Gene constructs, transformants, methods for producing isoprenoids, methods for producing pneumatic tires, and methods for producing rubber products

Abstract

To provide gene constructs containing promoter base sequences functional in roots of Taraxacum kok-saghyz (TKS).SOLUTION: The present invention relates to a gene construct containing the promoter base sequence of TkOSC2 gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a gene construct, a transformant, a method for producing an isoprenoid, a method for producing a pneumatic tire, and a method for producing a rubber product. [Background technology]

[0002] A promoter is a sequence upstream of a gene that controls the amount and site of expression of the gene.

[0003] In protein expression using transformed (genetically modified) plants, the expression level of the recombinant protein is controlled by the promoter, so the selection of the promoter is important for increasing the productivity of useful proteins and metabolites in the genetically modified plant (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-201357 Summary of the Invention [Problem to be solved by the invention]

[0005] The 35S promoter derived from the cauliflower mosaic virus is commonly used to create transformed (genetically modified) plants. However, it has been reported that the 35S promoter expresses proteins throughout the plant body. Expressing a gene throughout the plant body can cause poor growth and reduce productivity of the target substance. Therefore, to efficiently produce substances using genetically modified plants, it is necessary to select a promoter that allows the recombinant gene to function in the location where it is most effective.

[0006] When producing useful substances in plants whose roots are edible, such as sweet potato (for example, Patent Document 1), or in plants that produce rubber in the root lacteals, such as Taraxacum kokushi (TKS), it is important to make the recombinant gene function in the root.

[0007] Taraxacum kokkaiensis (Tarkasa kousa) (T. kousa) produces rubber in the lacteals of its roots. Therefore, when modifying the quality and quantity of rubber through genetic modification, it is effective to link the recombinant gene downstream of a promoter that functions in the roots and express it in the roots.

[0008] Although various promoters that function in roots have been reported, there are few reports of TKS promoters that function in roots.

[0009] The present invention aims to solve the above problems and to provide a gene construct containing a promoter sequence that functions in the roots of Taraxacum kokushikanensis (TKS). [Means for solving the problem]

[0010] As a result of intensive research to solve the above-mentioned problems, the inventors of the present invention found that the TkOSC2 gene is expressed in the roots of Taraxacum kokkaiensis. They then created a gene construct containing the base sequence of a promoter located upstream of the TkOSC2 gene, incorporated a target gene downstream of the base sequence of the promoter, and expressed it in Taraxacum kokkaiensis. They found that the target gene was expressed primarily in the roots, and thus completed the present invention.

[0011] That is, the present invention relates to a gene construct containing the promoter nucleotide sequence of the TkOSC2 gene. [Effects of the Invention]

[0012] According to the present invention, since the gene construct contains the base sequence of the promoter of the TkOSC2 gene, it is possible to provide a gene construct containing the base sequence of a promoter that functions in the roots of Taraxacum kokkaiensis (TKS).

[0013] The transformant of the present invention is a transformant transformed with the above-mentioned gene construct, and therefore the expression of a desired protein (e.g., a protein involved in polyisoprenoid biosynthesis) is enhanced in the roots of Taraxacum kokushikanensis (TKS).

[0014] The method for producing isoprenoids of the present invention is a method for producing isoprenoids using the transformant, and therefore, it is possible to produce isoprenoids more efficiently.

[0015] The method for producing a pneumatic tire of the present invention includes a step of producing an isoprenoid by the method for producing an isoprenoid, a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture, a raw tire molding step of molding a raw tire from the kneaded mixture, and a vulcanization step of vulcanizing the raw tire.Since pneumatic tires are produced from isoprenoids produced more efficiently, pneumatic tires using isoprenoids can be produced with good productivity.

[0016] The method for producing a rubber product of the present invention includes a step of producing an isoprenoid by the above-described method for producing an isoprenoid, a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture, a raw rubber product molding step of molding the kneaded mixture into a raw rubber product, and a vulcanization step of vulcanizing the raw rubber product.Since rubber products are produced from isoprenoids produced more efficiently, rubber products using isoprenoids can be produced with high productivity. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a photograph showing the state of staining in plant bodies of an example and a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0018] <Gene construct> The gene construct of the present invention contains the promoter nucleotide sequence of the TkOSC2 gene, making it possible to provide a gene construct containing a promoter nucleotide sequence that functions in the roots of Taraxacum kokkaiensis (TKS). The reason why the above effects are obtained in the present invention is not clear, but is presumed to be as follows.

[0019] The TkOSC2 gene is expressed in the roots of Taraxacum kokkai. Therefore, the promoter located upstream of the TkOSC2 gene (the TkOSC2 gene promoter) is a promoter that causes genes located downstream of the promoter to be expressed primarily in the roots. Therefore, when Taraxacum kokkai is transformed with a gene construct incorporating the nucleotide sequence of a promoter located upstream of the TkOSC2 gene and a gene of interest downstream of the promoter nucleotide sequence (i.e., a gene construct containing the TkOSC2 gene promoter and a gene of interest operably linked to the promoter), the gene of interest is thought to be expressed primarily in the roots. As described above, the TkOSC2 gene promoter has promoter activity that causes gene expression primarily in the roots.

[0020] As used herein, the term "TkOSC2 gene" refers to the OSC2 gene derived from Taraxacum kokkuri, which encodes an oxidized squalene cyclase (OSC). OSCs (Oxidosqualene Cyclases) are enzymes involved in the synthesis and modification of pentacyclic triterpenes, which function as antibacterial substances in the defense response against plant pathogens. As used herein, the promoter of the TkOSC2 gene refers to a sequence that is located upstream of the TkOSC2 gene and controls the expression site of the TkOSC2 gene.

[0021] As used herein, a promoter having promoter activity that causes gene expression primarily in roots means that when a desired gene is functionally linked to the promoter and introduced into a plant, the promoter has the activity of regulating gene expression so that the desired gene is expressed primarily in the roots. Here, expression of a gene primarily in the roots refers to a state in which the gene is not expressed at all or very little in parts of the plant other than the roots, and the gene is expressed essentially exclusively in the roots. Furthermore, as used herein, functionally linking a gene to a promoter means linking the gene sequence downstream of the promoter so that it is under the control of the promoter.

[0022] The gene construct of the present invention is not particularly limited as long as it contains the nucleotide sequence of the promoter of the TkOSC2 gene, and examples thereof include a circular recombinant plasmid (vector) and linear DNA. Of these, vectors are preferred because they are in a form that can be introduced into plants. Hereinafter, the case where the gene construct is a vector will be mainly described, but the gene construct is not limited to a vector.

[0023] The vector can be prepared by inserting the promoter sequence of the TkOSC2 gene into a vector generally known as a plant transformation vector by a conventionally known method. Examples of vectors that can be used to prepare the vector include pBI-based vectors, binary vectors such as pGA482, pGAH, and pBIG, intermediate plasmids such as pLGV23Neo, pNCAT, and pMON200, and pH35GS containing a GATEWAY cassette.

[0024] The vector may contain other nucleotide sequences as long as it contains the nucleotide sequence of the TkOSC2 gene promoter. In addition to the nucleotide sequence, a vector usually contains vector-derived sequences, including restriction enzyme recognition sequences, spacer sequences, marker gene sequences, and reporter gene sequences.

[0025] Examples of the marker gene include drug resistance genes such as a kanamycin resistance gene, a hygromycin resistance gene, and a bleomycin resistance gene. The reporter gene is introduced to confirm the expression site in the plant body, and examples of the reporter gene include a luciferase gene, a GUS (β-glucuronidase) gene, a GFP (green fluorescent protein), and an RFP (red fluorescent protein).

[0026] (TkOSC2 gene promoter) The promoter of the TkOSC2 gene is not particularly limited in origin, but is preferably derived from a plant, more preferably from a polyisoprenoid-producing plant, particularly preferably from a plant belonging to the genus Taraxacum, and more preferably from Taraxacum kok-saghyz (Russian dandelion). In this specification, the term "polyisoprenoid-producing plant" refers to a plant capable of producing polyisoprenoids, and specific examples thereof will be described later. Similarly, specific examples of plants belonging to the genus Taraxacum will also be described later.

[0027] The promoter of the TkOSC2 gene is preferably a DNA represented by any one of the following [A1] to [A3]. [A1] DNA consisting of the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 [A2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and has promoter activity that causes gene expression primarily in roots. [A3] DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and having promoter activity that causes gene expression mainly in roots.

[0028] "Hybridizing" as used herein refers to the process of hybridizing DNA to DNA having a specific base sequence or to a portion of the DNA. Therefore, the DNA having the specific base sequence or the base sequence of a portion of the DNA may be useful as a probe for Northern or Southern blot analysis, or may be DNA of a length that can be used as an oligonucleotide primer for PCR (Polymerase Chain Reaction) analysis. DNA used as a probe may be DNA of at least 100 bases, preferably at least 200 bases, and more preferably at least 500 bases, but may also be DNA of at least 10 bases, preferably at least 15 bases.

[0029] Methods for DNA hybridization experiments are well known and are described, for example, in Molecular Cloning, 2nd and 3rd editions (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), and Immunology methods manual, Academic Press (Molecular). Hybridization conditions can be determined and experiments can be performed according to many other standard textbooks.

[0030] Examples of stringent conditions include incubating a DNA-immobilized filter and probe DNA in a solution containing 50% formamide, 5x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA overnight at 42°C, followed by washing the filter in a 0.2x SSC solution at approximately 65°C, although less stringent conditions can also be used. Stringent conditions can be modified by adjusting the formamide concentration (lower formamide concentrations result in lower stringency), salt concentration, and temperature. Low stringency conditions include, for example, incubation overnight at 37°C in a solution containing 6x SSC (20x SSC is 3 mol / L sodium chloride, 0.2 mol / L sodium dihydrogen phosphate, 0.02 mol / L EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / L denatured salmon sperm DNA, followed by washing with a 1x SSC, 0.1% SDS solution at 50°C. Even lower stringency conditions include hybridization under the low stringency conditions described above using a solution with a high salt concentration (e.g., 5x SSC), followed by washing.

[0031] The various conditions described above can also be established by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to adapt the conditions.

[0032] It is also known that, like the DNA that can hybridize under stringent conditions, a promoter nucleotide sequence that shares a certain degree of sequence identity with the original nucleotide sequence may also have promoter activity. In order to maintain promoter activity, the sequence identity with the nucleotide sequence of nucleotides 1-2206 represented by SEQ ID NO: 1 is at least 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0033] The sequence identity of nucleotide sequences or amino acid sequences can be determined using the algorithms BLAST by Karlin and Altschul [Proc. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)].

[0034] Methods for confirming that DNA that hybridizes with the above-mentioned DNA under stringent conditions or DNA that has 80% or more sequence identity with the above-mentioned DNA is DNA that has promoter activity that primarily expresses genes in roots include, for example, conventionally known methods such as reporter assays using β-galactosidase, luciferase, or GFP (Green Fluorescent Protein) as reporter genes.

[0035] The nucleotide sequence of the TkOSC2 gene promoter can be identified by a conventional method, for example, by extracting genomic DNA from a growing plant using the CTAB (Cetyl Trimethyl Ammonium Bromide) method. Next, specific primers and random primers are designed based on the known nucleotide sequence of the TkOSC2 gene, and the extracted genomic DNA is used as a template for TAIL (Thermal Asymmetric Interlaced)-PCR to amplify the gene containing the TkOSC2 gene promoter and identify its nucleotide sequence.

[0036] The gene construct (the vector) preferably contains a promoter for the TkOSC2 gene and a gene of interest (a base sequence) operably linked to the promoter. By transforming Taraxacum kokkaiensis with a gene construct containing the promoter for the TkOSC2 gene and a gene of interest operably linked to the promoter, the gene of interest is expressed primarily in the roots.

[0037] More preferably, the gene construct (the vector) contains the TkOSC2 gene promoter and a gene (nucleotide sequence) operably linked to the promoter that encodes a protein involved in polyisoprenoid biosynthesis. By introducing such a gene construct (vector) into a plant to perform transformation, the gene contained in the gene construct (vector) that encodes a protein involved in polyisoprenoid biosynthesis is expressed primarily in the roots, thereby enabling the production of polyisoprenoids in the plant to be improved. This is presumably because, if the expression of a foreign gene introduced to improve latex productivity is promoted in parts other than the roots, it places a burden on the metabolism of the plant and latex production, resulting in adverse effects.

[0038] (Protein involved in polyisoprenoid biosynthesis) The gene encoding a protein involved in polyisoprenoid biosynthesis is preferably at least one gene selected from the group consisting of a gene encoding farnesyl diphosphate synthase, a gene encoding geranylgeranyl diphosphate synthase, a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a gene encoding isopentenyl diphosphate isomerase, a gene encoding cis-prenyltransferase, a gene encoding small rubber particle protein, and a gene encoding rubber elongation factor. Among these, at least one gene selected from the group consisting of a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a gene encoding isopentenyl diphosphate isomerase, a gene encoding cis-prenyltransferase, and a gene encoding small rubber particle protein is more preferred because it can further improve the production amount of polyisoprenoids, and the gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase or a gene encoding cis-prenyltransferase is even more preferred, with the gene encoding cis-prenyltransferase being particularly preferred.

[0039] The present inventors aimed to create a transformant with improved natural rubber production by enhancing a portion of the polyisoprenoid biosynthetic pathway, thereby increasing polyisoprenoid production. First, within the polyisoprenoid biosynthetic pathway, two pathways for biosynthesis of isoprenyl diphosphate (IPP), one of the key substances, are known: the mevalonate pathway (MVA pathway) and the non-mevalonate pathway (MEP pathway). Focusing on the MVA pathway, several proteins expected to play important roles were selected from among the various proteins involved in the polyisoprenoid biosynthetic pathway in order to enhance the MVA pathway and a portion of the downstream pathway.

[0040] Specifically, we selected seven proteins: farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS), which are prenyltransferases involved in the reaction of linking IPP to an allylic substrate and sequentially connecting isoprene units. These enzymes synthesize farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which are thought to be the starting substrates of natural rubber; 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which is the rate-limiting step in the MVA pathway; isopentenyl diphosphate isomerase (IPI), which is involved in the isomerization of IPP and dimethylallyl diphosphate (DMAPP); cis-prenyltransferase (CPT), which is thought to be involved in the chain elongation of isoprenoid compounds; and small rubber particle protein (SRPP) and rubber elongation factor (REF), which are known to be involved in the biosynthesis of polyisoprenoids. Then, by constructing a gene construct (vector) containing a base sequence in which each of the genes encoding these proteins was linked so that they were under the control of the TkOSC2 gene promoter, and by introducing the constructed gene construct (vector) into a plant, it was possible to increase the production of polyisoprenoids in the plant.

[0041] As described above, in the polyisoprenoid biosynthetic pathway, a reaction proceeds in which isopentenyl diphosphate (IPP) is sequentially linked to an allylic substrate. The enzymes that catalyze this reaction are collectively referred to as prenyltransferases, since they sequentially link isoprene units. In this specification, prenyltransferase refers to a generic term for enzymes that catalyze the condensation reaction between IPP and isoprenyl diphosphate (n isoprene units) (allylic substrate) to synthesize a new isoprenyl diphosphate (n+1 isoprene units) with one additional isoprene unit.

[0042] Prenyltransferases are a group of enzymes that synthesize various isoprenyl diphosphates, such as geranyl diphosphate (GPP: C10), farnesyl diphosphate (FPP: C15), geranylgeranyl diphosphate (GGPP: C20), geranylfarnesyl diphosphate (GFPP: C25), and hexaprenyl diphosphate (HPP: C30), which are the basic precursors of each terpenoid, by linking isoprene units. Farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are categorized as prenyltransferases.

[0043] In this specification, farnesyl diphosphate synthase (FPS) is an enzyme that catalyzes the biosynthesis of farnesyl diphosphate (FPP) using isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and geranyl diphosphate (GPP) as substrates. In addition, in this specification, geranylgeranyl diphosphate synthase (GGPS) is an enzyme that catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP) using isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), and farnesyl diphosphate (FPP) as substrates.

[0044] Furthermore, in this specification, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) is one of the rate-limiting enzymes in the mevalonate pathway (MVA pathway) and includes both 3-hydroxy-3-methylglutaryl-CoA reductase (NADPH) (EC 1.1.1.34) and 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.88). In addition, in this specification, isopentenyl diphosphate isomerase (IPP isomerase, IPI) is an enzyme that catalyzes the isomerization reaction between isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). Furthermore, as used herein, cis-prenyltransferase (cis-prenyltransferase, CPT) is an enzyme that catalyzes a reaction that elongates the chain length of an isoprenoid compound to a cis form. Here, as used herein, an isoprenoid compound refers to a compound having an isoprene unit (C5H8). Furthermore, a cis-isoprenoid is a compound having an isoprenoid compound in which the isoprene units are bonded in a cis form, and examples thereof include cis-farnesyl diphosphate, undecaprenyl diphosphate, and natural rubber. In addition, in this specification, Small Rubber Particle Protein (SRPP) refers to a rubber particle-binding protein that binds to rubber particles present in the latex of polyisoprenoid-producing plants such as Hevea brasiliensis. In addition, in this specification, Rubber Elongation Factor (REF) refers to a rubber particle-binding protein that binds to rubber particles present in the latex of polyisoprenoid-producing plants such as Hevea brasiliensis.

[0045] ((gene)) The genes encoding the farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, isopentenyl diphosphate isomerase, cis-prenyltransferase, small rubber particle protein, and rubber elongation factor are not particularly limited in origin, and are preferably derived from plants, more preferably from polyisoprenoid-producing plants. Among these, those derived from plants belonging to the genus Taraxacum are preferred, and those derived from Taraxacum kok-saghyz (Russian dandelion) are more preferred.

[0046] The gene encoding the cis-prenyltransferase preferably consists of DNA described in any one of [B1] to [B3] below. [B1] DNA consisting of the base sequence of base numbers 1-927 represented by SEQ ID NO: 2 [B2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1 to 927 represented by SEQ ID NO: 2 and encodes a protein having an enzyme activity that catalyzes a reaction that elongates the chain length of an isoprenoid compound in cis form. [B3] DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-927 represented by SEQ ID NO: 2, and encoding a protein having an enzyme activity that catalyzes a reaction that elongates the chain length of an isoprenoid compound in cis form.

[0047] The term "hybridize" as used herein is the same as that described above. The stringent conditions are also the same as those described above.

[0048] It is also known that the base sequence of a gene encoding a certain protein, such as the DNA hybridizable under stringent conditions, may have the same enzymatic activity if it has a certain degree of sequence identity with the original base sequence. In order to maintain enzymatic activity, the sequence identity with the base sequence of bases 1-927 represented by SEQ ID NO: 2 is at least 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0049] A conventionally known method can be used to confirm that DNA that hybridizes with the above-mentioned DNA under stringent conditions or DNA that has 80% or more sequence identity with the above-mentioned DNA is DNA that encodes a protein having a function such as a predetermined enzyme activity. For example, a method in which the target protein is expressed using a transformant containing a gene encoding the target protein using Escherichia coli or the like, and the presence or absence of the function of the target protein is determined by measuring the activity or the like using the respective activity measurement method.

[0050] Furthermore, methods for identifying the nucleotide sequence of the gene encoding the protein and the amino acid sequence of the protein can be conventionally known. For example, total RNA is extracted from a growing plant, mRNA is purified as needed, and cDNA is synthesized by reverse transcription. Next, degenerate primers are designed based on the amino acid sequence of a known protein corresponding to the target protein, and RT-PCR is performed to partially amplify DNA fragments and partially identify the sequence. Next, the full-length nucleotide sequence and amino acid sequence are identified by a method such as RACE. The RACE method (Rapid Amplification of cDNA Ends) is a method in which, when the nucleotide sequence of a cDNA is partially known, PCR is performed based on the nucleotide sequence information of the known region to clone the unknown region up to the end of the cDNA. This method allows full-length cDNA to be cloned by PCR without the need to prepare a cDNA library. It is preferable that the degenerate primers are prepared from plant-derived sequences having a sequence site highly common to the target protein. Furthermore, when the nucleotide sequence encoding the protein is known, a primer containing a start codon and a primer containing a stop codon can be designed from the known nucleotide sequence, and the full-length nucleotide sequence and amino acid sequence can be identified by performing RT-PCR using the synthesized cDNA as a template.

[0051] <Transformants> By transforming Taraxacum kokkuri using a gene construct containing the TkOSC2 gene promoter and a gene of interest operably linked to the promoter, i.e., by introducing the gene construct (vector) into the plant, an organism (transformant) transformed so that the gene of interest is expressed primarily in the roots can be obtained. In the transformant, the protein encoded by the gene of interest is expressed primarily in the roots, and thus the function of the protein, such as a specific enzymatic activity, is newly enhanced in the roots within the plant into which the gene construct (vector) has been introduced.

[0052] A gene construct (vector) containing the TkOSC2 gene promoter and a gene (nucleotide sequence) encoding a protein involved in polyisoprenoid biosynthesis operably linked to the promoter is introduced into a plant to perform transformation, i.e., by introducing the gene construct (vector) into a plant, a transformed organism (transformant) can be obtained in which the gene encoding the protein involved in polyisoprenoid biosynthesis contained in the gene construct (vector) is expressed primarily in the roots. In the transformant, the predetermined protein involved in polyisoprenoid biosynthesis is expressed primarily in the roots, and thereby the function of the predetermined enzyme activity, etc., of the protein is newly enhanced in the roots within the plant into which the gene construct (vector) has been introduced, thereby enhancing a part of the polyisoprenoid biosynthetic pathway, thereby resulting in an increase in the amount of polyisoprenoids produced in the plant.

[0053] The host used in the transformant is not particularly limited as long as it is a plant, but is preferably a plant capable of producing polyisoprenoids, and examples thereof include plants of the genus Hevea such as rubber tree (Hevea brasiliensis); plants of the genus Sonchus such as sowweed (Sonchus oleraceus), sowweed (Sonchus asper), and sowweed (Sonchus brachyotus); solidago altissima, goldenrod (Solidago virgaurea subsp. asiatica), mountain goldenrod (Solidago virgaurea subsp. leipcarpa), kirigamine goldenrod (Solidago virgaurea subsp. leipc arpaf. paludosa), giant goldenrod (Solidago virgaurea subsp. gigantea), and giant goldenrod (Solidago gigantea). genus Solidago, such as sunflower (Helianthus annuus), white-barked sunflower (Helianthus argophyllus), Helianthus atrorubens, dwarf sunflower (Helianthus debilis), little sunflower (Helianthus decapetalus), and giant sunflower (Helianthus giganteus); genus Helianthus, such as dandelion (Taraxacum), Siberian dandelion (Taraxacum venustum H. Koidz), Japanese dandelion (Taraxacum hondoense Nakai), Kanto dandelion (Taraxacum platycarpum Dahlst), Kansai dandelion (Taraxacum japonicum), and common dandelion (Taraxacum officinale genus Taraxacum, such as Ficus Weber, Taraxacum koksaghyz, and the like; fig (Ficus carica), rubber tree (Ficus elastica), and ficus pumila L.Ficus genus, such as Ficus erecta Thumb., Ficus ampelas Burm.f., Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa Lf., Ficus septica Burm.f., and Ficus benghalensis; Parthenium genus, such as guayule (Parthenium argentatum), Parthenium hysterophorus, and ragweed (Parthenium hysterophorus); lettuce (Lactuca serriola), and Ficus benghalensis. Among these, plants belonging to the genus Taraxacum are preferred, with Taraxacum kok-saghyz (Russian dandelion) being more preferred.

[0054] The gene construct (vector) can be introduced into a plant by any method for introducing DNA into plant cells, such as a method using Agrobacterium (Japanese Patent Laid-Open Nos. 59-140885, 60-70080, and WO 94 / 00977), electroporation (Japanese Patent Laid-Open No. 60-251887), or a method using a particle gun (gene gun) (Japanese Patent Nos. 2606856 and 2517813). Of these, it is preferable to use the Agrobacterium method to introduce the gene construct (vector) into a plant and produce a transformant. In this case, the gene construct (vector) is introduced into Agrobacterium, and the Agrobacterium is cultured and grown by a conventional method (for example, by shaking culture in YEB medium at a culture temperature of 20 to 35°C for 10 to 30 hours), and then the Agrobacterium is infected into a callus, a plant tissue fragment, or a young plant, thereby producing a transformant into which a predetermined gene contained in the gene construct (vector) has been introduced.

[0055] Agrobacterium bacteria containing the above-mentioned gene construct (vector) can be prepared by conventionally known methods, including, for example, a method in which a recombinant vector is prepared by inserting the nucleotide sequence of the promoter of the TkOSC2 gene and the nucleotide sequence of a gene encoding a protein involved in polyisoprenoid biosynthesis into a plasmid capable of homologous recombination with the T-DNA region of a Ti plasmid carried by Agrobacterium bacteria, and then introducing the vector into Agrobacterium bacteria; and a method in which a recombinant binary vector is prepared by inserting the nucleotide sequence of the promoter of the TkOSC2 gene and the nucleotide sequence of a gene encoding a protein involved in polyisoprenoid biosynthesis into the above-mentioned binary vector, and then introducing the vector into Agrobacterium bacteria. Furthermore, the Agrobacterium genus bacteria that can be used include Agrobacterium tumefaciens strains (C58, LBA4404, EHA101, EHA105, C58C1RifR, GV3101, etc.).

[0056] By the above-mentioned methods, the transformant (transformed plant cell) can be obtained.

[0057] The transformant may be a transformed plant into which the gene construct (vector) has been introduced. The transformed plant is not particularly limited as long as it is a plant containing transformed plant cells. For example, the transformed plant encompasses not only the transformed plant cells obtained by the above-described method, but also their progeny or clones, and further progeny plants obtained by subcultivating them. Once a transformed plant cell is obtained in which the nucleotide sequence of the promoter of the TkOSC2 gene and the nucleotide sequence of a gene encoding a protein involved in polyisoprenoid biosynthesis contained in the gene construct (vector) have been introduced into its genome, progeny or clones can be obtained from the transformed plant cell by sexual reproduction, asexual reproduction, tissue culture, cell culture, cell fusion, or the like. Furthermore, propagation materials (e.g., seeds, fruits, cuttings, tubers, tuberous roots, stumps, adventitious buds, adventitious embryos, callus, protoplasts, etc.) can be obtained from the transformed plant cell or its progeny or clone, and the plant can be mass-produced based on these materials.

[0058] Methods for regenerating plants (transformed plants) from transformed plant cells are known, for example, the method of Doi et al. (JP 2000-316403 A) for eucalyptus, the method of Fujimura et al. (Fujimura et al. (1995), Plant Tissue Culture Lett., vol. 2: p. 74-) for rice, the method of Shillito et al. (Shillito et al. (1989), Bio / Technology, vol. 7: p. 581-) for maize, the method of Visser et al. (Visser et al. (1989), Theor. Appl. Genet., vol. 78: p. 589-) for potato, and the method of Akama et al. (Akama et al. (1992), Plant Cell Rep., vol. 12: p. 7-) for Arabidopsis. Those skilled in the art will be able to regenerate plants from transformed plant cells by referring to these methods.

[0059] An example of a method for producing a transformed plant of the present invention will be specifically described below. One example of a method for producing a transformed plant of the present invention includes an infection step in which callus obtained by culturing plant-derived tissue under callus induction conditions for 5 to 9 weeks (induction step) is infected with Agrobacterium containing the vector; a selective culture step in which the vector-introduced callus is selectively grown; and a regeneration induction step in which somatic embryos are induced from the callus. In this production method, transformed plant cells (transformed callus) are produced by the infection step and the selective culture step, and then somatic embryos are induced from the callus by the regeneration induction step, and the somatic embryos are cultured to regenerate the callus into a plant, thereby producing a transformed plant. More specifically, a method for regenerating callus into a plant involves inducing somatic embryos from callus, culturing the somatic embryos to form shoots, and culturing the shoots to regenerate the callus into a plant.

[0060] More specifically, the method preferably comprises the following steps: an infection step of infecting callus obtained by culturing plant-derived tissue under callus induction conditions for 5 to 9 weeks with Agrobacterium containing the vector; a selective culture step of selectively growing the vector-introduced callus; a regeneration induction step of culturing the callus in a regeneration-inducing medium to form somatic embryos and shoots; and a rooting step of culturing the shoots in a rooting medium to cause them to root. More preferably, the method comprises the following steps: an infection step of infecting callus obtained by culturing plant-derived tissue under callus induction conditions for 5 to 9 weeks with Agrobacterium containing the vector; a selective culture step of selectively growing the vector-introduced callus; a regeneration induction step of culturing the callus in a regeneration-inducing medium to form somatic embryos and shoots; an elongation step of culturing the formed shoots in an elongation medium to elongate them; and a rooting step of culturing the elongated shoots in a rooting medium to cause them to root.

[0061] Each step of the method for producing a transformed plant can be carried out by a known method, for example, according to the method described in JP 2016-154455 A.

[0062] In the regenerated plant, expression of the gene for the protein of interest can be confirmed by well-known techniques, for example, by Western blot analysis.

[0063] <Method of producing isoprenoids> By introducing a gene construct (vector) containing the TkOSC2 gene promoter and a gene (nucleotide sequence) operably linked to the promoter that encodes a protein involved in polyisoprenoid biosynthesis, the gene contained in the vector encoding the protein involved in polyisoprenoid biosynthesis is expressed primarily in the roots, thereby improving polyisoprenoid production in the plant. Specifically, isoprenoids such as polyisoprenoids can be produced by culturing transformed plant cells obtained by the above-described method, callus obtained from the transformed plant cells, cells redifferentiated from the callus, etc. in an appropriate medium, or by growing transformed plants regenerated from the transformed plant cells, or plants obtained from seeds obtained from the transformed plants, etc. under appropriate cultivation conditions. In the transformed plant, the introduced protein enhances a portion of the polyisoprenoid biosynthetic pathway in the roots, allowing the protein (enzyme) to function, thereby improving the amount of biosynthesis of the compound in the roots and ultimately improving polyisoprenoid productivity in the plant. As described above, isoprenoids such as polyisoprenoids can be produced using the transformants.

[0064] In this specification, the term "isoprenoid compound" refers to a compound having an isoprene unit (C5H8), and the term "polyisoprenoid" is a general term for polymers composed of isoprene units (C5H8). Examples of polyisoprenoids include monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesterterpenes (C 25 ), triterpenes (C 30 ), tetraterpenes (C 40), natural rubber, farnesyl diphosphate, geranylgeranyl diphosphate, etc. Among these, polymers composed of isoprene units and having a weight-average molecular weight of 1,000 or more are preferred, polymers composed of isoprene units and having a weight-average molecular weight of 10,000 or more are more preferred, and polymers composed of isoprene units and having a weight-average molecular weight of 100,000 or more are even more preferred. The weight-average molecular weight (Mw) of the polyisoprenoid can be measured by gel permeation chromatography (GPC) under the following conditions (1) to (7). (1) Equipment: Tosoh HLC-8020 (2) Separation column: Tosoh GMH-XL (3) Measurement temperature: 40℃ (4) Carrier: Tetrahydrofuran (5)Flow rate: 0.6ml / min (6) Detector: Differential refraction, UV (7) Molecular weight standard: Standard polystyrene

[0065] In producing an isoprenoid (preferably a polyisoprenoid), the isoprenoid (preferably a polyisoprenoid) may be recovered from the transformant according to a known method. When the transformant is a plant, the isoprenoid (preferably a polyisoprenoid) may be recovered from the roots of the plant according to a known method.

[0066] Furthermore, the isoprenoid (preferably polyisoprenoid) obtained by the above-described method for producing an isoprenoid can be recovered as a solid by further subjecting it to the following solidification step.

[0067] In the solidification step, the solidification method is not particularly limited, and examples thereof include a method of adding an isoprenoid (preferably a polyisoprenoid) to a solvent that does not dissolve the isoprenoid (preferably a polyisoprenoid), such as ethanol, methanol, or acetone, or a method of adding an acid to an isoprenoid (preferably a polyisoprenoid) solution. By performing the solidification step, the rubber can be recovered as a solid. The obtained rubber can be dried before use, if necessary.

[0068] <Manufacturing methods for rubber products> The method for producing a rubber product includes a step of producing an isoprenoid (preferably a polyisoprenoid) by the method for producing an isoprenoid, a kneading step of kneading the isoprenoid (preferably a polyisoprenoid) obtained by the method for producing an isoprenoid with additives to obtain a kneaded mixture, a raw rubber product molding step of molding the kneaded mixture into a raw rubber product, and a vulcanization step of vulcanizing the raw rubber product.

[0069] The rubber product is not particularly limited as long as it can be produced using rubber (preferably natural rubber), and examples include pneumatic tires, rubber rollers, rubber fenders, gloves, and medical rubber tubes.

[0070] When the rubber product is a pneumatic tire, i.e., when the rubber product manufacturing method is a pneumatic tire manufacturing method, the raw rubber product molding step corresponds to a raw tire molding step of molding a raw tire from the kneaded mixture, and the vulcanization step corresponds to a vulcanization step of vulcanizing the raw tire. That is, the pneumatic tire manufacturing method is a pneumatic tire manufacturing method including: a step of manufacturing an isoprenoid (preferably a polyisoprenoid) by the isoprenoid manufacturing method, a kneading step of kneading the isoprenoid (preferably a polyisoprenoid) obtained by the isoprenoid manufacturing method with additives to obtain a kneaded mixture, a raw tire molding step of molding a raw tire from the kneaded mixture, and a vulcanization step of vulcanizing the raw tire.

[0071] (Kneading process) In the kneading step, the isoprenoid (preferably polyisoprenoid) obtained by the method for producing an isoprenoid is kneaded with additives to obtain a kneaded product.

[0072] The additives are not particularly limited, and additives used in the production of rubber products can be used. For example, when the rubber product is a pneumatic tire, examples include rubber components other than the isoprenoid (preferably polyisoprenoid), reinforcing fillers such as carbon black, silica, calcium carbonate, alumina, clay, and talc, silane coupling agents, zinc oxide, stearic acid, processing aids, various antioxidants, softeners such as oil, wax, vulcanizing agents such as sulfur, and vulcanization accelerators.

[0073] The kneading in the kneading step may be carried out using a rubber kneading machine such as an open roll, a Banbury mixer, or an internal kneader.

[0074] (Raw rubber product molding process (raw tire molding process in the case of tires)) In the raw rubber product molding process, a raw rubber product (a raw tire in the case of a tire) is molded from the kneaded material obtained in the kneading process. The method for molding the raw rubber product is not particularly limited, and any method commonly used for molding raw rubber products may be applied as appropriate. For example, when the rubber product is a pneumatic tire, the kneaded product obtained in the kneading step may be extruded to match the shapes of the respective tire components, molded in a conventional manner on a tire building machine, and the respective tire components may be bonded together to form a raw tire (unvulcanized tire).

[0075] (Vulcanization process) In the vulcanization step, the raw rubber product obtained in the raw rubber product molding step is vulcanized to obtain a rubber product. The method for vulcanizing the raw rubber product is not particularly limited, and any method commonly used for vulcanizing raw rubber products may be applied as appropriate. For example, when the rubber product is a pneumatic tire, the raw tire (unvulcanized tire) obtained in the raw rubber product molding step is vulcanized by heating and pressurizing it in a vulcanizer to obtain a pneumatic tire. [Example]

[0076] The present invention will be specifically described based on examples (Examples) that are considered to be preferable when carrying out the present invention, but the present invention is not limited to these examples.

[0077] First, an overview of each example and comparative example will be described below. Example 1: Recombinant TKS plant (proTkOSC2-GUS, using the promoter of the TkOSC2 gene) Comparative Example 1: Recombinant TKS plant (using the 35S promoter of cauliflower mosaic virus) Comparative Example 2: Non-recombinant TKS plant (control) In Example 1 and Comparative Example 1, a GUS gene, which is a marker gene, is linked downstream of the promoter to transform a plant, and the site where the marker gene is introduced is identified. When the GUS gene is introduced into a plant, the tissues where GUS is functioning turn blue when a specific substrate is added. Therefore, the parts where the gene is functioning turn blue, while the parts where it is not functioning do not. This makes it possible to determine where the gene is functioning.

[0078] Example 1 Step 1 (Promoter identification) Based on literature and the results of gene (RNA) expression analysis, we searched for genes that function in the roots and latex of Russian dandelion (TKS), and based on information from Putter et al. (Scientific Reports (2019)), we identified the TkOSC2 gene as a gene that functions in the roots. Based on information from the Genome Warehouse, we identified the promoter region of the TkOSC2 gene as the promoter sequence (DNA consisting of the base sequence 1-2206 represented by SEQ ID NO: 1), 1 kb upstream of the gene.

[0079] Step 2 (obtaining promoter DNA) The promoter DNA of TkOSC2 is obtained by artificial synthesis of nucleic acids.

[0080] Step 3 (Construction of recombinant vector) A cassette (proTkOSC2::GUS) linking the TkOSC2 promoter and marker gene (GUS) is inserted into a vector (pCAMBIA2300) to create a recombinant vector (pCAMBIA2300 / proTkOSC2-GUS-NOSter).

[0081] Step 4 (Recombining the recombinant vector into Agrobacterium (Agro)) The recombinant vector (pCAMBIA2300 / proTkOSC2-GUS-NOSter) is introduced into Agrobacterium by electroporation. The Agrobacterium containing the recombinant vector is recovered in SOC medium in the dark (28°C, 170 rpm, 1 hour), and then statically cultured in LB medium (50 mg / L kanamycin) (28°C, 3 days, in the dark). Transformed Agrobacterium is confirmed by PCR.

[0082] Step 5 (Growth of Agrobacterium) The transformed Agrobacterium is pre-cultured in the dark in LB medium (50 mg / L kanamycin) at 28°C, 170 rpm, for 1 day. The pre-cultured culture is then scaled up and cultured in LB medium (50 mg / L kanamycin). To scale up, the pre-cultured solution is diluted 10-fold with LB medium and cultured in the dark at 28°C, 170 rpm, and culture is continued until the turbidity of the bacterial solution reaches OD600 = 0.6.

[0083] Step 6 (Preparation of Agrobacterium stock solution) When the turbidity of the bacterial solution reaches OD600 = 0.6, centrifuge (room temperature, 4000 rpm, 10 minutes). Remove the supernatant and resuspend in an equal volume of MS medium to prepare the Agro-bacterial solution.

[0084] Step 7 (Preparation of Agroinfection Solution) Adjust the turbidity (OD600) of the stock solution of Agro bacteria prepared in step 6 to 0.01 to use as the Agro infection solution.

[0085] Step 8 (Preparing leaf segments for infection) Cut leaf segments measuring 0.2-0.5 cm from the leaves of a young T. koreana (T. sieboldii) culture strain using a scalpel. When preparing the leaf segments with a scalpel, place them on filter paper soaked in MS liquid medium to prevent them from drying out. Immediately immerse the prepared segments in the Agroinfection solution prepared in step 7.

[0086] Step 9 (Agroinfection of leaf segments) The leaf segments are immersed in the Agroinfection solution for 30 minutes to infect the leaf segments with Agrobacterium.

[0087] Step 10 (Co-cultivation of leaf segments and Agro The leaf segments immersed in the agroinfection solution are cultured in the dark (28°C, 3 days) on MS medium (1 mg / L benzyladenine (BA), 0.1 mg / L naphthaleneacetic acid (NAA)).

[0088] Step 11 (Washing the co-cultured leaf segments) The co-cultured leaf segments are washed with MS medium (25 mg / L meropene).

[0089] Step 12 (Recovery culture) The washed leaf segments are cultured in MS medium (0.5 mg / L BA, 0.1 mg / L NAA, 25 mg / L meropenem) (16-hour light period, 22°C, 1 week).

[0090] Step 13 (selective culture, callus formation) The recovered leaf segments are cultured in MS medium (0.5 mg / L BA, 0.1 mg / L NAA, 50 mg / L kanamycin, 25 mg / L meropenem) (16-hour photoperiod, 22°C, 2-3 weeks) to form callus.

[0091] Step 14 (Selective culture, callus cultivation, leaf and above-ground regeneration) The formed callus is cultured (16 hours photoperiod, 22°C, 6 weeks) on MS medium (0.5 mg / L BA, 0.1 mg / L NAA, 50 mg / L kanamycin, 25 mg / L meropenem) to regenerate the above-ground parts (elongate shoots).

[0092] Step 15 (Rooting the shoots to obtain recombinant TKS plants (proTkOSC2-GUS)) The elongated shoots are cultured in MS medium for rooting (16-hour photoperiod, 22°C, 4 weeks) to allow rooting, and recombinant TKS plants (proTkOSC2-GUS) are obtained.

[0093] Step 16 (Preparation of GUS activity detection solution) Prepare a substrate stock solution (20 mg X-Gluc dissolved in 1 mL dimethylformamide). Prepare an extraction buffer (50 mM sodium dihydrogen phosphate (pH 7), 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauroyl sarcosinate). Add 26 μL of the substrate stock solution to 1 mL of extraction buffer to prepare a 1 mM X-Gluc solution, which will serve as the GUS activity detection solution.

[0094] Step 17 (Infiltrating the GUS activity detection solution into the plant tissue by reducing the pressure) Cut the plant leaves into pieces approximately 0.5-1 cm square and the roots into pieces approximately 0.5-1 cm long, then place them in a microtube containing 500 μL of 1 mM X-Gluc solution and mix gently. Open the lid of the polycarbonate desiccator and place the rack containing the tubes inside. Leave the tube caps open. Close the lid of the polycarbonate desiccator and open the stopcock. Attach the piping hose from the vacuum pump to the stopcock and turn the power switch ON. Reduce the pressure for 5 minutes. After 5 minutes have passed, close the stopcock of the polycarbonate desiccator and turn the power switch of the vacuum pump OFF. Slowly remove the piping hose from the vacuum pump. Gradually open the stopcock of the polycarbonate desiccator. When the vacuum reaches 0 MPa, open the lid of the polycarbonate desiccator and remove the sample.

[0095] Step 18 (GUS staining of plant tissue) The sample is incubated at 37°C for the required time. The sample is then removed, the reaction mixture is extracted with a micropipette, and 1 mL of 70% ethanol is added. Repeat this process until the solution becomes clear.

[0096] Step 19 (Observation of GUS-stained tissue) Samples in which GUS staining (blue pigment) is observed are observed under a microscope. If GUS staining is faint inside the tissue, it is difficult to see from the outside, so slices are prepared and observed under a stereomicroscope. The tissue in which blue staining is observed is considered to be the functional site of the promoter.

[0097] (Comparative Example 1) The same procedure as in Example 1 was carried out, except that the promoter sequence of the TkOSC2 gene in Example 1 was replaced with the 35S promoter sequence of the cauliflower mosaic virus.

[0098] (Comparative Example 2) This is carried out by subjecting non-recombinant Taraxacum kokkaiensis (TKS) to the procedures in Example 1 from step 16 onwards.

[0099] The results of the Examples and Comparative Examples are shown in Figure 1. As shown in Figure 1, in the recombinant TKS plant of Example 1 (proTkOSC2-GUS, using the TkOSC2 gene promoter), staining was weak in the leaves but strong in the roots, indicating that the target gene is mainly expressed in the roots. On the other hand, in the recombinant TKS plant of Comparative Example 1 (using the 35S promoter of cauliflower mosaic virus), the entire plant was stained, indicating that the target gene was expressed throughout the entire plant. In the non-recombinant TKS plant of Comparative Example 2, no marker gene was introduced, and therefore no staining was observed anywhere in the plant body.

[0100] It has been found that a gene construct containing the promoter base sequence of the TkOSC2 gene makes it possible to provide a gene construct containing the promoter base sequence that functions (mainly) in the roots of Taraxacum kokkaiensis (TKS).

[0101] The present invention (1) relates to a gene construct containing the promoter sequence of the TkOSC2 gene.

[0102] The present invention (2) relates to the gene construct according to the present invention (1), wherein the promoter of the TkOSC2 gene comprises a DNA according to any one of [A1] to [A3] below. [A1] DNA consisting of the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 [A2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and has promoter activity that causes gene expression primarily in roots. [A3] DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and having promoter activity that causes gene expression mainly in roots.

[0103] The present invention (3) relates to a gene construct according to the present invention (1) or (2), which comprises a promoter of the TkOSC2 gene and a gene encoding a protein involved in polyisoprenoid biosynthesis operably linked to the promoter.

[0104] The present invention (4) relates to the gene construct according to the present invention (3), wherein the gene encoding a protein involved in polyisoprenoid biosynthesis is at least one gene selected from the group consisting of a gene encoding farnesyl diphosphate synthase, a gene encoding geranylgeranyl diphosphate synthase, a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a gene encoding isopentenyl diphosphate isomerase, a gene encoding cis-prenyltransferase, a gene encoding small rubber particle protein, and a gene encoding rubber elongation factor.

[0105] The present invention (5) relates to the gene construct according to the present invention (3), wherein the gene encoding a protein involved in polyisoprenoid biosynthesis is a gene encoding cis-prenyltransferase.

[0106] The present invention (6) relates to the gene construct according to the present invention (5), wherein the gene encoding the cis-prenyltransferase comprises a DNA according to any one of [B1] to [B3] below: [B1] DNA consisting of the base sequence of base numbers 1-927 represented by SEQ ID NO: 2 [B2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1 to 927 represented by SEQ ID NO: 2 and encodes a protein having an enzyme activity that catalyzes a reaction that elongates the chain length of an isoprenoid compound in cis form. [B3] DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-927 represented by SEQ ID NO: 2, and encoding a protein having an enzyme activity that catalyzes a reaction that elongates the chain length of an isoprenoid compound in cis form.

[0107] The present invention (7) relates to a transformant transformed with the gene construct according to any one of the present inventions (1) to (6).

[0108] The present invention (8) relates to the transformant according to the present invention (7), which is a plant belonging to the genus Taraxacum.

[0109] The present invention (9) relates to the transformant according to the present invention (7), which is Taraxacum kok-saghyz (Russian dandelion).

[0110] The present invention (10) relates to a method for producing an isoprenoid using the transformant according to any one of the present inventions (7) to (9).

[0111] The present invention (11) relates to a method for producing a pneumatic tire, including a step of producing an isoprenoid by the method for producing an isoprenoid according to the present invention (10), a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture, a raw tire molding step of molding a raw tire from the kneaded mixture, and a vulcanization step of vulcanizing the raw tire.

[0112] The present invention (12) relates to a method for producing a rubber product, including a step of producing an isoprenoid by the method for producing an isoprenoid according to the present invention (10), a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture, a raw rubber product molding step of molding the kneaded mixture into a raw rubber product, and a vulcanization step of vulcanizing the raw rubber product.

Claims

1. A gene construct containing the promoter sequence of the TkOSC2 gene.

2. 2. The gene construct according to claim 1, wherein the promoter of the TkOSC2 gene comprises a DNA according to any one of [A1] to [A3] below. [A1] DNA consisting of the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 [A2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and has promoter activity that causes gene expression primarily in roots. [A3] DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-2206 represented by SEQ ID NO: 1 and having promoter activity that causes gene expression mainly in roots.

3. 2. The gene construct according to claim 1, comprising a promoter for the TkOSC2 gene and a gene encoding a protein involved in polyisoprenoid biosynthesis operably linked to the promoter.

4. The gene construct according to claim 3, wherein the gene encoding a protein involved in polyisoprenoid biosynthesis is at least one gene selected from the group consisting of a gene encoding farnesyl diphosphate synthase, a gene encoding geranylgeranyl diphosphate synthase, a gene encoding 3-hydroxy-3-methylglutaryl CoA reductase, a gene encoding isopentenyl diphosphate isomerase, a gene encoding cis-prenyltransferase, a gene encoding small rubber particle protein, and a gene encoding rubber elongation factor.

5. 4. The gene construct according to claim 3, wherein the gene encoding a protein involved in polyisoprenoid biosynthesis is a gene encoding cis-prenyltransferase.

6. The gene construct according to claim 5, wherein the gene encoding the cis-prenyltransferase comprises a DNA according to any one of [B1] to [B3] below. [B1] DNA consisting of the base sequence of base numbers 1-927 represented by SEQ ID NO: 2 [B2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 1-927 represented by SEQ ID NO: 2 and encodes a protein having an enzyme activity that catalyzes a reaction that elongates the chain length of an isoprenoid compound to a cis form. [B3] DNA encoding a protein having an enzyme activity that catalyzes a reaction to elongate the chain length of an isoprenoid compound to a cis form, the DNA consisting of a base sequence having 80% or more sequence identity with the base sequence of base numbers 1-927 represented by SEQ ID NO:

2.

7. A transformant transformed with the gene construct according to any one of claims 1 to 6.

8. The transformant according to claim 7, which is a plant belonging to the genus Teraxacum.

9. The transformant according to claim 7, which is Taraxacum kok-saghyz (Russian dandelion).

10. A method for producing an isoprenoid using the transformant according to claim 7.

11. A method for producing a pneumatic tire, comprising: a step of producing an isoprenoid by the method for producing an isoprenoid according to claim 10; a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture; a raw tire molding step of molding a raw tire from the kneaded mixture; and a vulcanization step of vulcanizing the raw tire.

12. A method for producing a rubber product, comprising: a step of producing an isoprenoid by the method for producing an isoprenoid according to claim 10; a kneading step of kneading the obtained isoprenoid with additives to obtain a kneaded mixture; a raw rubber product molding step of molding a raw rubber product from the kneaded mixture; and a vulcanization step of vulcanizing the raw rubber product.

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