JP2009050190A - Method for extracting liposoluble physiologically active substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for extracting a trace of a liposoluble physiologically active substance from a single grain of seed. <P>SOLUTION: The method includes the step of treating seed powder and/or seed embryo powder with a cellulose hydrolase-containing buffer solution, and the step of extracting a liposoluble physiologically active substance. As the liposoluble physiologically active substance, ubiquinone 10 can be employed. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for extracting a fat-soluble physiologically active substance such as ubiquinone 10 from plant tissue.

  Ubiquinone 10 (hereinafter referred to as CoQ10) is an important substance related to the redox of the respiratory chain electron transport system, and has been the subject of research from both the basic and applied aspects. In recent years, not only the energy production activation effect but also the antioxidant function has attracted attention, and in addition to conventional pharmaceutical uses, it has spread to society as a supplement and cosmetics. In Japan, it was approved and sold as a metabolic cardiotonic drug in 1974, and analytical and quantitative methods for production, medical treatment, and nutrition have been studied. For this reason, the analysis and quantification of CoQ10 covers a wide variety such as yeast, animal-derived samples (serum etc.), vegetables, fish, meat and oil. The basic extraction method of CoQ10 was simple organic solvent extraction, and samples that could not be extracted as they were were extracted after being crushed in advance.

  On the other hand, a very small amount of CoQ10 and a relatively large amount of coenzyme Q9 (hereinafter referred to as CoQ9) exist in seeds such as rice. When this rice seed was used as a target, the conventional extraction and quantification methods could not efficiently extract and quantify CoQ10, not to mention CoQ10 even if a large amount of sample was used. For this reason, a considerably complicated extraction and quantification method using autoclave treatment and thin layer chromatography has been devised, and the recovery rate has been greatly improved by this method, enabling quantification (Non-patent Document 1). However, even with the method disclosed in Non-Patent Document 1, it is impossible to quantify fat-soluble physiologically active substances such as CoQ10 and CoQ9 from a small amount of sample such as one rice grain seed.

  On the other hand, in the extraction of oil from plant seeds (soybean, rapeseed, sunflower, sesame), preliminary extraction with water treatment, autoclave treatment, and enzyme treatment increases the efficiency of subsequent extraction with organic solvents. (Patent Document 1), and among these treatments, pretreatment with hydrolase was also used in the extraction of other functional substances from plant tissues (Patent Documents 2 and 3).

S. Takahashi et al. 2006, FEBS Lett., 580 (3), 955-959 Japanese Patent Laid-Open No. 2002-256281 JP 2005-27520 A JP 2002-348245 A

  However, the method disclosed in Patent Documents 1 to 3 does not assume a technical idea of extracting a fat-soluble physiologically active substance that is contained in a very small amount of seed such as CoQ10, and the fat-soluble substance is not considered. It was impossible to extract the physiologically active substance from one seed. In view of the above-described circumstances, an object of the present invention is to provide a method capable of extracting a trace amount of a fat-soluble physiologically active substance from one seed grain.

  As a result of diligent studies to achieve the above-mentioned object, the present inventors have examined various pretreatment operations to be performed before the organic solvent extraction operation, and seed powder and / or seeds by cellulose hydrolase (cellulase). The present inventors completed the present invention by finding that fat-soluble physiologically active substances such as CoQ10 can be extracted easily and in high yield from a small amount of sample by treating the embryo powder of the present invention.

That is, the present invention includes the following.
(1) A method for extracting a fat-soluble physiologically active substance, comprising a step of treating seed powder and / or a seed embryo powder with a buffer containing a cellulose hydrolase and a step of extracting a fat-soluble physiologically active substance.
(2) The method for extracting a fat-soluble physiologically active substance according to (1), wherein the seed powder and / or the embryo powder of the seed are derived from rice.
(3) The method for extracting a fat-soluble physiologically active substance according to (2), wherein the rice is a transformed rice that produces ubiquinone 10 as a fat-soluble physiologically active substance.
(4) The method for extracting a fat-soluble physiologically active substance according to (1), wherein the amount of the seed powder and / or the seed embryo powder is an amount corresponding to one seed.
(5) The method for extracting a fat-soluble physiologically active substance according to (1), wherein the step of extracting the fat-soluble physiologically active substance uses a low-polar organic solvent.
(6) The method for extracting a fat-soluble physiologically active substance according to (5), wherein the low-polar organic solvent is a saturated hydrocarbon having 5 to 8 carbon atoms.
(7) The method for extracting a fat-soluble physiologically active substance according to (1), wherein the fat-soluble physiologically active substance is ubiquinone and / or vitamin E.

  ADVANTAGE OF THE INVENTION According to this invention, the very outstanding method which can extract the fat-soluble physiologically active substance which exists in a very small amount in a seed from a trace amount sample, such as one seed grain, can be provided. According to the method for extracting a fat-soluble physiologically active substance according to the present invention, for example, a fat-soluble physiologically active substance can be extracted and quantified from a sample corresponding to one seed grain, so that the fat-soluble physiologically active substance present in the seed The substance can be analyzed with high accuracy. Moreover, by applying the method for extracting a fat-soluble physiologically active substance according to the present invention, the fat-soluble physiologically active substance present in the seed can be recovered with a very excellent yield.

  Hereinafter, a method for extracting a fat-soluble physiologically active substance according to the present invention will be described in detail with reference to the drawings.

  The method for extracting a fat-soluble physiologically active substance according to the present invention is a method for extracting a fat-soluble physiologically active substance present in a trace amount in seeds. Here, examples of the fat-soluble physiologically active substance include ubiquinones and fat-soluble vitamins.

  Ubiquinones are derivatives of 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone. As ubiquinones, coenzyme Q6, coenzyme Q7, coenzyme Q8, coenzyme Q9, coenzyme Q10, and the like are known depending on the number of side chain isoprene units repeated. Examples of the fat-soluble vitamins include vitamin A, vitamin D, vitamin E, and vitamin K.

  In the method for extracting a fat-soluble physiologically active substance according to the present invention, first, seeds collected from plants are accumulated, and these seeds, preferably embryos separated from these seeds, are pulverized. In the method for extracting a fat-soluble physiologically active substance according to the present invention, the plant is not particularly limited, and any of a wild-type plant and a transformed plant imparted with a desired character may be used. An example of a transformed plant is a transformed plant that has been genetically manipulated to increase the production of a specific substance. In particular, in the method for extracting a fat-soluble physiologically active substance according to the present invention, ddsA (decaprenyl diphosphate synthase) derived from gluconic acid bacteria (Gluconobacter suboxydans) is introduced so as to increase the production amount of CoQ10. It is preferable to target CoQ10 producing plants.

  The plant is not particularly limited, and for example, rice, tobacco, barley, wheat, bread wheat, rye, oats, pearl barley, sorghum, corn, millet, millet, millet, buckwheat, buckwheat, crap, sugarcane, sugar beet, eggplant, potato , Sweet potato, yam, taro, konjac, burdock, lotus root, cucumber, calla cucumber, loofah, burdock, taro, cassava, tomato, carrot, asparagus, pumpkin, radish, turnip, rape, canola, alalpha, pepper, capsicum , Broccoli, cauliflower, lettuce, pine, spinach, garlic, mugwort, okra, perilla, sesame, gardenia, horseradish, mustard, ginger, ginger, turmeric, saffron, onion, leek, garlic, bracken, zema , Soybean, adzuki bean, kidney bean, pea, broad bean, cowpea, velvet bean, crow pea, peanut, wolfberry, poppy, olive, mint, almond, pistachio, macadamia nut, peanut, date palm, coconut palm, sago palm, sugar palm, nippa palm, pineapple , Oyster, apple, plum, peach, loquat, pear, grape, cherry, strawberry, raspberry, blueberry, prune, pomegranate, fig, akebi, gummy, mangosteen, kiwifruit, melon, citrus, banana, papaya, mango, Arabidopsis thaliana , Nazuna, Inazuna, Lily, Rose, Sakura, Yamazakura, Button, Camellia, Sasanqua, Lavender, Fuji, Peonies, Violet, Pansy, Sweet pea, Daffodil, Hanashobu, Iris, Basho , Shabu, mizubasho, water lily, lotus, orchid, morning glory, hornbill, thistle, gerbera, dahlia, tulip, cosmos, sunflower, dandelion, chamomile, rosemary, physalis, hamanasu, matatabi, clover, lilac, cactus, yukinoshita, dokuda Tea, coffee, spruce, larch, red pine, bamboo, ginkgo, cedar, cypress, fir, rubber, beech, zelkova, camphor tree, maple buffalo, eucalyptus, maple, poplar, acacia, bay, cotton, rush, shiba, safflower, walnut, Avocado is mentioned, but rice is particularly preferable.

  The method for pulverizing seeds and / or embryos collected from these plants is not particularly limited, and examples include various pulverization methods such as a pulverization method using a mortar and a ball mill device. Can do.

  In the method for extracting a fat-soluble physiologically active substance according to the present invention, the powdered seeds and / or embryos are then treated with a buffer containing cellulose hydrolase. Here, cellulose hydrolase is synonymous with a group of enzymes called cellulases, and it can be derived from endoglucanase (EC 3.2.1.4) that cleaves from the inside of the molecule, and from either the reducing end or non-reducing end of the sugar chain. It is meant to include both exoglucanases (EC 3.2.1.91) that decompose and release cellobiose. As the cellulose hydrolase, for example, cellulase derived from Aspergillus niger can be used.

  Moreover, the buffer solution containing cellulose hydrolase means a solution maintained at a salt concentration and pH optimum for cellulose hydrolase. Therefore, if the cellulose hydrolase to be specifically used is determined, the composition and pH of the buffer solution can be appropriately selected by those skilled in the art.

  Furthermore, the treatment conditions when treating seeds and / or embryos powdered with a buffer containing cellulose hydrolase include reaction temperature, presence / absence of stirring, reaction time, and the like. These treatment conditions can be appropriately set according to the amount of powdered seeds and / or embryos and the type of cellulose hydrolase.

  In the method for extracting a fat-soluble physiologically active substance according to the present invention, the fat-soluble physiologically active substance is then extracted from the buffer solution after the treatment. In this extraction step, an extraction process using an organic solvent is performed. As the organic solvent, for example, it is preferable to use a low polar organic solvent such as pentane, hexane, heptane, and octane, and hexane is particularly preferable. The amount of the organic solvent is not particularly limited, but is 5 to 25 ml, preferably 10 to 20 ml, more preferably 12 to 16 ml, based on 20 mg of seed and / or embryo powder.

  By this extraction step, the fat-soluble physiologically active substance accumulated in the seed in the organic solvent layer can be extracted. The fat-soluble physiologically active substance extracted in the organic solvent layer can be recovered in a dry state by removing the organic solvent. Moreover, the organic solvent containing a fat-soluble physiologically active substance can also be preserve | saved as it is, and the fat-soluble physiologically active substance contained in the seed can also be quantified using the said organic solvent.

  Although it does not specifically limit when detecting the fat-soluble physiologically active substance extracted in the organic solvent, For example, a high performance liquid chromatography, a thin layer chromatography, a gas chromatography etc. are applicable. Among them, it is preferable to use high performance liquid chromatography.

  According to the method for extracting a fat-soluble physiologically active substance according to the present invention described above, the fat-soluble physiologically active substance is extracted from the organic solvent layer by treating seed and / or embryo powder with cellulose hydrolase. can do. In the method for extracting a fat-soluble physiologically active substance according to the present invention, the fat-soluble physiologically active substance is surely extracted even if the amount of powder to be treated with cellulose hydrolase is as small as a seed. be able to. Conventionally, there is no known method capable of extracting a fat-soluble physiologically active substance from one seed, and the method for extracting a fat-soluble physiologically active substance according to the present invention is applicable even when the amount of a powder sample is small. There is an advantage that can be done.

  Furthermore, the method for extracting a fat-soluble physiologically active substance according to the present invention can extract a fat-soluble physiologically active substance by a very simple operation. That is, in the method for extracting a fat-soluble physiologically active substance according to the present invention, it is possible to omit a processing step that is essential for a conventional method such as autoclaving.

  Hereinafter, although the extraction method of the fat-soluble physiologically active substance which concerns on this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, CoQ10 was extracted by the method for extracting a fat-soluble physiologically active substance according to the present invention using rice recombined to express CoQ10 in mitochondria. The rice production method was described in detail in Reference Experiment 1 described later. In this example, CoQ9 was extracted using wild-type rice that mainly produces CoQ9.

  First, brown rice collected from the transformed rice produced in Reference Experiment 1 was crushed in a mortar, and 100 mg or 20 mg of the obtained brown rice powder was put into a 1.5 ml Eppendorf tube, and pH 5.0 acetate buffer (1 ml) Each hydrolase (cellulase) dissolved in) was added (step 1). Next, each eppendorf tube was placed on its side so that it was completely immersed in a 37 ° C. bath, and allowed to stand for 24 hours (step 2). After standing for 24 hours, 4 ml of hexane was added to each solution transferred from the Eppendorf tube to another container, and the mixture was stirred for 30 seconds with a mixer (step 3). Thereafter, centrifugation was performed at 2000 rpm for 2 minutes, and 3 ml of the hexane layer was recovered (step 4). Furthermore, extraction and recovery were performed in the same manner with 4 ml of hexane twice (total 3 times in combination with Step 3) (Step 5). Finally, all the collected hexane layers were collected in an eggplant-shaped flask and concentrated to dryness with an evaporator (step 6).

  CoQ10 recovered in step 6 was analyzed by the following method. First, the hexane layer concentrated and dried in Step 6 was dissolved in 2 ml of a mixed solution of acetonitrile / methanol = 1/1, and 50 μl thereof was injected into the HPLC apparatus. The HPLC apparatus and analysis conditions are as follows.

Equipment: Liquid Chromatograph HP-1100 (Hewlett Packard)
Moving layer: MeOH / MeCN = 60/40 → (15min) → 90/10 (5min maintained)
Flow rate: 1.0ml
Detector: Variable wavelength UV-VIS (measurement wavelength 275nm)
Column: CAPCELL PACK C ₈ SHISEIDO TYPE: UG120 5mm SIZE: 4.6mmφ × 250mm

[Comparative Example 1]
In Comparative Example 1, CoQ10 was extracted and analyzed in the same manner as in Example 1 except that α-amylase was used instead of cellulase.

[Comparative Example 2]
In Comparative Example 2, CoQ10 was extracted and analyzed in the same manner as in Example 1 except that protease was used instead of cellulase.

[Comparative Example 3]
In Comparative Example 3, CoQ10 was extracted and analyzed in the same manner as in Example 1 except that lipase was used instead of cellulase.

[Comparative Example 4]
In Comparative Example 4, CoQ10 or CoQ9 was extracted by the following method using the transformed rice produced in Reference Experiment 1 or wild-type rice expressing CoQ9. In summary, the method of Comparative Example 4 is an extraction method using an organic solvent without pretreatment with an enzyme.
First, after peeling off the shell, the weighed brown rice was powdered (step 1). Next, 100 mg of brown rice powder was placed in a 10 ml test tube (step 2). Next, 5 ml of hexane was added to the test tube for extraction (step 3). Thereafter, centrifugation was performed at 2500 rpm for 5 minutes (step 4). After centrifugation, the hexane layer separated in the test tube was collected (step 5). Next, 5 ml of hexane was added again to the test tube after the hexane layer was collected, and re-extraction was performed (step 6). Thereafter, the mixture was centrifuged at 2500 rpm for 5 minutes (Step 7). After the centrifugation treatment, the hexane layer separated in the test tube was collected (step 8). Finally, all the collected hexane layers were collected in an eggplant type flask and concentrated to dryness with an evaporator (step 9).
In Comparative Example 4, CoQ10 or CoQ9 was performed in the same manner as in Example 1.

[Comparative Example 5]
In Comparative Example 5, CoQ9 was added in the same manner as Comparative Example 4 except that 1 ml of distilled water was added to the test tube after Step 2 of Comparative Example 4 and allowed to swell for 1 hour, and 4 ml of hexane was used in Step 3 of Comparative Example 4. Extracted and analyzed.

[Comparative Example 6]
In Comparative Example 6, CoQ9 was extracted and analyzed in the same manner as in Example 5 except that 1 ml of distilled water was added and the temperature for swelling for 1 hour was 80 ° C.

[Comparative Example 7]
In Comparative Example 7, CoQ9 was extracted and analyzed in the same manner as in Comparative Example 5 except that 1 ml of distilled water was added to swell for 1 hour, and 1 ml of 6% sulfuric acid aqueous solution was further added to set the swelling temperature to 80 ° C. However, in Comparative Example 7, Step 4 of Comparative Example 4 was excluded.

[Comparative Example 8]
In Comparative Example 8, 1 ml of distilled water was added into the test tube after Step 2 of Comparative Example 4 and autoclaved at 120 ° C. for 20 minutes. CoQ9 was extracted and extracted in the same manner as in Comparative Example 4 except that Step 4 was omitted. analyzed.

[Comparative Example 9]
Comparative Example 9 was the same as Comparative Example 4 except that 1 ml of distilled water and 1 ml of 6% sulfuric acid aqueous solution were added to the test tube after Step 2 of Comparative Example 4 and autoclaved at 120 ° C. for 20 minutes. CoQ9 was extracted and analyzed.

[Comparative Example 10]
In Comparative Example 10, CoQ9 was extracted and analyzed in the same manner as Comparative Examples 6 and 7, except that 1 μg of CoQ9 standard product was added to 100 mg of brown rice powder.

[Reference Experiment 1]
Hereinafter, as Reference Experiment 1, a method for producing recombinant rice producing CoQ10 will be described. For recombinant rice producing CoQ10, mitochondria from ddsA (decaprenyl diphosphate synthase: enzyme that synthesizes the side chain of CoQ10) derived from Gluconobacter suboxydans driven by the 35S promoter It has been reported that rice recombined so that it is expressed in E. coli contains almost no CoQ9 and high levels of CoQ10 (S. Takahashi et al. 2006, FEBS Lett. 580 (3), 955-959). On the other hand, the ALS promoter from rice has been shown to function strongly in immature seeds and actively dividing tissues (K. Osakabe et al. 2005, Molecular Breeding, 16, 313-320). Therefore, ddsA driven by the ALS promoter was expected to increase the CoQ10 content in seeds by being strongly expressed during the active period of coenzyme biosynthesis. Therefore, the inventors prepared a construct (ALSpro :: S14 :: ddsA :: P10T) in which a gene in which S14 Signal of the mitochondrial localization sequence and ddsA are fused is driven by an ALS promoter.

1. Construction of binary vector (pPALS-ddsA) with ddsA (Figures 1 and 2)
S14Signal, which is a 0044NA encoding mitochondrial localization signal peptide of rice mitochondrial ribosomal protein, is derived from Gluconobacter suboxydans, a type of acetic acid bacteria, and synthesizes the side chain of CoQ10 (10 units isoprene chain) The enzyme decaorenyl diphosphate synthase gene (ddsA, accession number AB006850) and two binary vectors developed for plant transformation (pPALS PSR-02 and PSR-03, http://www.kumiai-chem.co. jp / palselect / index.html) was used to create a binary vector pPALS PSR-03 with ALSpro :: S14 Signal :: ddsA :: P10T cassette.

  Specifically, as shown in FIG. The pUS14ddsA plasmid in which S14 Signal :: ddsA was incorporated was digested with Not I, smoothed with Klenow Fragment, and then digested with Sal I to excise the S14 Signal :: ddsA gene. The pUC18 plasmid was sequentially digested with Sal I / Sma I (blunt ends), ligated with the previous S14 signal :: ddsA gene, transformed into E. coli JM109 strain, and a single colony having the desired plasmid was selected (KLB- 161). On the other hand, in order to use P10T (riceprolamin 10 terminator gene) as a terminator, the pPALS PSR-03 binary vector was treated with Bam HI / Eco RI, and the ALS :: P10T fragment was excised, inserted into the same site of pUC19, Hind III / The ALS gene was removed by Sac I treatment, and P10T was placed on the pUC19 vector. This fragment and the S14 Signal :: ddsA fragment having the Hind III / Sac I site were ligated and transformed into E. coli strain JM109. At this stage, the S14 Signal :: ddsA :: P10T cassette was put on pUC19 (KLB-164).

  Next, the pUC19 vector in which the S14 Signal :: ddsA :: P10T cassette was incorporated (KLB-164 above) was subjected to Sal I treatment / Blp I treatment / blunting with T4 DNA polymerase / BAP treatment. On the other hand, after treating with Ahd I / Nco I from the pPALS PSR-02 binary vector, nucleotides were degraded from 5 ′ side to 3 ′ side by S1 nuclease. The fragment blunted with T4 DNA polymerase was ligated with blunt-ended KLB-164 and transformed into Escherichia coli HB101. The obtained colonies were screened by PCR (OsALS-7 / ddsA 3A primer set, described below), and colonies that produced PCR products as long as possible were picked up. As a result, the obtained ALS promoter (ALSpro) fragment was 294 bp shorter on the upstream side and 24 bp on the downstream side compared to the ALS promoter on PSR-02, but it was judged that the function as a promoter was not impaired. did. The ALSpro :: S14 Signal :: ddsA :: P10T cassette was excised with Hind III / Eco RI and blunted with T4 DNA polymerase. On the other hand, pPALS PSR-03 binary vector was cleaved with Eco RI, blunted with T4 DNA polymerase, treated with BAP, ligated with the previous cassette, and transformed into E. coli DH5α strain (KLB-180) (Fig. 2) .

  The entire base sequence of the obtained KLB-180 is shown in SEQ ID NO: 1 and FIGS. 3-1 to 3-10. In FIGS. 3-1 to 3-10, the gene sequence is underlined by a single line. Moreover, in FIGS. 3-1 to 3-10, the gene name is shown at the start point of the gene sequence. In FIGS. 3-1 to 3-10, only the ddsA gene is shown by a double line. In FIGS. 3-1 to 3-10, the Sac I site of the pPALS-ddsA vector is represented as the first base. From this result, it was judged that the target fragment was amplified in all the primer sets.

2. Introduction of pPALS-ddsA into Agrobacterium
The above binary vector having the ALS promoter :: S14 :: ddsA :: P10 terminator cassette introduced into DH5α (KLB-180) was introduced into EHA101 strain (KLB-131) using the three-parent mating method as follows. did.

  That is, first, Agrobacterium EHA101 strain, E. coli DH1 strain, and E. coli HB101 strain were suspended in 5 ml of LB liquid medium (with antibiotics added). At this time, Escherichia coli was cultured at 37 ° C. and Agrobacterium was cultured at 28 ° C. Next, 1.5 ml of each bacterial solution was transferred to an Eppendorf tube and collected by centrifugation (4 ° C., 15000 rpm, 3 minutes). Thereafter, the supernatant was discarded, and 1 ml of LB liquid medium (without antibiotics) was added and suspended. The cells were collected again by centrifugation (4 ° C., 15000 rpm, 3 minutes). In addition, suspension with LB liquid medium and collection by centrifugation were repeated once more.

  Next, the supernatant was discarded, and 200 μl of LB liquid medium was added and suspended. Thereafter, the cells were inoculated on LB agar medium (without antibiotics) and cultured at 28 ° C. overnight. All grown colonies were scraped off and suspended in 500 μl of LB liquid medium (without antibiotics). 100 μl of each suspension was applied to LB agar medium (containing 12.5 ppm rifampicin, 25 ppm chloramphenicol, 50 ppm kanamycin, 50 ppm tetracycline) and cultured at 28 ° C. Two to three days after the start of the culture, colonies that had grown were picked up and confirmed by PCR (part of the T-DNA region). Several colonies selected by PCR were again streaked and purified. Further, colony PCR was performed to confirm that the binary vector was introduced into the microbial cells.

3. Rice transformation by Agrobacterium with pPALS-ddsA <Pre-culture of rice seed>
Rice (Nipponbare) seeds from which rice husks had been removed were placed in a 50 ml falcon tube, and the following operations were all performed in a clean bench. After washing gently with 70% ethanol, the rinse was removed by rinsing with sterile water. 1/2 diluted sodium hypochlorite solution was added and shaken for 15 minutes. The sodium hypochlorite solution was discarded and washed with sterilized water until no bubbling occurred. Seeds were put on sterilized filter paper to remove moisture. The sterilized seeds were placed on a callus induction medium (N6D medium) described in Table 1 with the embryos facing upward (12-16 grains / dish) and cultured at 33 ° C. in the light for 5 days.

<Pre-culture of Agrobacterium>
3 days before infection, Agrobacterium with pPALS-ddsA (EHA101) contains rifampicin (12.5mg / L), chloramphenicol (25mg / L), tetracycline (50mg / L), kanamycin (50mg / L) It was applied to LB solid medium and cultured at 24 ° C. in the dark for 3 days. In addition, since this Agrobacterium did not grow well on AB medium, Agrobacterium grown on LB medium was used.

<Agrobacterium infection and co-culture>
40 ml of AAM solution was placed in a 50 ml Falcon tube, and acetosyringone was added to 30 mg / L. The pre-cultured Agrobacterium was scraped about 1/4 mm with a sterilized micropartel and suspended in an AAM solution to which acetosyringone was added. At this time, pipetting was performed well with a Komagome pipette so that no Agrobacterium lumps remained. From the seeds cultured for 5 days, well-developed callus derived from the scutellum was selected, and the shoot and endosperm part were removed and placed in a new 50 ml Falcon tube. Add the Agrobacterium suspension to the Falcon tube containing the scutellum-derived callus, mix gently by inversion for 1.5 minutes, and then place it on the coculture medium (2N6-AS medium) so that the calli do not contact each other. Co-cultured for 3 days in the dark.

<Removal and selection of Agrobacterium>
The callus co-cultured was transferred to a 50 ml Falcon tube and washed 7-8 times with sterile water. Furthermore, the callus was washed with sterilized water containing 500 mg / L of carbenicillin. After removing the washing liquid as much as possible with a Komagome pipette or the like, the callus was transferred onto a sterilized filter paper to remove excess water. The callus was placed on a selective medium (N6D medium) containing carbenicillin 400 mg / L and 0.5 μM pyriminobac (hereinafter referred to as PM) (14 callus / petri dish) and cultured at 33 ° C. for 1 month in the light. During this period, the cells were transplanted to a new selection medium every two weeks.

<Redifferentiation of transformants>
On the selection medium, calli that were actively growing were transplanted to a regeneration medium without PM. The re-differentiated plant was further grown on a hormone-free medium to promote root growth, transplanted to soil and matured. The T0 generation of transformed plants was self-pollinated to obtain T1 seeds.

4). Confirmation of gene transfer by PCR
DNA was extracted from callus according to the protocol using DNeasy Plant Mini Kit, and the transgene was confirmed by PCR (FIG. 4). A PCR reaction was performed using 5 μL of the extracted DNA solution under the following conditions.

DNA solution: 5 μL
Sense primer (25 pmol/l): 2μl
Antisense primer (25 pmol/l): 2μl
Distilled water: 16μl
Ready to go PCR bead (Amersham): 1

  The reaction temperature cycle during PCR was maintained at 94 ° C. for 10 min, followed by 25 cycles of 94 ° C. for 30 s, 55 ° C. for 1 min and 72 ° C. for 1 m30 s, and then maintained at 72 ° C. for 7 min. .

Moreover, the primer used when confirming each area | region into which T-DNA area | region was introduce | transduced is shown in FIG.
ALS-RspJ (sense primer) 5'-TTGTTGGATATCATCGTCCCGCAC-3 '(SEQ ID NO: 4)
OsALS1 (Antisense primer) 5'-CTGGCTACTCCATAAACCGTAG-3 '(SEQ ID NO: 5)
OsALS2 (sense primer) 5'-GTCCACGACTAGTCCATGATTT-3 '(SEQ ID NO: 6)
OsALS3 (antisense primer) 5'-GTGGCTATGTGTATGCAGTTC-3 '(SEQ ID NO: 7)
OsALS7 (sense primer) 5'-CTCATCTTGCGCTGCGTTTGT-3 '(SEQ ID NO: 8)
pTN51 (antisense primer) 5'-TCAGGGATGTGACATTGCTCTTGC-3 '(SEQ ID NO: 9)
3-1-4 (sense primer) 5'-AGGTGTCACAGTTGTTG-3 '(SEQ ID NO: 10)
ALS-Rsp2 (antisense primer) 5'-AGTCCTGCCATCACCATCCAG-3 '(SEQ ID NO: 11)
Cal pro 1S (sense primer) 5'-TGAAGGTGTGTATTTGTCC-3 '(SEQ ID NO: 12)
Cal pro 1A (antisense primer) 5′-GCCTTCTCTGTATAACCTG-3 ′ (SEQ ID NO: 13)
pTN7 (sense primer) 5'-GTGCAATGTTTCGAGGAGC-3 '(SEQ ID NO: 14)
pTN9 (antisense primer) 5′-CACGTTTGATCGAGGCACTGA-3 ′ (SEQ ID NO: 15)
pTN17 (antisense primer) 5'-TCCATGTAGATTTCCCGG-3 '(SEQ ID NO: 16)
pTN27 (sense primer) 5′-GTTGTGGATACCTCGCGGAA-3 ′ (SEQ ID NO: 17)
ddsA 2S (sense primer) 5′-CATTCATACCGCCACACTGCT-3 ′ (SEQ ID NO: 18)
ddsA 3A (antisense primer) 5′-CCGTGAATGACTTCAAGATAGCG-3 ′ (SEQ ID NO: 19)
ddsA 3S (sense primer) 5′-TGGGAGCGCGTCATTGGAGAAG-3 ′ (SEQ ID NO: 20)
pTN3 (antisense primer) 5′-GCACACGATAGTATGCAACACC-3 ′ (SEQ ID NO: 21)
pTN53 (sense primer) 5'-GCCATCCGACGGATGATGTTTA-3 '(SEQ ID NO: 22)
pTN54 (antisense primer) 5'-GGACGTGAATGTAGACACGTCG-3 '(SEQ ID NO: 23)

In addition, PCR was similarly performed using the following primers for confirming the remaining Agrobacterium.
PTIB-1 (sense primer) 5'-TTGCGCTGCTTTGGCAAATGACGG-3 '(SEQ ID NO: 24)
PTIB-2 (antisense primer) 5'-TATGAGGCGCATCGTCGGATCAGT-3 '(SEQ ID NO: 25)

  About 40 days after transformation with Agrobacterium, DNA was extracted from 3 calli grown on the selective medium, and PCR and electrophoresis were performed. As a result, the target gene was integrated. (FIGS. 5 and 6). From the photographs of lanes 2, 6 and 10 in FIG. 5 and lane 6 in FIG. 6, the ddsA of the T-DNA region, the lanes 3, 7 and 11 in FIG. 5 and the lane 4 in FIG. 6 from the P10 terminator, FIG. DNA corresponding to the ALS promoter from lanes 2, 3 and 5 and a part of lanes 5 to S14 in FIG. 6 were amplified by PCR, and a band corresponding to the desired number of bases was confirmed. In addition, since no amplification of the band was observed in the PCR using the primer set (PTIB1 / PTIB2) for confirming the presence of the helper plasmid from lane 11 in FIG. 6, the amplified T-DNA region was transfected. It was a result, and it was shown that it was not derived from the remaining Agrobacterium. From the above results, introduction of the T-DNA region into rice cultured cells was confirmed.

5). Confirmation of gene transfer by in vivo ALS assay A part of callus (about 25 mg) that was thought to have grown by gene transfer after selection for about 40 days was converted to 500 μM 1,1-cyclopropanedicarboxylic acid (CPCA) and 0.25 μM pyriminoback. It was placed on (PM) -containing N6D medium (see Table 1), and gene transfer was confirmed by the following in vivo ALS test.

  Specifically, first, 50 mg of callus was placed on an N6D medium containing 500 μM CPCA and 0.25 μM PM, and allowed to stand at 30 ° C. in the dark for 1 day. In the control, 0.25 μM PM was removed from the medium. Next, the callus was removed from the petri dish and placed in a 1.5 ml microtube. 220 μl of distilled water containing 0.025% Triton X-100 was added to the 1.5 ml microtube, and the mixture was allowed to stand at 60 ° C. for 5 minutes.

  Thereafter, the mixture was extracted with ultrasonic waves (frequency: 40 kHz) for 15 minutes, and then 200 μl of the supernatant was transferred to another 1.5 ml microtube. 20 μl of 5% (v / v) sulfuric acid was added thereto, and the mixture was allowed to stand at 60 ° C. for 30 minutes. Next, 100 μl of 5% (w / v) α-naphthol solution dissolved in 100 μl of 0.5% (w / v) creatine solution and 2.5N NaOH solution was added and allowed to stand at 37 ° C. for 15 minutes. Subsequently, gene transfer was confirmed by measuring absorbance at 525 nm.

  An N6D medium containing 500 μM CPCA and 0.25 μM PM was prepared as follows. CHU powder (1 bag), myo-inositol (100 mg), nicotinic acid (0.5 mg), pyridoxine hydrochloride (0.5 mg), thiamine hydrochloride (1 mg), 2,4-D (2 mg), casamino acid (300 mg) , Glycine (2 mg), L-proline (2.8 g), sucrose (30 g), gellite (4 g) dissolved in 1 liter of distilled water and autoclaved, then the medium was cooled to 55 ° C and 500 µM CPCA and 0.25 µM PM was added. 30 ml of the medium was dispensed into the petri dish to prepare an N6D medium containing 500 μM CPCA and 0.25 μM PM.

  As a result of in vivo ALS test for all 28 individuals with confirmed growth, color development was not observed in the non-transformed callus in the presence of 0.25 μM pyriminobac, whereas red color development was observed in all 28 individuals. It was confirmed that the gene was introduced. Table 2 shows the amount of acetoin accumulated in each transformed callus (average of triplicates ± standard deviation). The difference in the amount of acetoin accumulation among individuals was considered to reflect the difference in the expression level depending on the copy number and position effect of the transgene in the transformant.

Twenty-eight transformants in which gene transfer was confirmed by callus were transplanted to a regeneration medium containing no pyriminobac. As a result, subdivided individuals were obtained for all but one individual. When the re-differentiated plant body grew to a plant height of about 5 cm on a hormone-free medium, the gene introduction into the plant body was confirmed by PCR. Specifically, after extracting DNA from leaves, two primer sets of 3-1-4 / pTN3 and ddsA3S / pTN3 are used to amplify the connection between ddsA and p10 terminator. PCR was performed. As a result, the target band was confirmed in all individuals (FIG. 7). The redifferentiated plants were sequentially cultivated in an isolated greenhouse. The heading was confirmed in all plants in an average of 2 months from the start of cultivation in the isolated greenhouse. Progeny seeds (T ₁ ) were collected from these plants.

[Results and discussion]
Extraction and quantification of CoQ10 in seeds collected from recombinant rice producing CoQ10 were performed by the method described in Example 1 described above. The result is shown in FIG. Table 3 shows the number of test seeds of each recombinant individual, the separation ratio of CoQ10-accumulated seeds, and the content of seeds with the largest amount of CoQ10 accumulation.

All 17 individuals tested showed accumulation of CoQ10 and separation according to Mendelian rule. Among them, ALSCoQ-6 showed the most accumulation of CoQ10 (10.5 μg / g). Moreover, the dispersion | variation in CoQ10 accumulation | storage for every grain of each individual | organism | solid is shown in FIG. It was considered that the CoQ10 content in the seed in which the most accumulation of each individual was observed corresponds to the accumulation in the homozygous individual.
As is clear from the above results, the method of Example 1 enables the quantification of CoQ10 in _one seed, and it has been clarified that the accumulated amount of CoQ10 can be determined in T1 seed by this method. .

  Meanwhile, Table 4 shows the results of extracting CoQ9 from wild-type rice by the method described in Example 1 and the results of extracting CoQ9 from wild-type rice by the methods of Comparative Examples 1 to 3.

  From the results shown in Table 4, endogenous CoQ9 can be quantified with high extraction efficiency in the experimental group (Example 1) using cellulase. On the other hand, it was revealed that the extraction efficiency of CoQ9 was low even when the brown rice powder was treated with α-amylase, protease or lipase as in Comparative Examples 1 to 3.

  On the other hand, Table 5 shows the results of extracting CoQ10 from the transformed rice produced in Reference Experiment 1 using the method described in Comparative Example 4.

  As can be seen from Table 5, it was found that most CoQ10 accumulated in most individuals compared to non-transformants. Moreover, the chart of the result of having analyzed CoQ10 about the non-transformed callus and transformed callus No5 shown in Table 5 is shown in FIG.10 and FIG.11, respectively. Comparison of these FIG. 10 and FIG. 11 also suggested that CoQ10 was significantly increased in the transformants.

  However, the results shown in Table 5 are cases where a large amount (100 mg or more) of brown rice powder is used, and it is impossible to quantify CoQ10 or CoQ9 in one grain of brown rice (15 mg to 25 mg). In contrast, the method described in Example 1 revealed that CoQ10 in one grain (15 mg to 25 mg) of brown rice could be quantified as shown in FIG. 10 and Table 3. In addition, Table 6 and FIG. 12 show the results of extracting and quantifying CoQ9 in one grain of brown rice from wild-type rice by the method described in Example 1. In addition, Takahashi et al.'S autoclave method and TLC method (S. Takahashi et al. 2006, FEBS Lett. 580 (3), 955-959) can recover CoQ9 almost completely with 100 mg of brown rice powder. Has been reported to be possible. In this report, the CoQ9 content of rice grains (variety: Nipponbare) quantified by this method is 4.2-6.0 μg / g.

  From the results shown in Table 6 and FIG. 12, it was revealed that according to the method described in Example 1, CoQ9 can be quantified with good reproducibility from the amount of brown rice powder corresponding to one grain of brown rice. Next, in the method described in Example 1, the amount of enzyme (number of units) required when 20 mg of brown rice powder (amount corresponding to one brown rice) was used as a material was examined. The results are shown in Table 7.

表７に示した結果から、実施例１に記載の方法において20ユニット以上のセルラーゼを用いれば再現性良くCoQ9が定量できることが判明した。

From the results shown in Table 7, it was found that CoQ9 can be quantified with good reproducibility by using 20 units or more of cellulase in the method described in Example 1.

  Table 8 and FIG. 13 show the results of extracting and quantifying CoQ9 by the method described in Comparative Example 4 using 100 mg of brown rice powder for the method described in Example 1 as described above.

  As shown in Table 8 and FIG. 13, in the method of Comparative Example 4, it was possible to quantify only 1/10 or less of the reported value, and it became clear that the extraction efficiency was extremely poor. Further, the extraction efficiency was not improved even by the method described in Comparative Example 5 or Comparative Example 6. Therefore, as Comparative Example 10, a CoQ9 addition recovery test was conducted for the purpose of improving extraction efficiency. 1 mg of CoQ9 standard product was added to 100 mg of brown rice powder (which contains 0.42 to 0.6 μg of CoQ9), and the method described in Comparative Example 6 in which extraction efficiency was poor and an improved method using an acid Table 9 shows the results of extracting and quantifying CoQ9 by the method described in Comparative Example 7.

  From the results shown in Table 9, it was considered that the extraction rate was slightly improved by the method described in Comparative Example 7 performed as Comparative Example 10. That is, it was considered that the acid treatment increases the extraction efficiency. On the other hand, since autoclave treatment was known to increase extraction efficiency, it was examined whether the quantitative value would change between the method without acid treatment (Comparative Example 8) and the method with treatment (Comparative Example 9). The results are shown in Table 10 and FIG. 14 (Comparative Example 9 only).

  As shown in Table 10 and FIG. 14, the quantitative value of Comparative Example 8 was not significantly different from Comparative Example 7 in which no autoclave treatment was used. However, as described in Comparative Example 9 in which acid treatment and autoclave treatment were used in combination, In the method, the quantitative value increased and the extraction efficiency was considered to have improved. However, as shown in Table 10, the reproducibility was poor (in other words, data fluctuation was large). This poor reproducibility was assumed to be due to the degradation of CoQ9 under acidic conditions. Thus, unlike the method described in Example 1, it can be concluded that CoQ9 and CoQ10 cannot be measured quantitatively by the method of extracting CoQ9 and CoQ10 by acid treatment.

It is a schematic diagram for demonstrating the integration scheme to PSR-03 of Ole18 pro :: S14 :: ddsA :: P10ter cassette. It is a schematic diagram for demonstrating the integration scheme to ASR pro :: S14 :: ddsA :: P10ter cassette in PSR-03. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a sequence diagram showing the entire base sequence of KLB-180. It is a schematic diagram for demonstrating the amplification position of the binary vector construct used for transformation, and the primer set used for PCR. It is an electrophoresis photograph which shows the confirmed result of the transgene by PCR using DNA extracted from the transformed callus. It is an electrophoresis photograph which shows the confirmed result of the transgene by PCR using DNA extracted from the transformed callus. It is an electrophoresis photograph which shows the confirmed result of the transgene by PCR using DNA extracted from the leaf of the transformed plant. 2 is a chart showing the results of HPLC for analyzing CoQ9 and CoQ10 extracted by the method described in Example 1. FIG. 2 is a scatter diagram of the amount of CoQ10 accumulated in one T1 seed of each line measured by the method described in Example 1. FIG. 6 is a chart showing the results of HPLC analysis of CoQ9 and CoQ10 for non-transformed callus by the method described in Comparative Example 4. 6 is a chart showing the results of HPLC analysis of CoQ9 and CoQ10 for transformed callus No5 by the method described in Comparative Example 4. 2 is a chart showing the results of HPLC analysis of CoQ9 in one brown rice of wild-type rice by the method described in Example 1. 6 is a chart showing the results of HPLC analysis of CoQ9 in 100 mg of wild-type rice brown rice powder by the method described in Comparative Example 4. 10 is a chart showing the results of HPLC analysis of CoQ9 in 100 mg of wild-type rice brown rice powder by the method described in Comparative Example 9.

Claims (7)

Treating the seed powder and / or seed embryo powder with a buffer containing cellulose hydrolase, and extracting the fat-soluble physiologically active substance,
Extraction method of fat-soluble physiologically active substance.   The method for extracting a fat-soluble physiologically active substance according to claim 1, wherein the seed powder and / or the seed embryo powder is derived from rice.   The method for extracting a fat-soluble physiologically active substance according to claim 2, wherein the rice is a transformed rice that produces ubiquinone 10 as a fat-soluble physiologically active substance.   The method for extracting a fat-soluble physiologically active substance according to any one of claims 1 to 3, wherein the amount of the seed powder and / or the seed embryo powder is an amount corresponding to at least one seed.   The method for extracting a fat-soluble physiologically active substance according to any one of claims 1 to 4, wherein the step of extracting the fat-soluble physiologically active substance uses a low-polar organic solvent.   6. The method for extracting a fat-soluble physiologically active substance according to claim 5, wherein the low-polar organic solvent is a saturated hydrocarbon having 5 to 8 carbon atoms.   The method for extracting a fat-soluble physiologically active substance according to any one of claims 1 to 6, wherein the fat-soluble physiologically active substance is ubiquinone and / or vitamin E.

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