JP2006212019A - Method for producing ubiquinone-10 using plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant producing ubiquinone-10 in large amounts and to provide a method for producing ubiquinone-10 by using such plant and to provide a dietary supplement, a supplement and a food additive each containing ubiquinone-10 produced by the plant or the method. <P>SOLUTION: A plant transformed with an expression cassette in which a decaprenyl diphosphate synthase derived from Gluconobacter suboxydans is operatively linked with a mitochondrial targeting sequence is prepared and ubiquinone-10 is produced by using the plant. The food and the cosmetic each contain ubiquinone-10 derived from the plant. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing ubiquinone-10 using a plant, a recombinant plant used in such a production method, and an expression cassette used for producing such a recombinant plant.

  Ubiquinone is one of the components of an electron transport system, and is a compound in which an isoprenoid (a natural organic compound having a basic unit of isopentenyl diphosphate (IPP) having 5 carbon atoms) is added to a quinone skeleton, It is a biological component having a structure of 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone and playing an important role also called coenzyme Q (coenzyme Q = CoQ). Naturally, homologues from ubiquinone-5 to ubiquinone-12 exist mainly depending on the number of isoprenoid units in the side chain.

  It is known that ubiquinone exists in animal and plant tissues and microorganisms and plays an important physiological and biochemical function. Ubiquinone is a vitamin-like substance, also known as an antioxidant, and it is also skin aging, immune deficiency, heart disease, periodontal disease, chronic fatigue syndrome, degenerative brain, neuromotor dysfunction, diabetes, stroke, arteriosclerosis It has been confirmed to be effective for cancer, hypertension, hypotension, brain infarction, allergic diseases and the like. In addition to being used for the treatment of congestive heart failure, it is also known for its effects on heart palpitations, shortness of breath, swelling of legs and face, coldness, etc. (as a review on ubiquinone) Non-patent documents 1 to 3).

  However, ubiquinone-10 (UQ-10) is the only ubiquinone that has been recognized as an effective pharmaceutical product. Ubiquinone-10 is synthesized in the body, but since it decreases due to aging, fatigue, and stress, it often needs to be replenished. Therefore, it is currently included in cosmetics and sports drinks as well as pharmaceuticals, and sold as a component of health foods and dietary supplements.

  Ubiquinone-10 is currently extracted and purified from solanaceous plants, yeast or microbial cells, but its production cannot keep up with demand, and in addition to conventional methods, development of more efficient production methods is required. It was.

  The isoprenoid that forms the side chain of ubiquinone is a natural organic compound having a basic unit of isopentenyl diphosphate (IPP) having 5 carbon atoms, and there are many in nature. Besides being used as the side chain of ubiquinone, it is also known as carotenoid, natural rubber, and prenylated protein. In biosynthesis in vivo, new IPP is bound to basic IPP and FPP (phanesyl diphosphate), and isoprenoids with long chain lengths are gradually synthesized.

  A decaprenyl diphosphate synthase gene (SEQ ID NO: 5) cloned from the bacterium Gluconobacter suboxydans is known to produce ubiquinone-10 when expressed in E. coli (Patent Document 1, Non-Patent Document 3). ). However, when ubiquinone-10 is produced in bacteria such as Escherichia coli, it is necessary to purify ubiquinone-10 when producing nutritional supplements, supplements, foods, beverages, food supplements, etc. containing ubiquinone-10. A great deal of money and labor is required. On the other hand, when it is expressed in a plant that can be used for food, the cost and labor for such purification can be reduced. However, there is no report of production of ubiquinone-10 by expressing the bacterial gene in plants.

  On the other hand, in eukaryotes, production of ubiquinone-10 using recombinant fission yeast has been attempted (Patent Document 2). In Patent Document 2, S. cerevisiae is a fission yeast. cloning and sequencing the pombe decaprenyl synthase gene and using the fusion gene linked to the mitochondrial translocation signal, S. cerevisiae cerevisiae is transformed. However, production of ubiquinone-10 in recombinant yeast cells has not been confirmed. Furthermore, it is suggested that the enzyme activity is low in the recombinant yeast cell described in Patent Document 2. In contrast, yeast expressing the ddsA gene from Gluconobacter produced ubiquinone-10. Therefore, until now, no method has been established for producing ubiquinone-10 using recombinant cells other than E. coli and yeast.

Therefore, it is desired to provide a method for enhancing ubiquinone-10 production and a plant with enhanced ubiquinone-10 production by transforming the plant.
JP-A-10-57072 JP-A-9-173076 Makoto Kawamukai, Biosynthesis and New Physiological Functions of Ubiquinone, Chemistry and Biology, 40 (5); 172-178 Makoto Kawamukai, J. et al. Biosci. Bioeng. 94, 511-517 (2002) Okada et al. (Eur. J. Biochem. 255, 52-59 (1998))

  An object of the present invention is to provide a method for producing ubiquinone-10 in a recombinant plant and a recombinant plant for producing ubiquinone-10. It is also an object of the present invention to provide an expression cassette used for preparing the recombinant plant of the present invention. Furthermore, it is also an object of the present invention to provide compositions, dietary supplements, supplements and pharmaceutical compositions comprising ubiquinone-10 produced by the plants of the present invention.

  The present invention has solved the above-mentioned problem of providing a plant that produces ubiquinone-10 by targeting a decaprenyl diphosphate synthase gene to mitochondria and / or the Golgi apparatus in a plant and expressing it.

Accordingly, the present invention provides the following.
1. A plant transformed with an expression cassette expressing polyprenyl diphosphate synthase.
2. Item 2. The plant according to Item 1, wherein the polyprenyl diphosphate synthase is decaprenyl diphosphate synthase.
3. Item 2. The plant according to Item 1, wherein the decaprenyl diphosphate synthase is an enzyme derived from bacteria.
4). Item 4. The plant according to Item 3, wherein the decaprenyl diphosphate synthase is an enzyme derived from Gluconobacter suboxydans.
5. Item 3. The plant according to Item 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid that hybridizes with the nucleic acid shown in SEQ ID NO: 5 under stringent conditions.
6). Item 3. The plant according to Item 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having one or several deletions, additions or substitutions in the nucleic acid sequence shown in SEQ ID NO: 5.
7). Item 3. The plant according to Item 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having a sequence identity higher than 80% with the nucleic acid sequence shown in SEQ ID NO: 5.
8). Item 3. The plant according to Item 2, wherein the decaprenyl diphosphate synthase has an amino acid sequence having one or several deletions, additions, or substitutions in the amino acid sequence shown in SEQ ID NO: 6.
9. Item 3. The plant according to Item 2, wherein the decaprenyl diphosphate synthase has an amino acid sequence identity higher than 80% with the amino acid sequence shown in SEQ ID NO: 6.
10. Item 3. The plant according to Item 2, wherein the nucleic acid encoding decaprenyl diphosphate synthase is operably linked to a nucleic acid encoding a mitochondrial targeting sequence.
11. A nucleic acid encoding the mitochondrial targeting sequence, a nucleic acid encoding a fragment of rice RPS10 protein (SEQ ID NO: 2), a nucleic acid encoding a fragment of RPS14 protein (SEQ ID NO: 4), a nucleic acid encoding a fragment of RPS11 protein, 11. The plant according to item 10, selected from the group consisting of a nucleic acid encoding a fragment of ATPase β subunit protein (SEQ ID NO: 10) and a nucleic acid encoding a fragment of ATPase γ subunit protein (SEQ ID NO: 12). .
12 Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2).
13. Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). .
14 The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice Item 11. The plant according to Item 10, encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of the RPS10 protein (SEQ ID NO: 2).
15. Item 10. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding the amino acid sequence from positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). Plant.
16. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice RPS10 protein (sequence). Item 11. The plant according to item 10, which has the amino acid sequence of a fragment located within positions 1 to 56 of number 2).
17. Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2).
18. Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4).
19. Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence of positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). .
20. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice The plant according to item 10, which is encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of the RPS14 protein (SEQ ID NO: 4).
21. Item 11. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity higher than 80% with a nucleic acid sequence encoding amino acid sequence 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). Plant.
22. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions, or substitutions in the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice RPS14 protein ( Item 11. The plant according to Item 10, which has the amino acid sequence of a fragment located within positions 1 to 48 of SEQ ID NO: 4).
23. Item 11. The plant according to Item 10, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4).
24. The plant according to item 2, wherein the polyprenyl diphosphate synthase is operably linked to a Golgi apparatus targeting sequence.
25. 25. The plant according to item 24, wherein the nucleic acid encoding the Golgi apparatus targeting sequence is a nucleic acid encoding a fragment of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
26. Item 25. The plant according to Item 24, wherein the Golgi apparatus targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
27. The Golgi apparatus targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. The plant according to item 24.
28. The Golgi apparatus targeting sequence may be one or several deletions, additions, or additions to the nucleic acid sequence encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. A plant according to item 24, encoded by a nucleic acid having a substitution, or a fragment of a nucleic acid sequence encoding the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
29. The Golgi apparatus targeting sequence is encoded by a nucleic acid having a sequence that is 80% identical to the nucleic acid sequence encoding the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. The plant according to item 24.
30. The Golgi apparatus targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). Or the plant according to item 24, which has the amino acid sequence of a fragment within positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
31. Item 25. The plant according to Item 24, wherein the Golgi apparatus targeting sequence has an amino acid sequence that is 80% identical to the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
32. Item 2. The plant according to Item 1, wherein the polyprenyl diphosphate synthase gene is operably linked to a seed-specific promoter.
33. Item 1. The plant is selected from the group consisting of plant cells, plant cultured cells, plant seeds, regenerated plant bodies, plant callus, plant tissue, leaves, stems, roots, flowers, seedlings, algae, and moss. Plant.
34. The plant according to item 1, which is a monocotyledonous plant.
35. The plant according to item 1, which is a dicotyledonous plant.
36. 36. The seed of a plant according to any one of items 1, 32, 34, or 35.
37. 36. The plant according to item 35, wherein the dicotyledonous plant is selected from the group consisting of rice, corn, oats, wheat, barley, buckwheat, judodama, oats, Indian millet, banana, and sugar cane.
38. The plant according to item 36, wherein the monocotyledonous plant is selected from the group consisting of soybean, tomato, potato, sweet potato, almond, pistachio nut, peanut, hazelnut, walnut, cashew nut, and sesame.
39. An expression cassette for expressing polyprenyl diphosphate synthase in plants.
40. 40. An expression cassette according to item 39, wherein the polyprenyl diphosphate synthase is decaprenyl diphosphate synthase.
41. 40. The expression cassette according to item 39, wherein the decaprenyl diphosphate synthase is a bacterial enzyme.
42. Item 42. The expression cassette according to Item 41, wherein the decaprenyl diphosphate synthase is an enzyme derived from Gluconobactersuboxydans.
43. 41. The expression cassette according to item 40, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid that hybridizes with the nucleic acid shown in SEQ ID NO: 5 under stringent conditions.
44. The decaprenyl diphosphate synthase is encoded by a nucleic acid having one or several deletions, additions or substitutions in the nucleic acid sequence shown in SEQ ID NO: 5, or a fragment of the nucleic acid sequence shown in SEQ ID NO: 5 The expression cassette described in 1.
45. 41. An expression cassette according to item 40, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having 80% higher sequence identity with the nucleic acid sequence shown in SEQ ID NO: 5.
46. The decaprenyl diphosphate synthase has an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence of a fragment in the sequence shown in SEQ ID NO: 6. 41. An expression cassette according to item 40.
47. 41. The expression cassette according to item 40, wherein the decaprenyl diphosphate synthase has an amino acid sequence identity higher than 80% with the amino acid sequence shown in SEQ ID NO: 6.
48. 41. The expression cassette of item 40, wherein the nucleic acid encoding decaprenyl diphosphate synthase is operably linked to a nucleic acid encoding a mitochondrial targeting sequence.
49. The nucleic acid encoding the mitochondrial targeting sequence is a nucleic acid encoding a fragment of rice RPS10 protein (SEQ ID NO: 2), a nucleic acid encoding a fragment of RPS14 protein (SEQ ID NO: 4), RPS11 protein, ATPase β subunit protein 49. The expression cassette of item 48, selected from the group consisting of: and ATPase γ subunit protein.
50. 49. The expression cassette according to item 48, wherein the mitochondrial targeting sequence is a fragment consisting of an amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2).
51. 49. The expression according to item 48, wherein the mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). cassette.
52. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice 49. An expression cassette according to item 48, which is encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of the RPS10 protein (SEQ ID NO: 2).
53. 49. The item 48, wherein the mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding the amino acid sequence from positions 1 to 56 of the rice RPS10 protein (SEQ ID NO: 2). Expression cassette.
54. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice RPS10 protein (sequence). 49. An expression cassette according to item 48, which has the amino acid sequence of a fragment within positions 1 to 56 of number 2).
55. 49. The expression cassette of item 48, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2).
56. Item 49. The expression cassette according to Item 48, wherein the mitochondrial targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4).
57. 49. The expression according to item 48, wherein the mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). cassette.
58. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice 49. An expression cassette according to item 48, encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of the RPS14 protein (SEQ ID NO: 4).
59. 49. The item 48, wherein the mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity higher than 80% with a nucleic acid sequence encoding amino acids 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). Expression cassette.
60. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice RPS14 protein (sequence). 49. An expression cassette according to item 48, which has the amino acid sequence of a fragment within positions 1 to 48 of number 4).
61. 49. An expression cassette according to item 48, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4).
62. 40. The expression cassette of item 39, wherein the polyprenyl diphosphate synthase is operably linked to a Golgi targeting sequence.
63. 63. An expression cassette according to item 62, wherein the nucleic acid encoding the Golgi apparatus targeting sequence is a nucleic acid encoding a fragment of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
64. 63. The expression cassette according to Item 62, wherein the Golgi apparatus targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
65. The Golgi apparatus targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. The expression cassette of item 62.
66. The Golgi apparatus targeting sequence may be one or several deletions, additions, or additions to the nucleic acid sequence encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 65. Expression cassette according to item 62, encoded by a nucleic acid having a substitution, or a fragment of a nucleic acid sequence encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). .
67. The Golgi apparatus targeting sequence is encoded by a nucleic acid having a sequence that is 80% identical to the nucleic acid sequence encoding the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. The expression cassette of item 62.
68. The Golgi apparatus targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). Or an expression cassette according to item 62, which has the amino acid sequence of a fragment within positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8).
69. 63. An expression cassette according to item 62, wherein the Golgi apparatus targeting sequence has an amino acid sequence 80% identical to amino acid sequences 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). .
70. 40. The expression cassette of item 39, wherein the polyprenyl diphosphate synthase gene is operably linked to a seed specific promoter.
71. 71. A plant comprising the expression cassette according to item 70.
72. 72. Seed of plant according to item 71.
73. A method for producing ubiquinone-10 using a plant, the method comprising:
(1) obtaining the plant described in item 1; and (2) culturing the plant;
Including.
74. A method for producing ubiquinone-10 using a plant, the method comprising:
(1) introducing the expression cassette according to item 37 into a plant;
(2) culturing a plant into which the expression cassette has been introduced;
Including.
75. A plant tissue containing ubiquinone-10 obtained from the plant according to item 1.
76. A plant cell containing ubiquinone-10 obtained from the plant according to item 1.
77. A food comprising the plant-derived ubiquinone-10 according to item 1.
78. Furthermore, the foodstuff of item 77 containing the plant-derived component of item 1.
79. Item 79. The food according to Item 78, wherein the plant-derived component according to Item 1 is a protein.
80. A composition comprising the plant-derived ubiquinone-10 according to item 1.
81. The composition according to item 80, further comprising a plant-derived component according to item 1.
82. 82. The composition according to item 81, wherein the plant-derived component according to item 1 is a protein.
83. A dietary supplement comprising the plant-derived ubiquinone-10 according to item 1.
84. 84. A dietary supplement according to item 83, further comprising a plant-derived component according to item 1.
85. 85. The dietary supplement according to item 84, wherein the plant-derived component according to item 1 is a protein.
86. A cosmetic comprising the plant-derived ubiquinone-10 according to item 1.
87. The cosmetic according to Item 86, further comprising a plant-derived component according to Item 1.
88. Item 88. The cosmetic according to Item 87, wherein the plant-derived component according to Item 1 is a protein.
89. A beverage comprising the plant-derived ubiquinone-10 according to item 1.
90. 90. A beverage according to item 89, further comprising a plant-derived component according to item 1.
91. Item 91. The beverage according to Item 90, wherein the plant-derived component according to Item 1 is a protein.
92. A leaf comprising the plant-derived ubiquinone-10 according to item 1.
93. An embryo comprising the plant-derived ubiquinone-10 according to item 1.
94. A fruit comprising the plant-derived ubiquinone-10 according to item 1.
95. A stem comprising the plant-derived ubiquinone-10 according to item 1.
96. A root comprising the plant-derived ubiquinone-10 according to item 1.
97. A flower comprising the plant-derived ubiquinone-10 according to item 1.
98. A seed comprising the plant-derived ubiquinone-10 according to item 1.
99. Rice bran comprising the plant-derived ubiquinone-10 according to item 1.
100. A rice cell or tissue comprising ubiquinone-10.
101. 100. The cell or tissue according to item 100, which is a seed.
102. 100. The cell or tissue according to item 100, which is brown rice.

  According to the present invention, there are provided methods for producing ubiquinone-10 using plants, recombinant plants used in such production methods, and expression cassettes used to produce such recombinant plants. In addition, compositions, dietary supplements, supplements, and pharmaceutical compositions comprising ubiquinone-10 produced by the plants of the present invention are also provided.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  Listed below are definitions of terms particularly used in the present specification.

  As used herein, “plant” is a general term for organisms belonging to the plant kingdom, characterized by chlorophyll, hard cell walls, the presence of abundant and permanent embryonic tissue, and organisms that are not capable of motility. . Typically, a plant refers to a flowering plant having cell wall formation and anabolism by chlorophyll. “Plant” includes monocotyledonous and dicotyledonous plants, as well as algae, ferns and mosses. Monocotyledonous plants include gramineous plants. Preferred monocotyledonous plants include corn, sugarcane, wheat, rice, oat, barley, sorghum, corn, oats, rye, Indian millet, banana and millet, more preferably corn, wheat, rice and sugarcane. However, it is not limited to these. Examples of dicotyledonous plants include cruciferous plants, legumes, eggplants, cucurbits and convolvulaceae, but are not limited thereto. Examples of dicotyledonous plants include, but are not limited to, soybean, tomato, potato, sweet potato, almond, pistachio nut, peanut, hazelnut, walnut, cashew nut, and sesame. Unless otherwise indicated, a plant means any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include fruits, seeds, roots, leaves, stems, flowers, and embryos. Examples of plant cells include callus and suspension culture cells. When using rice, rice bran or embryos of rice can also be used as a source of recombinantly produced ubiquinone-10. In certain embodiments, a plant can refer to a plant individual.

  Examples of gramineous plants include plants belonging to Oryza, Triticum, Hordeum, Secale, Saccharum, Sorghum, or Zea, including, for example, rice, wheat, barley, rye, sugar cane, sorghum, corn, and the like.

  As used herein, the term “modified plant” refers to a plant in which at least a part of the structure and / or function of genomic information is changed as compared with a naturally occurring plant. Such a modified plant can be produced, for example, by transformation of a wild type plant, crossing with a plant obtained by transformation, suppression of gene expression by antisense nucleic acid and suppression of gene expression by RNA interference, etc. The method for producing the modified plant is not limited to these.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the DNA is replaced with 2'-O-propylribose Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)). The term “nucleic acid” is also used herein interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”. As used herein, “homology” of genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.

  As used herein, the term “highly stringent conditions” refers to allowing hybridization of DNA strands that are highly complementary in sequence and excluding significantly mismatched DNA hybridization. Refers to the conditions designed. Hybridization stringency is primarily determined by temperature, ionic strength, and concentration of denaturing agents (eg, formamide). Examples of “highly stringent conditions” for hybridization and washing are 0.015 M sodium chloride at 65-68 ° C., 0.0015 M sodium citrate or 0.015 M sodium chloride at 42 ° C., 0.0015 M Sodium citrate and 50% formamide. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory, 1989); Anderson et al., Nucleic Acid Hybrid.

  More stringent conditions (eg, higher temperature, lower ionic strength, higher formamide or other denaturing agents) can also be used, but the rate of hybridization will be affected. Other agents may be included in the hybridization and wash buffers for the purpose of reducing non-specific and / or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, although other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach Chapter 4 (IRL Press Limited).

Factors affecting the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C.) = 81.5 + 16.6 (log [Na +]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na +] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is (guanine in the hybrid + Percent cytosine) base. For incompletely matched hybrids, the melting temperature is lowered by about 1 ° C. for every 1% mismatch.

  The term “moderately stringent conditions” refers to conditions under which DNA duplexes with a higher degree of base pair mismatch can be formed than can occur under “highly stringent conditions”. Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride at 50-65 ° C., 0.0015 M sodium citrate or 0.015 M sodium chloride at 37-50 ° C., 0.0015 M Sodium citrate and 20% formamide. Illustratively, “moderately stringent conditions” of 50 ° C. in 0.015 M sodium ion allows for a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there is no absolute distinction between “highly stringent conditions” and “moderately stringent conditions”. For example, with 0.015M sodium ion (no formamide), the melting temperature of a perfectly matched length of DNA is about 71 ° C. Using a wash at 65 ° C. (with the same ionic strength), this allows a mismatch of about 6%. To capture farther related sequences, one skilled in the art can simply lower the temperature or increase the ionic strength.

A good evaluation of the melting temperature in 1 M NaCl * for oligonucleotide probes up to about 20 nt is given by:
Tm = 2 ° C per AT base pair + 4 ° C per GC base pair
* 6 × Sodium ion concentration in salt sodium citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using Purified Genes 683 (Brown and Fox, 1981).

  High stringency wash conditions for an oligonucleotide are usually at a temperature between 0 ° C. and 5 ° C. below the Tm of that oligonucleotide in 6 × SSC, 0.1% SDS.

  In the present specification, comparison of base sequence identity and calculation of homology are calculated using default parameters using the BLAST program which is a sequence analysis tool.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA can also be an aspect of expression. More preferably, such a polypeptide form may be post-translationally processed.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to in this specification are in L form. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

  In the present specification, a “corresponding” amino acid has or is predicted to have the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison in a protein molecule or polypeptide molecule. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity.

  In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.

  Targeting a desired protein to the mitochondria in a plant can, for example, operate a nucleic acid encoding a mitochondrial targeting sequence from any mitochondrial protein (eg, a plant mitochondrial protein) with a nucleic acid encoding the desired protein. This is possible by connecting them as possible.

  As used herein, “mitochondrial targeting sequence” refers to a sequence that transports an operably linked protein to the mitochondria. As used herein, “mitochondrial targeting ability” refers to the ability to transport operably linked proteins to the mitochondria. Examples of mitochondrial targeting sequences include, but are not limited to, mitochondrial targeting sequences of mitochondrial ribosomal proteins and ATP synthase subunits. Examples of the mitochondrial ribosomal protein as a source of targeting sequences to mitochondria in the present invention include rice RPS10 protein (SEQ ID NO: 2) (FIG. 1), RPS14 protein (SEQ ID NO: 4) (FIG. 2), and RPS11. Examples include, but are not limited to, mitochondrial targeting sequences derived from proteins, ATPase β subunit proteins, and ATPase γ subunit proteins. As a mitochondrial targeting sequence of RPS10 protein, for example, a polypeptide comprising an amino acid sequence at positions 1 to 56 can be used. A polypeptide having one or several amino acid substitutions, additions or deletions in these mitochondrial targeting sequences and having mitochondrial targeting ability can also be used in the present invention. In addition, fragments of these mitochondrial targeting sequences can also be used. Nucleic acids that hybridize under stringent conditions with nucleic acids encoding these mitochondrial targeting sequences can also be used. Furthermore, sequences having one or several deletions, additions and substitutions in these amino acid sequences and / or nucleic acid sequences can also be used. Sequences homologous to these sequences can also be used. As the mitochondrial targeting sequence of RPS14 protein, for example, a polypeptide comprising an amino acid sequence at positions 1 to 48 can be used. A polypeptide having one or several amino acid substitutions, additions or deletions in these mitochondrial targeting sequences and having mitochondrial targeting ability can also be used in the present invention. In addition, fragments of these mitochondrial targeting sequences can also be used. Nucleic acids that hybridize under stringent conditions with nucleic acids encoding these mitochondrial targeting sequences can also be used. Furthermore, sequences having one or several deletions, additions and substitutions in these amino acid sequences and / or nucleic acid sequences can also be used. Sequences homologous to these sequences can also be used.

  Targeting a desired protein to the Golgi apparatus in a plant, for example, makes a nucleic acid encoding a Golgi targeting sequence from any Golgi protein in the plant operable with a nucleic acid encoding the desired protein. Is possible.

  As used herein, a “Golgi apparatus targeting sequence” refers to a sequence that transports an operably linked protein to the Golgi apparatus. As used herein, “Golgi apparatus targeting ability” refers to the ability to transport operably linked proteins to the Golgi apparatus. Examples of the Golgi apparatus targeting sequence include, but are not limited to, the Golgi apparatus targeting sequence of N-acetylglucosaminyltransferase protein. Examples of the Golgi apparatus protein as a source of the Golgi apparatus targeting sequence in the present invention include a mitochondrial targeting sequence derived from tobacco N-acetylglucosaminyltransferase protein (SEQ ID NO: 8) (FIG. 3). However, it is not limited to these. As the Golgi targeting sequence of tobacco N-acetylglucosaminyltransferase, for example, a polypeptide comprising an amino acid sequence at positions 1 to 77 can be used. A polypeptide having one or several amino acid substitutions, additions or deletions in these mitochondrial targeting sequences and having the ability to target the Golgi apparatus can also be used in the present invention. Furthermore, fragments of these Golgi targeting sequences can also be used. In addition, nucleic acids that hybridize under stringent conditions with nucleic acids encoding these Golgi apparatus targeting sequences can also be used. Furthermore, sequences having one or several deletions, additions and substitutions in these amino acid sequences and / or nucleic acid sequences can also be used. Sequences homologous to these sequences can also be used.

Examples of the decaprenyl diphosphate synthase that can synthesize 10 units of a ubiquinone side chain derived from bacteria include, but are not limited to, an enzyme encoded by a gene derived from Gluconobacter suboxydans (SEQ ID NO: 5). Polypeptides that have one or several amino acid substitutions, additions, or deletions in the amino acid sequence of these decaprenyl diphosphate synthases and that have enzyme activity can also be used in the present invention. is there. Furthermore, fragments of the amino acid sequences of these enzymes can also be used. In addition, nucleic acids that hybridize under stringent conditions with nucleic acids encoding these enzyme proteins can also be used. Furthermore, sequences having one or several deletions, additions and substitutions in these amino acid sequences and / or nucleic acid sequences can also be used. Sequences homologous to these sequences can also be used. The terms “transformation” and “gene transfer” are used interchangeably herein. “Transformation” means that a genotype of a plant cell or plant tissue is changed by introducing a foreign nucleic acid containing a gene into the plant cell or plant tissue.

  “Transformant” refers to all or part of an organism such as a cell produced by transforming a host cell. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, transformed callus, transformed plant, etc., depending on the subject, and includes all of these forms herein. May refer to a particular form in a particular context.

  In the present specification, “transgenic plant” and “recombinant plant” refer to a plant into which a specific gene is incorporated.

  In the present specification, plant cultivation can be performed by any method known in the art. Examples of plant cultivation methods include, for example, Experimental Protocols for Model Plants—Edited by Rice and Arabidopsis- ”: Cell Engineering Separate Volume Plant Cell Engineering Series 4; Rice Cultivation Method (Member Toshi Okuno) pp. 28-32, and cultivation method of Arabidopsis (Yasuo Niwa) pp. 33-40 (supervised by Isao Shimamoto, Kiyotaka Okada), and those skilled in the art can easily carry out the invention, and therefore need not be described in detail herein.

  The host plant cell for obtaining the transformant is not particularly limited as long as it expresses a polypeptide that retains the physiological activity of the foreign gene (for example, decaprenyl diphosphate synthase). Various host plant cells used can be used. For example, rice, cucumber, sugar cane, oats, corn, wheat, barley, buckwheat, juzudama, oats, barnyard grass, soybeans, almonds, pistachio nuts, peanuts, hazelnuts, walnuts, cashew nuts, sesame seeds, Indian millet, bananas, tomatoes, Examples include potato, sweet potato, cyanobacterium, chlorella and sugar cane. Preferred plant cells are plants that produce ubiquinone-9 but not ubiquinone-10. Examples of such plants include, but are not limited to, wheat, barley, buckwheat, juzudama, oats, and barnyard grass. In addition, plants that mainly express ubiquinone-9 and produce a relatively small amount of ubiquinone-10 and plants that mainly express ubiquinone-10 and simultaneously produce a relatively small amount of ubiquinone-9 are also objects of the present invention. be able to. This is because it is possible to enhance the production of ubiquinone-10 by supplementing exogenous decaprenyl diphosphate synthase. Examples of such plants include, but are not limited to, leek, bell pepper, soybean, almond, pistachio nut, peanut, hazelnut, walnut, cashew nut, and sesame.

  As long as the polypeptide derived from the cell obtained in the present invention has substantially the same action as the naturally occurring polypeptide, one or more amino acids in the amino acid sequence are substituted, added and / or deleted. Alternatively, the sugar chain may be substituted, added and / or deleted.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, the binding site of a ligand molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in ligand binding capacity). It is well known. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, orthologs and paralogs may also be useful in the present invention.

  “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  A nucleic acid encoding such a protein can be obtained by a well-known PCR method, or can be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number is a function (for example, cancer marker, neurological disease) in a variant having the substitution, addition or deletion. As long as the marker etc.) is retained. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  General molecular biology techniques that can be utilized in the present invention include Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, etc. can be easily implemented by those skilled in the art.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Examples of such vectors include those containing a promoter at a position suitable for transcription of the polynucleotide of the present invention, which is capable of autonomous replication in a host cell.

  “Expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. The regulatory element can preferably include a terminator and a selectable marker. It is well known to those skilled in the art that the type of expression vector and the type of regulatory element used can vary depending on the host plant cell.

  “Expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. The regulatory element may preferably include a terminator. It is well known to those skilled in the art that the type of regulatory element used in the expression cassette can vary depending on the host plant cell.

  “Recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence into a target cell. Examples of such vectors include those that are capable of autonomous replication in prokaryotic host cells and that contain a promoter at a position suitable for transcription of the polynucleotide of the present invention.

  Examples of the “recombinant vector” for plant cells include vectors derived from vectors such as Ti plasmid, Ri plasmid, tobacco mosaic virus vector, and geminivirus vector. As long as the direct introduction method such as the particle gun method or electroporation is used, these vectors need not be used.

  As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription when RNA polymerase binds. is there. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene.

  A “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene. Examples of the terminator include, but are not limited to, CaMV35S terminator, terminator of nopaline synthase gene (Tnos), and terminator of tobacco PR1a gene. As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription when RNA polymerase binds. is there. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region is usually upstream of the structural gene, but preferably the putative promoter region is within about 2 kbp upstream from the first exon translation start point.

  In the present specification, when used for expression of a gene, “site specificity” generally means a part of an organism (eg, a plant) (eg, in the case of a plant, a root, a stem, a stem, a leaf, a flower, a seed, Germ, embryo, fruit, etc.) the specificity of the gene expression. “Time specificity” refers to the specificity of expression of a gene according to the stage of development of an organism (for example, a plant) (for example, the growth stage for a plant and the number of days of germination after germination). Such specificity can be introduced into a desired organism by selecting an appropriate promoter.

  In the present specification, the expression of the promoter of the present invention is “constitutive” means that in almost all tissues of an organism, an almost constant amount regardless of whether the organism is growing or juvenile or mature. The property expressed in Specifically, when Northern blot analysis is performed under the same conditions as in the examples of the present specification, for example, the same or corresponding at any time point (for example, 2 points or more (for example, 5th day and 15th day)) Expression is said to be constitutive by definition of the present invention when almost the same level of expression is observed at any of the sites. Constitutive promoters are thought to play a role in maintaining homeostasis of organisms in normal growth environments. The expression of the promoter of the present invention being “stress responsive” refers to the property that the expression level changes when at least one stress is applied to an organism. In particular, the property of increasing the expression level is called “stress inducibility”, and the property of decreasing the expression level is called “stress suppression”. The expression of “stress suppression” is a concept that overlaps with “constitutive” expression because it is premised on that expression is observed in a normal state. These properties can be determined by extracting RNA from any part of the organism and analyzing the expression level by Northern blot analysis or quantifying the expressed protein by Western blot. Plants or plant parts (specific cells, tissues, etc.) transformed with a vector incorporating a stress-inducible promoter together with a nucleic acid encoding the polypeptide of the present invention use a stimulating factor having inducible activity of the promoter. Thus, a specific gene can be expressed under certain conditions.

  An “enhancer” can be used to increase the expression efficiency of a target gene. When used in plants, an example of an enhancer is an enhancer region containing an upstream sequence in the CaMV35S promoter. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but there may be a sequence intervening between the promoter and the structural gene. It does not necessarily have to be placed adjacent to the structural gene. It should be noted that the targeting sequence for the organelle is arranged such that the reading frame of the gene encoding the protein is in frame.

  The presence of the introduced gene can be confirmed by a PCR method, a Southern blot method or the like. Expression of the introduced gene can be detected by Northern blotting or RT-PCR. If necessary, the expression of a protein that is a gene product can be confirmed by, for example, Western blotting.

(General techniques used in this specification)
Unless otherwise specifically indicated, the techniques used herein are within the scope of the art, unless otherwise indicated, glycoscience, microfluidics, microfabrication, organic chemistry, biochemistry, genetic engineering, Well-known conventional techniques in molecular biology, microbiology, genetics and related fields are used. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein.

  Molecular biological techniques, biochemical techniques, microbiological techniques, and glycoscience techniques used in this specification are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J .; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approc, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Experimental Medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, etc., which are described in this specification Related parts (which may be all) are incorporated by reference.

(Meal supplement)
Ubiquinone-10 of the present invention can be used as a dietary supplement (diet food). Ubiquinone-10 as a dietary supplement is an antioxidant, skin aging, immunodeficiency, heart disease, periodontal disease, chronic fatigue syndrome, degenerative brain, neuromotor dysfunction, diabetes, stroke, arteriosclerosis, cancer, It is expected to have an effect on hypertension, hypotension, cerebral infarction, allergic diseases and the like. In addition, the effects of improving congestive heart failure, palpitation / shortness of breath, swelling of legs and face, and coldness as effects of enhancing the ability of the heart are also predicted.

  When used as a dietary supplement for healthy individuals, the standard is about 30 mg / day. When used for patients with heart disease, about 100 mg / day or more is usually used. However, the amount can vary depending on the condition of the individual taking the dietary supplement.

  When ubiquinone-10 is used as a dietary supplement, purified ubiquinone-10 is fat-soluble, so it may be used after being dissolved in, for example, soybean oil. Or you may enclose the powdered ubiquinone-10 in a capsule. Furthermore, you may add and use for a drink. When the ubiquinone-10 of the present invention is used as a dietary supplement, a vitamin such as vitamin E may be added as necessary.

  For example, ubiquinone-10 dietary supplement can be prepared, for example, by the following formulation, but is not limited to: purified fish oil containing DHA, soy lecithin, beeswax, vitamin E (nutrient requirements) 302%) as a ratio, and tablets can be prepared using gelatin, glycerin, and caramel color as an encapsulating agent, and used as a dietary supplement.

  As another example, ubiquinone-10 dietary supplement can be prepared by, for example, the following formulation, but is not limited thereto: lactose, emulsifier, soybean extract, hawthorn extract, Paste, fine silicon dioxide, niacinamide, vitamin E, glycerin, colorant (titanium dioxide), vitamin B6, vitamin B2, carotene pigment and gelatin.

(Prescription)
The present invention also provides a method of treating and / or preventing a disease or disorder (eg, heart disease) by administering an effective amount of a therapeutic agent to a subject. By therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).

  The therapeutic agent is considered to be a medical practice standard (GMP = good) taking into account the clinical condition of the individual patient (especially the side effects of the therapeutic agent alone treatment), the site of delivery, the method of administration, the dosage regimen and other factors known to those skilled in the art. Formulate and dose in a manner consistent with medical practice. Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

  The therapeutic agent can be administered orally. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant.

  Generally, formulations are prepared by contacting the therapeutic agent uniformly and intimately with liquid carriers or finely divided solid carriers or both. Next, if necessary, the product is shaped into the desired formulation. As examples of such carrier vehicles, non-aqueous vehicles such as soybean oil and ethyl oleate are useful herein.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration Represents institutional approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.

  The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. The combinations can be administered, for example, either simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual . Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.

(Ubiquinone-10 intake)
Ubiquinone-10 of the present invention can be used in prevention, treatment and prognosis of various diseases and conditions.

  Examples of the diseases and conditions for which ubiquinone-10 of the present invention can be used include prevention and improvement of Alzheimer Parkinson's disease and Huntington's disease. Because many of the diseases related to the brain are thought to be caused by the fact that active oxygen damages nerve tissue, ubiquinone-10 plays a role in protecting mitochondria in brain cells. This is because it is thought to have a function of preventing brain diseases.

  The ubiquinone-10 of the present invention can be used for cancer prevention / improvement. Ubiquinone-10 levels in cancer patients are known to be very low. Therefore, there are many test results of applying ubiquinone-10 to cancer treatment. There is a report that ubiquinone-10 administration stopped cancer progression and alleviated symptoms. In some cases, the tumor disappeared completely after 2 months in the case of patients who received ubiquinone-10 at an increased dose of 390 mg per day. It is also actually used in medical practice.

  Ubiquinone-10 of the present invention can also be used as an anti-aging agent. Because ubiquinone-10 is a powerful antioxidant, anti-aging effects by preventing cell oxidation are expected. In fact, there is an experimental result that 30% of mice administered with ubiquinone-10 survived about 2 months longer than mice not administered.

  The ubiquinone-10 of the present invention can also be used to reduce wrinkles. In fact, the effect of improving wrinkles by applying to the skin has been confirmed.

  The ubiquinone-10 of the present invention can be used to improve obesity. Ubiquinone-10 is known to increase metabolic rate and contribute to weight loss. In some obese patients, ubiquinone-10 in the body is deficient and supplementation has been found to increase the burning rate of calories. In an experiment in which 100 mg of ubiquinone-10 was ingested for 8 to 9 weeks in obese patients, the weight loss of ubiquinone-10 deficient was more than twice that of normal ubiquinone-10 levels. Has been reported.

  The ubiquinone-10 of the present invention can also be used to improve chronic fatigue syndrome. Chronic fatigue syndrome does not get tired and causes symptoms such as insomnia, slight fever, and migraine. These symptoms are thought to be caused by some sort of failure in the energy production system in mitochondria. In fact, improvement in exercise tolerance and recovery time has been reported by administration of ubiquinone-10.

  Furthermore, ubiquinone-10 of the present invention is thought to be useful for strengthening the immune system, improving gingivitis / periodontal disease, postponing the onset of AIDS, preventing arteriosclerosis, and improving diabetes.

  In various diseases and conditions, ubiquinone-10 dietary supplements can be taken with reference to the following indicators:

  Whether or not ubiquinone-10 is deficient can be determined by collecting blood. In general, the average plasma ubiquinone-10 level in healthy individuals is 0.8 μg / ml. If it is lower than this value, it may be necessary to supplement ubiquinone-10. The appropriate dose depends on its form (capsule, oil-based soft capsule, etc.) when taken as a health food. It also depends on the deficiency level of the intake, dietary habits, various other factors, and symptoms / purposes. About 30 to 300 mg / day is generally used as a daily intake as a dietary supplement. For example, about 30 to about 60 mg / day when used for general health maintenance and anti-aging, about 60 to about 90 mg for improving endurance and physical strength, and about 60 when there is a sign of a specific disease / symptom. About 150 mg, and about 90 mg or more is a guideline for extreme exercise, but is not limited thereto.

  In addition, for individuals with specific diseases / symptoms, the following dosage is a guideline: bronchial asthma 30 mg / day; cirrhosis 100 mg / day; diabetes 100 mg / day; periodontal disease 100 mg / day; fatigue 300 mg / day; High cholesterol 300 mg / day; hypertension 75-360 mg / day; lung cancer 90-390 mg / day; heart disease 75-600 mg / day; and Parkinson's disease 400-800 mg / day. It should be noted that the treatment period required to observe the change and the post-treatment interval at which a response occurs can vary depending on the desired effect.

(Transformation)
In order to carry out transformation, generally, a method of directly introducing a gene into a plant (direct gene transfer method) or a method of indirectly transferring a gene into a plant (indirect gene transfer method) Is done.

  To date, as an indirect gene transfer method, a method using Agrobacterium has been widely used. For example, a method of infecting callus obtained after 3 weeks of culturing mature rice seeds with Agrobacterium (see Hiei et al., Plant Journal, 6: 271-282, 1994) or 4-5 after germination A method (see Tanaka et al., Japanese Patent No. 3141084) capable of rapidly obtaining a transformant by infecting sun seeds with Agrobacterium can be mentioned.

  On the other hand, as direct gene transfer methods, particle gun method (Christou, P. et al., Bio / Technology, 9: 957-962, 1991), polyethylene glycol method (Datta, SK et al., Bio / Technology, 8: 736-740, 1990) and electroporation (see Shimamoto, K. et al. Nature, 338: 274-276, 1989) and the like have been used for the production of transformants. Electroporation refers to a method of physically making a small hole in a plant cell using a direct current high voltage pulse and then introducing a gene into the cell from there. In addition, immature embryos are used for gene introduction into wheat (see JT Weeks et al., Plant. Physiol. 102: 1077-1084, 1993).

(Site-specific production of ubiquinone-10)
The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

(Example 1: Construction of decaprenyl diphosphate synthase gene (ddsA) linked to mitochondrial targeting sequence or Golgi targeting sequence)
A plasmid was constructed in which the ddsA gene having the nucleic acid sequence of SEQ ID NO: 5 was linked to a mitochondrial targeting sequence or a Golgi targeting sequence. As the mitochondrial targeting sequence, S14, which is a mitochondrial targeting sequence derived from RPS14 of SEQ ID NO: 4 (Kubo et al., Plant Science 164 (2003) 1047-1055; corresponding to amino acid residues 1 to 48 of SEQ ID NO: 4) Was used. The Golgi targeting sequence includes CTS (Essl et al., FEBS letters, 453 (1999) 169), which is a Golgi targeting signal at the N-terminus of tobacco N-acetylglucosaminyltransferase I (GnT1) of SEQ ID NO: 8. ˜173; 1 to 77 of SEQ ID NO: 8 were used. These targeting sequences are operably linked to the decaprenyl diphosphate synthase gene (ddsA) from Gluconobacter suboxydans (Okada et al. (Eur. J. Biochem. 255, 52-59 (1998))) and constitutive promoters The pUS14ddsA plasmid and the pUCTddsA plasmid were constructed by ligating between the CaMV35S promoter and the NOS terminator. The HindIII / EcoRI fragment of each plasmid was ligated to the multiple cloning site of pCAMBIA1301, a binary vector. A schematic diagram of the various plasmids constructed is shown in FIG.
(Example 2: Confirmation of expression of ddsA gene)
Rice was transformed with the various plasmids prepared in Example 1. Specifically, Agrobacterium (Agrobacterium tumefaciens) EHA105 strain was transformed by electroporation, and Agrobacterium was used according to the transformation method described in WO01 / 06844 to transform a plant. The transformed rice was selected in a medium containing hygromycin (50 mg / L). The introduction of the ddsA gene into plant cells was confirmed by the PCR amplification experiment or Southern hybridization shown below (data not shown).

Rice genomic DNA (4 μg) was digested with HindIII, subjected to 0.9% agarose electrophoresis, and blotted on Hybond-N ⁺ membrane (Amarsham Pharmacia) on the principle of capillary (Sambrook, Fritsch and Maniatis, Molecular Cloning: AL). Manual (2nd edition, Cold Spring Harbor Laboratory, 1989)). A 0.3 kb fragment of the ddsA gene (5 ′ part of the coding region) was used as a probe. The labeling of the probe was performed according to the manual of the manufacturer, Roche. Hybridization and washing were performed as described in Sambrook (supra) and subsequent reactions were performed according to the instructions of Roche. The final wash was performed at 68 ° C. using 0.1 × SSC and 0.1% SDS. As a result, a clone in which the target plasmid was incorporated into the chromosomal DNA was found, and the clone was used in the subsequent experiments.
(Example 3: Preparation of anti-G. Saboxydans DDSA antibody)
The entire coding region of the ddsA gene was amplified by PCR and ligated into the pGEX4T-3 vector (Amersham Pharmacia). E. coli JM109 was transformed. Expression of the GST / DDSA fusion protein was induced by the addition of 1 mM IPTG and purified according to the method of Asano et al. (Plant Cell (2002) 14, 619-628). The purified fusion protein was injected into rabbits 6 times at 2 week intervals. As a result, anti-G. saboxydans DDSA antibody was confirmed.
(Example 4: Confirmation of DDSA protein expression)
To the leaves of the recombinant plant, an extraction buffer solution (50 mM Tris-HCl, pH 6.8, 2% SDS, 6% 2-mercaptoethanol, 10% glycerol) having a volume 8 times the weight of the leaves is added to obtain a fine powder. Crushed into. The suspension was centrifuged at 12,000 rpm for 5 minutes at 4 ° C. and the supernatant was collected. 10 μg of total protein from each transformant was separated on a 12.5% polyacrylamide gel and transferred to an Immobilon PVDF membrane (Millopore).

  Western blotting was performed using the anti-DDSA antibody prepared by the method of Example 3. Lane 1 in FIG. 7 is a non-transformed plant-derived protein, lanes 2 to 4 are plant-derived proteins transformed with a plasmid expressing DDSA fused with S14, and lanes 5 to 7 are A plant-derived protein transformed with a plasmid expressing DDSA fused with CTS. As shown in FIG. 7, the expression of each fusion protein was confirmed.

Depending on the expression level of the protein, four groups of transformed plants expressing the S14-DDSA fusion protein (±; very low expression level, L; low expression level, M; moderate expression level, H; high expression level) ). ± was 15 individuals, L was 8 individuals, M was 9 individuals, and H was 9 individuals.
(Example 5: Extraction and measurement of ubiquinone)
Ubiquinone was extracted according to a method slightly modified from the method of Okada et al. Rice leaves were crushed in a mortar while adding liquid nitrogen, and 1.0 g was weighed into a 15 ml tube. After adding 2 ml of 3% H ₂ SO ₄ and stirring with a vortex mixer for several seconds, the cap was removed and the lid was covered with aluminum foil. Autoclave was performed at 120 ° C. for 30 minutes. 4 ml of 14% NaOH was added. Next, an autoclave was performed at 120 ° C. for 15 minutes. Immediately cooled in ice water. 3 ml of hexane: isopropanol (5: 1) was added and stirred with a vortex mixer for 1 minute. Centrifugation was performed at 2000 rpm at room temperature for 2 minutes. 1.5 ml of the supernatant was dispensed into a 2 ml tube. While heating, vacuum drying was performed for 10 minutes. The dried product was suspended in 20 μl of chloroform: methanol (2: 1) and spotted on a TLC plate. 5 μl of ubiquinone-10 standard (1 mg / ml) was also spotted on another plate.

  TLC development was performed for about 1 hour and 20 minutes using 100% benzene as a solvent. The portion at the same position as the standard ubiquinone-10 spot was cut out with a cutter by ultraviolet irradiation, and placed in a 1.5 ml tube. Hexane 200 μl and isopropanol 200 μl were added, and the mixture was stirred with a vortex mixer for 1 minute. The mixture was centrifuged at 13,000 rpm and room temperature for 1 minute, and the supernatant was transferred to a 1.5 ml tube. The solid was removed by vacuum drying for 10 minutes, suspending in 200 μl of degassed 100% ethanol and filtering through a filter. The suspension was analyzed by HPLC. Analysis of HPLC was performed by using ethanol as a solvent and using a C18 reverse phase column (YMC-Pack ODS-A; 150 mm × 60 mm) (flow rate = 1 ml / min, detection at 275 nm).

  As a result, as shown in FIG. 8, in the untransformed rice (FIG. 8A), there are many ubiquinone-9 and few ubiquinone-10, whereas transformed plants expressing S14: ddsA (FIG. 8). In 8B), almost no ubiquinone-9 was observed, and a large amount of ubiquinone-10 was observed. This indicates that ubiquinone-10 can be produced in large quantities in plants by ligating a decaprenyl diphosphate synthase gene to a mitochondrial targeting sequence and expressing it in plants. Ubiquinone-6 was added as an internal standard for measurement.

To confirm the molecular weight of each ubiquinone, a liquid chromatography-MS (LC-MS) using an LCT LC-MS system (Micromass) with an atmospheric pressure chemical ionization (APCI) source was run at 1.0 ml / min. Performed at a flow rate [Inertsil ODS3 column (GL Sciences); 3 μm; 4.6 mm × 150 mm]. After injection onto a column equilibrated with mobile phase (methanol / isopropanol (3: 1, v / v)), the column was eluted for 30 minutes with a constant composition mobile phase. The molecular weight of coenzyme Q eluted from 8.5 minutes in FIG. 8A and the molecular weight of ubiquinone eluted from 10.2 minutes in FIG. 8B were measured by LC-MS and are shown in FIGS. 9A and 9B, respectively. These charts demonstrate that the 8.5 minute peak is ubiquinone 9 [m / z 818 (M + Na) ⁺ ] and the 10.2 minute peak is ubiquinone 10 [m / z 886 (M + Na) ⁺ ]. .

Recombinant plants expressing ddsA produced ubiquinone 10 (FIG. 10). When ubiquinone (oxidized form) is reduced, it is converted to ubiquinol (reduced form), and as a result, ultraviolet absorption at 275 nm disappears. Therefore, the ultraviolet absorption peak at 275 nm that disappears upon reduction indicates the presence of ubiquinone.
(Example 6: Extraction and measurement of ubiquinone-10 from brown rice)
A progeny of rice produced in Example 2 was prepared, and brown rice was obtained from the progeny.

  Using the rice brown rice, ubiquinone was extracted and measured according to the method described in Example 5. The result is shown in FIG.

  FIG. 11 shows the results of production amounts of ubiquinone-9 and ubiquinone-10 in untransformed plants and brown rice of recombinant plants expressing S14-DDSA. Ubiquinone-6 was added during measurement as an internal standard.

  As is clear from this result, production of ubiquinone-10 was not confirmed in untransformed rice brown rice, but in rice brown rice expressing S14-DDSA, a larger amount than in ubiquinone-9 was observed. Of ubiquinone-10 was confirmed.

  This example demonstrates that the rice brown rice provided by the present invention is a good source of ubiquinone-10.

FIG. 1 shows the nucleic acid and amino acid sequences of the mitochondrial targeting sequence of rice RPS10. FIG. 2 shows the nucleic acid and amino acid sequences of the mitochondrial targeting sequence of rice RPS14. FIG. 3 shows the nucleic acid and amino acid sequences of the Golgi targeting sequence of tobacco N-acetylglucosaminyltransferase I. FIG. 4 shows the nucleic acid and amino acid sequences of the mitochondrial targeting sequence of the rice ATPase β subunit protein. FIG. 5 shows the nucleic acid and amino acid sequences of the mitochondrial targeting sequence of the Arabidopsis ATPase γ subunit protein. FIG. 6 is an expression cassette used for producing the recombinant plant of the present invention. In the figure, T35S is a terminator of CaMV35S gene; HygR is a hygromycin resistance gene; P35S is a CaMV35S promoter; Tnos is a terminator of nopaline synthase; Is the mitochondrial targeting sequence of RPS14 protein (amino acids 1 to 48); CTS is the Golgi targeting signal (positions 1 to 77) at the N-terminus of tobacco N-acetylglucosaminyltransferase I (GnT1). Promoter is the CaMV35S promoter; Intron-GUS is the GUS gene containing introns; LB is the left border sequence; RB is the right border sequence Show. FIG. 7 shows the results of detection of fusion protein expression shown in Example 4. FIG. 8 shows the production amounts of ubiquinone-9 and ubiquinone-10 in a non-transformed plant (A) and a plant (B) expressing S14-DDSA (a fusion protein of S14 and DDSA). is there. FIG. 9 shows the molecular weights of ubiquinone-9 (A) produced in wild type plants and ubiquinone-10 (B) produced in S14: ddsA plants. FIG. 10 is a comparison of ubiquinone production in recombinant plants expressing S14-DDSA at various expression levels. FIG. 11 shows that ubiquinone-10 is hardly detected from untransformed rice brown rice (upper left), but from the brown rice of rice expressing S14-DDSA (lower left, lower center is an enlarged view). This is a result showing that -10 was detected and ubiquinone 10 was produced.

SEQ ID NO: 1: Nucleic acid sequence (genomic sequence) of rice RPS10 gene.
SEQ ID NO: 2: Amino acid sequence of rice RPS10. The amino acid sequence from position 1 to position 56 is a mitochondrial targeting sequence.
SEQ ID NO: 3: Nucleic acid sequence (cDNA sequence) of rice RPS14 gene.
SEQ ID NO: 4: Amino acid sequence of rice RPS14. The amino acid sequence from positions 1 to 48 is a mitochondrial targeting sequence.
SEQ ID NO: 5: Nucleic acid sequence (cDNA sequence) of the Gluconobacter suboxydans ddsA gene.
SEQ ID NO: 6: Amino acid sequence of decaprenyl diphosphate synthase derived from Gluconobacter suboxydans SEQ ID NO: 7: Nucleic acid sequence (cDNA sequence) encoding tobacco N-acetylglucosaminyltransferase I protein
SEQ ID NO: 8: Amino acid sequence of tobacco N-acetylglucosaminyltransferase I protein. The amino acid sequence from position 1 to position 77 is the Golgi targeting sequence.
SEQ ID NO: 9: Nucleic acid sequence (cDNA sequence) encoding rice ATPase β subunit protein.
SEQ ID NO: 10: Amino acid sequence of rice ATPase β subunit protein. The amino acid sequence from position 1 to position 85 is a mitochondrial targeting sequence.
SEQ ID NO: 11: Nucleic acid sequence (cDNA sequence) encoding an Arabidopsis ATPase γ subunit protein.
SEQ ID NO: 12: Amino acid sequence of Arabidopsis ATPase γ subunit protein. The amino acid sequence from positions 1 to 42 is a mitochondrial targeting sequence.

Claims (102)

A plant transformed with an expression cassette expressing polyprenyl diphosphate synthase. The plant according to claim 1, wherein the polyprenyl diphosphate synthase is decaprenyl diphosphate synthase. The plant according to claim 1, wherein the decaprenyl diphosphate synthase is an enzyme derived from bacteria. The plant according to claim 3, wherein the decaprenyl diphosphate synthase is an enzyme derived from Gluconobacter suboxydans. The plant according to claim 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid that hybridizes with the nucleic acid shown in SEQ ID NO: 5 under stringent conditions. The plant according to claim 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having one or several deletions, additions or substitutions in the nucleic acid sequence shown in SEQ ID NO: 5. The plant according to claim 2, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having a sequence identity higher than 80% with the nucleic acid sequence shown in SEQ ID NO: 5. The plant according to claim 2, wherein the decaprenyl diphosphate synthase has an amino acid sequence having one or several deletions, additions, or substitutions in the amino acid sequence shown in SEQ ID NO: 6. The plant according to claim 2, wherein the decaprenyl diphosphate synthase has an amino acid sequence identity higher than 80% with the amino acid sequence shown in SEQ ID NO: 6. The plant of claim 2, wherein the nucleic acid encoding decaprenyl diphosphate synthase is operably linked to a nucleic acid encoding a mitochondrial targeting sequence. A nucleic acid encoding the mitochondrial targeting sequence, a nucleic acid encoding a fragment of rice RPS10 protein (SEQ ID NO: 2), a nucleic acid encoding a fragment of RPS14 protein (SEQ ID NO: 4), a nucleic acid encoding a fragment of RPS11 protein, 11. The nucleic acid encoding a fragment of an ATPase β subunit protein (SEQ ID NO: 10) and a nucleic acid encoding a fragment of an ATPase γ subunit protein (SEQ ID NO: 12) according to claim 10. plant. 11. The plant according to claim 10, wherein the mitochondrial targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). 11. The mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence from positions 1 to 56 of a rice RPS10 protein (SEQ ID NO: 2). plant. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice The plant according to claim 10, which is encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence from position 1 to position 56 of the RPS10 protein (SEQ ID NO: 2). 11. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding the amino acid sequence from position 1 to position 56 of rice RPS10 protein (SEQ ID NO: 2). The described plant. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice RPS10 protein (sequence). The plant according to claim 10, which has the amino acid sequence of a fragment located within positions 1 to 56 of number 2). 11. The plant according to claim 10, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). The plant according to claim 10, wherein the mitochondrial targeting sequence is a fragment consisting of an amino acid sequence of positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). 11. The mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence from position 1 to position 48 of a rice RPS14 protein (SEQ ID NO: 4). plant. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice The plant according to claim 10, which is encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence from position 1 to position 48 of the RPS14 protein of (SEQ ID NO: 4). 11. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding an amino acid sequence from position 1 to position 48 of rice RPS14 protein (SEQ ID NO: 4). The described plant. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions, or substitutions in the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice RPS14 protein ( The plant according to claim 10, which has the amino acid sequence of a fragment located within positions 1 to 48 of SEQ ID NO: 4). 11. The plant according to claim 10, wherein the mitochondrial targeting sequence has an amino acid sequence identity higher than 80% with the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). The plant of claim 2, wherein the polyprenyl diphosphate synthase is operably linked to a Golgi targeting sequence. 25. The plant of claim 24, wherein the nucleic acid encoding the Golgi apparatus targeting sequence is a nucleic acid encoding a fragment of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. The plant according to claim 24, wherein the Golgi apparatus targeting sequence is a fragment consisting of an amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). The Golgi apparatus targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). The plant according to claim 24. The Golgi apparatus targeting sequence may be one or several deletions, additions, or additions to the nucleic acid sequence encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. Plant according to claim 24, encoded by a nucleic acid having a substitution or a fragment of a nucleic acid sequence encoding the amino acid sequence from position 1 to position 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). . The Golgi apparatus targeting sequence is encoded by a nucleic acid having a sequence that is 80% identical to the nucleic acid sequence encoding the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). The plant according to claim 24. The Golgi apparatus targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). Or a plant according to claim 24, which has the amino acid sequence of a fragment within positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 25. The plant of claim 24, wherein the Golgi apparatus targeting sequence has an amino acid sequence that is 80% identical to the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). . The plant of claim 1, wherein the polyprenyl diphosphate synthase gene is operably linked to a seed-specific promoter. The plant according to claim 1, wherein the plant is selected from the group consisting of plant cells, plant culture cells, plant seeds, regenerated plant bodies, plant callus, plant tissue, leaves, stems, roots, flowers, sprout, algae, and moss. The described plant. The plant according to claim 1, which is a monocotyledonous plant. The plant according to claim 1, which is a dicotyledonous plant. 36. A plant seed according to any one of claims 1, 32, 34, or 35. 36. The plant of claim 35, wherein the dicotyledonous plant is selected from the group consisting of rice, corn, oats, wheat, barley, buckwheat, judodama, oats, Indian millet, banana, and sugar cane. 37. The plant of claim 36, wherein the monocotyledonous plant is selected from the group consisting of soybean, tomato, potato, sweet potato, almond, pistachio nut, peanut, hazelnut, walnut, cashew nut, and sesame. An expression cassette for expressing polyprenyl diphosphate synthase in plants. 40. The expression cassette of claim 39, wherein the polyprenyl diphosphate synthase is decaprenyl diphosphate synthase. 40. The expression cassette of claim 39, wherein the decaprenyl diphosphate synthase is a bacterial enzyme. 42. The expression cassette according to claim 41, wherein the decaprenyl diphosphate synthase is an enzyme derived from Gluconobactersuboxydans. 41. The expression cassette according to claim 40, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid that hybridizes with the nucleic acid shown in SEQ ID NO: 5 under stringent conditions. The decaprenyl diphosphate synthase is encoded by a nucleic acid having one or several deletions, additions or substitutions in the nucleic acid sequence shown in SEQ ID NO: 5, or a fragment of the nucleic acid sequence shown in SEQ ID NO: 5. 40. Expression cassette according to 40. 41. The expression cassette of claim 40, wherein the decaprenyl diphosphate synthase is encoded by a nucleic acid having a sequence identity as high as 80% with the nucleic acid sequence set forth in SEQ ID NO: 5. The decaprenyl diphosphate synthase has an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence of a fragment in the sequence shown in SEQ ID NO: 6. 41. An expression cassette according to claim 40. 41. The expression cassette of claim 40, wherein the decaprenyl diphosphate synthase has an amino acid sequence identity greater than 80% with the amino acid sequence set forth in SEQ ID NO: 6. 41. The expression cassette of claim 40, wherein the nucleic acid encoding decaprenyl diphosphate synthase is operably linked to a nucleic acid encoding a mitochondrial targeting sequence. The nucleic acid encoding the mitochondrial targeting sequence is a nucleic acid encoding a fragment of rice RPS10 protein (SEQ ID NO: 2), a nucleic acid encoding a fragment of RPS14 protein (SEQ ID NO: 4), RPS11 protein, ATPase β subunit protein 49. The expression cassette of claim 48, selected from the group consisting of: and ATPase gamma subunit protein. 49. The expression cassette according to claim 48, wherein the mitochondrial targeting sequence is a fragment consisting of the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2). 49. The mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence from position 1 to position 56 of a rice RPS10 protein (SEQ ID NO: 2). Expression cassette. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice 49. The expression cassette of claim 48, encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence from position 1 to position 56 of the RPS10 protein (SEQ ID NO: 2). 49. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding the amino acid sequence from position 1 to position 56 of rice RPS10 protein (SEQ ID NO: 2). The expression cassette as described. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 56 of rice RPS10 protein (SEQ ID NO: 2), or rice RPS10 protein (sequence). 49. The expression cassette of claim 48, having the amino acid sequence of a fragment within positions 1 to 56 of number 2). 49. The expression cassette of claim 48, wherein the mitochondrial targeting sequence has an amino acid sequence identity greater than 80% with the amino acid sequence from position 1 to position 56 of the rice RPS10 protein (SEQ ID NO: 2). 49. The expression cassette according to claim 48, wherein the mitochondrial targeting sequence is a fragment consisting of an amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4). 49. The mitochondrial targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence from position 1 to position 48 of a rice RPS14 protein (SEQ ID NO: 4). Expression cassette. The mitochondrial targeting sequence is a nucleic acid having one or several deletions, additions, or substitutions in the nucleic acid sequence encoding the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice 49. The expression cassette of claim 48, encoded by a fragment of a nucleic acid sequence encoding the amino acid sequence from position 1 to position 48 of the RPS14 protein (SEQ ID NO: 4). 49. The mitochondrial targeting sequence is encoded by a nucleic acid having a sequence identity greater than 80% with a nucleic acid sequence encoding an amino acid sequence from position 1 to position 48 of rice RPS14 protein (SEQ ID NO: 4). The expression cassette as described. The mitochondrial targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 48 of rice RPS14 protein (SEQ ID NO: 4), or rice RPS14 protein (sequence). 49. The expression cassette of claim 48, having the amino acid sequence of a fragment within positions 1 to 48 of number 4). 49. The expression cassette of claim 48, wherein the mitochondrial targeting sequence has an amino acid sequence identity greater than 80% with an amino acid sequence from position 1 to position 48 of rice RPS14 protein (SEQ ID NO: 4). 40. The expression cassette of claim 39, wherein the polyprenyl diphosphate synthase is operably linked to a Golgi targeting sequence. 63. The expression cassette of claim 62, wherein the nucleic acid encoding the Golgi apparatus targeting sequence is a nucleic acid encoding a fragment of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. The expression cassette according to claim 62, wherein the Golgi apparatus targeting sequence is a fragment consisting of an amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). The Golgi apparatus targeting sequence is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. The expression cassette of claim 62. The Golgi apparatus targeting sequence may be one or several deletions, additions, or additions to the nucleic acid sequence encoding the amino acid sequence of positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. Expression according to claim 62, encoded by a nucleic acid having a substitution or a fragment of the nucleic acid sequence encoding the amino acid sequence from position 1 to position 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). cassette. The Golgi apparatus targeting sequence is encoded by a nucleic acid having a sequence that is 80% identical to the nucleic acid sequence encoding the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. The expression cassette of claim 62. The Golgi apparatus targeting sequence is an amino acid sequence having one or several deletions, additions or substitutions in the amino acid sequence at positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. An expression cassette according to claim 62, which has the amino acid sequence of a fragment within positions 1 to 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). 63. Expression according to claim 62, wherein the Golgi apparatus targeting sequence has an amino acid sequence that is 80% identical to the amino acid sequence from position 1 to position 77 of tobacco N-acetylglucosaminyltransferase I protein (SEQ ID NO: 8). cassette. 40. The expression cassette of claim 39, wherein the polyprenyl diphosphate synthase gene is operably linked to a seed specific promoter. 71. A plant comprising the expression cassette of claim 70. 72. The seed of the plant of claim 71. A method for producing ubiquinone-10 using a plant, the method comprising:
(1) obtaining a plant according to claim 1; and (2) culturing the plant;
Including. A method for producing ubiquinone-10 using a plant, the method comprising:
(1) introducing the expression cassette according to claim 37 into a plant;
(2) culturing a plant into which the expression cassette has been introduced;
Including. A plant tissue comprising ubiquinone-10 obtained from the plant according to claim 1. A plant cell comprising ubiquinone-10 obtained from the plant according to claim 1. A food comprising the plant-derived ubiquinone-10 according to claim 1. Furthermore, the foodstuff of Claim 77 containing the plant-derived component of Claim 1. The food according to claim 78, wherein the plant-derived component according to claim 1 is a protein. A composition comprising the plant-derived ubiquinone-10 according to claim 1. The composition according to claim 80, further comprising a plant-derived component according to claim 1. 82. The composition of claim 81, wherein the plant-derived component of claim 1 is a protein. A dietary supplement comprising the plant-derived ubiquinone-10 according to claim 1. The dietary supplement according to claim 83, further comprising the plant-derived component according to claim 1. 85. The dietary supplement according to claim 84, wherein the plant-derived component of claim 1 is a protein. A cosmetic comprising the plant-derived ubiquinone-10 according to claim 1. Furthermore, the cosmetics of Claim 86 containing the plant-derived component of Claim 1. The cosmetic according to claim 87, wherein the plant-derived component according to claim 1 is a protein. A beverage comprising the plant-derived ubiquinone-10 according to claim 1. 90. The beverage according to claim 89, further comprising a plant-derived component according to claim 1. The beverage according to claim 90, wherein the plant-derived component according to claim 1 is a protein. A leaf comprising the plant-derived ubiquinone-10 according to claim 1. An embryo comprising the plant-derived ubiquinone-10 according to claim 1. A fruit comprising the plant-derived ubiquinone-10 according to claim 1. A stem comprising the plant-derived ubiquinone-10 according to claim 1. A root comprising the plant-derived ubiquinone-10 according to claim 1. A flower comprising the plant-derived ubiquinone-10 according to claim 1. A seed comprising the plant-derived ubiquinone-10 according to claim 1. Rice bran comprising the plant-derived ubiquinone-10 according to claim 1. A rice cell or tissue comprising ubiquinone-10. 101. The cell or tissue of claim 100, which is a seed. 101. The cell or tissue according to claim 100, which is brown rice.