Method for producing polyisoprenoid, method for producing pneumatic tire, and method for producing rubber product

Abstract

To provide a polyisoprenoid production method capable of suitably producing polyisoprenoid even when using a callus.SOLUTION: A method for producing polyisoprenoid comprises using a transformed callus into which a gene encoding a cis-prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein are introduced, to produce polyisoprenoid.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for producing polyisoprenoids, a method for producing pneumatic tires, and a method for producing rubber products.

Background Art

[0002] Currently, natural rubber (a type of polyisoprenoid) used in industrial rubber products is obtained by cultivating rubber-producing plants such as Hevea brasiliensis of the Euphorbiaceae family and Ficus elastica of the Moraceae family, biosynthesizing natural rubber in the laticifer cells of the plant body, and manually collecting the natural rubber from the plant.

[0003] Currently, natural rubber used in industrial rubber products has para rubber tree as almost the only source of collection. Para rubber tree is a plant that can grow only in limited regions such as Southeast Asia and South America. Conventionally, by applying ethephon or methyl jasmonate to para rubber tree, induction of laticifer formation has been caused to increase the production of natural rubber. Furthermore, para rubber tree requires about 7 years to become a mature tree from which rubber can be collected, and the period during which natural rubber can be collected is limited to 20 to 30 years. In the future, an increase in the demand for natural rubber is expected, mainly in developing countries. However, due to the above reasons, it is difficult to significantly increase the production of natural rubber from para rubber tree. Therefore, depletion of natural rubber resources is a concern, and a stable supply source of natural rubber other than mature para rubber trees and improvement of the production efficiency of natural rubber in para rubber trees are desired.

[0004] Natural rubber has isopentenyl diphosphate (IPP) as its basic unit and has a cis-1,4-polyisoprene structure. It is considered that cis-type prenyltransferase (CPT) is related to the biosynthesis of natural rubber due to its structure. For example, the presence of multiple CPTs has been confirmed in Ficus elastica, and Hevea Rubber transferase 1 (HRT1), Hevea Rubber transferase 2 (HRT2), etc. are known (see, for example, Non-Patent Documents 1 and 2). Also, in Taraxacum brevicorniculatum, a kind of dandelion, it is known that the rubber synthesis amount decreases by suppressing the expression level of CPT (see, for example, Non-Patent Document 3).

[0005] In addition, as research on proteins involved in the biosynthesis of natural rubber, Rubber Elongation Factor (REF) and Small Rubber Particle Protein (SRPP) have attracted attention (see, for example, Non-Patent Documents 4 and 5). However, the relationship between these proteins and CPT has not been elucidated. On the other hand, it has been suggested that Nogo-B receptor (NgBR) is involved in the biosynthesis of dolichol in human CPT (see, for example, Non-Patent Document 6).

[0006] Also, methods for increasing the production of natural rubber from Ficus elastica have been studied, but since the rubber synthesis mechanism in Ficus elastica has not been elucidated, transgenic Ficus elastica with enhanced expression of genes in the known monomer (isopentenyl diphosphate) synthesis pathways (mevalonate pathway (MVA pathway) and non-mevalonate pathway (MEP pathway)) has been proposed (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Literature

[0008]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Summary of the Invention

Problems to be Solved by the Invention

[0009] As described above, it is desired to develop a stable natural rubber supply source other than mature Hevea brasiliensis trees and improve the production efficiency of natural rubber in Hevea brasiliensis. However, at present, there are many unclear parts in the natural rubber biosynthesis mechanism, especially its regulatory mechanism, and there is still much room for improvement for a significant increase in the production of natural rubber.

[0010] As an efficient production method for useful substances of plants, the use of plant cultured cells such as callus cells and hairy root cells has been considered. The use of plant cultured cells has the merit that useful substances contained in plants can be stably produced by managing cells and producing substances in an artificially controlled environment. However, in the secondary metabolites of plants such as natural rubber, it is cited as a problem that the production efficiency is low under normal growth conditions. Therefore, devices such as adding a drug that induces the production of secondary metabolites to the medium and culturing plant cultured cells have been made.

[0011] Callus derived from rubber-producing plants such as callus derived from Ficus elastica does not produce much isoprenoid (including rubber). This is presumably because callus is composed of undifferentiated cells, and unlike primary metabolites, which are metabolites necessary for survival, the transcriptional level of genes for synthesizing secondary metabolites such as isoprenoid is low.

[0012] In particular, in the production of natural rubber, natural rubber production in plants is carried out in special cells called laticifer cells. Therefore, it is considered that the transcriptional level of many genes related to natural rubber production is low in undifferentiated callus.

[0013] As a result of the study by the present inventors, it has been clarified that genes related to NR synthesis such as REF and HRT1 (cis-type prenyltransferase (CPT) family proteins), which have a high transcriptional level in the laticifers (tissues that synthesize natural rubber) of Ficus elastica, are hardly transcribed in Ficus elastica callus.

[0014] An object of the present invention is to provide a method for producing polyisoprenoid that can suitably produce polyisoprenoid even when callus is used, and solves the above problems.

Means for Solving the Problems

[0015] In order to solve the above problems, the inventors attempted to increase the synthesis amount of isoprenoid synthesis (natural rubber) in callus by introducing genes related to natural rubber into the callus.

[0016] For natural rubber synthesis, three types of proteins are considered important: HRT1 (a cis-type prenyltransferase (CPT) family protein) that directly synthesizes natural rubber, HRBP (a Nogo-B receptor (NgBR) family protein) that assists the activity of HRT1, and REF (a Rubber Elongation Factor (REF) family protein) that is necessary for forming rubber particles. In particular, since HRT1 requires the assistance of HRBP to exhibit activity, it is considered necessary to introduce genes in a set of HRT1 and HRBP.

[0017] However, when the inventors confirmed the amount of each gene in the callus, it was found that the transcriptional amount of HRT1 was very low as expected, while HRBP showed a high transcriptional amount even in the callus. From this, it was considered that the introduction of HRBP into the callus was unnecessary and that the introduction of only HRT1 was sufficient. However, when only HRT1 was introduced into the callus, the generation of rubber particles could not be confirmed. Therefore, it was found that the introduction of only HRT1 into the callus was insufficient for improving the efficiency of polyisoprenoid (rubber) production in the callus. Similarly, when only REF was introduced into the callus, the generation of rubber particles could not be confirmed.

[0018] On the one hand, when REF was introduced into callus simultaneously with HRT1, the generation rate of rubber particles increased, and it was newly revealed that the introduction of HRT1 and REF into callus in combination is important for polyisoprenoid (rubber) production in callus. In particular, in callus which is undifferentiated cells, it is considered an efficient method for natural rubber production in callus that polyisoprenoid production normally carried out in laticifer cells can be reproduced only by increasing the transcription of these two genes. Thus, the inventors completed the present invention by finding that polyisoprenoids can be preferably produced by the obtained transformed callus by introducing a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein into callus. That is, the present invention relates to a method for producing polyisoprenoids, characterized by producing polyisoprenoids using transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein have been introduced.

Advantages of the Invention

[0019] According to the present invention, since it is a method for producing polyisoprenoids, characterized by producing polyisoprenoids using transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein have been introduced, even when callus is used, a method for producing polyisoprenoids capable of preferably producing polyisoprenoids can be provided.

[0020] The method for manufacturing a pneumatic tire of the present invention includes a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid of the present invention and an additive to obtain a kneaded product, a green tire forming step of forming a green tire from the kneaded product, and a vulcanization step of vulcanizing the green tire. Therefore, since a pneumatic tire is manufactured from a polyisoprenoid obtained by a method with high production efficiency during polyisoprenoid production, plant resources can be effectively utilized, and a pneumatic tire can be manufactured in consideration of the environment.

[0021] The method for manufacturing a rubber product of the present invention includes a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid of the present invention and an additive to obtain a kneaded product, a green rubber product forming step of forming a green rubber product from the kneaded product, and a vulcanization step of vulcanizing the green rubber product. Therefore, since a rubber product is manufactured from a polyisoprenoid obtained by a method with high production efficiency during polyisoprenoid production, plant resources can be effectively utilized, and a rubber product can be manufactured in consideration of the environment.

Brief Description of the Drawings

[0022]

Figure 1

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Figure 6

Modes for Carrying Out the Invention

[0023] The method for producing a polyisoprenoid of the present invention is characterized in that a polyisoprenoid is produced using a transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein have been introduced. In addition, the production method of the present invention may include other steps as long as it includes the step of producing a polyisoprenoid using the transformed callus, and each step may be performed once or may be repeatedly performed a plurality of times.

[0024] Since the method for producing a polyisoprenoid of the present invention is carried out using a transformed callus into which a specific gene has been introduced, unlike the conventional method of administering a drug, once the gene is introduced, the effect of the gene introduction is exerted by the mechanism inherent in the organism, so a continuous effect can be expected without subsequent continuous treatment.

[0025] First, a transformed callus (also referred to as the transformed callus according to the present embodiment) into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein have been introduced will be described. The transformed callus according to the present embodiment has a gene encoding a foreign cis-type prenyltransferase (CPT) family protein and a gene encoding a foreign Rubber Elongation Factor (REF) family protein.

[0026] Here, in the present specification, "foreign" means artificially introduced, and a foreign gene means a gene artificially introduced.

[0027] By introducing a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein into callus, callus transformed to express a cis-type prenyltransferase (CPT) family protein and a Rubber Elongation Factor (REF) family protein (transformed callus) can be produced. In the transformed callus, since the CPT family protein and the REF family protein are co-expressed, they function together with the Nogo-B receptor (NgBR) family protein that the callus originally has, and it is expected that the activity of the CPT family protein will be stabilized and enhanced. As a result, the transformed callus co-expressing the CPT family protein and the REF family protein continuously enhances rubber synthesis activity, and by using the transformed callus to produce polyisoprenoid, it is expected that the production amount of polyisoprenoid can be suitably increased.

[0028] The transformed callus according to this embodiment preferably has a gene encoding a foreign cis-type prenyltransferase (CPT) family protein and a gene encoding a foreign Rubber Elongation Factor (REF) family protein, but does not have a gene encoding a foreign Nogo-B receptor (NgBR) family protein. The callus before transformation already sufficiently has a gene encoding the Nogo-B receptor (NgBR) family protein, and further, by introducing a gene encoding the Nogo-B receptor (NgBR) family protein, the Nogo-B receptor (NgBR) family protein may be overexpressed, and conversely, the production efficiency during polyisoprenoid production may decrease, but this can be prevented.

[0029] Also, the more the number of introduced genes increases (the longer it becomes), the worse the efficiency of gene introduction into cells. Therefore, from the perspective of the efficiency of gene introduction into cells, it is easier to obtain target cells (callus into which genes have been introduced) when the number of genes is smaller, and it is easier to enable polyisoprenoid (rubber) production in callus. Thus, from such a perspective as well, it is preferable that the transformed callus according to this embodiment does not have a gene encoding a foreign Nogo-B receptor (NgBR) family protein.

[0030] The origin of the gene encoding the above cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein is not particularly limited, but it is preferably derived from a rubber-producing plant, and more preferably derived from a plant belonging to at least one genus selected from the group consisting of the genus Hevea, the genus Taraxacum, and the genus Parthenium. Also, it is preferable that the origin of the gene encoding the above cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein is the same as the origin of the callus into which the gene is introduced. Among them, it is more preferably derived from at least one plant selected from the group consisting of Para rubber tree, Russian dandelion, and guayule, and particularly preferably derived from Para rubber tree. Most preferably, both are derived from Para rubber tree.

[0031] The rubber-producing plants are not particularly limited. For example, Hevea genus such as Hevea brasiliensis; Sonchus genus such as Sonchus oleraceus, Sonchus asper, Sonchus brachyotus; Solidago genus such as Solidago altissima, Solidago virgaurea subsp. asiatica, Solidago virgaurea subsp. leipcarpa, Solidago virgaurea subsp. leipcarpa f. paludosa, Solidago virgaurea subsp. gigantea, Solidago gigantea Ait. var. leiophylla Fernald; Helianthus genus such as Helianthus annuus, Helianthus argophyllus, Helianthus atrorubens, Helianthus debilis, Helianthus decapetalus, Helianthus giganteus; Taraxacum genus such as Taraxacum, Taraxacum venustum H.Koidz, Taraxacum hondoense Nakai, Taraxacum platycarpum Dahlst, Taraxacum japonicum, Taraxacum officinale Weber, Taraxacum koksaghyz; Ficus genus such as Ficus carica, Ficus elastica, Ficus pumila L., Ficus erecta Thumb., Ficus ampelas Burm.f.) Ficus species such as Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa L.f., Ficus septica Burm.f., Ficus benghalensis; Parthenium species such as Parthenium argentatum, Parthenium hysterophorus, Parthenium hysterophorus; Lactuca serriola, Ficus benghalensis, etc. can be mentioned.

[0032] In addition, in this specification, the cis-type prenyltransferase (CPT) family protein is an enzyme that catalyzes the reaction of extending the chain length of isoprenoid compounds in a cis-type manner. Specifically, for example, in plants, polyisoprenoids are biosynthesized through the polyisoprenoid biosynthesis pathway as shown in Figure 1. Among this pathway, the CPT family protein is considered to be an enzyme that catalyzes the reaction in the portion surrounded by the dotted frame in Figure 1. A characteristic of the CPT family protein is that it has an amino acid sequence contained in the Cis IPPS domain (NCBI Accession No. cd00475). Examples of the above CPT family proteins include CPT (HRT1, HRT2, CPT3-5) derived from rubber tree, AtCPT1-9 derived from Arabidopsis thaliana, CPT1-3 derived from lettuce, and CPT1-3 derived from Russian dandelion.

[0033] In this specification, the Nogo-B receptor (NgBR) family protein is a protein that binds to the membrane through one or more transmembrane regions on the N-terminal side and has a function of interacting with CPT family proteins or other proteins on the C-terminal side, and assists the function by retaining the CPT family protein on the membrane. The characteristics of the above NgBR family protein are that it has a transmembrane domain on the N-terminal side and has an amino acid sequence contained in the Cis IPPS superfamily domain (NCBI Accession No. COG0020) on the C-terminal side. Examples of the above NgBR family protein include NgBR (HRBP) derived from Para rubber tree, LEW1 derived from Arabidopsis thaliana, LsCPTL1-2 derived from lettuce, and TbRTA derived from dandelion.

[0034] In this specification, the Rubber Elongation Factor (REF) family protein is a rubber particle-binding protein that binds to rubber particles present in the latex of rubber-producing plants such as Hevea brasiliensis, and contributes to the stabilization of rubber particles. Furthermore, although the produced polyisoprenoid is stored in the rubber particles, the REF family protein also contributes to the formation of these rubber particles. The characteristics of the above REF family protein are that it has an amino acid sequence contained in the REF superfamily domain (NCBI Accession No. pfam05755). Examples of the above REF family protein include REF, Small Rubber Particle Protein (SRPP), etc.

[0035] Specific examples of the above CPT family protein include the following [1]. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2

[0036] In addition, it is known that a protein may have its original function even if it contains substitution, deletion, insertion, or addition of one or more amino acids in the original amino acid sequence. Therefore, the following [2] is also included as a specific example of the CPT family protein. [2] A protein consisting of a sequence containing substitution, deletion, insertion, and / or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2, and having an enzyme activity that catalyzes the reaction of extending the chain length of an isoprenoid compound in a cis form

[0037] In addition, in order to maintain the function as the CPT family protein, in the amino acid sequence represented by SEQ ID NO: 2, preferably one or more amino acids, more preferably 1 to 58 amino acids, still more preferably 1 to 44 amino acids, even more preferably 1 to 29 amino acids, particularly preferably 1 to 15 amino acids, most preferably 1 to 6 amino acids, and even most preferably 1 to 3 amino acids of substitution, deletion, insertion, and / or addition are included. It is preferably an amino acid sequence.

[0038] As an example of amino acid substitution, conservative substitution is preferred, and specifically, substitution within the following groups in parentheses is included. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

[0039] In addition, it is known that a protein having an amino acid sequence with high sequence identity to the original amino acid sequence may also have a similar function. Therefore, the following [3] is also included as a specific example of the CPT family protein. [3] A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and having an enzyme activity that catalyzes the reaction of extending the chain length of an isoprenoid compound in a cis form

[0040] In order to maintain the function as the above-mentioned CPT family protein, the sequence identity with the amino acid sequence represented by SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0041] The sequence identity of amino acid sequences and nucleotide sequences can be determined using the algorithms BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul.

[0042] As a method for confirming that the protein has the above enzyme activity, for example, a conventionally known method can be used. For example, using Escherichia coli or the like, a transformant into which a gene encoding the target protein is introduced is used to express the target protein, and the presence or absence of the function of the target protein is measured by each activity measurement method, such as activity measurement.

[0043] Specific examples of the above-mentioned NgBR family protein include the following [4]. [4] A protein consisting of the amino acid sequence represented by SEQ ID NO: 4

[0044] It is also known that a protein may have its original function even if it contains substitution, deletion, insertion, or addition of one or more amino acids in the original amino acid sequence. Therefore, specific examples of the above-mentioned NgBR family protein also include the following [5]. [5] A protein consisting of a sequence containing substitution, deletion, insertion, and / or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 4, and having a function of binding to a membrane with one or more transmembrane regions on the N-terminal side and interacting with other proteins on the C-terminal side

[0045] In order to maintain the function as the above-mentioned NgBR family protein, in the amino acid sequence represented by SEQ ID NO: 4, preferably one or more amino acids, more preferably 1 to 52 amino acids, still more preferably 1 to 39 amino acids, even more preferably 1 to 26 amino acids, particularly preferably 1 to 13 amino acids, most preferably 1 to 6 amino acids, and even most preferably 1 to 3 amino acids, a substitution, deletion, insertion, and / or addition of amino acids are preferably included.

[0046] As an example of amino acid substitution, conservative substitution is preferred, and specifically, substitution within the following groups in parentheses can be mentioned. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

[0047] Also, as described above, it is known that a protein having an amino acid sequence with a high sequence identity to the original amino acid sequence may also have a similar function. Therefore, as a specific example of the above-mentioned NgBR family protein, the following [6] is also included. [6] A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4, binding to the membrane with one or more transmembrane regions on the N-terminal side, and having a function of interacting with other proteins on the C-terminal side

[0048] In order to maintain the function as the above-mentioned NgBR family protein, the sequence identity with the amino acid sequence represented by SEQ ID NO: 4 is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0049] As a method for confirming that it is the above-mentioned NgBR family protein, for example, a conventionally known method can be used. For example, a method of identifying the amino acid sequence and confirming whether it has the amino acid sequence included in the Cis IPPS superfamily domain (NCBI Accession No. COG0020) can be mentioned.

[0050] Specific examples of the above REF family protein include the following [7]. [7] A protein consisting of the amino acid sequence represented by SEQ ID NO: 6

[0051] In addition, it is known that a protein may have its original function even when it contains substitution, deletion, insertion, or addition of one or more amino acids in the original amino acid sequence. Therefore, the following [8] is also included as a specific example of the above REF family protein. [8] A rubber particle-binding protein consisting of a sequence containing substitution, deletion, insertion, and / or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 6 and binding to rubber particles present in latex

[0052] In order to maintain the function as the above REF family protein, in the amino acid sequence represented by SEQ ID NO: 6, it is preferably an amino acid sequence containing substitution, deletion, insertion, and / or addition of preferably one or more amino acids, more preferably 1 to 28 amino acids, still more preferably 1 to 21 amino acids, still more preferably 1 to 14 amino acids, particularly preferably 1 to 7 amino acids, most preferably 1 to 3 amino acids, and even most preferably 1 amino acid.

[0053] As an example of amino acid substitution, conservative substitution is preferred, and specifically, substitution within the following groups in parentheses can be mentioned. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

[0054] Also, as described above, it is known that proteins having an amino acid sequence with a high sequence identity to the original amino acid sequence may have the same function. Therefore, the following [9] is also cited as a specific example of the REF family protein. [9] A rubber particle-binding protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6 and binding to rubber particles present in latex

[0055] In order to maintain the function as the REF family protein, the sequence identity with the amino acid sequence represented by SEQ ID NO: 6 is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0056] As a method for confirming that it is the REF family protein, for example, a conventionally known method can be used. For example, a method of identifying an amino acid sequence and confirming whether it has an amino acid sequence included in the REF superfamily domain (NCBI Accession No. pfam05755) can be cited.

[0057] Specific examples of the gene encoding the CPT family protein include the following [1] or [2]. [1] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 [2] DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and encodes a protein having an enzyme activity that catalyzes a reaction for extending the chain length of an isoprenoid compound in a cis form

[0058] As used herein, "hybridize" refers to the step of hybridizing DNA having a specific base sequence or a part of the DNA to the DNA. Therefore, the DNA having the specific base sequence or the base sequence of a part of the DNA may be useful as a probe for Northern or Southern blot analysis, or may be DNA having a length that can be used as an oligonucleotide primer for PCR (Polymerase Chain Reaction) analysis. Examples of DNA used as a probe include DNA of at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more, but may also be DNA of at least 10 bases or more, preferably 15 bases or more.

[0059] Methods for DNA hybridization experiments are well known. For example, in addition to those described in Molecular Cloning, 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press (Molecular), hybridization conditions can be determined and experiments can be conducted according to a number of other standard textbooks.

[0060] The above stringent conditions include, for example, incubating a filter immobilized with DNA and probe DNA overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg / ml of denatured salmon sperm DNA, and then washing the filter, for example, in a 0.2×SSC solution at about 65°C. However, lower stringent conditions can also be used. Changes in stringent conditions can be achieved by adjusting the concentration of formamide (lowering the concentration of formamide results in lower stringency), and by changing the salt concentration and temperature conditions. Examples of low stringency conditions include incubating overnight at 37°C in a solution containing 6×SSCE (20×SSCE 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 / ml of denatured salmon sperm DNA, and then washing with a 1×SSC, 0.1% SDS solution at 50°C. Further lower stringent conditions can include, after performing hybridization using a solution with a high salt concentration (e.g., 5×SSC) under the above-described low stringency conditions, the conditions for washing.

[0061] The various conditions described above can also be set by adding or changing the blocking reagent used to suppress the background of the hybridization experiment. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions to adapt the conditions.

[0062] Examples of DNA that can hybridize under the stringent conditions described above include DNA consisting of a nucleotide sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1 when calculated based on the above parameters using programs such as BLAST and FASTA.

[0063] As a method for confirming that DNA that hybridizes with the above-described DNA under stringent conditions is DNA encoding a protein having a predetermined enzyme activity, a conventionally known method can be used. For example, using Escherichia coli or the like, the target protein is expressed by a transformant into which a gene encoding the target protein has been introduced, and the presence or absence of the function of the target protein is measured by each activity measurement method, such as activity measurement.

[0064] Specific examples of the gene encoding the above-described NgBR family protein include the following [3] or [4]. [3] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 [4] DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and encodes a protein having a function of binding to a membrane through one or more transmembrane regions on the N-terminal side and interacting with other proteins on the C-terminal side

[0065] "Hybridize" as used herein is the same as described above. Also, the above-described stringent conditions are the same as described above.

[0066] Examples of DNA that can hybridize under the stringent conditions described above include DNA consisting of a nucleotide sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3 when calculated based on the above parameters using programs such as BLAST and FASTA.

[0067] As a method for confirming that the DNA that hybridizes with the above-described DNA under stringent conditions is DNA encoding an NgBR family protein, a conventionally known method can be used. For example, when the DNA is translated into an amino acid sequence, a method of confirming whether it has an amino acid sequence contained in the Cis IPPS superfamily domain (NCBI Accession No. COG0020) can be mentioned.

[0068] Specific examples of the gene encoding the above REF family protein include the following [5] or [6]. [5] DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5 [6] DNA that hybridizes with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a rubber particle-binding protein that binds to rubber particles present in latex

[0069] "Hybridize" as used herein is the same as described above. Also, the above stringent conditions are the same as described above.

[0070] Examples of DNA that can hybridize under the stringent conditions described above include DNA consisting of a nucleotide sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5 when calculated based on the above parameters using programs such as BLAST and FASTA.

[0071] As a method for confirming that the DNA that hybridizes with the above-described DNA under stringent conditions is a DNA encoding a REF family protein, a conventionally known method can be used. For example, when the DNA is translated into an amino acid sequence, a method of confirming whether it has an amino acid sequence contained in the REF superfamily domain (NCBI Accession No. pfam05755) can be mentioned.

[0072] In addition, as a method for identifying the amino acid sequence and base sequence of the above protein, a conventionally known method can be used. For example, total RNA is extracted from growing plants, mRNA is purified as necessary, and cDNA is synthesized by reverse transcription reaction. Next, based on the amino acid sequence of a known protein corresponding to the target protein, degenerate primers are designed, RT-PCR is performed, a DNA fragment is amplified partially, and the sequence is identified partially. Then, the RACE method or the like is performed to identify the full-length base sequence and amino acid sequence. The RACE method (Rapid Amplification of cDNA Ends method) is a method of cloning the unknown region up to the cDNA end by performing PCR based on the base sequence information of the known region when the base sequence of cDNA is partially known. It is a method by which full-length cDNA can be cloned by the PCR method without preparing a cDNA library. Note that the degenerate primers are preferably prepared from a plant-derived sequence having a sequence site highly homologous to the above target protein. In addition, when the base sequence encoding the above protein is known, primers containing a start codon and primers containing a stop codon are designed from the known base sequence, and RT-PCR is performed using the synthesized cDNA as a template to identify the full-length base sequence and amino acid sequence.

[0073] The origin of the callus into which the gene encoding the cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein are introduced is not particularly limited, but it is preferably derived from a rubber-producing plant, and more preferably derived from a plant belonging to at least one genus selected from the group consisting of the genus Hevea, the genus Taraxacum, and the genus Parthenium. Among them, it is even more preferably derived from at least one plant selected from the group consisting of Hevea brasiliensis, Taraxacum kok-saghyz, and Parthenium argentatum, and particularly preferably derived from Hevea brasiliensis. Also, it is preferable that the gene encoding the cis-type prenyltransferase (CPT) family protein, the gene encoding the Rubber Elongation Factor (REF) family protein, and the origin of the callus into which the gene is introduced are the same.

[0074] The transformed callus according to this embodiment has a gene encoding a foreign cis-type prenyltransferase (CPT) family protein and a gene encoding a foreign Rubber Elongation Factor (REF) family protein, but may also have other foreign genes.

[0075] The origin of the above-mentioned other foreign genes is not particularly limited, but it is preferably derived from the rubber-producing plants described above.

[0076] The above-mentioned other foreign genes may be any genes without any limitation. However, from the perspective of enhancing rubber synthesis ability, it is preferably a gene encoding a protein originally present on rubber particles in rubber-producing plants. The protein present on rubber particles may be a protein that binds to the membrane surface of rubber particles, a protein that binds so as to be inserted into the membrane of rubber particles, or a protein that binds to the above-mentioned membrane and forms a complex to exist on the membrane surface. Further, the above-mentioned other foreign genes may be a drug resistance gene (NPTII gene) or a fluorescent protein (GFP gene).

[0077] Examples of the protein originally present on rubber particles in the above-mentioned rubber-producing plants include β-1,3-glucanase, Hevein, and the like.

[0078] The method for preparing callus and the method for introducing a gene into callus are not particularly limited and may be carried out by known methods. For example, the method for preparing callus from plant tissue pieces may be carried out by the methods described in WO2012 / 099100, JP-A-2017-055670, and the like. Further, for example, the introduction of a gene into callus may be carried out by the method described in JP-A-2020-005592 and the like.

[0079] As a method for preparing the above-described transformed callus, specifically, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and a DNA containing the nucleotide sequence represented by SEQ ID NO: 5 are inserted downstream of the promoter of an appropriate expression vector using an appropriate restriction enzyme or the like to prepare a recombinant DNA. Then, by introducing the recombinant DNA into a host callus cell compatible with the expression vector, a transformed callus cell can be obtained. Alternatively, a recombinant DNA is prepared using an expression vector into which a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 has been inserted downstream of the promoter using an appropriate restriction enzyme or the like, and an expression vector into which a DNA containing the nucleotide sequence represented by SEQ ID NO: 5 has been inserted downstream of the promoter using an appropriate restriction enzyme or the like, and the transformed callus cell can also be obtained by introducing the recombinant DNA into a host callus cell compatible with the expression vector.

[0080] As the above-described expression vector, one that can autonomously replicate in the above-described host callus cell or can be integrated into the chromosome and contains a promoter at a position where the above-described recombinant DNA can be transcribed can be used.

[0081] Examples of the above-described expression vector include pBI-based vectors, Ti plasmids, tobacco mosaic virus vectors, and the like.

[0082] As the above-described promoter, any promoter that functions in plant cells can be used. Examples thereof include the 35S promoter of cauliflower mosaic virus (CaMV), the rice actin 1 promoter, the nopaline synthase gene promoter, the 35S promoter of tobacco mosaic virus, and the actin gene promoter derived from rice.

[0083] As the method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing DNA into host plant cells. For example, the method using Agrobacterium (Japanese Patent Laid-Open No. 59-140885, Japanese Patent Laid-Open No. 60-70080, WO94 / 00977), the electroporation method (Japanese Patent Laid-Open No. 60-251887), the method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), the method using metal crystals (whiskers) (Japanese Patent Laid-Open No. 2020-005592), etc. can be mentioned.

[0084] By culturing the transformed callus according to this embodiment in an appropriate medium, the gene encoding the cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein introduced into the transformed callus according to this embodiment are expressed, and it becomes possible to produce polyisoprenoids with the transformed callus. Thereby, even when callus is used, polyisoprenoids can be suitably produced.

[0085] The method for culturing the callus is not particularly limited, and it may be carried out by a known method. For example, the callus can be cultured by the methods described in the publications cited for the method for preparing the callus and the method for introducing the gene into the callus.

[0086] In the production method of this embodiment, after producing polyisoprenoids with the transformed callus according to this embodiment, a step of recovering the polyisoprenoids may be carried out as necessary.

[0087] The method for recovering polyisoprenoids from callus is not particularly limited and may be performed by known methods. For example, as described in WO2012 / 099100, callus is recovered from the medium, frozen in liquid nitrogen, then freeze-dried to remove the moisture in the callus. Next, the callus from which the moisture has been removed is ground in a mortar, and then polyisoprenoids may be extracted with an organic solvent using a Soxhlet extraction apparatus. The obtained polyisoprenoid (natural rubber) may be used after drying if necessary.

[0088] In this specification, polyisoprenoid is a general term for polymers composed of isoprene units (C5H8). Examples of polyisoprenoids include sesquiterpenes (C 25 ), triterpenes (C 30 ), tetraterpenes (C 40 ), polymers such as natural rubber. Also, in this specification, isoprenoid means a compound having an isoprene unit (C5H8) and is a concept that includes polyisoprenoids.

[0089] (Method for manufacturing rubber products) The method for manufacturing a rubber product according to this embodiment includes a kneading step of kneading the polyisoprenoid obtained by the method for manufacturing a polyisoprenoid according to the above-described embodiment and an additive to obtain a kneaded product, a raw rubber product molding step of molding a raw rubber product from the kneaded product, and a vulcanization step of vulcanizing the raw rubber product.

[0090] The rubber product is not particularly limited as long as it can be manufactured using rubber (preferably natural rubber). Examples include pneumatic tires, rubber rollers, rubber fenders, gloves, medical rubber tubes, and the like.

[0091] When the rubber product is a pneumatic tire, that is, when the method for manufacturing the rubber product of the present embodiment is the method for manufacturing the pneumatic tire of the present embodiment, the above-mentioned green rubber product forming step corresponds to a green tire forming step of forming a green tire from the above-mentioned kneaded material, and the above-mentioned vulcanization step corresponds to a vulcanization step of vulcanizing the above-mentioned green tire. That is, the method for manufacturing a pneumatic tire of the present embodiment includes a kneading step of kneading the polyisoprene obtained by the method for manufacturing the above-mentioned polyisoprene and an additive to obtain a kneaded material, a green tire forming step of forming a green tire from the above-mentioned kneaded material, and a vulcanization step of vulcanizing the above-mentioned green tire.

[0092] <Kneading step> In the kneading step, the polyisoprene obtained by the method for manufacturing the above-mentioned polyisoprene and an additive are kneaded to obtain a kneaded material.

[0093] The additive is not particularly limited, and additives used in the manufacture of rubber products can be used. For example, when the rubber product is a pneumatic tire, for example, rubber components other than the above-mentioned polyisoprene, reinforcing fillers such as carbon black, silica, calcium carbonate, alumina, clay, talc, etc., silane coupling agents, zinc oxide, stearic acid, processing aids, various anti-aging agents, softeners such as oils, waxes, vulcanizing agents such as sulfur, vulcanization accelerators, etc. can be mentioned.

[0094] The kneading in the kneading step may be performed using a rubber kneading device such as an open roll, a Banbury mixer, or a closed kneader.

[0095] <Green rubber product forming step (green tire forming step in the case of a tire)> In the green rubber product forming step, a green rubber product (a green tire in the case of a tire) is formed from the kneaded material obtained in the kneading step. The method for forming a raw rubber product is not particularly limited, and a method used for forming a raw rubber product may be appropriately applied. For example, when the rubber product is a pneumatic tire, the kneaded product obtained by the kneading step is extruded according to the shape of each tire member, formed by a normal method on a tire molding machine, and each tire member is bonded together to form a green tire (unvulcanized tire).

[0096] <Vulcanization step> In the vulcanization step, a rubber product is obtained by vulcanizing the raw rubber product obtained by the raw rubber product forming step. The method for vulcanizing a raw rubber product is not particularly limited, and a method used for vulcanizing a raw rubber product may be appropriately applied. For example, when the rubber product is a pneumatic tire, a pneumatic tire is obtained by heating and pressurizing the green tire (unvulcanized tire) obtained by the raw rubber product forming step in a vulcanizer to vulcanize it.

Example

[0097] Based on the examples, the present invention will be specifically described, but the present invention is not limited thereto.

[0098] The outlines of the examples and comparative examples will be described. Example: Callus into which both HRT1 and REF were introduced Comparative Example 1: Callus into which both HRT1 and HRBP were introduced Comparative Example 2: Callus into which only REF was introduced Note that the origin of the introduced gene and the callus used are both Paragonum nox.

Table 1

[0099] Hereinafter, the details of the experimental methods of the examples and comparative examples will be described.

[0100] 〔Acquisition of gene〕 According to the method described in the examples of JP-A-2017-012058, the CPT gene (HRT1), NgBR gene (HRBP), and REF gene derived from *Paragonimus westermani* were obtained. For the obtained genes, their sequences were identified, and the full-length nucleotide sequences and amino acid sequences were identified. The nucleotide sequence of HRT1 is shown in SEQ ID NO: 1. The amino acid sequence of HRT1 is shown in SEQ ID NO: 2. Also, the nucleotide sequence of HRBP is shown in SEQ ID NO: 3. The amino acid sequence of HRBP is shown in SEQ ID NO: 4. Also, the nucleotide sequence of REF is shown in SEQ ID NO: 5. The amino acid sequence of REF is shown in SEQ ID NO: 6.

[0101] [Construction of Vector] As the introduction vector, the pRI-201-AN vector was used as the basic vector. REF was introduced into MCS1 of the pRI201 vector, and HRT1 and / or HRBP were introduced into MCS2. For any of the genes, the 35S promoter, AtADH 5’UTR, target gene, and HSP terminator of pRI201-AN were used as the gene expression cassette. Also, when introducing two or more gene expression cassettes, they were introduced in the same orientation. As the plasmids for introduction, pRI201-REF, pRI201-HRT1-HRBP, and pRI201-HRT1-REF were prepared.

[0102] [Preparation of *Paragonimus westermani* Callus (Induction Step)] The leaves of *Paragonimus westermani* were washed with running water for 15 minutes and sterilized with 70% ethanol for 1 minute. The sterilized leaves were sterilized while stirring in a 1% sodium hypochlorite solution for 15 minutes. After sterilization, the leaves were washed 3 times with sterilized water and the moisture was blotted with sterilized paper. After making incisions with a scalpel in the vein part of the leaves, they were placed on the CIM medium (MS medium containing 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 2 mg / L BA (benzyladenine), 3 mass% sucrose, and 0.22 mass% gellan gum) so that the surface was in close contact. They were cultured at 25°C in the dark, and the callus obtained after 2 weeks was transferred to a new CIM medium to obtain callus (cultured cells).

[0103] [Differentiation of Callus (Size Adjustment Step)] A stainless-steel mesh with a hole size of 1.0 mm was placed on a sterilized beaker, and the callus was transferred onto the mesh. Then, the callus was gently stroked with a spoon, passed through the mesh, and small cell masses were obtained. These small cell masses (cultured cell masses) were suspended in a small amount (about 25 mL) of liquid medium 1 (MS medium containing 30 mg / L sucrose and 2 mg / L 2,4-D) to obtain a cultured cell mass suspension (callus suspension) (size of the cultured cell mass: 0.8 mm).

[0104] [Preparation of whisker suspension] 5 mg of potassium titanate whiskers (product LS20 manufactured by Titanium Industry Co., Ltd., average fiber diameter: 0.2 - 0.6 μm, average fiber length: 10 - 20 μm) were placed in a 1.5 ml tube, 0.5 ml of ethanol was added, and after leaving it overnight, the ethanol was completely evaporated to obtain sterilized whiskers. 1 ml of sterilized water was added to the tube containing these whiskers, stirred well, then centrifuged at 3000 rpm for 5 minutes, and the supernatant water was discarded to wash the whiskers. After performing this washing operation 3 times, 0.5 ml of the above liquid medium 1 was added into the same tube to obtain a whisker suspension.

[0105] [Preparation of transgene] As the transgene, the previously prepared plasmids (pRI201-REF, pRI201-HRT1-HRBP, pRI201-HRT1-REF) were used. The plasmids were dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at a concentration of 1 mg / ml and used as the transgene solution.

[0106] [Mixing of cultured cells and whiskers] 250 μl of the cultured cell mass suspension obtained above was added to the tube containing the whisker suspension obtained above, stirred, and then centrifuged at 1,000 rpm for 10 seconds to precipitate the callus and whiskers, and the supernatant was discarded to obtain a mixture of callus and whiskers.

[0107] [Mixing of transgene] To 20 μl of the introduced gene solution obtained above (containing 20 μg of plasmid), 10 μl of the above liquid medium 1 was added and mixed. After that, it was added to the tube containing the above mixture of callus and whiskers, and the mixture to which callus, whiskers, and the introduced gene were added was shaken well to obtain a mixture (cultured cell - whisker dispersion).

[0108] [Step of attaching whiskers to cultured cells] Next, the tube containing this mixture (cultured cell - whisker dispersion) (the mass ratio of cultured cells to whiskers in the cultured cell - whisker dispersion (mass of cultured cells / mass of whiskers): 50, the mass ratio of plasmid to cultured cells in the cultured cell - whisker dispersion (mass of plasmid / mass of cultured cells): 12500, the total concentration of cultured cells and whiskers in the cultured cell - whisker dispersion: 0.5% by mass) was centrifuged at 18,000×g for 5 minutes (temperature: 4°C). After centrifugation, it was shaken well again. The operations of centrifugation and shaking well again were performed 3 times to attach whiskers to the cultured cells.

[0109] [Gene introduction step] The tube containing the mixture (cultured cell - whisker dispersion after centrifugation) obtained in the previous step was placed in the bath of an ultrasonic generator (bath type: using water as the medium) so that the tube was sufficiently immersed. It was irradiated with ultrasonic waves at a frequency of 40 kHz and an intensity of 0.25 W / cm 2 for 1 minute. After irradiation, it was allowed to stand at 4°C for 30 minutes to damage the cultured cells with the whiskers and introduce foreign genes into the cells.

[0110] [Cell recovery step] The callus into which the gene was introduced (the mass ratio of whiskers to cultured cells (mass of whiskers / mass of cultured cells): 0.02) was added to a 3.5 - cm petri dish, 3 ml of MS medium was added, and it was cultured at 28°C in the dark using a rotary shaker (50 rpm) for 1 week (168 hours) to recover the damaged cells and obtain dividing cells. At this time, the whiskers were not removed by washing or the like.

[0111] [Selection of transformed cells] The obtained divided cells were cultured in MS medium containing kanamycin (5 mg / L) at 23°C in the dark to select transformed calli and non-transformed calli.

[0112] 〔Cell staining〕 The obtained transformants were made into fine cell clumps by liquid culture. The callus clumps after selection were placed in 3 mL of MS liquid medium and cultured with stirring at 25°C and 100 rpm in the dark. On the 10th to 14th day after the start of culture, the cells were transferred to 25 mL of fresh MS liquid medium. On the 10th to 14th day after subculture, the cells were subcultured into fresh medium, and the culture scale was scaled up to 50 mL and 100 mL, and finally the cells were cultured in 200 mL of MS liquid medium. After the start of subculture at the 200 mL scale, 100 μL of the culture solution was collected every 14th day to collect the cells. 1 μL of Nile Red solution (1 mg / mL DMSO) was added to the culture solution containing the cells, and the cells were stained by standing at room temperature for 30 minutes. After the cells were precipitated by centrifugation, the supernatant was collected. 100 μL of 100 mM Tris-HCl (pH 7.5) was added to the remaining cells, and the cells were gently washed and then precipitated again by centrifugation, and the supernatant was discarded. After washing the cells in the same way once more, the stained cells were resuspended in 100 μL of 100 mM Tris-HCl (pH 7.5).

[0113] 〔Cell observation〕 Observation of the cells was carried out using a microscope (Olympus SZX16). Observation of the stained cells was carried out using a fluorescence filter (SZX2-FGFP (excitation wavelength (Ex) 460 - 490 nm, fluorescence wavelength (Em) 510 nm or more)).

[0114] 〔Calculation of the ratio of calli forming rubber particles〕 The ratio of calli forming rubber particles was calculated by the following formula. The larger the ratio of calli forming rubber particles, the higher the production efficiency during the production of polyisoprenoid, which means that polyisoprenoid can be preferably produced. Rubber particle formation rate (%) = Number of cells in which formation of rubber particles was confirmed / Total number of observed cells × 100 Here, the number of cells in which the formation of rubber particles could be confirmed was counted as 1 if rubber particles could be confirmed even once, not limited to the number of rubber particles observed in one cell.

[0115] 〔Confirmation of gene expression level〕 The amount of gene transcription in cells was measured using real-time PCR. Single-stranded cDNA was obtained from rubber tree callus using Rever Tra Ace qPCR RT Master Mix (TOYOBO). The following reaction solution was prepared using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio).

Table 2

Table 3

Table 4

[0116] 〔Test results〕

Table 5

[0117] The observation results of the cells of the transformed callus of the example are shown in Figure 2. Furthermore, the results of plotting the increase amount of HRT1 and the rubber particle formation rate are shown in Figure 3. From Figure 3, a positive correlation is seen between the increase amount of HRT1 and the rubber particle formation rate. However, in some samples, rubber particle formation was observed despite a small increase amount of HRT1.

[0118]

Table 6

[0119] The observation results of the cells of the transformed callus of Comparative Example 1 are shown in Fig. 4. Although particulate matter can be seen even when the rubber particle formation rate is 0%, since the luminance is low, there was an impression that many of them were not counted as rubber particles. For example, several particulate matters can be seen in the cell mass in the upper right of the photograph in Fig. 4A, but since they did not meet the luminance requirements, they were not counted as rubber particles.

[0120]

Table 7

[0121] The observation results of the cells of the transformed callus of Comparative Example 2 are shown in Fig. 5.

[0122] An enlarged view of the cells of the transformed callus of the Example is shown in Fig. 6A, and an enlarged view of the cells of the transformed callus of the Comparative Example is shown in Fig. 6B. Among the particles (arrows, rubber particles) visible in the photograph of Fig. 6A, HRT1 is producing the content (polyisoprenoid (rubber)), and REF and the lipid membrane are thought to form the outside of the particles. In the callus of the Example where the expression levels of both HRT1 and REF are high, generation of particles as shown in Fig. 6A can be confirmed, but in the callus of the Comparative Example where the expression level of either one is low, almost no particles are observed as shown in Fig. 6B.

[0123] It is presumed that CPT synthesizes rubber (polyisoprenoid) and REF forms particles in which rubber accumulates. By introducing the gene encoding the CPT family protein and the gene encoding the REF family protein, the amount of HRT1 mRNA in the callus increased up to 11-fold at most, and REF increased up to 167-fold at most. Since there is a moderate correlation between the amount of HRT1 mRNA and the ratio of callus that has become able to produce rubber (Fig. 3), it is considered that HRT1 synthesizes rubber (polyisoprenoid). No correlation was found between the amount of REF mRNA and the ratio of callus that has become able to produce rubber. Presumably, REF is considered to be involved in particle formation. Therefore, even if only REF is highly expressed, it is presumed that empty particles are formed.

[0124] From the comparison between FIG. 2 and FIGS. 4 and 5, the following was revealed. The significant difference in cell size between FIG. 2 (Example) and FIG. 4 (Comparative Example 1) is the transcription level of REF. In FIG. 2, since the transcription level of REF was sufficient, membrane particles were formed, and polyisoprenoids synthesized by CPT were accumulated therein, and thus those on the particles strongly stained with Nile Red were observed. However, in FIG. 4, since the transcription level of REF was insufficient, the formation of membrane particles was not promoted, and it is considered that those on the particles were not observed. On the other hand, in FIG. 5 (Comparative Example 2), although the transcription level of REF is high, particles containing polyisoprenoids were not observed. This is presumably because the amount of HRT (CPT) producing polyisoprenoids was small, so even if membrane particles were formed, polyisoprenoids were not accumulated therein and thus were not stained with Nile Red. From these facts, it is considered that both CPT and REF need to be present at high transcription levels in polyisoprenoid production in callus.

[0125] From the above examples, it was found that polyisoprenoids can be preferably produced even when using callus by producing polyisoprenoids with a transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein are introduced.

[0126] The present invention (1) relates to a method for producing polyisoprenoids, characterized by producing polyisoprenoids with a transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein are introduced.

[0127] The present invention (2) relates to a method for producing a polyisoprenoid according to the present invention (1), wherein the transformed callus does not have a gene encoding a foreign Nogo-B receptor (NgBR) family protein.

[0128] The present invention (3) relates to a method for producing a polyisoprenoid according to the present invention (1) or (2), wherein at least one selected from the group consisting of a gene encoding the cis-type prenyltransferase (CPT) family protein and a gene encoding the Rubber Elongation Factor (REF) family protein is derived from a rubber-producing plant.

[0129] The present invention (4) relates to a method for producing a polyisoprenoid according to the present invention (1) or (2), wherein at least one selected from the group consisting of a gene encoding the cis-type prenyltransferase (CPT) family protein and a gene encoding the Rubber Elongation Factor (REF) family protein is derived from a plant belonging to at least one genus selected from the group consisting of the genus Hevea, the genus Taraxacum, and the genus Parthenium.

[0130] The present invention (5) relates to a method for producing a polyisoprenoid according to the present invention (1) or (2), wherein at least one selected from the group consisting of a gene encoding the cis-type prenyltransferase (CPT) family protein and a gene encoding the Rubber Elongation Factor (REF) family protein is derived from at least one plant selected from the group consisting of Hevea brasiliensis (Para rubber tree), Taraxacum koksaghyz (Russian dandelion), and Parthenium argentatum (guayule).

[0131] The present invention (6) relates to a method for producing a polyisoprenoid according to any one of the present inventions (1) to (5), wherein the transformed callus is derived from a rubber-producing plant.

[0132] The present invention (7) relates to a method for manufacturing a pneumatic tire, which includes a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid according to any one of the present disclosures (1) to (6) and an additive to obtain a kneaded product, a green tire forming step of forming a green tire from the kneaded product, and a vulcanization step of vulcanizing the green tire.

[0133] The present invention (8) relates to a method for manufacturing a rubber product, which includes a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid according to any one of the present disclosures (1) to (6) and an additive to obtain a kneaded product, a green rubber product forming step of forming a green rubber product from the kneaded product, and a vulcanization step of vulcanizing the green rubber product.

[0134] (Sequence Listing Free Text) SEQ ID NO: 1: Nucleotide sequence of a gene encoding HRT1 derived from Para rubber tree SEQ ID NO: 2: Amino acid sequence of HRT1 derived from Para rubber tree SEQ ID NO: 3: Nucleotide sequence of a gene encoding HRBP derived from Para rubber tree SEQ ID NO: 4: Amino acid sequence of HRBP derived from Para rubber tree SEQ ID NO: 5: Nucleotide sequence of a gene encoding REF derived from Para rubber tree SEQ ID NO: 6: Amino acid sequence of REF derived from Para rubber tree

Claims

1. A method for producing polyisoprenoid, characterized by producing polyisoprenoid using a transformed callus into which a gene encoding a cis-type prenyltransferase (CPT) family protein and a gene encoding a Rubber Elongation Factor (REF) family protein are introduced.

2. The method for producing polyisoprenoid according to claim 1, wherein the transformed callus does not have a gene encoding a foreign Nogo-B Receptor (NgBR) family protein.

3. The method for producing polyisoprenoid according to claim 1, wherein at least one selected from the group consisting of the gene encoding the cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein is derived from a rubber-producing plant.

4. The method for producing polyisoprenoid according to claim 1, wherein at least one selected from the group consisting of the gene encoding the cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein is derived from a plant belonging to at least one genus selected from the group consisting of the genus Hevea, the genus Taraxacum, and the genus Parthenium.

5. The method for producing polyisoprenoid according to claim 1, wherein at least one selected from the group consisting of the gene encoding the cis-type prenyltransferase (CPT) family protein and the gene encoding the Rubber Elongation Factor (REF) family protein is derived from at least one plant selected from the group consisting of Hevea brasiliensis (Para rubber tree), Taraxacum kok-saghyz (Russian dandelion), and Parthenium argentatum (guayule).

6. The method for producing polyisoprenoid according to claim 1, wherein the transformed callus is derived from a rubber-producing plant.

7. A method for manufacturing a pneumatic tire, comprising a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid according to any one of claims 1 to 6 and an additive to obtain a kneaded product, a green tire molding step of molding a green tire from the kneaded product, and a vulcanization step of vulcanizing the green tire.

8. A method for manufacturing a rubber product, comprising a kneading step of kneading a polyisoprenoid obtained by the method for manufacturing a polyisoprenoid according to any one of claims 1 to 6 and an additive to obtain a kneaded product, a green rubber product molding step of molding a green rubber product from the kneaded product, and a vulcanization step of vulcanizing the green rubber product.

Images