JP2010046043A - Transgenic plant, method for producing the same and method for producing ubiquinone 10 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To highly accumulate CoQ10 (ubiquinone 10) in the seeds of a plant. <P>SOLUTION: The transgenic plant is provided by introducing an expression cassette having the promotor of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene capable of expressing under the control of the above promotor, and accumulating the ubiquinone 10 in the seeds. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a transformed plant for accumulating ubiquinone 10 (also referred to as coenzyme Q10 (CoQ10)) in seeds, a method for producing the same, and a method for producing ubiquinone 10 using the transformed plant.

  Ubiquinone 10 (hereinafter sometimes referred to as CoQ10) is an important substance related to the oxidation-reduction 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. Efforts have been made to accumulate CoQ10 in rice seeds that hardly accumulate CoQ10, and a relatively large amount of CoQ10 has been successfully accumulated in seeds of genetically modified rice (Patent Document 1: Japanese Patent Laid-Open No. 2006-2006). 212019 publication, non-patent document 1: S. Takahashi et al., FEBS Letters, 580, 955-959 (2006)). A sequence encoding the mitochondrial localization signal peptide of rice mitochondrial ribosomal protein in the decaprenyl diphosphate synthase gene of the enzyme that synthesizes the side chain of CoQ10 (10 units of isoprene chain), derived from gluconic acid bacteria, a kind of acetic acid bacteria By linking and expressing decaprenyl diphosphate synthase in rice mitochondria, CoQ10 has been successfully accumulated in rice seeds (Patent Document 2: United States Patent 6,103,488, Non-Patent Document 2: Okada, K., et al., Eur. J. Biochem. 255: 52-59 (1998)). However, the content of CoQ10 accumulated in the seeds has not yet reached a practical level even when the seeds are edible and CoQ10 is taken from the seeds and when CoQ10 is extracted from the seeds. .

  In order to calculate the content necessary for practical use, it is necessary to set the daily intake of CoQ10 as a supplement, but to date there is no value based on scientific evidence. Therefore, as a result of investigating the daily required intake recommended for CoQ10 supplement marketed as a reference value, it was recommended in the range of approximately 30 mg to 300 mg. On the other hand, the monthly consumption of rice per capita in 2007 is about 4900g (US net http://www.komenet.or.jp/). If this is converted to 30 days a month, it will be about 160g per day. If the daily required intake is set to 30 mg and this required amount is supplemented by the daily rice consumption, it is necessary that the rice contains CoQ10 in an amount of about 190 μg / g. On the other hand, CoQ10 is also contained in other foods (Non-patent Document 3: Kamei M., et al., Int. J. Vitam. Nutr. Res. 56: 57-63 (1986)) It can be considered that it is not necessary to replenish only with CoQ10 reinforced rice. Therefore, if it is assumed that half (15 mg) of the daily intake requirement (30 mg) is consumed in rice containing CoQ10, the amount of CoQ10 of 95 μg / g needs to be contained in the rice.

Japanese Unexamined Patent Publication No. 2006-212019 United States Patent 6,103,488 S. Takahashi et al., FEBS Letters, 580, 955-959 (2006) Okada, K., et al., Eur. J. Biochem. 255: 52-59 (1998) Kamei M. et al., Int. J. Vitam. Nutr. Res. 56: 57-63 (1986)

  Therefore, in view of the above-described practical use, the present invention provides a transformed plant capable of highly accumulating CoQ10 in plant seeds, a method for producing the same, and a method for producing ubiquinone 10 using the transformed plant. With the goal.

  In order to achieve the above-described object, the present inventors have established a new transformation vector for the purpose of accumulating more CoQ10 than recombinant rice produced by the method described in Non-Patent Document 1. By producing and using this, it succeeded in accumulating more CoQ10 in the seed than before, and completed the present invention.

The present invention includes the following.
(1) A transformed plant that accumulates ubiquinone 10 in seeds by introducing an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter.

  (2) The transformed plant according to (1), wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.

(3) The transformed plant according to (1), wherein a mutant acetolactate synthase gene is further introduced as a selection marker.
(4) The transformed plant according to (1), which is transformed rice.

  (5) A method for producing a transformed plant for accumulating ubiquinone 10 in seeds, wherein an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter is introduced. .

  (6) The method for producing a transformed plant according to (5), wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.

(7) The method for producing a transformed plant according to (5), wherein the mutant acetolactate synthase gene is further introduced as a selection marker, and the individual into which the expression cassette is introduced is selected by a pyrimidinylcarboxy herbicide. .
(8) The method for producing a transformed plant according to (5), which is transformed rice.

  (9) a step of collecting seeds from a transformed plant obtained by introducing an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter; A process for extracting ubiquinone 10 contained in seeds.

  (10) The method for producing ubiquinone 10 according to (9), wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.

  (11) The method for producing ubiquinone 10 according to (9), wherein the transformed plant is further introduced with a mutant acetolactate synthase gene as a selection marker.

  (12) The method for producing ubiquinone 10 according to (9), wherein the transformed plant is transformed rice.

  According to the present invention, it is possible to provide a transformed plant capable of accumulating a significantly larger amount of ubiquinone 10 in the seed than in the past and a method for producing the same. By using the transformed plant and the method for producing the same according to the present invention, a large amount of ubiquinone 10 which is a useful substance can be produced, and the production amount of ubiquinone 10 per unit acreage can be greatly improved. it can.

  Hereinafter, a transformed plant according to the present invention, a method for producing the same, and a method for producing ubiquinone 10 will be described in detail with reference to the drawings.

  The plant to be transformed in the present invention is not particularly limited, for example, rice, tobacco, barley, wheat, bread wheat, rye, oats, sorghum, sorghum, corn, millet, millet, buckwheat, buckwheat, bonito, Kudzu, sugar cane, 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 , Alfalfa, pepper, capsicum, cabbage, broccoli, cauliflower, lettuce, pine, spinach, garlic, mugwort, okra, perilla, sesame, gardenia, horseradish, mustard, ginger, ginger, turmeric, saffron, tama Geek, leek, garlic, bracken, spring, soybean, azuki bean, kidney bean, pea, broad bean, cowpea, velvet bean, peanut, groundnut, groundnut, wolfberry, poppy, olive, mint, almond, pistachio, macadamia nut, peanut, date palm, Coconut palm, Sago palm, Sugar palm, Nippa palm, Pineapple, Oyster, Apple, Ume, Peach, Loquat, Pear, Grape, Cherry, Strawberry, Raspberry, Blueberry, Prune, Pomegranate, Fig, Akebi, Gummy, Mangosteen, Kiwifruit, Melon, Citrus, Banana, Papaya, Mango, Arabidopsis, Nazuna, Inazuna, Lily, Rose, Sakura, Yamazakura, Button, Camellia, Sazanka, Lavender, Fuji, Peonies, Violet, Pansy, Sweetpea, Su Sen, Hanashobu, Iris, Basho, Shobu, Oshibasho, Water Lily, Lotus, Orchid, Morning Glory, Buttercup, Thistle, Gerbera, Dahlia, Tulip, Cosmos, Sunflower, Dandelion, Chamomile, Rosemary, Physalis, Hamanas, Matava, Clover , Cactus, saxifrage, dokudami, tea, coffee, spruce, larch, red pine, bamboo, ginkgo, cedar, cypress, fir, rubber, beech, zelkova, camphor, maple buffalo, eucalyptus, maple, poplar, acacia, bay bay, rush , Shiba, safflower, walnut, and avocado, with rice being particularly preferred.

  The expression cassette introduced into the plant to be transformed has a peptide elongation factor gene promoter and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter.

  Here, the peptide elongation factor gene is also called a translation elongation factor, and is a gene that encodes a protein involved in peptide chain synthesis in a protein biosynthesis system. Peptide elongation factor genes include the above-described plant-derived peptide elongation factor genes, mammalian-derived peptide elongation factor genes, filamentous fungal-derived peptide elongation factor genes, bacterial-derived peptide elongation factor genes, and yeast-derived peptide elongation factor genes. Are known.

  The peptide elongation factor gene promoter means a region located upstream in the transcription direction of the peptide elongation factor gene described above and controlling the expression of the peptide elongation factor gene. As the promoter of the peptide elongation factor gene, any promoter may be used as long as it controls the expression of the peptide elongation factor gene described above. For example, as a peptide elongation factor gene promoter, rice-derived peptide elongation factor gene 1α promoter, rice-derived peptide elongation factor gene 1β promoter, maize-derived peptide elongation factor gene 1α promoter, Arabidopsis derived peptide elongation factor gene 1α promoter , Arabidopsis derived peptide elongation factor gene 1β promoter, human derived polypeptide chain elongation factor gene 1α promoter, red bread mold derived peptide elongation factor EF-1α promoter, E. coli derived tufA gene promoter, yeast derived peptide elongation factor EF-1α A promoter or the like can be used.

  As an example, the base sequence of the rice-derived peptide elongation factor gene 1α promoter is shown in SEQ ID NO: 1. In addition, the base sequences of the promoters of the above-mentioned various organism-derived peptide elongation factor genes can be easily obtained from public databases storing base sequence information such as Genbank.

  The base sequence shown in SEQ ID NO: 1 or the base sequence registered as a promoter of a peptide elongation factor gene in a public database consists of a base sequence in which one or more bases are deleted, substituted, added or inserted. A polynucleotide that functions as a promoter can be used in the present invention as a promoter of a peptide elongation factor gene. Here, the plurality of bases means, for example, 2 to 250 bases, preferably 2 to 125 bases, more preferably 2 to 50 bases, and most preferably 2 to 25 bases.

  Further, it can hybridize under stringent conditions to the base sequence shown in SEQ ID NO: 1 or a complementary strand of a base sequence registered as a promoter of a peptide elongation factor gene in a public database, and A polynucleotide that functions as a promoter can be used in the present invention as a promoter of a peptide elongation factor gene. Here, stringent conditions can be easily set with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition).

  In particular, when the subject of transformation is rice, it is preferable to use a rice-derived peptide elongation factor gene promoter, and it is also preferable to use a plant-derived peptide elongation factor gene promoter for plants other than rice.

  The decaprenyl diphosphate synthase gene is also called a decaprenyl diphosphate synthase gene and means a gene encoding an enzyme having an activity of synthesizing 10 units of isoprene chain. The decaprenyl diphosphate synthase gene is not particularly limited, and a conventionally known gene can be used. For example, as the decaprenyl diphosphate synthase gene, a ddsA (decaprenyl diphosphate synthase) gene derived from Gluconobacter suboxydans can be used. The decaprenyl diphosphate synthase gene includes, for example, Agrobacterium-derived decaprenyl diphosphate synthase gene, fission yeast-derived dps1 gene, human-derived DPS1 gene, in addition to the ddsA gene derived from gluconic acid bacteria , Decaprenyl diphosphate synthase gene from Aspergillus clavatas, decaprenyl diphosphate synthase gene from Bleomyces albus, decaprenyl diphosphate synthase gene from Leukospodium skoti, decaprenyl from rhodobacter capsulatus Uses diphosphate synthase gene, decaprenyl diphosphate synthase gene from Cytoela complicator, decaprenyl diphosphate synthase gene from Paracoccus denitrificans, etc. can do.

  As an example, the base sequence of the decaprenyl diphosphate synthase gene (ddsA gene) derived from Gluconobacter suboxydans is shown in SEQ ID NO: 2, and the amino acid sequence of the decaprenyl diphosphate synthase encoded by the ddsA gene is sequenced The number 3 is shown. In addition, the base sequence and amino acid sequence of the decaprenyl diphosphate synthase genes derived from various organisms described above can be easily obtained from public databases storing base sequence information such as Genbank and Swiss-Prot.

  In addition, the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence registered as a decaprenyl diphosphate synthase in a public database consists of an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted. A protein exhibiting decaprenyl diphosphate synthase activity can be used in the present invention as decaprenyl diphosphate synthase. A plurality of amino acids here means, for example, 2 to 30 amino acids, preferably 2 to 20 amino acids, more preferably 2 to 15 amino acids, and most preferably 2 to 7 amino acids.

  Further, it can hybridize under stringent conditions to the base sequence shown in SEQ ID NO: 2 or the complementary strand of the base sequence registered as a decaprenyl diphosphate synthase gene in a public database, and A polynucleotide encoding a protein having decaprenyl diphosphate synthase activity can be used in the present invention as a decaprenyl diphosphate synthase gene. Here, stringent conditions can be easily set with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition).

  On the other hand, an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter includes a selection marker gene and an expression regulatory region in addition to the promoter and gene. You may have. As the selection marker gene, a conventionally known one can be used. For example, examples of selection marker genes include auxotrophic marker genes, drug resistance marker genes, and herbicide resistance marker genes. In particular, as a selection marker gene, it is preferable to use a gene encoding a mutant acetolactate synthase capable of imparting resistance to pyrimidinylcarboxy-based herbicides such as bispyribac-sodium, pyrithiobac-sodium and pyriminobac. This mutant acetolactate synthase gene can be appropriately used with reference to Japanese Patent No. 4091429 and US Pat. No. 7,119,256.

  Examples of the expression regulatory region include a region encoding a peptide that imparts localization specific to an organ and a transcription termination region (terminator gene). For example, examples of the expression regulatory region include a gene encoding a mitochondrial localization signal peptide or an endoplasmic reticulum localization signal peptide. In particular, it is preferable to use a mitochondrial localization signal peptide gene as the expression regulatory region. In other words, by accumulating the above-described decaprenyl diphosphate synthase in mitochondria, the amount of CoQ10 accumulated in seeds can be greatly improved.

  By introducing the expression cassette configured as described above into a plant to be transformed, a transformed plant that highly accumulates CoQ10 in seeds can be produced. Any conventionally known technique can be applied as a technique for introducing an expression cassette into a plant. For example, a technique of introducing a foreign gene into a target plant cell using Agrobacterium tumefaciens can be used.

  Alternatively, the expression cassette may be driven into a conventionally known expression vector, and the above-described decaprenyl diphosphate synthase gene may be introduced into a plant as an expression vector that can be expressed under the control of a peptide elongation factor gene promoter. Specifically, the above-described expression cassette is inserted into a binary vector containing the T-DNA sequence of Agrobacterium Ti plasmid. This Ti plasmid is transformed into E. coli and the like. Next, a binary vector grown in Escherichia coli or the like is transformed into Agrobacterium containing a helper plasmid. Then, after the target plant is infected with this Agrobacterium, the transformed plant is identified. When the identified transformed plant is a cultured cell, the plant cell can be regenerated into a complete plant by a conventionally known technique.

  In addition, the expression cassette described above or a vector containing the expression cassette may be directly introduced into a plant using a conventionally known technique. Furthermore, a polyethylene glycol method, an electroporation method, a particle gun method, or the like can be used as a method for transformation using the above-described expression cassette or a vector containing the expression cassette.

  The transformed plant produced as described above has a high accumulation of CoQ10 in its seeds. Therefore, seeds collected from transformed plants can be used as supplements or cosmetics in addition to pharmaceutical applications as seeds with high accumulation of CoQ10, or after being subjected to various processing. Alternatively, CoQ10 may be produced by separating and purifying CoQ10 from seeds collected from the transformed plant.

  As a method for producing CoQ10 from seeds, seeds collected from a transformed plant are pulverized and then extracted with an organic solvent. Here, the method for pulverizing the seed is not particularly limited, and various pulverization methods such as a method of pulverizing using a mortar and a method of pulverizing using a ball mill apparatus can be exemplified. In addition, prior to the extraction step with an organic solvent, water treatment, autoclave treatment, and enzyme treatment may be performed in advance. For example, examples of the enzyme treatment include a method of treating powdered seeds 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.

  In the extraction process of CoQ10 using an organic solvent, as the organic solvent, for example, a low-polar organic solvent such as pentane, hexane, heptane, and octane is preferably used, 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 with respect to 20 mg of seed powder.

  By the above method, CoQ10 can be produced from the seeds of the above-described transformed plant. The manufactured CoQ10 can be used as a supplement or a cosmetic product in addition to a pharmaceutical use without any limitation. In particular, in the seed of the transformed plant described above, for example, a seed of a transformed plant in which a decaprenyl diphosphate synthase gene is introduced under the control of a normal constitutive expression vector (for example, S35 promoter derived from cauliflower mosaic virus) In comparison, the accumulated amount of CoQ10 is significantly increased. Therefore, by using the above-described transformed plant, the production amount of CoQ10 per unit acreage can be greatly improved.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

<Materials and methods>
1. Binary vector plasmid for rice transformation
1-1 Binary vector plasmid used by Takahashi et al.
The binary vector plasmid construct used in the method of Takahashi et al. (S. Takahashi et al., FEBS Letters, 580, 955-959 (2006)) is shown in FIG. A rice seed homozygote (S14ddsA91-1) transformed with Agrobacterium having this binary vector plasmid was used as a comparative control in the following experiments.

1-2 Binary vector plasmid with ddsA driven by ALS promoter The method for preparing this binary vector plasmid was reported in Japanese Patent Application No. 2007-218986. The construct is shown in FIG. In Japanese Patent Application No. 2007-218986, the CoQ10 content of a progeny seed (T ₁ ) of rice transformed with Agrobacterium having this binary vector plasmid was described. In the present specification, the CoQ10 content of the progeny seeds is described.

1-3 Binary vector plasmid with ddsA driven by EF promoter Rice longevity factor (EF) β promoter, S14Signal, which is a mitochondrial localization signal peptide of rice mitochondrial ribosomal protein, Glucon, a kind of acetic acid bacteria Gluconobacter suboxydans decaprenyl diphosphate synthase gene (ddsA, accession number AB006850), P10T (riceprolamin 10 terminator gene) and binary vector developed for plant transformation (pPALS PSR-03 (Kumiai chemistry) The binary vector pPALS R-3 having the EF-1β pro :: S14 Signal :: ddsA :: P10T cassette was produced using the same).

  Specifically, it was produced by the method shown in FIGS. 3-1 and 3-2. The pUC18 vector in which the 35Spro :: S14 Signal :: ddsA :: NOSter cassette was incorporated was treated with Not I / blunted with Klenow Fragment, followed by Sal I treatment to isolate the S14 Signal :: ddsA portion. On the other hand, the pUC18 vector was treated with Sal I / Sma I, ligated with an S14 Signal :: ddsA fragment having a blunt end and a Sal I protruding end, and transformed into E. coli strain HB101. At this stage, the S14 Signal :: ddsA cassette was put on pUC18 [pUC18 (S14 :: ddsA)].

  On the other hand, pPALS R-3 was treated with Bam HI / Eco RI, and the ALS :: P10T portion was excised, incorporated into the Bam HI / Eco RI site on the pUC19 vector, and transformed into E. coli strain HB101. At this stage, the ALS :: P10T cassette was put on pUC19 [pUC19 (ALS :: P10T)].

  The previous pUC18 (S14 :: ddsA) vector was treated with Hind III / Sac I to excise the S14 Signal :: ddsA portion. Next, the pUC19 (ALS :: P10T) vector was also treated with Hind III / Sac I to remove the ALS portion, and the S14 Signal :: ddsA fragment was ligated and transformed into E. coli strain HB101. At this stage, the S14 Signal :: ddsA :: P10T cassette was put on pUC19 [pUC19 (S14 :: ddsA :: P10T)].

  The pUC19 (S14 :: ddsA :: P10T) vector was treated with Blp I / blunted with T4 DNA polymerase and then treated with Sal I. On the other hand, pUC19 vector (KLB-150) in which EF-1β promoter was incorporated was treated with Bgl II / blunted with T4 DNA polymerase and then treated with Sal I to obtain an EF-1β promoter fragment. The plasmid was ligated with a pUC19 (S14 :: ddsA :: P10T) vector having an end and transformed into E. coli strain HB101. At this stage, the EF-1βpro :: S14 Signal :: ddsA :: P10T cassette was put on pUC19 (KLB-175).

Next, EF: S14: ddsA: P10-1 (5′-AAAAAA GAATTC CCGATGTTCAATGACCGAACGA-3 ′) using the pUC19 vector (KLB-175) in which the EF-1βpro :: S14 Signal :: ddsA :: P10T cassette was incorporated as a template Underlined Eco RI recognition sequence) / EF: S14: ddsA: P10-2 (5'-AAAAAA CCTGCAGG TGTACAGCATGGCTTTCAAGAGGG-3 ', underlined Sse8387I recognition sequence) Reaction conditions, 98 ° C. (5 min) → [98 ° C. (30 sec) → 60 ° C. (30 sec) → 72 ° C. (75 sec), 25 cycles] → 72 ° C. (7 min)}, P10 terminator from 5′-end of EF-1β promoter The region leading to the 3′-end of was amplified. Since these primers were given Eco RI and Sse8387I recognition sequences, the PCR product and the pPALS R-3 vector were digested with the same restriction enzymes, ligated, and introduced into E. coli strain HB101. At this stage, EF-1βpro :: S14 Signal :: ddsA :: P10T cassette was put on pPALS R-3 (KLB-183). The entire base sequence of the obtained KLB-183 is shown in SEQ ID NO: 1.

2. Introduction of Binary Vector with ddsA Driven by EF Promoter into Agrobacterium The method for preparing Agrobacterium competent cells for electroporation is shown below.

  Agrobacterium (EHA105 strain) was pre-cultured at 27 ° C. for 24 hours in 1 ml of YM liquid medium (containing 12.5 ppm rifampicin and 25 ppm chloramphenicol). 1 ml of the preculture was added to 150 ml of YM liquid medium (containing 12.5 ppm rifampicin and 25 ppm chloramphenicol) in a 300 ml Erlenmeyer flask, and main culture was performed at 27 ° C. for 24 hours (OD600 = 1.0 to 1.5) . The culture solution was transferred to a 500 ml centrifuge tube and centrifuged at 10,000 rpm and 4 ° C. for about 20 minutes to precipitate the cells. Thereafter, the supernatant was discarded, 40 ml of ice-cooled 10% glycerol (sterilized) was added on ice, the cells were resuspended, and then centrifuged at 10,000 rpm and 4 ° C. for 12 minutes. After performing this operation twice, add 20 ml of ice-cold 10% glycerol (sterilized) on ice, resuspend the cells, transfer to a 50 ml centrifuge tube, and transfer to 10000 rpm at 4 ° C. Centrifuge for minutes. After centrifugation, the supernatant was discarded and suspended in 2 ml of ice-cold 10% glycerol, and 20 μl of the suspension was dispensed into a 1.5 ml Eppendorf tube and stored at −80 ° C.

  A method for selecting electroporation and pPALS vector-introduced Agrobacterium is shown below.

pPALS vector plasmid (100 ng) was added to 20 μl of Agrobacterium competent cells for electroporation dissolved on ice and mixed gently. The competent cell mixture was transferred to an ice-cooled cuvette and electroporated using Gene Pulser Xcell (manufactured by Bio-Rad Laboratories) under the conditions of 25 μF, 1.8 kV, and 200Ω. Then, immediately 1ml of YM liquid medium (Medium 1L in the 0.4 g yeast the Extract (DIFCO Co.), 10g mannitol, 0.1g NaCl, 0.1g MgSO 4, .pH containing 0.5g K ₂ HPO ₄ .3H ₂ O Was adjusted to 7.0.) Was added to the cuvette and mixed, and the whole amount was transferred to a 1.5 ml Eppendorf tube, and cultured with shaking at 27 ° C. and 250 rpm for 3 hours. After completion of the culture, the whole amount was applied to a YM agar medium (containing 12.5 ppm rifampicin, 25 ppm chloramphenicol, 50 ppm tetracycline), and cultured with shaking at 25 ° C. for 4 days.

3. Rice transformation with Agrobacterium carrying a binary vector with ddsA driven by EF promoter (sterilization of seeds)
Rice seeds were sterilized with 70% ethanol for 30 seconds, followed by 2.5% sodium hypochlorite solution for 20 minutes, and then washed with sterile water.

(Pre-culture of rice seeds infected with Agrobacterium)
Sterilized rice seeds were placed on N6D medium containing 2,4-D, pre-cultured for 5 days under light conditions at 34 ° C., and subjected to infection with Agrobacterium.

(Agrobacterium preculture)
Three days before infection, Agrobacterium was applied to AB medium containing rifampicin (25 mg / L), chloramphenicol (25 mg / L), and kanamycin (50 mg / L), and cultured at 24 ° C in the dark for 3 days .

(Agrobacterium infection and co-culture)
The shoot and endosperm portion of the scutellum-derived callus derived from the rice seeds cultured for 5 days were removed and temporarily placed in N6D medium. 40 ml of AAM solution was placed in a 50 ml falcon tube, and acetosyringone DMSO solution was added to 20 mg / l (final concentration). The acetosyringone solution was prepared by dissolving 0.1 g of acetosyringone in 1 ml of DMSO and sterilizing by filter. The pre-cultured Agrobacterium was scraped about 1/4 mm with sterilized microspartel, suspended in AAM solution, and gently pipetted with a pipette so that no Agrobacterium lumps remained. Callus temporarily placed in N6D medium was placed in a new 50 ml falcon tube, and the above-mentioned Agrobacterium suspension was added thereto, and the mixture was gently mixed by inversion for 1.5 minutes. Then, the suspension was poured into a sterilized tea strainer with callus. After excess water was removed from the tea strainer containing the callus on a sterilized Kim towel, it was placed on the 2N6-AS medium so that the callus did not touch each other, and co-cultured for 3 days in the dark at 24 ° C.

(Agrobacterium removal and selection)
The callus co-cultured with Agrobacterium for 3 days was transferred to a 50 ml Falcon tube and washed with sterile water containing carbenicillin (500 mg / l) until the washing solution was no longer cloudy. The callus was transferred to a sterilized Kimwipe to remove excess water, and then placed on N6D medium containing 400 mg / l carbenicillin and 0.5 μM pyriminobag (hereinafter referred to as PM) so that there were 14 calli on one plate. Thereafter, the cells were cultured at 34 ° C. in a light place for one month. In addition, subculture was performed when 2 weeks passed.

(Redifferentiation and potting of transformants)
Callus that grew on the N6D selection medium were transplanted to a regeneration medium (RE-III) containing no 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. The composition of the medium used is shown in Table 1.

*以外の成分濃度はmg/Lで示した。

Ingredient concentrations other than * are shown in mg / L.

4. Confirmation of introduction of T-DNA region into rice
DNA was extracted from leaves using DNeasy Plant Mini Kit (Qiagen) and subjected to PCR analysis. As a forward primer (3-1-4, 5′-AGGTGTCACAGTTGTTG-3 ′: SEQ ID NO: 6) and a reverse primer (pTN3, 5′-GCACACGATAGTATGCAACACC-3 ′) to confirm the junction of ALS and P10 terminator: SEQ ID NO: 7) was used. In order to confirm the junction between ddsA and p10 terminator, pTN3 was used as a reverse primer (ddsA3S, 5′-TGGGAGCGCGTCATTGGAGAAG-3 ′: SEQ ID NO: 8). The PCR reaction was carried out at 95 ° C. for 2 minutes, followed by 35 cycles of 95 ° C. for 30 seconds, 54 ° C. for 1 minute and 72 ° C. for 1 minute, and the final extension reaction was carried out at 72 ° C. for 10 minutes.

5. Extraction and quantification of coenzyme Q10 One brown rice was crushed in a mortar, then placed in a 1.5 ml Eppendorf tube, and cellulase (from Aspergillus nigar, SIGMA, dissolved in pH 5.0 acetate buffer) ) Was added. After the addition, the eppendorf tube was placed on a side so that the Eppendorf tube could be completely immersed in a 37 ° C. bath, and then allowed to stand for 24 hours with light shielding. After completion of the reaction, vitamin K1 was added as an internal standard substance, 4 ml of hexane was added in the dark, and the mixture was stirred for 30 seconds and then centrifuged at 2000 rpm for 2 minutes to recover 3 ml of the hexane layer. Furthermore, extraction was performed in the same manner with 4 ml of hexane twice (total 3 times). The extracted hexane layer was collected in an eggplant-shaped flask and concentrated to dryness with an evaporator. The residue was dissolved in 2 ml of a mixed solution of acetonitrile: methanol = 1: 1, and 50 μl thereof was injected into HPLC. HPLC analysis was performed using a CAPCELL PACK C8 (manufactured by Shiseido) column with HP-1100 (manufactured by Hewlett Packard). A methanol / acetonitrile mixed solution was used as the mobile phase, and the methanol: acetonitrile (60:40) was changed from methanol: acetonitrile (90:10) over 15 minutes at a flow rate of 1.0 ml / min. Coenzyme Q10 was detected under the condition of variable wavelength UV-VIS (measurement wavelength: 275 nm).

6. In vivo ALS assay using rice leaf and callus Rice leaf was treated according to the following method, and ALS activity was measured. First, about 150 mg of leaves were collected from the transformed rice, finely chopped, and placed in a petri dish. Treatment solution (mixed salt for 25% MS medium, 500μM (final concentration) 1,1-cyclopropanedicarboxilic acid (CPCA), 5μg Triton X-100, 0.5μM (final concentration) pyrimino back (PM) was added at 30 ℃, bright After the reaction, the treated leaves were placed in a 1.5 ml microtube, and 200 μl of distilled water containing 0.025% Triton X-100 was added. Extracted with sonic waves (frequency 40 kHz) for 20 minutes, transferred 200 μl of supernatant to another tube, added 20 μl of 5% (v / v) sulfuric acid and allowed to stand at 60 ° C. for 30 minutes, then 100 μl of 0.5 Add 100 μl of 5% (w / v) 1-naphthol solution (prepared when used) dissolved in 2% (w / v) creatine solution and 2.5N NaOH solution and let stand at 37 ° C for 30 minutes. It was measured.

  In the case of callus, ALS activity was measured by the following method. A part of the transformed callus (about 50 mg) was placed on an N6D medium containing 500 μM 1,1-cyclopropanedicarboxylic acid (CPCA) and 0.5 μM PM, and allowed to stand at 30 ° C. for 24 hours in the dark. After the reaction, the treated callus was placed in a 1.5 ml microtube, and 220 μl of distilled water containing 0.025% Triton X-100 was added. After leaving still at 60 degreeC for 5 minutes, it extracted for 25 minutes with the ultrasonic wave (frequency 40kHz). 200 μl of the supernatant was transferred to another tube, 20 μl of 5% (v / v) sulfuric acid was added, and the mixture was allowed to stand at 60 ° C. for 30 minutes. Then, 100 μl of 0.5% (w / v) creatine solution and 100 μl of 5% (w / v) 1-naphthol solution (prepared when used) dissolved in 2.5N NaOH solution were added and left at 37 ° C. for 30 minutes. Absorbance at 530 nm was measured.

  An N6D medium containing 500 μM CPCA and 0.5 μM PM was prepared according to the following operation. CHU powder (1 bag), myo-inositol 100mg, nicotinic acid 0.5mg, pyridoxine hydrochloride 0.5mg, thiamine hydrochloride 1mg, 2,4-D 2mg, casamino acid 300mg, glycine 2mg, L-proline 2.8g, sucrose 30g Then, 4 g of gellite was dissolved in 1 L of distilled water and adjusted to pH 5.8. After autoclaving, the medium was cooled to 55 ° C. and 500 μM CPCA (final concentration) and 0.5 μM PM (final concentration) were added. 30 ml of the medium was dispensed into the petri dish to prepare an N6D medium containing 500 μMCPCA and 0.25 μM PS.

7. Sensitivity test of transformed rice to pyrithiobac sodium salt (PS) The seeds of transformed rice were immersed in 5% sodium perchlorate and subsequently washed several times with distilled water. After washing, it was allowed to germinate by being immersed in distilled water at 27 ° C. for 3 days. The germinated seeds were sown in a Hoagland medium containing a predetermined concentration of PS, cultivated at 27 ° C. for 7 days, and the growth was observed after 7 days.

8. RT-PCR analysis Rice (ALSCoQ6-4 and ALSCoQ28-5) transformed with binary vector with ddsA driven by the above ALS promoter, wild-type rice as negative target, PSR-04-7-9 as positive target (Transformation homozygote that drives W548L / S627I two-point mutant rice ALS with ALS promoter) was cultivated in an isolated greenhouse and RNeasy Plant Mini Kit (Qiagen) was obtained from 4 leaves of plants grown at 4-5 leaf stage. To extract total RNA. Using PrimeScript RT reagent Kit (Takara Bio), cDNA was synthesized from 2 μg of total RNA by reverse transcription reaction. The reverse transcription reaction was carried out at 37 ° C. for 15 minutes, followed by heat inactivation of the reverse transcriptase at 85 ° C. for 5 seconds. Quantitative RT-PCR was performed using SYBR premix Ex Taq (Takara Bio) using Thermal Cycler Dice TP800 (Takara Bio). The reaction conditions were initial denaturation at 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 5 seconds and 60 ° C. for 30 seconds. Actin1 gene was used as an internal standard gene. The primer sets used are described below.
ALS gene (Accession number: AB049822) Amplification primer (134 bp DNA fragment)
Forward primer (ALS-q1S: 5'-GTGCACATTGACATTGATC-3 ': SEQ ID NO: 9)
Reverse primer (ALS-q1A: 5'-ATCAGAACTTGTCTTTGTTGTGCTC-3 ': SEQ ID NO: 10)
ddsA gene (Accession number: AB006850) Amplification primer (101 bp DNA fragment)
Forward primer (ddsA-q1S: 5'-TGGAGCGGTTTGGCACCAATC-3 ': SEQ ID NO: 11)
Reverse primer (ddsA-q1A: 5'-ATCACCAACGGTCTTGCCCAAA-3 ': SEQ ID NO: 12)
Actin-1 gene (Accession number: AK100267) Amplification primer (116 bp DNA fragment)
Forward primer (Act-q1S: 5'-CCCAAGAATGCTAAGCCAAGAG-3 ': SEQ ID NO: 13)
Reverse primer (Act-q1A: 5'-TGATAACAGATAGGCCGGTTGAA-3 ': SEQ ID NO: 14)

<Result>
1. Result of rice transformed with binary vector with ddsA driven by ALS promoter
1-1. CoQ10 content in T1 seeds CoQ was extracted from 10 or more seeds of each recombinant plant and quantified by HPLC. Table 2 shows the content of CoQ10 in the seeds with the largest accumulation of CoQ10. All 22 individuals tested showed accumulation of CoQ10, of which ALSCoQ-6 showed the most accumulation of CoQ10 (10.5 μg / g dry weight).

1-2 CoQ10 content of T2 seed
The T ₁ seeds were sown in pots ALSCoQ-6 and ALSCoQ-28, was grown in an isolated greenhouse. When the plant height grew to about 10 cm, the transgene was confirmed by PCR with the 3-1-4 / pTN3 primer set that amplifies the junction of ALS and P10 terminator described in the experimental method. As a result, a DNA fragment of the desired size was detected by PCR, and 6 individuals (ALSCoQ-6-1 to 6) determined to be homozygous or heterozygous for ALSCoQ-6 and 1 individual (ALSCoQ) determined to be a null individual. -6-7) and was selected to T ₁ plants ALSCoQ-28 homo- or heterozygous been judged 6 individuals (ALSCoQ-28-1~6), isolated greenhouse and transplanted into 1/5000 Wagner pots in the cultivation, to obtain T ₂ seeds. Callus was induced from the obtained seeds and in vivo ALS assay was performed in the presence of 0.5 μM PM. As a result, all callus derived from the seeds of ALSCoQ-6-4 and ALSCoQ-28-5 showed PM resistance and thus were judged to be homozygous. On the other hand, all callus derived from ALSCoQ-6-7 seeds judged to be null individuals from the PCR results showed PM sensitivity. All other individuals were judged to be heterozygous because callus showing PM resistance and callus showing sensitivity were separated at a ratio of 3: 1 (Table 3).

Next, ALSCoQ-6-4 and 28-5 determined to be homozygous, ALSCoQ-6-1, 6-2 and 28-1 determined to be heterozygous, ALSCoQ-6- determined to be a null individual The CoQ10 content in 7 seeds was quantified (FIG. 4). As a result, ALSCoQ-6-4 and 28-5 were found to be homozygous by confirming the accumulation of CoQ10 in all seeds tested. The accumulation of CoQ10 in other individuals was consistent with the results of the in vivo ALS assay. That is, lines determined to be heterozygotes (ALSCoQ-6-1, 6-2, 28-1) were separated into seeds that accumulated CoQ10 and seeds that did not accumulate, and lines that were determined to be null (ALSCoQ-6- In 7), no accumulation of CoQ10 was observed in all seeds. ALSCoQ-6-4 and 28-5, which were found to be homozygotes, also showed variation in CoQ10 content among seeds, but averaged 9.9 μg / g dry weight and 9.5 μg / g dry, respectively. has accumulated CoQ10 of weight, content predicted by T ₁ seeds; showed values close to (ALSCoQ6,10.5 μg / g dry weight ALSCoQ28,8.1 μg / g dry weight) ( Fig. 4). This content is transformants overexpressing the ddsA gene 35S promoter were generated by Takahashi et al. Method, similarly with the seeds of highest CoQ10 T ₂ homozygotes of accumulated strains with (S14ddsA91-1) It was almost the same as the result (11.2μg / g dry weight) measured by this method (Table 2). In addition, the result shown as a table | surface in FIG. 4 is an average value +/- standard deviation of 8 grains.

1-3. PC herbicide sensitivity test of T ₂ homozygote
In order to examine the sensitivity of ALSCoQ-6-4 and ALSCoQ-28-5 to ALS inhibitors, each seed was sown in a medium containing pyrithiobac sodium salt (hereinafter referred to as PS) and cultivated for 7 days. Later, growth was observed. As a result, growth inhibition was observed even at the lowest concentration tested (0.01 μM) compared to the group without the addition of a drug, and strong growth inhibition was observed in the foliage and roots in the presence of 0.1 μM PS. Growth was completely suppressed in the presence of 1 μM or more of PS. The sensitivity of these transformants to drugs was the same as that of wild-type rice (FIGS. 5-1 and 5-2). As a result, it was considered that the selection marker gene introduced into rice simultaneously with the ddsA gene was not expressed in the plant body by the callus-specific promoter.

1-4. ALS and ddsA expression in T ₂ homozygotes
In order to confirm the expression of S627I OsALS protein in ALSCoQ-6-4 and ALSCoQ-28-5, an in vivo ALS assay using leaves was performed. As a result, red color development was observed in the PM-free group, whereas red color development was not observed in the 0.15 μM PM-added group in which endogenous ALS was inhibited, as in the case of wild-type rice. In addition, PSR04-7-9 (transformed homozygote in which W548L / S627I OsALS was driven by an ALS promoter), which was similarly tested, showed red coloration even in the PM addition group. From these results, it was confirmed that ALSCoQ-6-4 and ALSCoQ-28-5 did not express S627I OsALS protein in plants (Table 4).

  Next, the expression of ALS gene and ddsA gene was confirmed using real-time PCR. As a result, the positive target PSR04-7-9 showed about 7 times the expression of the ALS gene as compared with the wild type, whereas the expression of the ALSCoQ-6 and 28 ALS genes was not different from the wild type. Therefore, it was revealed that the mutant ALS gene was not expressed even at the RNA level. On the other hand, expression of the ddsA gene in the wild type and PSR04-7-9 was not confirmed, whereas significant expression was confirmed in ALScoQ-6 and 28. Therefore, it was clarified that the ddsA gene was expressed in the transformant and CoQ10 was accumulated (FIG. 6). In addition, the bar graph shown in FIG. 6 shows the average of 3 independent tests, and the error bar shows the standard deviation.

2. Result of rice transformed with binary vector with ddsA dried by EF promoter
2-1 Rice transformation with R-3 with EF promoter :: S14 :: ddsA :: P10 terminator cassette
Rice transformation was performed with R-3 having an EF promoter :: S14 :: ddsA :: P10 terminator cassette, and selection was performed in N6D medium containing 0.5 μM pyriminobac (PM). As a result, it was considered that 57% callus (72 out of 126 calli derived from 126 seeds) grew and the gene was introduced after selection for about one and a half months. All the callus in which proliferation was observed moved to the regeneration medium, and finally 58 lines of redifferentiated individuals (regeneration efficiency 64%) were obtained. All of these showed growth of shoots and roots on hormone-free medium. PCR was performed using a primer set (3-1-4 / pTN3) that amplifies between ALS and PT10 terminator in any 30 individuals and a primer set (ddsA 3S / pTN3) that amplifies between ddsA and PT10 terminator. The PCR products (428 bp and 289 bp) corresponding to the desired number of bases were confirmed in all individuals (FIGS. 7 and 8).

  In FIG. 7, LB means left border, RB means right border, callus sp pro means callus specific promoter, S627IOsALS means S627I mutant ALS gene, P10T means 10Kda prolamin terminator. Meaning, EFpro means rice EF promoter, S14 means mitochondrial translocation signal of rice rps14 gene, DdsA means decaprenyl diphosphate synthase. In FIG. 7, the bold line indicates a region amplified by PCR to confirm T-DNA introduced into the genomic DNA.

  In FIG. 8, M lane indicates a 100 bp DNA marker, No. 1-6 lane indicates transformed rice selected with 0.5 μM pyriminobac (PM), No. 7 lane indicates wild type rice, 'Lane indicates a 200 bp DNA marker.

2-2. T ₁ transformants contemporary plants measured production of CoQ10 content in seeds were grown in an isolated greenhouse were transplanted to 1/5000 Wagner pots were seed the T ₁ seed. The obtained T ₁ seeds were quantified by HPLC after extracting CoQ for each 10 grains of each line. As a result, wild-type rice contains CoQ9 and a small amount of CoQ10, whereas in the transformant, all the analyzed lines (22 lines) have much less CoQ9 and CoQ9 in contrast to the wild type. It was confirmed that more CoQ10 was accumulated (Fig. 9). In FIG. 9, A indicates the wild type, and B indicates the transformant. In each line, variation was observed in the accumulation of CoQ10 for each grain, and there were seeds in which no accumulation of CoQ10 was observed (FIG. 10). In addition, the result shown in FIG. 10 is the result of quantifying the seeds of 10 grains from each line. This variation is caused by three T ₁ seeds: a homozygote (homo) having a pair of introduced genes at the same locus, a heterozygote (hetero) having only one gene, or a null having no gene at all. As described above, it was determined that the CoQ10 content in the seeds where the most accumulation of each line was observed was equivalent to the accumulation in the final homo individuals. Therefore, comparing the seeds with the highest accumulation of CoQ10 in each line, 3 lines (EFCoQ-21, 26, 28) that accumulated more than 20μg / g dry weight were confirmed (Table 5). ).

  CoQ10 content (24.2 μg / g dry weight) of EFCoQ-21 with the highest accumulation of CoQ10 was confirmed in the lines (ALSCoQ-6 and S14ddsA91-1) that showed the highest accumulation in transformants driven by ALS or CaMV35S promoter. ) 2.2 to 2.3 times. Moreover, since the CoQ10 content in the transformant driven with the EF promoter was generally higher than that of the transformant driven with the ALS promoter, it became clear that the CoQ10 content can be increased by using the EF promoter (FIG. 11). ).

  In FIG. 11, A is the result of quantification for each grain in 22 lines of transformants into which the ALSpro :: S14 :: ddsA cassette was introduced. Moreover, in FIG. 11, B is the result of quantifying each grain in 22 lines of transformants into which the EFpro :: S14 :: ddsA cassette was introduced.

<Discussion>
It has been shown that the rice ALS promoter is strongly expressed in immature and vigorous organs such as immature seeds (Osakabe et al., Mol. Breed. 16: 313-320 (2005)). On the other hand, the biosynthesis of CoQ is thought to be promoted by organs and tissues with high cell division. Therefore, the accumulated amount of CoQ10 in seeds is expressed by the CaMV35S promoter, which is widely used in plant transformation, because the ddsA gene whose expression is controlled by the ALS promoter is efficiently expressed during the active period of CoQ10 biosynthesis. It was expected to increase compared to the case where expression was controlled. However, when the rice ALS promoter was used, it was not possible to increase the CoQ10 content in the homozygous seeds, and CoQ10 almost equal to that obtained when the CaMV35S promoter was accumulated in the seeds.

On the other hand, among the rice transformed with the binary vector having the ddsA gene whose expression was controlled by the EF promoter, EFCoQ-21, which showed the highest accumulation of CoQ10, contained about 24 μg of CoQ10 per 1 g of seed. Although this is a result of T ₁ seeds, as described above, it was considered that there was no significant difference from the accumulated amount of homozygote. This accumulated amount is equivalent to about twice the amount of recombinant rice produced by Takahashi et al., And if the ddsA gene whose expression is controlled by the EF promoter is used, more CoQ10 is accumulated in rice seeds than in the conventional method. It became clear that we could do it.

  In the future, the following methods can be considered as methods for further increasing the CoQ10 content. So far, it has been reported that the introduction of polyprenyltransferase (PPT) from yeast increases the CoQ10 content in leaves by about 6-fold (Ohara et al., Plant J. 40: 734-743 (2004)). Since PPT is a rate-determining enzyme for CoQ synthesis that catalyzes the binding of polyprenyl diphosphate and 4-hydroxybenzoic acid (Fig. 13), an increase in CoQ10 content can be expected by introducing the ppt gene simultaneously with the ddsA gene. . PPT also exhibits broad substrate specificity for prenyl diphosphates of different lengths (Suzuki et al., Biosci. Biotechnol. Biochem. 58: 1814-1819 (1994)), so that decaprenyl diphosphate in rice It is also possible that CoQ10 content may be increased by searching for PPT that efficiently catalyzes the binding of 4-hydroxybenzoic acid. Since rice PPT has been reported recently (Ohara K et al., Plant Cell Physiol. 47: 581-590 (2006)), it is possible to further improve the amount of CoQ10 accumulated by co-expression with ddsA. It is believed that there is.

It is a schematic diagram which shows the construction of the binary vector plasmid used by the method of Takahashi et al. (S. Takahashi et al., FEBS Letters, 580, 955-959 (2006)). It is a schematic diagram showing a construct of a binary vector plasmid having a ddsA gene under the control of rice ALS promoter expression. FIG. 3 is a schematic diagram showing a construct of a binary vector plasmid having a ddsA gene under the expression control of the rice elongation factor (EF) β promoter. FIG. 3 is a schematic diagram showing a construct of a binary vector plasmid having a ddsA gene under the expression control of the rice elongation factor (EF) β promoter. It is a characteristic figure which shows CoQ10 content of the T2 seed transformed with the binary vector plasmid which has a ddsA gene under the expression control of a rice ALS promoter. It is a characteristic view which shows the result of the PS sensitivity test in the plant body transformed by the binary vector plasmid which has a ddsA gene under the expression control of a rice ALS promoter. It is a characteristic view which shows the result of the PS sensitivity test in the plant body transformed by the binary vector plasmid which has a ddsA gene under the expression control of a rice ALS promoter. It is a characteristic diagram which shows the result of quantitative RT-PCR with respect to ALS gene and ddsA gene in the transformant transformed with the binary vector plasmid which has ddsA gene under the expression control of a rice ALS promoter. FIG. 3 is a schematic diagram showing a region amplified by PCR for confirming vector introduction when transformed with a binary vector plasmid having a ddsA gene under the expression control of rice elongation factor (EF) β promoter. It is an electrophoresis photograph showing the results of PCR for confirming the introduction of a vector when transformed with a binary vector plasmid having a ddsA gene under the control of the expression of rice elongation factor (EF) β promoter. It is a characteristic figure which shows the result which confirmed the accumulation | storage of CoQ9 and CoQ10 by HPLC about the plant and wild type which were transformed with the binary vector plasmid which has a ddsA gene under the expression control of a rice elongation factor (EF) (beta) promoter. It is a characteristic view showing the result of confirming the accumulation of CoQ10 by HPLC on T1 seeds transformed with a binary vector plasmid having the ddsA gene under the expression control of the rice elongation factor (EF) β promoter. T1 seed transformed with a binary vector plasmid having ddsA gene under the control of rice ALS promoter expression and T1 seed transformed with a binary vector plasmid having ddsA gene under the control of rice elongation factor (EF) β promoter expression It is a characteristic view which shows the result of having compared the amount of CoQ10 accumulation about. It is a characteristic view which shows the biosynthetic pathway of CoQ10 and isoprenoid.

Claims (12)

  A transformed plant in which an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter is introduced, and ubiquinone 10 is accumulated in seeds.   The transformed plant according to claim 1, wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.   The transformed plant according to claim 1, wherein a mutant acetolactate synthase gene is further introduced as a selection marker.   The transformed plant according to claim 1, which is transformed rice.   A method for producing a transformed plant that accumulates ubiquinone 10 in seeds, wherein an expression cassette having a promoter of a peptide elongation factor gene and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter is introduced.   6. The method for producing a transformed plant according to claim 5, wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.   6. The method for producing a transformed plant according to claim 5, wherein a mutant acetolactate synthase gene is further introduced as a selection marker, and an individual into which the expression cassette is introduced with a pyrimidinylcarboxy herbicide is selected.   6. The method for producing a transformed plant according to claim 5, wherein the plant is transformed rice. A step of collecting seeds from a transformed plant obtained by introducing an expression cassette having a peptide elongation factor gene promoter and a decaprenyl diphosphate synthase gene that can be expressed under the control of the promoter;
And a step of extracting ubiquinone 10 contained in the collected seed.   The method for producing ubiquinone 10 according to claim 9, wherein the expression cassette further has a region encoding a mitochondrial localization signal peptide upstream of the decaprenyl diphosphate synthase gene.   The method for producing ubiquinone 10 according to claim 9, wherein the transformed plant is further introduced with a mutant acetolactate synthase gene as a selection marker.   The method for producing ubiquinone 10 according to claim 9, wherein the transformed plant is transformed rice.

Images