JP2008035831A - Plant with heightened productivity at useful part, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant with heightened productivity at a useful part the, and to provide a method for producing the plant. <P>SOLUTION: The transformed plant is transformed by at least one kind selected from the group consisting of (a) a polynucleotide encoding an isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase, and has hightened productivity at the useful part. The method for producing the plant with hightended productivity at the useful part, the plant with hightened productivity at the useful part, the useful part, the method for heightening the productivity of the useful part of the plant, the kit for producing the plant with hightened productivity at the useful part are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention mainly relates to a plant in which the productivity of useful parts is enhanced and a method for producing the plant.

Isoprene is known as a carbon number 5 terpenoid released in large amounts from many higher plants, particularly deciduous broad-leaved trees such as eucalyptus and poplar. In higher plant cells, isoprene is mediated by a non-mevalonate pathway in chloroplasts. It is biosynthesized from dimethylallyl pyrophosphate by the action of isoprene synthase. Although the annual total amount of isoprene released from plants at the global level is enormous and reaches 5 × 10 ¹⁴ gyr ⁻¹ in terms of carbon, its significance for plants remains unclear.

  The main object of the present invention is to provide a plant in which productivity of useful parts is enhanced and a method for producing the plant.

  The present inventors started research with the primary purpose of elucidating a part of the physiological role of isoprene production and release in plants, and the amount of isoprene released into the atmosphere is small while earnest research is repeated. The weight, size and number of transgenic Arabidopsis leaves obtained by introducing the gene of an enzyme that catalyzes the reaction leading to isoprene production are increased compared to wild-type Arabidopsis thaliana. As a result, the present invention has been completed.

The present invention mainly relates to the following matters.
[Claim 1]
(A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A transformed plant that has been transformed with at least one selected from the group consisting of and improved the productivity of useful parts.
[Item 2] The transformed plant according to Item 1, having at least one characteristic selected from the group consisting of the following (1) to (3):
(1) The expression level of at least one selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase is higher than that of the wild type,
(2) the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase is higher than that of the wild type, and
(3) The amount of isoprene released is greater than that of the wild type.
[Item 3] The transformed plant according to Item 1 or 2, wherein the amount of isoprene released is 150 to 3000% of the wild type.
[Claim 4]
Item 4. The useful part is at least one selected from the group consisting of leaves, stems, stems, roots, fruits, flowers, seeds, bark and underground stems, or a part thereof, Transformed plants.
[Item 5] The transformed plant according to any one of Items 1 to 4, further having at least one characteristic selected from the group consisting of the following (A) to (G):
(A) Pesticide resistance is higher than wild type,
(B) Heavy metal resistance is higher than wild type,
(C) pest resistance is higher than wild type,
(D) low temperature tolerance is higher than wild type,
(E) salt tolerance is higher than wild type,
(F) Dry tolerance is higher than wild type and
(G) The weak light resistance is higher than the wild type.
[Item 6] At least selected from the group consisting of food, food or food composition production, cosmetic production, pharmaceutical production, industrial product production, appreciation, forest regeneration, desert regeneration, and phytomediation Item 6. The transformed plant according to any one of Items 1 to 5, which is used for one kind of use.
[Item 7] The transformed plant according to any one of Items 1 to 6, which is a seed, a seedling, a young seedling, a middle seedling, an adult seedling, or a plant in a growth process therebetween.
[Section 8]
(A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
Item 8. The transformed plant according to any one of Items 1 to 7, wherein at least one selected from the group consisting of is homozygous and integrated in a cell chromosome.
[Claim 9]
(I) (a) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A method for producing a plant with enhanced productivity of useful parts, comprising the step of obtaining a transformant by introducing at least one selected from the group consisting of:
[Section 10]
Furthermore, (II) a step of performing at least one selected from the group consisting of the following (i) to (iv):
(I) For at least one of the weight, size and number of useful parts, the transformant obtained in step (I) is compared with the wild type, and at least one of the weight, size and number of useful parts is Selecting a transformant with a larger than wild type,
(Ii) For at least one expression level selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type And selecting a transformant whose expression level is higher than that of the wild type,
(Iii) For the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type Comparing the type and selecting a transformant in which the expression level of the mRNA is higher than that of the wild type, and
(Iv) For the amount of isoprene released, the transformant obtained in the step (I) is compared with the wild type, and a transformant having a larger amount of isoprene released than the wild type is selected.
Item 10. A method for producing a plant with enhanced productivity of the useful part according to Item 9.
[Item 11] A plant produced by the method according to Item 9 or 10, wherein productivity of useful parts is enhanced.
[Item 12] A useful part of the plant according to item 11.
[Claim 13]
(A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A method for increasing the productivity of a useful part of a plant, comprising a step of introducing at least one selected from the group consisting of:
[Section 14]
(A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A kit for producing a plant with an enhanced productivity of useful parts, comprising a plant expression plasmid having at least one selected from the group consisting of:
[Item 15] The transformed plant according to any one of Items 1 to 8, which is further transformed with a polynucleotide encoding (d) isopentenyl diphosphate Δ-isomerase.
[Item 16] The useful part productivity according to Item 9, further comprising the step of obtaining a transformant by introducing (d) a polynucleotide encoding isopentenyl diphosphate Δ-isomerase into a plant cell. A method of producing a plant with enhanced squeeze.
[Item 17] A plant produced by the method according to item 16, wherein productivity of useful parts is enhanced.
[Item 18] A useful part of the plant according to item 17.
[Item 19] The method for enhancing the productivity of a useful part of a plant according to Item 13, further comprising the step of (d) introducing a polynucleotide encoding isopentenyl diphosphate Δ-isomerase into the plant cell.
[Item 20] In order to produce a plant with an enhanced productivity of the useful part according to Item 14, further comprising (d) a plant expression plasmid having a polynucleotide encoding isopentenyl diphosphate Δ-isomerase Kit.
[Item 21] The plant with enhanced productivity of the useful part according to Item 14, wherein the plant expression plasmid according to Item 14 further comprises (d) a polynucleotide encoding isopentenyl diphosphate Δ-isomerase. Kit for making.

  Hereinafter, the present invention will be described in more detail.

Isoprene (2-methyl-1,3-butadiene) is a structural unit of terpenes (isoprenoids) present in many plants, and is represented by the structural formula CH ₂ ═C (CH ₃ ) CH═CH ₂ . It is a hydrocarbon having a conjugated double bond. It is known that isoprene is released in many plants, and in particular, it is released in large quantities from broad-leaved trees such as eucalyptus and poplar. Isoprene is also produced in conifers and herbs, but usually releases less than broad-leaved trees. Examples of species with low isoprene release include plants of legumes, grasses, and pine familys.

  Isoprene is produced by oxidatively dephosphorylating dimethylallyl pyrophosphate (DMAPP), which is a precursor of isoprene. Isoprene synthase catalyzes this reaction. DMAPP is produced by isomerization of isopentenyl pyrophosphate (IPP) by the action of isopentenyl diphosphate Δ-isomerase. The isomerization reaction by isopentenyl diphosphate Δ-isomerase occurs bi-directionally between DMAPP and IPP. That is, DMAPP and IPP are interconverted by isopentenyl diphosphate Δ-isomerase. There are two types of pathways for biosynthesizing IPP / DMAPP, the conventionally known “mevalonate pathway” and the unexplained “non-mevalonate pathway (1-deoxyxylulose pathway)”. From the study of The mevalonate pathway is thought to be possessed by almost all organisms. The rate-limiting step of the mevalonate pathway is a reaction in which hydroxymethylglutamyl coenzyme A (HMG-CoA) is reduced to mevalonate, which is thought to be the origin of the name.

  On the other hand, terpenoids, steroids, carotenoids, natural rubber and the like are biosynthesized from IPP or DMAPP via geranyl pyrophosphate.

  That is, there are roughly two pathways: a pathway that synthesizes isoprene from IPP via DMAPP, and a pathway that goes from IPP or DMAPP to geranyl pyrophosphate. When the former is rate-limiting, a large amount of isoprene is synthesized and released into the atmosphere. When the latter is rate-limiting, isoprene is not produced and other secondary metabolites are synthesized in large quantities.

  The reaction of the mevalonate pathway is as follows.

  DMAPP produced by the reaction 7 is dephosphorylated by isoprene synthase to produce isoprene. On the other hand, IPP or DMAPP undergoes secondary metabolism to produce complex compounds such as terpenoids.

  The present invention is selected from the group consisting of (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. The transformed plant is transformed with at least one selected from the above, and the productivity of the useful part is increased.

  (A) Isoprene synthase is an enzyme that catalyzes a reaction of synthesizing isoprene from DMAPP.

  Examples of the isoprene synthase include those derived from all species that produce isoprene synthase, among which, for example, those derived from higher plants or fern plants are preferable, and in particular, broadleaf trees, ferns, conifers, etc. Those derived from species with a large amount of isoprene released are preferred. In particular, the willow family (for example, poplar), the myrtaceae family (for example, eucalyptus), the beech family (for example, Quercus kunugi, the Quercus genus Naragashi), the fern family (for example, fern) and the like, which release a large amount of isoprene, are preferable. As an example, the amino acid sequence of Populus alba isoprene synthase is SEQ ID NO: 4 (Genbank accession number: BAD98243), and the amino acid sequence of the leguminous kudzu (Pueraria montana var. Lobata) is SEQ ID NO: 10 (Genbank accession number: AAQ84170). The isoprene synthase includes, for example, about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about 96% with the amino acid sequence shown in SEQ ID NO: 4 or 10. More preferably, an isoprene synthase having an amino acid sequence having a homology of about 98% or more is included.

  As the polynucleotide encoding isoprene synthase, the polynucleotide encoding the isoprene synthase is preferable (here, the codon corresponding to each amino acid may be any). As an example of a polynucleotide encoding isoprene synthase, SEQ ID NO: 3 shows the nucleotide sequence of poplar isoprene synthase gene (Genbank accession number: AB198180), and SEQ ID NO: 9 shows the nucleotide sequence of genus isoprene synthase gene (Genbank accession). Session number: AY316691). The polynucleotide encoding isoprene synthase has about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably the nucleotide sequence shown in SEQ ID NO: 3 or 9. A polynucleotide having a base sequence having about 96% or more, more preferably about 98% or more identity is included.

  (B) 5-phosphomevalonate kinase (EC 2.7.4.2) is an enzyme that catalyzes the reaction 5 (5-phosphomevalonate + ATP → 5-diphosphomevalonate + ADP).

  Examples of 5-phosphomevalonate kinase include those derived from all species that produce 5-phosphomevalonate kinase. Among them, for example, yeast, bacteria, plants, those derived from animals, particularly yeasts, plants, bacteria The thing of origin can be used conveniently. As an example, the amino acid sequence of 5-phosphomevalonate kinase of yeast (Saccharomyces cerevisiae) is shown in SEQ ID NO: 6 (Genbank accession number: AAA34596). 5-phosphomevalonate kinase includes, for example, about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about 96% with the amino acid sequence shown in SEQ ID NO: 6. More preferably, 5-phosphomevalonate kinase having an amino acid sequence having a homology of about 98% or more is included.

  As the polynucleotide encoding 5-phosphomevalonate kinase, the above-mentioned polynucleotide encoding 5-phosphomevalonate kinase is suitable (here, any codon corresponding to each amino acid may be used). As an example of a polynucleotide encoding 5-phosphomevalonate kinase, the nucleotide sequence of a 5-phosphomevalonate kinase gene of yeast (Saccharomyces cerevisiae) is shown in SEQ ID NO: 5 (Genbank accession number: M63648). The polynucleotide encoding 5-phosphomevalonate kinase has a nucleotide sequence of SEQ ID NO: 5 of about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably A polynucleotide having a base sequence having about 96% or more, more preferably about 98% or more identity is included.

  (C) Diphosphomevalonate decarboxylase (EC 4.1.1.33) is an enzyme that catalyzes reaction 6 (5-diphosphomevalonate + adenosine triphosphate → IPP + carbon dioxide + phosphate + ADP).

  Examples of the diphosphomevalonate decarboxylase include those derived from all species that produce diphosphomevalonate decarboxylase. Among them, for example, yeasts, bacteria, plants, animals, particularly yeasts, bacteria, plants, Can be preferably used. As an example, the amino acid sequence of a yeast (Saccharomyces cerevisiae) diphosphomevalonate decarboxylase is shown in SEQ ID NO: 8 (Genbank accession number: CAA66158). The diphosphomevalonate decarboxylase is, for example, about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about 96% or more with the amino acid sequence shown in SEQ ID NO: 8. More preferably, diphosphomevalonate decarboxylase having an amino acid sequence having a homology of about 98% or more is included.

  The polynucleotide encoding the diphosphomevalonate decarboxylase is preferably the polynucleotide encoding the diphosphomevalonate decarboxylase (wherein the codon corresponding to each amino acid may be any). As an example of a polynucleotide encoding diphosphomevalonate decarboxylase, the nucleotide sequence of the diphosphomevalonate decarboxylase gene of yeast (Saccharomyces cerevisiae) is shown in SEQ ID NO: 7 (Genbank accession number: X97557). The polynucleotide encoding diphosphomevalonate decarboxylase has a base sequence represented by SEQ ID NO: 7 of about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about A polynucleotide having a base sequence having an identity of 96% or more, more preferably about 98% or more is included.

  The transformed plant of the present invention comprises (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. It can be obtained by introducing at least one selected from the group (hereinafter also referred to as the polynucleotides (a) to (c)) into plant cells.

  According to one embodiment of the present invention, in addition to the polynucleotides (a) to (c), (d) a polynucleotide encoding isopentenyl diphosphate Δ-isomerase may be introduced into a plant cell. (D) Isopentenyl diphosphate Δ-isomerase (EC 5.3.3.2) is an enzyme that bidirectionally catalyzes the isomerization reaction between IPP and DMAPP.

  For example, by introducing a polynucleotide encoding (d) isopentenyl diphosphate Δ-isomerase together with a polynucleotide encoding isoprene synthase, a series of pathways of IPP → DMAPP → isoprene is promoted. As a result, it is considered that the productivity of the useful portion can be increased efficiently. In addition, for example, (b) a polynucleotide encoding 5-phosphomevalonate kinase and / or (c) a polynucleotide encoding diphosphomevalonate decarboxylase and (d) a polynucleotide encoding isopentenyl diphosphate Δ-isomerase Introducing the reaction 5 and / or the reaction 6 → IPP → DMAPP can promote the series of pathways, and the reaction of DMAPP → isoprene can be promoted. It is thought that productivity can be improved efficiently.

  Examples of isopentenyl diphosphate Δ-isomerase include those derived from all species that produce isopentenyl diphosphate Δ-isomerase. Among them, for example, those derived from yeast, bacteria, plants, animals, especially Those derived from yeast, bacteria and plants can be preferably used. As an example, the amino acid sequence of isopentenyl diphosphate Δ-isomerase of Saccharomyces cerevisiae is shown in SEQ ID NO: 12 (Genbank accession number: AAA34708). Isopentenyl diphosphate Δ-isomerase includes, for example, about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably the amino acid sequence shown in SEQ ID NO: 12. Isopentenyl diphosphate Δ-isomerase having an amino acid sequence having a homology of about 96% or more, more preferably about 98% or more is included.

  As the polynucleotide encoding isopentenyl diphosphate Δ-isomerase, the polynucleotide encoding the isopentenyl diphosphate Δ-isomerase is preferable (wherein the codons corresponding to each amino acid are any). May be) As an example of a polynucleotide encoding isopentenyl diphosphate Δ-isomerase, the base sequence of the isopentenyl diphosphate Δ-isomerase gene of yeast (Saccharomyces cerevisiae) is shown in SEQ ID NO: 11 (Genbank accession number: J05090). . The polynucleotide encoding isopentenyl diphosphate Δ-isomerase has a nucleotide sequence of SEQ ID NO: 11 of about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, More preferably, a polynucleotide having a base sequence having an identity of about 96% or more, more preferably about 98% or more is included.

  Various operations such as transformation and screening of the polynucleotide (d) can be performed in the same manner as the polynucleotides (a) to (c) described below. In the transformation, the polynucleotide (d) may be inserted into an expression plasmid together with the polynucleotides (a) to (c) and introduced into plant cells, or (a) to (c). In addition to the polynucleotide, it may be inserted into an expression plasmid and introduced into a plant cell. Further, the order of introduction of the polynucleotide (d) and the introduction of the polynucleotides (a) to (c) is not limited.

  The polynucleotides (a) to (c) can be obtained by conventional methods such as PCR, LAMP, EMSA, and automatic synthesis.

  For example, in the PCR method, basically, a reaction solution containing a polynucleotide that can be a template, a forward primer and a reverse primer, a DNA polymerase, dNTP mixture (a mixture of deoxynucleoside triphosphates), and a PCR buffer is subjected to temperature changes. It is a method of amplifying a specific polynucleotide sandwiched between a forward primer and a reverse primer by repeatedly subjecting it to the following three steps: (1) denaturation (2) annealing (3) extension. For example, in the case of amplifying a polynucleotide encoding isoprene synthase, for example, a genomic DNA, genomic library, cDNA library or the like prepared from an organism that expresses an isoprene synthase gene is used as a template that can be a template. be able to. The forward primer and the reverse primer can be designed based on, for example, an about 10-30 bp nucleotide sequence located at or around the 5 ′ end of the isoprene synthase gene and at or around the 3 ′ end, such as automatic synthesis. It can be synthesized by the conventional method. When the sequence to be amplified does not include the start codon, a forward primer is designed so as to include the start codon in-frame, or the start codon is introduced into the expression vector described later in-frame. Examples of the DNA polymerase include commercially available heat-resistant polymerases such as Pfu (manufactured by Promega), Taq (manufactured by TOYOBO), KOD (manufactured by TOYOBO), Vent (manufactured by NEB), ExTaq (manufactured by TaKaRa), PlatinumPfx ( Invitrogen) and the like can be suitably used. As the dNTP mixture, a commercially available mixture of dATP, dCTP, dGTP and dTTP can be suitably used. The PCR buffer is appropriately selected according to the DNA polymerase to be used, and a commercially available product or a DNA polymerase accessory can be suitably used. The PCR reaction can be performed according to conventional procedures or according to the instructions of the DNA polymerase. Those skilled in the art can appropriately change the PCR reaction conditions such as temperature, time, number of cycles, reaction composition, and the like.

  The polynucleotides (a) to (c) are preferably introduced into plant cells as an expression plasmid linked to an expression vector.

  As the expression vector, a vector that can autonomously replicate in a host or can be integrated into a chromosome and contains a promoter at a position that enables transcription of a polynucleotide is preferably used. The 5 'end of the polynucleotide amplified by PCR is linked to the 3' end of the promoter directly or in-frame via an appropriate sequence (for example, a restriction enzyme recognition site). The expression vector may further contain a plant cell enhancer (for example, EL2 enhancer, CaMV35S enhancer, etc.), a selection marker gene, a labeling tag, and the like. A stop codon is not necessarily required, but is preferably arranged immediately below the structural gene. In addition, with reference to the amino acid sequence, the polynucleotide can be linked in-frame to an expression promoter, enhancer, initiation codon, and the like. Various base sequences and amino acid sequences can be obtained from search systems such as Genbank and EMBL. The amino acid sequence can also be determined by a conventional method such as a system that estimates an amino acid sequence based on a base sequence, N-terminal analysis, C-terminal analysis, or the like.

  The promoter is not particularly limited as long as it promotes the expression of isoprene synthase, 5-phosphomevalonate kinase, and / or diphosphomevalonate decarboxylase in the host cell. For high expression in higher plants, a constitutive expression promoter such as EL2 promoter, CaMV35S promoter, Cab promoter, RuBisCo promoter, PR1 promoter, ubiquitin promoter and the like can be used. Further, isoprene synthase, 5-phosphomevalonate kinase, and / or diphosphomevalonate decarboxylase is selected from the group consisting of specific organs of plants (for example, leaves, stems, stems, roots, fruits, flowers, seeds, bark and rhizomes) Promoters that are specifically expressed in at least one of the above may be used. Examples of such promoters include cocoon-specific promoter RA8 (Special Table 2002-528125), flower organ-specific promoter RPC213 (International Publication WO99 / 43818), mesophyll cell-specific promoter rbcS (Plant Physiol. 102,991- 1000 (1993)) can be used, but is not limited thereto.

  The plant cell (host) is not particularly limited because it is appropriately selected according to the purpose and use, but is preferably a higher plant cell. Although it does not specifically limit as a higher plant, Gramineae, Leguminosae, Lepidoptera, Brassicaceae, Orchidaceae, Lilyaceae, Iridaceae, Asteraceae, Azalea, Rosaceae, Pinaceae, Camphoraceae, Walnutaceae, Willow Plants such as the family and the pine family are included. Specific examples of higher plants include rice, millet, millet, millet, sugarcane, sorghum, corn, buckwheat, wheat, barley, rye, triticale, oats, oats, tartary buckwheat, cucumber, etc .; soybean, azuki bean, peas, Beans or nuts such as broad beans, kidney beans, alfalfa, peanuts, walnuts, almonds, poppies, coffee, pistachios, macadamia; sweet potatoes, potatoes, yams, taros, cassava, potatoes such as yams, taros; sesame, rapeseed, safflower, Oily plants such as sunflower, coconut, palm, olive, cottonseed, cacao, safflower, avocado; carrot, radish, turnip, radish, lotus root, burdock, onion, leek, celery, leek, honey bee, garlic, chingensai Chinese cabbage, spinach, Mizuna, parsley, lettuce, cabbage, broccoli, asparagus, cauliflower, pepper, pepper, cucumber, cucumber, bitter gourd, zucchini, watermelon, tomato, pumpkin, eggplant, ginger, ginger, garlic, sea cucumber, komatsuna, etc. Vegetables: orange, grapefruit, lemon, grape, muscat, peach, plum, apricot, apple, pear, oyster, plum, fig, pomegranate, strawberry, melon, pineapple, mango, mangosteen, papaya, acerola, kiwi, blueberry, raspberry Fruits such as cedar, cypress, cherry, cedar, cypress, pine, willow, giraffe, maple, poplar, oak, elm, pecan, teak, cherry, ume, maple, etc .; Arabidopsis, rose, carnation Tulip, Eustoma, Gentian, Petunia, Gypsophila, Nadesico, Lily, Violet, Pansy, Lavender, Gerbera, Cosmos, Chrysanthemum, Geranium, Periwinkle, Begonia, Cyclamen, Nilebergia, Periwinkle, Tenjikuaoi, Orchid, Asian Sunflower, Ivy, Ivy Plants such as, but not limited to, areca, Indian rubber, orchid orchid, sansevieria, chevrela, spatiphyllum, dracaena, neoregelia, pachira, benjamin, potos, monstera, poinsettia, anemone, kyoto.

  Plant cells may or may not originally have an isoprene synthase gene, a 5-phosphomevalonate kinase gene, and / or a diphosphomevalonate decarboxylase gene. A significant increase in the productivity of the useful portion can be achieved by transforming the species in which the abundance of these genes is originally low or absent with the polynucleotides (a) to (c) according to the present invention. Examples of the species in which the abundance of the isoprene synthase gene, 5-phosphomevalonate kinase gene, and / or diphosphomevalonate decarboxylase gene is originally low or absent are, for example, legumes, gramineae, oleaceae, cruciferous, mulberry, Asteraceae, oleaceae, etc. are considered.

  The plant cell may be a modified plant cell such as a plant cell into which a polynucleotide other than the polynucleotides (a) to (c) is introduced or a plant cell modified by hybridization. By using plant cells modified in this way, further advantageous properties can be imparted to the transformed plant of the present invention.

  As a method for introducing the polynucleotides (a) to (c) or the expression plasmid containing the same into plant cells, conventional methods such as lithium acetate method, electroporation method, Agrobacterium method, gene gun (particle) Cancer) method, calcium phosphate method, protoplast method, Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979), and Molecular Cloning 2nd ed. (Sambrook, J., Fritsch, EF, Maniatis, T. Cold Spring Harbor Laboratory Press 1989), Plant Molecular Biology Manual R. and B. Gel R. Can be suitably used the method described in (incorporated herein in its entirety). Furthermore, an individual into which an expression plasmid has been introduced is usually selected. Selection of an individual into which an expression plasmid has been introduced can be performed by a conventional method, for example, selection by a drug-containing medium, selection by a hormone-containing medium, or selection by auxotrophy. Furthermore, using conventional methods such as genomic PCR, RT-PCR, Northern hybridization, Western analysis, etc., integration of the polynucleotides (a) to (c) above into plant cell chromosomes, isoprene synthase, 5-phosphomevalonic acid Expression of mRNA or protein of kinase or diphosphomevalonate decarboxylase can be confirmed. In this way, transformed cells into which the polynucleotides (a) to (c) have been introduced can be obtained.

  Furthermore, if necessary, a transformed plant can be obtained by differentiating and growing the transformed cells into which the polynucleotides (a) to (c) have been introduced. For example, plant cells can be differentiated and grown from plants to plants by transferring them to a medium containing plant hormones. The method of differentiation and growth is incorporated herein by reference in its entirety, “Growth & Differentiation in Plants, 3rd Edition by PF Walling & IDJ Phillips, 1981, ISBN 4-7622-6365-6. [83.5] (Plant Growth and Differentiation (above) PF Wearling, IDJ Phillips, translated by Masaki Furuya) and “Growth & Differentiation in Plants, 3rd Edition by PF” Walling & IDJ Phillips, 1981, ISBN 4-7622-7366-X [83.11] (Plant growth and differentiation (bottom), PF Waring, IDJ. By Philips Masaki Furuya translation supervisor) "can be referred to.

  Further, the transformant thus obtained may be screened.

The screening is performed, for example, by performing at least one selected from the group consisting of the following (i) to (iv):
(I) For at least one of the weight, size and number of useful parts, the obtained transformant is compared with the wild type, and at least one of the weight, size and number of the useful parts is larger than the wild type Selecting a transformant,
(Ii) For at least one expression level selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the obtained transformant is compared with the wild type, and the expression Selecting a transformant whose amount is higher than the wild type,
(Iii) For the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the obtained transformant is compared with the wild type, Selecting a transformant in which the expression level of the mRNA is higher than that of the wild type, and
(Iv) For the amount of isoprene released, the obtained transformant is compared with the wild type, and a transformant with a larger amount of isoprene released than the wild type is selected.

  “Transformation” generally refers to a genetic phenomenon in which DNA isolated from one cell is taken up by another cell and recombines with the cell chromosome (Tokyo Chemistry Doujin Dictionary 2nd edition, 416). Pp. 417), which means that DNA molecules including plasmids and genes bound thereto are directly introduced into cells (Iwanami Biology Dictionary, 4th edition, pages 380-381). In this document, transformation means introducing a polynucleotide (eg, DNA, mRNA) into a cell, and at this time, the polynucleotide may or may not recombine with a cell chromosome. In order to stably express the phenotype and the genotype, it is desirable that the oligonucleotide undergoes recombination with the cell chromosome, and that the recombination with the cell chromosome is desirably homozygous.

  “Plants” include plants in any growth process, for example, seeds, seedlings, young seedlings, medium seedlings, adult seedlings, fruiting, or plants in the process of growth therebetween. The “plant” also includes plant parts, unless otherwise specified, and includes, for example, roots, shoots, leaves, stems, trunks, fruits, flowers, seeds, bark, and underground stems. “Cells” include cells (eg, cultured cells), cell aggregates (eg, callus, tissue section, somatic embryo), and individual cells in a plant.

“Transformants” include transformed cells and transformed plants. A "transformant", the (a) ~ the polynucleotide of (c) not only transformants T ₀ generation obtained by introducing into a plant cell, its progeny (T ₁ generation, T ₂ Generation and subsequent generations). It is desirable that the transformant is homozygous and has the polynucleotides (a) to (c) in the cell chromosome so that its progeny can stably express the phenotype and genotype.

  In the thus obtained transformed plant of the present invention, productivity of useful parts is enhanced.

  The useful part is not particularly limited because it varies depending on the purpose and application, and includes all organs of plants, for example, a group consisting of leaves, stems, stems, roots, fruits, flowers, seeds, bark and underground stems. It is at least one kind selected from more, or a part thereof. Organ names may vary depending on the type of plant, but these differently named organs are referred to by common names such as leaves, stems, stems, roots, fruits, flowers, seeds, bark and rhizomes. It should be understood to correspond to an organ. For example, in the case of Gramineae, it is understood that berries and grains correspond to fruits, and in the case of legumes, legumes correspond to fruits.

  Stems include above-ground stems, underground stems, rhizomes, tubers, flower axes, and the like.

  Roots include succulent roots, hairy roots, strut roots and the like.

  A fruit includes a fruit that includes a fruit that has developed a ovary and a fruit that includes a tissue that has developed from an organ other than the ovary, regardless of the developmental origin of the flower organs that make up the fruit.

  Some of the fruits include ovary, fruit buds, gargle, total capsule, ovary wall, outer pericarp, mesocarp, inner pericarp, pear fruit and cucumber fruit. The fruit also includes fruit pods.

  A part of the flower includes a flower coat, a corolla, a petal, a gargle, a total envelope, a floral pattern, a stamen, a pistil, a pollen, an ovule, a sub-crown, and the like.

  As for the names of plant organs, for example, the primary color Makino Japanese plant picture book I (issued September 6, 1999, published by Kitatakakan Co., Ltd., author Makita Tomitaro) and the primary color Makino Japanese plant picture book II (May 2000) May 10th, issue place Hokuryukan Co., Ltd., author Tomita Makino).

  Increased productivity of the useful part means that the weight (fresh weight, dry weight), surface area, volume, number, size, length, width, etc. of the useful part or a part thereof are increased. The increase in the productivity of the useful part means, for example, at least one item selected from the following group: (1) the overall weight increases, (2) the above-ground part weight increases, (3 ) Increased leaf weight, (4) increased leaf number, (5) increased leaf size (length or width), (6) increased leaf thickness, (7) stem Or the stem weight increases, (8) the stem or stem thickness increases, (9) the stem or stem length increases, (10) the root weight increases, (11) the root length. (12) increase in fruit weight, (13) increase in fruit number, (14) increase in fruit weight (15) increase in flower weight, (16) number of flowers Increase, (17) increase petal weight, (18) increase number of petals, (19) increase petal size, (20) petal wall thickness To a large, (21) the weight of the seeds is increased, an increase in the number of (22) seeds, (23) the weight of the bark increases, increasing the thickness of (24) bark.

  The isoprene synthase-transformed plant of the present invention has, for example, at least one characteristic selected from the following group: (1) The total weight is 200% or more of the wild type, preferably 250% or more. 2) The weight of the above-ground part is 200% or more of the wild type, preferably 250% or more. (3) The weight of the leaf is 200% or more of the wild type, preferably 250% or more. (4) The number of leaves. Is 130% or more of the wild type, preferably 150% or more. (5) The leaf size (length or width) is 130% or more, preferably 150% or more of the wild type. (6) The thickness is 130% or more of the wild type, preferably 150% or more. (7) The weight of the stem or stem is 200% or more of the wild type, preferably 300% or more. (8) The thickness of the stem or stem. More than 150% of the wild type (diameter or circumference) is preferred Or 200% or more, (9) stem or stem length of 150% or more of the wild type, preferably 200% or more, and (10) root weight of 200% or more of the wild type, preferably 300%. (11) The root length is 150% or more of the wild type, preferably 200% or more. (12) The fruit weight is 200% or more of the wild type, preferably 300% or more. 13) The number of fruits is 200% or more of the wild type, preferably 300% or more, (14) The weight of the pulp is 200% or more, preferably 300% or more of the wild type, (15) The weight of the flower 200% or more of the wild type, preferably 300% or more. (16) The number of flowers is 200% or more of the wild type, preferably 300% or more. (17) The petal weight is 200% or more of the wild type. , Preferably 300% or more, (1 ) The number of petals is 200% or more of the wild type, preferably 300% or more. (19) The petal size (circumference) is 200% or more of the wild type, preferably 300% or more. (20) The petal thickness is 200% or more of the wild type, preferably 300% or more. (21) The seed weight is 200% or more of the wild type, preferably 300% or more. (21) The number of seeds is wild. It is 200% or more of the mold, preferably 300% or more.

  As used herein, the wild type refers to (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. It means a plant body or plant cell that is not transformed with at least one selected from the group consisting of

  Increased productivity of the useful part is attributed to either an increase in cell number or an increase in cell size, or both an increase in cell number and an increase in cell size. Therefore, the number of cells in the transformed plant of the present invention can be, for example, 130% of the wild type, preferably 200% or more, more preferably 300% or more, and / or individual cells in the transformed plant of the present invention. The cell size can be, for example, 130% of the wild type, preferably 200% or more, more preferably 300% or more.

  The transformed plant of the present invention has an isoprene release amount of, for example, about 150 to 3000%, preferably about 200 to 2000%, preferably about 300 to 1500%, more preferably about 500 to 1000% of the wild type. possible. Transformed plants that release such an amount of isoprene have high productivity of useful parts and can be suitably used for various applications.

The transformed plant of the present invention may further have at least one characteristic selected from the following groups (A) to (G):
(A) Pesticide resistance is higher than wild type,
(B) Heavy metal resistance is higher than wild type,
(C) pest resistance is higher than wild type,
(D) low temperature tolerance is higher than wild type,
(E) salt tolerance is higher than wild type,
(F) Dry tolerance is higher than wild type and
(G) The weak light resistance is higher than the wild type.

  For example, when the bark or pericarp of a plant becomes thicker as the productivity of useful parts increases, or the amount of various substances such as hormones in the pathway leading to isoprene synthesis changes, the above (A) to ( It is conceivable that the tolerance of G) is imparted.

  (A) Plants with higher pesticide resistance than wild type or (B) transformed plants with higher heavy metal resistance than wild type are suitable as, for example, plants for phytomediation of pesticides or heavy metal contaminated soil. (C) A transformed plant having higher pest resistance than the wild type is suitable as a plant for cultivation in a tropical or temperate region, for example. (D) A transformed plant having a low temperature tolerance higher than that of the wild type is suitable, for example, as a plant for cultivation in a cold region. (E) A transformed plant having higher salt tolerance than the wild type is suitable as, for example, a plant for cultivation in a salt damage area or a coastal area, and a plant for salt water cultivation. (F) Transformed plants having higher drought tolerance than wild type are suitable as, for example, desert regeneration plants, plants for cultivation in dry regions, and foliage plants. (G) A transformed plant having a low light resistance higher than that of the wild type is suitable as, for example, a plant for cultivation and a foliage plant in an area where the sunshine hours are short.

  Of course, a transformed plant not having the above resistances (A) to (G) is also included in the transformed plant of the present invention.

  The transformed plant of the present invention comprises food, food or food composition production, cosmetic production, pharmaceutical production, industrial product production, appreciation, forest regeneration, desert regeneration and phytomediation. It can be used for at least one application selected from the group.

  Edible transformed plants are usually taken as is or processed and consumed by animals including humans. The kind of plant is not particularly limited as long as it is an edible species including an ingestible part.

  A transformed plant for producing a food or food composition is usually left as it is or processed, or only specific components are extracted and added to the food or food composition. The kind of plant is not particularly limited as long as it is an edible species including an ingestible part. The food or food composition also includes non-human animal food such as pet food and livestock feed.

  A transformed plant for producing a cosmetic product is usually extracted with a specific component or extract and added to the cosmetic product. Cosmetic products include skin care products, nail care products, hair care products and the like. Examples of the specific component or extract include, but are not limited to, vitamins, amino acids, hyaluronic acid, collagen, isoflavones, herbal extracts and the like as whitening components, moisturizing components, antioxidant components, sedative components, and the like. The type of plant varies depending on the desired component or extract and is not particularly limited.

  In a transformed plant for producing a pharmaceutical product, only a specific component or extract is usually extracted and added to the pharmaceutical product. Not only active ingredients of pharmaceuticals, but also refreshing agents, binders, sweeteners, disintegrants, lubricants, coloring agents, fragrances, stabilizers, preservatives, sustained release regulators, surfactants, solubilizers, wetting agents It may be added as a nutritional supplement. The type of plant varies depending on the specific component or extract and is not particularly limited.

  A transformed plant for producing an industrial product is usually used as it is or processed, or a specific component is extracted and used as a raw material or member for an industrial product. For example, trees can be processed and used as raw materials or members for buildings, furniture, crafts, disposable chopsticks, and the like. For example, starch can be extracted from potatoes such as potato, sweet potato and cassava, or cereals such as rice, wheat and corn, and used as a raw material for various industrial products. In addition, for example, cellulose, rubber, resin, and the like can be extracted from trees or herbs and used as raw materials for transportation equipment, electrical products, textile products, and the like. It can be used as a raw material for chemicals, disinfectant, fuel, etc. by producing ethanol by fermenting cereals such as sugar cane, sweet potato, barley and corn, or glucose of potatoes such as potato and sweet potato. Since the kind of plant changes with purposes, uses, etc., it is not specifically limited.

  Transformed plants for viewing are usually distributed as they are or arranged in a shape suitable for viewing, and are mainly used for viewing in homes, stores, offices, and the like. As a kind of plant, for example, a foliage plant native to the tropics, subtropics, or dry zone, a plant that grows flowers or fruits, and the like are preferable, but not limited thereto. As the foliage plant, the above-mentioned herbs can be preferably exemplified.

  Transformed plants for reforestation are usually planted on land where reforestation is desired at the growth stage of seeds to medium seedlings. The type of plant is not particularly limited, and is preferably a type that can grow under the weather conditions of the land where reforestation is desired. In addition, when the forest is destroyed due to farmland reclamation, etc., it is desirable to select the same type as before the forest was destroyed so that it can be restored to the original forest. When the forest is destroyed, it is desirable to select a type that can grow in the changed environment.

The present invention also includes step (I) (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. The present invention provides a method for producing a plant in which the productivity of useful parts is increased, which comprises the step of obtaining a transformant by introducing at least one selected from the group consisting of: The method may further include the step of (d) obtaining a transformant by introducing a polynucleotide encoding isopentenyl diphosphate Δ-isomerase into a plant cell. The method may further include a step of growing into a plant at a desired growth stage, if necessary. The growth method is as described above. The method may further include the step of (II) performing at least one selected from the group consisting of the following (i) to (iv):
(I) For at least one of the weight, size and number of useful parts, the transformant obtained in step (I) is compared with the wild type, and at least one of the weight, size and number of useful parts is Selecting a transformant with a larger than wild type,
(Ii) For at least one expression level selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type And selecting a transformant whose expression level is higher than that of the wild type,
(Iii) For the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type Comparing the type and selecting a transformant in which the expression level of the mRNA is higher than that of the wild type, and
(Iv) Regarding the amount of isoprene released, the transformant obtained in the step (I) is compared with the wild type, and a transformant having a larger amount of isoprene released than the wild type is selected.

  (I) is a highly reliable method because it directly compares the productivity of useful parts. In the examples described later, it was confirmed that the productivity of useful parts was significantly increased in transformed plants in which the amount of isoprene synthase mRNA expression or protein was higher than that of the wild type and the amount of isoprene released was higher than that of the wild type. Therefore, (ii) to (iv) are also suitable as a method for selecting an individual whose productivity of the useful portion is enhanced. (Ii) and (iii) are advantageous in that they can be carried out by conventional methods, and can be carried out even if the transformant has not grown to a stage where a useful part is produced.

  The present invention also provides a plant having an enhanced productivity of the useful part obtained by the method for producing a plant having an enhanced productivity of the useful part, and a useful part thereof. The plant in which the productivity of the useful part is enhanced and the useful part are as described above.

  The present invention also provides (I) (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase, There is provided a method for increasing the productivity of a useful part of a plant, comprising the step of introducing at least one selected from the group consisting of: The method may further include the step of (d) introducing a polynucleotide encoding isopentenyl diphosphate Δ-isomerase into a plant cell.

  The present invention also includes (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. Provided is a kit for producing a plant with an enhanced productivity of a useful portion, comprising a plant expression plasmid having at least one selected from the above. As the plant expression plasmid, those described above can be used. Here, the plant expression plasmid is not particularly limited as long as the isoprene synthase can be expressed in the plant, and it does not matter whether the isoprene synthase is expressed in organisms other than plants (for example, microorganisms and animals). The plant expression plasmid in the kit may further have (d) a polynucleotide encoding isopentenyl diphosphate Δ-isomerase. Alternatively, the kit may further comprise (d) a plant expression plasmid having a polynucleotide encoding isopentenyl diphosphate Δ-isomerase.

For each operation in the book, Molecular Cloning which is incorporated in its entirety in this _{document: A laboratory Mannual, 2 nd Ed} . (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Each operation may be modified or omitted as appropriate, and necessary operations may be added.

  In this book, various reactions, phenomena, metabolic pathways, etc. are explained, but these explanations do not exclude other possibilities. The present invention is not bound by theory.

  Moreover, those skilled in the art will be able to make various modifications without departing from the spirit of the present invention.

  According to the present invention, a transformed plant in which the productivity of useful parts is increased and a method for producing the same are provided. Furthermore, the present invention also provides a method for producing a plant with enhanced productivity of useful parts, a plant with enhanced productivity of useful parts by the method, and useful parts of the plants. Furthermore, the present invention also provides a method for increasing the productivity of useful parts of a plant and a kit for producing a plant with enhanced productivity of useful parts.

  According to the present invention, selected from the group consisting of (a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) a polynucleotide encoding diphosphomevalonate decarboxylase. In the transformant obtained by transforming with at least one of the above, the productivity of the useful portion thereof is significantly enhanced as compared with the wild type.

  Examples of the present invention will be described below. These examples are for explaining the present invention and do not limit the present invention.

Plant material and growth conditions Populus alba is subcultured in 1% agar medium with Murashige-Skoog (MS) (sucrose 3%, inositol 0.01%, indole butyric acid final concentration 4 μM, pH 5.6-5.8). The planting was carried out about once every two months. The culture conditions were 25 ° C. and long-day conditions (16 hours light period (120 μmolm ⁻² s ⁻¹ ), 8 hours dark period). The amount of the medium was 100 ml per culture flask (900 ml). At the time of planting, the stem portion cut out so as to contain a few side buds was transplanted to a new medium.

The plant body of Arabidopsis (ecotype Col-0) is in an artificial meteorological apparatus set at 21 ° C. and long day conditions (16 hours light period (120 μmol ⁻² s ⁻¹ ), 8 hours dark period) in the case of soil conditions. Grown on. When growing under aseptic conditions, the seed surface is sterilized with a seed sterilization solution (30% [v / v] sodium hypochloride, 0.02% Triton X-100) for 20 minutes, and then washed four times with sterile water. The seeds were sown in 1/2 MS (0.5% sucrose, pH 5.8, 0.8% agar) medium.

Extraction of total RNA from Populus alba Total RNA was extracted according to the protocol of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The sample is P.I. Samples of alba (about 5 cm plant height) green leaves sampled and frozen in liquid nitrogen were used.

P. Reverse Transcription Reaction Using Alba Total RNA 2.4 μg of the total RNA was subjected to a reverse transcription reaction using SuperScript III RNase H ^- reverse transcriptase Kit (Invitrogen, Carlsbad, Calif., USA).

Total RNA (2.4 μg) 12 μl (up with RNase free water)
50 μM oligo (dT) ₂₀ 1 μl
1 mM 10 mM dNTP Mix
14 μl

The above reaction mixture was heated at 65 ° C. for 5 minutes, then transferred to ice, and 5 × First-Strand Buffer 4 μl, 0.1 M DTT 1 μl, RNaseOUT ^™ RNase inhibitor 1 μl, SuperScript ^™ III reverse transfer at 60 ° C. for 60 minutes. Then, reverse transcription reaction was performed. At that time, the reaction solution was first warmed up in a hot water bath and transferred to an air incubator to prevent the reaction solution from evaporating. Thereafter, the enzyme was inactivated by heating in a heat block (70 ° C.) for 15 minutes. After returning to room temperature, 1 μl of RNase H was added to the reaction solution and incubated at 37 ° C. for 20 minutes to decompose the template RNA. It was confirmed that β-actin (beta-actin) as a positive control was amplified using the reverse transcription reaction product as a template.

P. by RT-PCR. Isolation of alba isoprene synthase (PaIspS) gene Reversed using the primer pair (Fw.1, Rv.1) designed based on the sequence of the hybrid poplar (P. tremula x P. alba) isoprene synthase gene RT-PCR was performed with KOD-Plus-DNA polymerase (TOYOBO, Osaka, Japan) using the copy product as a template.

Fw.1: 5'- ggggacaagtttgtacaaaaaagcaggcttc atggcaactgaattattgtgcttgc -3 '
(SEQ ID NO: 1)
Rv.1: 5'- ggggaccactttgtacaagaaagctgggtc ttatctctcaaagggtagaataggctctg -3 '
(SEQ ID NO: 2)
(Underlined sequence is attB site of GATEWAY system for subcloning)

10 x KOD plus buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
50 μM primer (Fw.1, Rv.1) 1 μl each for RT product 1 μl
1 U / μl KOD-Plus-DNA polymerase 1 μl
Measure up to 50 μl with H ₂ O

PCR program
# 1: 95 ° C 4min
# 2: 95 ° C for 1 min
53 ° C 30 sec
68 ℃ 2min
(30 cycles)
# 3: 68 ° C 5min
# 4: 4 ° C ∞

After confirming that a band of the desired size was amplified by agarose electrophoresis, it was subcloned into pDONR221 by BP recombination using GATEWAY ^™ system (Invitrogen), and the sequence was confirmed by sequencing. The obtained construct was designated as pDONR221-PaIspS.

Construction of PaIspS Constitutive Expression Construct Using pDONR221-PaIspS as an entry vector, PaIspS full-length cDNA was transferred to pGWB2 which is a binary destination vector for constitutive expression in plants using GATEWAY ^™ system. The obtained construct was designated as pGWB2-PaIspS. This construct was introduced into Agrobacterium tumefaciens GV3101 (pMP90) by electroporation (GIBCO (Invitrogen), Carlsbad, CA, USA).

Transformation of Arabidopsis thaliana by inflorescence penetrating method (1) Preparation of plant body About one month after sowing, the primary inflorescence of an individual whose primary inflorescence became several centimeters was cut out to induce a secondary inflorescence. Individuals about one week after the primary inflorescence was cut off were used for transformation.
(2) Preparation of Agrobacterium suspension Agrobacterium tumefaciens GV3101 (pMP90) having pGWB2-PaIspS was inoculated into an LB medium containing an antibiotic, and OD ₆₀₀ was changed from 1.2 to 1.5 at 25 ° C. and 200 rpm. Cultured until complete. The culture solution is centrifuged at 4800 rpm for 20 minutes, the supernatant is removed, a 5% sucrose aqueous solution is added so that the OD ₆₀₀ is about 0.8, the cells are suspended, and silwet L-77 (Nihon Unicar) is added to this solution. Was added to a final concentration of 0.05%.
(3) Transformation After turning the plant upside down and immersing it in an Agrobacterium suspension for about 10 seconds, the wild Arabidopsis thaliana strain (Col-0) was tilted sideways and dried for about 10 minutes. This plant was put in a 2 L conical beaker, covered with a wrap and kept in a high humidity state, transferred to an artificial meteorograph and placed for one day. The plant body was taken out of the container, and was allowed to grow for about two weeks after being given water, and then dried to harvest the seed.

Selection of transformants (1) Selection of T ₁ seeds Seeds obtained by transformation were ½ MS (0.5% sucrose, pH 5.8, 0.8% agar, 50 μg / ml kanamycin, 0.05% PPM) plate was seeded with about 2000, and after low temperature treatment for 2 nights, it was grown with an artificial meteorograph. Twenty-nine kanamycin-resistant individuals were transplanted into soil, and when rosette leaves became 8 or more, one of them was sampled and T-DNA was confirmed by genomic PCR.
(2) To confirm the expression of PaIspS gene introduced selection of _{T 2} individuals, it was decided to perform northern analysis using the _{T 2} generation seedling. The sample was using the _{T 2} generation seedling of 2 weeks after sowing. Total RNA was extracted, 7.5 μg of total RNA sample was transferred to HybondN +, and hybridization was performed. The membrane was shaken in 2 × SSC / 0.1% SDS for 15 minutes at room temperature. The washings were discarded and shaken at 60 ° C. for 15 minutes in 2 × SSC / 0.1% SDS preheated to 60 ° C. Further, the washing solution was discarded and the mixture was shaken in 0.2 × SSC / 0.1% SDS at 60 ° C. for 20 minutes. After this washing, the membrane wrapped in the wrap was exposed to BAS imaging plate and X-ray film (2 hours and 24 hours), and the signal was detected.

  As described above, total RNA was extracted from seedlings of Populus alba, and a full-length cDNA of a PaIspS candidate gene was obtained by RT-PCR. This cDNA was expressed using an E. coli expression system, and enzyme activity was measured using various prenyl diphosphates as substrates. Here, the enzyme activity was measured by adding E. coli into which a full-length cDNA of PaIspS candidate gene was introduced into a culture medium for E. coli containing various prenyl diphosphates, and cultivating it with GC using the headspace of this culture solution as a sample. The concentration of isoprene was measured. The obtained product was identified as isoprene, and it was confirmed that this cDNA encodes isoprene synthase (primary screening of cDNA).

  CDNA subjected to primary screening using E. coli was introduced into Arabidopsis thaliana by the Agrobacterium method, and secondary screening was performed by confirming the expression of PaIspS gene and isoprene release by Northern analysis (FIG. 1a, b).

  In Arabidopsis thaliana about 4 weeks after sowing, phenotypic analysis was performed under normal growth conditions. As a result, the high-expressing plant body tended to have an increased leaf size and number compared to the wild type (Fig. 2a). As a result of the actual measurement, the number of rosette leaves of the plant body that highly expressed isoprene synthase was increased 1.3 to 1.5 times on average as compared to the wild type (Fig. 2b). Furthermore, the size of the leaf was also increased 1.3 to 1.4 times in the plants with high expression in both length and width (Fig. 2c, d). As a result, the total amount of fresh weight of the rosette leaves in the plant body was increased 1.9 to 2.2 times in the highly expressing plant body (FIG. 2e). There was no difference in the timing of flower stems.

  It is considered that either the increase in the number of cells or the increase in the cell size, or both the increase in the number of cells and the increase in the cell size contribute to the number and size of the rosette leaves.

  Therefore, in order to investigate the cause of the increase in the size and number of rosette leaves in high-expressing plants, the size of the epidermal cells in the rosette leaves was tested. For the test, rosette leaves immediately after germination and rosette leaves about 4 weeks after sowing were used.

  The size of the rosette leaf epidermal cells immediately after germination was examined. When comparing leaves of the same size, it was found that the high-expressing plant body had a smaller cell size and a larger number than the wild strain. This shows that the number of cells in leaves of the same size is higher in high-expressing plants, and this increase in the number of cells ultimately leads to an increase in the size and number of rosette leaves. The possibility was suggested. The increase in the number of cells suggests that the rate of cell division may be increased.

  On the other hand, the size of the epidermal cells of rosette leaves about 4 weeks after sowing was examined. When comparing leaves of the same area, it was found that the high-expressing plant body had a larger cell size and a smaller number than the wild strain. This indicates that the number of cells in leaves of the same area is less in high-expressing plants, and this increase in cell size may eventually lead to an increase in the size and number of rosette leaves. Sex was suggested. The increase in cell size suggests the possibility of increased cell enlargement.

  Thus, an increase in cell number was confirmed in rosette leaves immediately after germination, whereas an increase in cell size was confirmed in rosette leaves about 4 weeks after sowing.

  The reason why such a phenomenon occurs may be related to the biosynthesis of isoprene from IPP or DMAPP. That is, there is a possibility that the balance of various plant hormones biosynthesized from DMAPP or IPP, such as gibberellin, cytokinin, brassinosteroid, etc. has been altered, affecting cell division, hypertrophy and the like. In other words, it is possible that isoprene itself does not directly affect the cell number or cell size, but indirectly. Although not reported so far, isoprene itself may be a signal that may promote cell division and hypertrophy.

  In recent years, the problem of food shortages due to a decrease in the agricultural population, a decrease in farmland, and an increase in the population has become serious. According to the present invention, the yield of the edible part of the plant can be increased, and food can be efficiently produced, so that the problem of food shortage can be improved.

  In recent years, the problem of depletion of natural resources has become serious. According to the present invention, the yield of useful parts containing a large amount of industrial products and fuel raw materials can be increased, and industrial products and fuels (for example, biomass ethanol that can be used as petroleum substitute fuel) are efficiently produced from plants. This can improve the problem of depletion of natural resources.

FIG. 1a shows the expression level of PalsPs mRNA. It can be determined by Northern analysis that the asterisk is a high expression strain. FIG. 1 b shows the amount of isoprene released from Arabidopsis thaliana with high expression of PalsPs. The amount of isoprene released by PalsPs-highly expressed Arabidopsis thaliana varied depending on the individual, but all showed a tendency to be higher than that of the wild type. The amount of isoprene released was about 200 to 1000%. FIG. 2a is a photograph of rosette leaves. Apparently, the number, length, and area of leaves in the Arabidopsis thaliana strain with high expression of PalsPs are larger than those in the wild strain. FIG. 2b shows the number of rosette leaves. It can be seen that the number of leaves in the PalsPs high expression Arabidopsis thaliana strain is greater than that in the wild strain. FIG. 2c shows the length of the rosette leaves. It can be seen that in the Arabidopsis thaliana strain with high expression of PalsPs, the leaf length is increased compared to the wild strain. FIG. 2d shows the width of the rosette leaf. It can be seen that the leaf size of the PalsPs high-expressing Arabidopsis thaliana strain is larger than that of the wild type. FIG. 2e shows the fresh weight of rosette leaves. It can be seen that the fresh weight of the leaf is increased in the Arabidopsis thaliana strain with high expression of PalsPs than in the wild strain.

SEQ ID NO: 1 is a primer.
SEQ ID NO: 2 is a primer.

Claims (14)

(A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A transformed plant that has been transformed with at least one selected from the group consisting of and improved the productivity of useful parts. The transformed plant according to claim 1, having at least one characteristic selected from the group consisting of the following (1) to (3):
(1) The expression level of at least one selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase is higher than that of the wild type,
(2) the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase is higher than that of the wild type, and
(3) The amount of isoprene released is greater than that of the wild type. The transformed plant according to claim 1 or 2, wherein the amount of isoprene released is 150 to 3000% of the wild type.   The useful portion is at least one selected from the group consisting of leaves, stems, stems, roots, fruits, flowers, seeds, bark and underground stems, or a part thereof. Transformed plants. Furthermore, the transformed plant in any one of Claims 1-4 which has at least 1 characteristic selected from the group which consists of following (A)-(G):
(A) Pesticide resistance is higher than wild type,
(B) Heavy metal resistance is higher than wild type,
(C) pest resistance is higher than wild type,
(D) low temperature tolerance is higher than wild type,
(E) salt tolerance is higher than wild type,
(F) Dry tolerance is higher than wild type and
(G) The weak light resistance is higher than the wild type. At least one application selected from the group consisting of food, food or food composition manufacture, cosmetic manufacture, pharmaceutical manufacture, industrial product manufacture, appreciation, forest regeneration, desert regeneration and phytomediation The transformed plant according to any one of claims 1 to 5, which is used in the above. The transformed plant according to any one of claims 1 to 6, which is a seed, a seedling, a young seedling, a middle seedling, an adult seedling, or a plant body in a growth process therebetween. (A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
The transformed plant according to any one of claims 1 to 7, wherein at least one selected from the group consisting of is homozygous and integrated in a cell chromosome. (I) (a) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A method for producing a plant with enhanced productivity of useful parts, comprising the step of obtaining a transformant by introducing at least one selected from the group consisting of: Furthermore, (II) a step of performing at least one selected from the group consisting of the following (i) to (iv):
(I) For at least one of the weight, size and number of useful parts, the transformant obtained in step (I) is compared with the wild type, and at least one of the weight, size and number of useful parts is Selecting a transformant with a larger than wild type,
(Ii) For at least one expression level selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type And selecting a transformant whose expression level is higher than that of the wild type,
(Iii) For the expression level of at least one mRNA selected from the group consisting of isoprene synthase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase, the transformant obtained in the step (I) and the wild type Comparing the type and selecting a transformant in which the expression level of the mRNA is higher than that of the wild type, and
(Iv) For the amount of isoprene released, the transformant obtained in the step (I) is compared with the wild type, and a transformant having a larger amount of isoprene released than the wild type is selected.
A method for producing a plant with enhanced productivity of a useful part according to claim 9. A plant produced by the method according to claim 9 or 10, wherein productivity of useful parts is enhanced. A useful part of the plant of claim 11. (A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A method for increasing the productivity of a useful part of a plant, comprising a step of introducing at least one selected from the group consisting of: (A) a polynucleotide encoding isoprene synthase,
(B) a polynucleotide encoding 5-phosphomevalonate kinase, and
(C) a polynucleotide encoding diphosphomevalonate decarboxylase,
A kit for producing a plant with an enhanced productivity of useful parts, comprising a plant expression plasmid having at least one selected from the group consisting of:

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