invention. Here, the functional component-enriched rice refers to rice in which essential amino acids and / or vitamins and / or minerals are enhanced compared to wild-type or existing varieties.

FIG. 2 is a process diagram showing a method for constructing a plant expression vector pCAMBIA GluB-1 :: GAD2ΔC31 according to the present invention. It is an electrophoretic diagram which shows the result of having confirmed the integration of the foreign gene in the plant body of T1 generation by PCR method. It is the electrophoretic diagram which confirmed the expression at the transcription level of GAD2 (DELTA) C31 gene in the immature seed of T1 generation by RT-PCR. It is a figure which shows the result of the fluorescence analysis of the analysis of GABA content extracted from T1 generation brown rice, and the Western analysis of GAD2 (DELTA) C31 protein. It is the chart analyzed using the automatic amino acid analyzer of the free amino acid extracted from the brown rice of T1 generation. The results of amino acid analysis of wild-type brown rice and recombinant brown rice are compared and summarized in a table. It is the figure which clarified the localization of GAD2 (DELTA) C31 protein and GABA in recombinant brown rice with the antihistochemical method using the anti- GAD2 antibody and the anti- GABA antibody. It is the table | surface which calculated | required the result of having analyzed the various components contained in wild type brown rice and recombinant brown rice quantitatively. It is the result of having performed the GABA analysis of the brown rice in the progeny (T2 generation and T3 generation). It is the figure which investigated and compared GABA content in brown rice and its polished rice. It is the figure which showed the result of having analyzed the free amino acid content of the polished rice in T4 generation of recombinant Nipponbare with the automatic amino acid analyzer. It is the result of having analyzed various components in white rice other than a free amino acid. It is the figure which showed the long-term change of the blood pressure at the time of orally administering GABA reinforcement | strengthening rice (white rice) to the hypertensive rat SHR / Izm. It is the figure which showed the mode of the change of the blood pressure for 24 hours at the time of orally administering GABA reinforcement | strengthening rice (white rice) to rat SHRSP. It is the figure which measured the change of water immersion and GABA, changing an additive. In addition, since it is not the precise quantitative evaluation using an automatic amino acid analyzer, it differs from FIG. 10 as an absolute value.

Claims (4)

Glutamic acid comprising DNA encoding the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids of the amino acid sequence shown in SEQ ID NO: 2 An endosperm GABA-accumulating transformed rice having fertility, into which a DNA encoding an amino acid sequence having decarboxylase activity has been introduced.   The transformed rice according to claim 1, wherein the DNA encoding the amino acid sequence is linked to the downstream side of the promoter region of the rice glutelin gene.   White rice obtained from the transformed rice according to claim 1 or 2 and having a blood pressure lowering action, containing at least 30 mg / 100 g of GABA. The promoter region of the rice glutelin gene;
A glutamic acid decarboxylation comprising a DNA encoding the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing substitution, deletion, insertion, addition or inversion of one or several amino acids of the amino acid sequence shown in SEQ ID NO: 2 DNA encoding an amino acid sequence having enzyme activity ;
A rice vector which has a gene linked to and increases the GABA content of the endosperm.