JP2005087036A - Cacao-originated n-methyltransferase and polynucleotide encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a theobromine-accumulating N-methyltransferase capable of being utilized as an enzyme for industry, foods, or medical treatments, to provide a method for modifying the caffeine biosynthetic metabolism of a caffeine or theobromine-producing plant, plant tissue or plant cell to efficiently produce a caffeine metabolic system compound, to provide a method for modifying the production ratio of caffeine metabolic system compound groups, to provide a new polypeptide capable of being utilized for the methods, and to provide a polypeptide encoding the same. <P>SOLUTION: The polypeptide is a new N-methyltransferase originated from cacao. The N-methyltransferase is produced with a microorganism or plant transformed with a vector containing a polypeptide encoding the polypeptide. The anti-sense RNA of the polypeptide is introduced into a plant to inhibit the expression of N-methyltransferase. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention produces theobromine or a precursor thereof using cocoa-derived N-methyltransferase, a polynucleotide encoding the enzyme, a microorganism and a plant transformed with the polynucleotide, the plant, or a cultured cell or tissue thereof. And a method of changing the amount of theobromine or a precursor thereof by a nucleic acid structure containing an antisense sequence of the polynucleotide.

Caffeine is contained in plants belonging to the genus Camellia sinensis such as tea ( Camelia sinensis ), plants belonging to the genus Caffeidae such as coffee ( Coffea arabica ), or plants belonging to the genus Mochinoki such as mate ( Ilex paraguariansis ) Purine alkaloids that are used as pharmaceutical raw materials and food additives.

In addition, cocoa ( Theobroma cacao ) is known as a seed that accumulates theobromine (3,7-dimethylxanthine), which is a precursor of caffeine. There is a small amount of caffeine in cacao, but theobromine is the main purine alkaloid. Since theobromine has a low central excitatory effect, it is known that its use causes a relaxing effect. Because cacao does not accumulate caffeine and accumulates a significant amount of theobromine, the relaxation effect of ingesting chocolate, which is a processed cacao product, has attracted attention.

It has been clarified by ¹⁴ C-tracer experiments that caffeine is biosynthesized from xanthosine through theobromine through a three-step N-methylation reaction (see Non-Patent Document 1). The enzyme activity that catalyzes N-methylation was first reported in 1975 in a study using a crude extract of tea leaves (see Non-Patent Document 2). On the other hand, purification of methyltransferase was attempted with coffee, but the purity of the purification was extremely low (see Non-Patent Document 3). In Cha, there is a report of partial purification of methyltransferase, but the enzyme protein has not been isolated so far (see Non-Patent Document 4).

Under such circumstances, the amino acid sequence of an enzyme involved in purine alkaloid biosynthesis and the DNA encoding the amino acid sequence have been clarified one after another by Kato et al. For example, caffeine synthase, that is, N-methyltransferase that catalyzes the two-stage methylation reaction of 7-methylxanthine to theobromine to caffeine, which is the final reaction of caffeine biosynthesis, can be obtained from tea ( Camellia sinensis ) and coffee ( Coffea arabica ) (see Patent Document 1 and Patent Document 2). Further, theobromine synthase, that is, N-methyltransferase that catalyzes one-step methylation of 7-methylxanthine to theobromine has been isolated from coffee ( Coffea arabica ) (see Patent Document 3).
However, for cocoa, the isolation and identification of N-methyltransferase involved in purine alkaloid biosynthesis and the presence of genes have never been disclosed so far.

Japanese Patent Laid-Open No. 2001-37490 JP 2002-85072 A JP 2003-88372 A Takeo Suzuki et al., “Phytochemistry” (UK), 1992, Vol. 31, No. 8, p. 2575-2584 Takeo Suzuki et al., “Biochemical Journal” (UK), 1975, 146, p. 87-96 Paulo Mazzafera et al., “Phytochemistry” (UK), 1994, 37, 6, p. 1577-1584 Misako Kato et al., “Physiologia Plantarum” (Denmark), 1996, Vol. 98, p. 629-636

  An object of the present invention is to provide a theobromine-accumulating N-methyltransferase, which is one of the enzymes constituting a cocoa-derived purine alkaloid biosynthesis system, a polynucleotide encoding the same, a vector using the same, and the like. . Furthermore, by using them, it is possible to provide microorganisms that efficiently produce theobromine-accumulating N-methyltransferase that can be used as industrial, food, or medical enzymes, and cafes for plants, plant tissues, or plant cells. An object of the present invention is to provide a method for modifying the biosynthetic metabolism of caffeine-related compounds to modify the production ratio of caffeine metabolic compounds.

As a result of earnest research, the present inventors prepared a primer based on the binding region of S-adenosylmethionine conserved in caffeine synthase derived from the previously isolated tea ( Camellia sinensis ). The inventors succeeded in isolating a new homologous gene from cacao ( Theobroca cacao ) by the RACE (rapid amplification of cDNA ends) method using primers.
Next, the isolated DNA was incorporated into a vector and then introduced into E. coli to express a large amount of the protein derived from the DNA. When the enzymatic properties of the obtained expressed protein were examined, N-methyltransferase activity was observed, and it was confirmed that the DNA was a gene encoding N-methyltransferase.
Further, as a result of examining the substrate specificity of the obtained cocoa-derived N-methyltransferase, it was confirmed that it was a novel theobromine synthase having an extremely high activity of synthesizing theobromine from 7-methylxanthine, and the DNA was cocoa theobromine. The gene was found to be able to promote accumulation. Based on the above findings, the present inventors have completed the present invention.

Accordingly, the present invention provides (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, or (b) one or several amino acids deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2. Or a modified polypeptide comprising an added amino acid sequence and having N-methyltransferase activity.
According to a preferred embodiment of the polypeptide of the present invention, it is derived from cocoa (Theobroma cacao).

The present invention also relates to a polynucleotide encoding the polypeptide.
The present invention also relates to (i) a polynucleotide comprising a base consisting of Nos. 48 to 1139 in the base sequence represented by SEQ ID NO: 1,
(Ii) including a base sequence in which one or several bases are deleted, substituted, or added in the bases of Nos. 48 to 1139 in the base sequence represented by SEQ ID NO: 1, and N-methyl Hybridizes under stringent conditions with a polynucleotide encoding a polypeptide having transferase activity, or (iii) a polynucleotide consisting of nucleotides 48 to 1139 in the nucleotide sequence represented by SEQ ID NO: 1; And a polynucleotide encoding a polypeptide having N-methyltransferase activity.
The present invention also relates to an expression vector comprising the polynucleotide and a structure for expressing a polypeptide encoded by the polynucleotide and having an N-methyltransferase activity in cells of microorganisms and / or plants.
The present invention also relates to a polynucleotide having a base sequence complementary to the base sequence of the polynucleotide or a partial sequence thereof, and a polypeptide having N-methyltransferase activity in a microorganism and / or plant cell. The present invention relates to an expression vector having a configuration for suppressing expression.
The present invention also relates to a microorganism transformed with the expression vector.
The present invention also relates to a method for producing a polypeptide, comprising a step of culturing the microorganism and a step of collecting the polypeptide from a culture obtained by the culture.

The present invention also relates to a plant cell, plant tissue, or plant that has been transformed with or infected with the expression vector.
The present invention also relates to a method for producing a plant secondary metabolite using the polypeptide or the plant cell, plant tissue, or plant.
According to a preferred embodiment of the production method of the present invention, the plant secondary metabolite is one or more compounds selected from xanthosine, 7-methylxanthosine, 7-methylxanthine, theobromine, theophylline, and caffeine.
According to another preferred embodiment of the production method of the present invention, the plant belongs to the genus Camellia , the plant belongs to the genus Coffea , the plant belongs to the genus Cola, or the genus Erythroxylum. A plant belonging to the genus Ilex , a plant belonging to the genus Neea , a plant belonging to the genus Firmiana , a plant belonging to the genus Paulinia , or a plant belonging to the genus Theobroma .

  According to the present invention, it is possible to efficiently produce a theobromine accumulating N-methyltransferase that can be used as an industrial, food, or medical enzyme. It is also possible to modify the production ratio of caffeine metabolic system compounds by modifying caffeine biosynthetic metabolism in caffeine-related substance-producing plants, plant tissues, or plant cells.

The N-methyltransferase of the present invention is a polypeptide derived from cocoa ( Theobroma cacao ) having 7-methylxanthine N3 methyltransferase activity, 3-methylxanthine N1 methyltransferase activity, and paraxanthine N3 methyltransferase activity.

  As the N-methyltransferase of the present invention, (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (preferably a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2), (b) SEQ ID NO: 2 A modified polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added and having N-methyltransferase activity, or (c) SEQ ID NO: 2 And an amino acid sequence having a homology of 80% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more), and N -Homologous polypeptides having methyltransferase activity.

Table 1 shows the results of comparing the homology of the amino acid sequence of the N-methyltransferase of the present invention (SEQ ID NO: 2) with known plant-derived N-methyltransferase. For calculation of homology, GENETYX MAC ver. 9.0 was used. The homology between the N-methyltransferase of the present invention and the known N-methyltransferase is 54.6% at the maximum, and it is clear that the N-methyltransferase of the present invention is a novel enzyme.

  In the “modified polypeptide” of (b), the number of amino acids involved in the modification is not particularly limited as long as the modified polypeptide has N-methyltransferase activity, but preferably 1 to about 30, more preferably 1. -10, more preferably 1-3.

  In addition, N-methyltransferase activity can be measured by the method shown in the Example, for example. The N-methyltransferase of the present invention is characterized by extremely high substrate specificity for 7-methylxanthine, which is a precursor of theobromine. Hereinafter, N-methyltransferase derived from cocoa may be referred to as “cocoa-derived theobromine synthase” or “BTS1” in the present specification.

The N-methyltransferase of the present invention can be preferably extracted from the leaf part, bean part, or stem part of cacao ( Theobroma cacao ), but the vector can be expressed using the polynucleotide shown below. Can be obtained by transforming a microbial host such as E. coli and collecting the expressed protein.

  Examples of the polynucleotide encoding the N-methyltransferase of the present invention include a polynucleotide having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2. Further, in the amino acid sequence represented by SEQ ID NO: 2, a base encoding an altered polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and having N-methyltransferase activity Also included are polynucleotides having sequences. In the present specification, the term “polynucleotide” includes both DNA and RNA.

The polynucleotide of the present invention is typically selected from the following group:
(I) a polynucleotide comprising a sequence consisting of bases 48 to 1139 in the base sequence represented by SEQ ID NO: 1 (preferably comprising bases 48 to 1139 in the base sequence represented by SEQ ID NO: 1 A polynucleotide comprising a sequence),
(Ii) an N-methyltransferase containing a base sequence in which one or several bases are deleted, substituted, or added in the bases of Nos. 48 to 1139 in the base sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a polypeptide having activity, and (iii) a polynucleotide consisting of nucleotides 48 to 1139 in the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, and N A polynucleotide encoding a protein having methyltransferase activity.

  In the base sequence (ii), the number of bases that can be deleted, substituted or added is preferably 1 to 90, more preferably 1 to 30, and still more preferably 1 to 9. The mutation as described above may occur in nature or may be artificially caused by site mutation in the base sequence.

  Hybridization under “stringent conditions” in (iii) above can be performed, for example, by the method described in Molecular Cloning: Cold Spring Harbor Laboratory Press, Current Protocols in Molecular Biology; Wiley Interscience. Examples include a GeneImage system (Amersham). Specifically, hybridization can be performed by the following operation.

The membrane to which the DNA or RNA molecule to be tested is transcribed is hybridized with a labeled probe in a protocol-specific hybridization buffer according to the product protocol. The composition of the hybridization buffer consists of 0.1% by weight sodium dodecyl sulfate (SDS), 5% by weight dextran sulfate, 1/20 volume of blocking reagent attached to the kit, and 2-7 × SSC. Blocking reagents include, for example, 100 × Denhardt's solution, 2% (weight / volume) bovine serum albumin (BSA), 2% (weight / volume) Ficoll ^™ 400, and 2% (weight / volume) Polyvinyl pyrrolidone prepared at a 5-fold concentration can be diluted to 1/20 and used. 20 × SSC is a 3 mol / L sodium chloride, 0.3 mol / L citric acid solution, and SSC is more preferably used at a concentration of 3 to 6 × SSC, more preferably 4 to 5 × SSC.

  The hybridization temperature is in the range of 40 to 80 ° C., more preferably 50 to 70 ° C., and still more preferably 55 to 65 ° C. After incubation for several hours to overnight, the plate is washed with a washing buffer. The washing temperature is preferably room temperature or higher, more preferably the temperature during hybridization. The composition of the wash buffer is 6 × SSC + 0.1 wt% SDS solution, preferably 4 × SSC + 0.1 wt% SDS solution, more preferably 2 × SSC + 0.1 wt% SDS solution, even more preferably 1 × SSC + 0.1 wt% SDS solution, most preferably 0.1 × SSC + 0.1 wt% SDS solution. By washing the membrane with such a washing buffer, the DNA molecule or RNA molecule hybridized with the probe can be identified using the label used for the probe.

The polynucleotide of the present invention may be naturally derived, fully synthesized, or synthesized using part of the naturally derived polynucleotide. The polynucleotide of the present invention can be obtained, for example, by PCR technology using an oligonucleotide that specifically hybridizes to a polynucleotide encoding N-methyltransferase as a primer [plant PCR experiment protocol (cell engineering separate volume, plant cell engineering series 2). ) Shujunsha (1995)] can be used to isolate the cells producing the N-methyltransferase of the present invention. Specifically, a linker is bound to cDNA synthesized from mRNA, and PCR is performed between the polynucleotide encoding the amino acid sequence constituting N-methyltransferase and the cDNA bound to the linker, etc. Can be isolated. Further, as shown in the Examples, S-adenosylmethionine (hereinafter referred to as SAM) preserved in caffeine synthase or theobromine synthase derived from previously isolated tea ( Camelia sinensis ) or coffee ( Coffea arabica ). The gene can be isolated from the cocoa ( Theobroma cacao ) by the RACE method using a primer prepared based on the binding region of

  A method for constructing an expression vector containing a polynucleotide encoding the N-methyltransferase of the present invention, a method for producing a transformed plant, and a method for suppressing the expression of N-methyltransferase by the RNAi method are as follows.

The expression vector of the present invention includes
(A) (i) a polynucleotide of the present invention, and (ii) a structure for expressing a polypeptide having N-methyltransferase activity encoded by the polynucleotide in a microorganism and / or plant cell. An expression vector, and (b) (i) a polynucleotide having a base sequence complementary to the base sequence of the polynucleotide of the present invention or a partial sequence thereof, and (ii) in a microorganism and / or plant cell, An expression vector having a configuration for suppressing the expression of a polypeptide having N-methyltransferase activity is included.

  “Configuration for expressing a polypeptide having N-methyltransferase activity encoded by a polynucleotide of the present invention in cells of microorganisms and / or plants”, or “Nucleotide in cells of microorganisms and / or plants, As the “configuration for suppressing the expression of a polypeptide having methyltransferase activity”, for example, a nucleic acid construct generally used as an expression cassette in the field of genetic engineering can be used. For example, a promoter that can be transcribed in microorganisms and / or plant cells, or a terminator sequence including a polyadenylation site necessary for stabilization of a transcript can be used.

In order to express DNA encoding the N-methyltransferase of the present invention or an antisense RNA thereof in microorganisms or plant cells, for example,
(I) a promoter capable of transcription in microorganisms or plant cells,
(Ii) all or part of the N-methyltransferase gene DNA linked in the sense or antisense direction downstream of the promoter, and (iii) stabilization of the transcript linked downstream of the gene DNA as necessary. An expression cassette containing a terminator sequence containing the necessary polyadenylation site can be introduced into a microorganism or plant cell.

  Here, as the template of antisense RNA, for example, all or a part of DNA encoding N-methyltransferase, or a base sequence in which DNA hybridizing under stringent conditions is linked in the antisense direction, or A nucleotide sequence in which all or part of RNA encoding N-methyltransferase, or RNA that hybridizes with it under stringent conditions is linked in the antisense direction, and produces caffeine or a precursor thereof. Examples thereof include those having a function of inhibiting the expression of N-methyltransferase in a host cell when expressed in the host cell.

  Here, a part of the DNA encoding N-methyltransferase is an inhibitory mRNA obtained on the basis of a sequence complementary to the part, that is, when an antisense RNA is formed in a host cell. This is the part that binds to mRNA for expressing N-methyltransferase in the host cell and inhibits the expression of N-methyltransferase in the host cell. As this part, it is a part required for formation of such antisense mRNA, for example, the part of the length of at least 14 bases can be mentioned.

As DNA or RNA that can be used as a template for forming antisense mRNA, for example, it has complementarity to all or part of the sequence consisting of bases 48 to 1139 in the base sequence of SEQ ID NO: 1. DNA or RNA that is present. DNA or RNA having complementarity with all or part of the mutant sequence that can hybridize with these base sequences under stringent conditions can also be used for such purposes.
These inhibitory DNAs or RNAs do not necessarily have to encode the N-methyltransferase of the present invention.

The expression cassette may contain a promoter for constitutively or inducibly expressing the inserted DNA.
Examples of the promoter for constant expression include cauliflower mosaic virus 35S promoter and rice actin promoter.

  In addition, as a promoter for inducible expression, it is known that it is expressed by external factors such as infection or invasion of filamentous fungi, bacteria, or viruses, low temperature, high temperature, dryness, anaerobic conditions, or spraying of specific compounds. And the like promoters. Examples of such promoters include rice chitinase gene promoters expressed by infection or invasion of filamentous fungi, bacteria, or viruses, tobacco PR protein gene promoters, rice lip19 gene induced by low temperature, and the like. Promoter, promoter of Arabidopsis thaliana “HSP18.2” gene induced by high temperature, promoter of rice “lab” gene induced by desiccation, promoter of corn alcohol dehydrogenase gene derived from anaerobic conditions, etc. It is done. In addition, the rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and the rice "rab" gene promoter is also induced by spraying the plant hormone abscisic acid. .

  Furthermore, examples of the promoter for expressing the DNA inserted into the expression cassette include a method of isolating and utilizing an N-methyltransferase promoter. As an example of a specific method for isolating a promoter, a genomic DNA fragment is selected by using a hybridization technique using all or part of the DNA of an N-methyltransferase gene as a probe, and the upstream DNA of the gene is identified. The method of doing can be mentioned.

  In order to prepare for the introduction of the recombinant DNA in the expression cassette into the plant, many cloning vectors containing a replication signal of E. coli and a marker gene for selecting transformed bacterial cells can be used. Examples of such vectors include pBR322, pUC vectors, M13mp vectors, and the like. The desired sequence can be introduced into the vector at an appropriate restriction enzyme cleavage site. Restriction enzyme cleavage site analysis, gel electrophoresis, and other biochemical-molecular biological methods are commonly used to characterize the resulting plasmid DNA. After each operation, the plasmid DNA can be cleaved and bound to another DNA. The sequence of each plasmid DNA can be cloned into the same plasmid or another plasmid.

Various techniques can be used to introduce an expression cassette into a plant host cell. These techniques, for example, transformation of plant cells with T-DNA using Agrobacterium tumefaciens as a transforming factor (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (Agrobacterium rhizogenes), direct introduction into protoplasts (e.g. , Injection method or electroporation method), or particle gun method, or other possibilities.

  There is no specially required vector for direct introduction into protoplasts. For example, a simple plasmid such as a pUC derivative can be used. Depending on the method of introducing the gene of interest into the plant cell, other DNA sequences may be required. For example, when a Ti or Ri plasmid is used for transformation of a plant cell, at least the rightmost sequence (usually the sequences at both ends) of the T-DNA region of the Ti and Ri plasmid is used as the adjacent region of the gene to be introduced. Must be connected so that

When Agrobacterium is used for transformation, it is necessary to clone the expression cassette to be introduced into a special plasmid, that is, an intermediate vector or a binary vector.
Intermediate vectors are not replicated in Agrobacterium. The intermediate vector is transferred into Agrobacterium by helper plasmid or electroporation. Since the intermediate vector has a region homologous to the sequence of T-DNA, the intermediate vector is incorporated into an Agrobacterium Ti or Ri plasmid by homologous recombination. Agrobacterium used as a host must contain a vir region. Usually, the vir region is contained in the Ti or Ri plasmid, and T-DNA can be transferred to plant cells by its function.

  On the other hand, since a binary vector can be replicated and maintained in Agrobacterium, when incorporated into Agrobacterium by a helper plasmid or electroporation, the host vir region causes the binary vector Of T-DNA can be transferred to plant cells.

  In addition, intermediate vectors or binary vectors containing the expression cassette thus obtained, and microorganisms such as Escherichia coli and Agrobacterium are also objects of the present invention.

  As a microorganism for introducing a polynucleotide encoding the N-methyltransferase of the present invention to express a large amount of N-methyltransferase protein, for example, various microorganisms used for protein expression in the field of genetic engineering are used. For example, bacteria (for example, E. coli or Bacillus subtilis) or eukaryotic microorganisms can be mentioned. Examples of the eukaryotic microorganism include cultured cells derived from animals (for example, mammals, birds, or insects), cultured cells derived from plants, and fungi (for example, yeast).

In addition, the DNA encoding the N-methyltransferase of the present invention is used in the sense or antisense form for the purpose of modifying the host cell metabolism to improve the productivity of a specific compound or to modify the production ratio of a specific group of compounds. Any plant that can produce caffeine, theobromine, or a precursor thereof can be used as the plant to be incorporated in the plant. Among them, plants belonging to the genus Camellia (for example, tea), coffee of Rubiaceae A plant belonging to the genus ( Coffea ) (for example, coffee), a plant belonging to the genus Cola ( Cola ), for example, a plant belonging to the genus Erythroxylum , a plant belonging to the genus Ilex , Neea ) A plant belonging to the genus, Firmian a ) plants belonging to the genus, plants belonging to the genus Paulinia , plants belonging to the genus Theobroma, and the like.

  The transformed plant cell can be converted into a plant body through a regeneration process. The method of regeneration varies depending on the type of plant cell. For example, Fujimura et al. (Plant Tissue Culture Lett., 1985, Vol. 2, p. 74-75) is used for rice, and Shirito et al. (Bio / Technology, for maize). 1989, 7, p. 581-587), or the method of Akama et al. (Plant Cell Rep., 1992, 12, p. 7-11) and the like in Arabidopsis thaliana.

In the present invention, the “plant” refers to the whole or a part of an organism individual classified as a plant (for example, leaves, stems, roots, flowers, fruits, seeds, etc.).
The plant produced by these methods changes the expression level of the N-methyltransferase protein of the present invention as compared to a wild-type plant producing caffeine, theobromine or a precursor thereof, and the host plant Changes in the amount of caffeine metabolite compounds produced due to alterations in the metabolism of caffeine and changes in the production ratio of caffeine metabolite compounds due to alterations in host plant metabolism occur. The transgenic plant thus obtained is the subject of the present invention. In addition to the said plant body, a plant tissue, a plant cell, etc. are contained in the plant said by this invention.

  In addition, in recent years, research on post-translational gene silencing of plants has made it possible to suppress the expression of target genes using the defense mechanisms inherent to plants against foreign nucleic acids such as viruses. (Cell, 1998, 95, p. 177-187; Chemistry and Biology, 1999, 37, p. 532-534; Protein Nucleic Acid Enzyme, 1999, 44, p. 1396-1397) ). According to this, when a DNA virus or RNA virus enters a plant, the plant transcribes the average RNA from these templates, and the transcript of the sequence originally possessed by the plant and the sequence-specific double-stranded RNA. Form. This double-stranded RNA can be suppressed by RNase, thereby suppressing the expression of the target gene (Cell, 1999, 96, p. 303-306). One of the important features of this method is that the target plant does not necessarily have to be transformed with the sequence whose expression is to be suppressed. In addition, a further feature of the present method is that if the target nucleic acid is introduced into a part of a plant (such as lower leaves) by infection or the like, the effect spreads over the entire plant body.

A specific expression suppression method is, for example,
(I) a double-stranded RNA containing the entire sequence of the N-methyltransferase gene or a part of the sequence, or all or a portion of the sequence that hybridizes under stringent conditions, or (ii) N-methyl Agrobacterium having double-stranded DNA containing the entire sequence of the transferase gene or a part of the sequence, or all or a part of the sequence that hybridizes with them under stringent conditions is infected to the lower leaves of the plant. Here, the partial sequence of the N-methyltransferase gene refers to an inhibitory mRNA obtained on the basis of a sequence complementary to the portion, that is, an aberrant RNA, formed in the host cell. This is the part that binds to mRNA for expressing N-methyltransferase in the host cell and inhibits the expression of N-methyltransferase in the host cell. This part is a part necessary for the formation of such an aberrant mRNA, and can include, for example, a part having a length of at least 14 bases.

  The plant body subjected to these methods changes the expression level of the N-methyltransferase protein of the present invention as compared to a wild type plant body that produces caffeine, theobromine, or a precursor thereof, and the host plant. Changes in the amount of caffeine metabolite compounds produced due to alterations in the metabolism of caffeine and changes in the production ratio of caffeine metabolite compounds due to alterations in host plant metabolism occur. The plants thus obtained are the subject of the present invention. The plant referred to in the present invention also includes specific tissues or cells of plants such as leaves, flowers, fruits and seeds.

  Caffeine and theobromine synthesis by cultivating plant cells or plant tissues that have been transformed with the above-constructed vector or infected with the above-constructed vector, or by similarly cultivating transformed plants. The production of secondary metabolites related to the system and the modification of the composition of secondary metabolites produced by these transformants can be performed. Examples of the secondary metabolite include at least one compound selected from the group consisting of xanthosine, 7-methylxanthosine, 7-methylxanthine, theobromine, theophylline, and caffeine.

Examples of the plant cell, plant tissue, or plant source used for transformation include plants belonging to the genus Camellia , plants belonging to the genus Coffee ( Coffea ), plants belonging to the genus Cola ( Cola ), Erythroxylum) plants belonging to the genus Ilex (Ilex) plant belonging to the genus Neea (NEEA) plant belonging to the genus, Sterculiaceae (Firmiana) plant belonging to the genus Porinia (Paullinia) plant belonging to the genus or Kakaonoki (Theobroma) to the genus The plant to which it belongs can be mentioned. Among others, Theaceae camellia (Camellia) plant belonging to the genus (eg, tea), Rubiaceae coffee (Coffea) plant belonging to the genus (for example, coffee), Sterculiaceae Koranoki (Cola) plant belonging to the genus (for example, Koranoki), Or a plant belonging to the genus Theobroma is preferable.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Synthesis of single-stranded cDNA for cloning of N-methyltransferase (1) Isolation of total RNA 1 g of young cacao leaves ( Theobroma cacao ) was disrupted with pestle and mortar in the presence of liquid nitrogen did. After sublimation of liquid nitrogen, mixing of 2% CTAB (Cetyltrimethylammonium bromide), 0.1 mol / L-Tris (pH 9.5), 20 mmol / L-EDTA, 1.4 mol / L-NaCl, and 5% β-mercaptoethanol The solution was dissolved in 5 mL and left at 65 ° C. for 10 minutes. An equal volume of chloroform solution [chloroform / isoamyl alcohol (24: 1)] was added and stirred to make a total volume of about 10 mL. Centrifugation was performed at 15,000 rpm for 10 minutes, the aqueous layer was taken, 2.5 mL of 10 mol / L lithium chloride solution was added, and the mixture was allowed to stand at −20 ° C. for 2 hours. Centrifugation is performed at 4 ° C. and 15,000 rpm for 10 minutes, the supernatant is removed, and the precipitate is added to 1 mL of TE solution [composition: 10 mmol / L-Tris, 1 mmol / L-EDTA-Na (pH 8.0)]. Dissolved and centrifuged again. The supernatant was collected, 1 mL of a TE saturated phenol solution was added and mixed by inversion, followed by centrifugation at 15,000 rpm for 10 minutes. Furthermore, phenol / chloroform was added to the supernatant and stirred, followed by centrifugation at 15,000 rpm for 10 minutes. The aqueous layer was collected, and an equal amount of the chloroform solution [chloroform / isoamyl alcohol (24: 1)] was added and stirred. Centrifugation was performed at 15,000 rpm for 10 minutes, the aqueous layer was collected, a 1/4 volume of 10 mol / L lithium chloride solution was added, and the mixture was allowed to stand at −20 ° C. for 2 hours. Centrifugation was performed at 15,000 rpm for 10 minutes, 70% ethanol was added to the resulting precipitate, and centrifugation was performed again. The precipitate was dried up with a vacuum pump and dissolved in 200 μL of water to obtain a fraction of total RNA.

(2) cDNA synthesis required for 3′-end cloning by 3′-RACE method In the 3′-RACE method, a commercially available set (3′-Full RACE Core Set; TAKARA) was used. Primers not in the sequence listing are those attached to this set. Using the total RNA (1 μg) heat-treated at 65 ° C. for 5 minutes as a template, an oligo dT-3-site adapter primer (oligo dT-3-sites Adapter Primer) according to the method described in the instruction manual of the above set Was used to synthesize cDNA by reverse transcription reaction. The composition of the reaction solution is as follows (Table 2). A reaction product is obtained under the conditions of reacting this reaction solution at 30 ° C./10 minutes, 50 ° C./30 minutes, and 95 ° C./5 minutes using a commercially available amplification system (Gene Amp PCR System 2400: Perkin Elmer). It was.

Example 2: Cloning of N-methyltransferase gene by 3'-RACE method The following reaction solution was prepared using the single-stranded cDNA prepared in Example 1 (2) as a template (Table 3). A primer TCS-SAM1 (SEQ ID NO: 3) was designed based on the binding region of S-adenosylmethionine conserved in tea-derived caffeine synthase TCS1 (SEQ ID NO: 2 in JP-A-2001-37490), and PCR was performed. It was used for the reaction. This reaction solution was subjected to a reaction at 95 ° C./1 minute using a commercially available amplification system (Gene Amp PCR System 2400: Perkin Elmer), followed by 94 ° C./1 minute, 57 ° C./1 minute, and 72 ° C./1 minute. PCR was performed under the condition of reacting 30 cycles for 30 seconds to obtain a reaction product.
TCS-SAM1: GAYTTRGGITGYKCIKCIGGICCIAAYAC (29mer) (SEQ ID NO: 3)

Example 3: Subcloning into a plasmid vector Using 0.8% agarose gel, electrophoresis of reaction products was carried out in TAE buffer (composition: 40 mmol / L-Tris, 40 mmol / L acetic acid, 1 mmol / L-EDTA). The obtained target product band was cut out, and DNA was collected from the gel using a commercially available DNA purification reagent (GENE CLEAN: Funakoshi). The recovered DNA was ligated to pT7blue vector (Novagen) and then transformed into E. coli DH5α. After color selection using X-gal, liquid culture was performed in an LB medium containing ampicillin, and a plasmid was extracted by the alkali-SDS method. The presence or absence of the insert was confirmed by agarose electrophoresis.

Example 4: Determination of nucleotide sequence Primer extension was performed in the following termination mix reaction solution (Tables 4 and 5) using the isolated plasmid. The reaction conditions were 98 ° C / 5 minutes, followed by 25 cycles of 98 ° C / 1 minute, 50 ° C / 45 seconds, and 72 ° C / 1.5 minutes, followed by reaction at 72 ° C / 1 minute. .

After completion of the reaction, 8 μL of a reaction stop solution was added to each tube. After heat denaturation at 98 ° C. for 5 minutes and the sample was rapidly cooled in ice, it was analyzed using a Shimadzu DSQ-2000L (S) system to determine the base sequence. The reagent attached to ThermoSequenase Cycle Sequencing Kit was used. The obtained base sequence is shown below.
GACATTGGGGTGTTCGGCGGGGCGAATACTTTCACTGTGATGTCAACAGTGATAGAGAGCACAAGGGACAAATGCAGTGAATTGAACTGGCAGATGCCAGAAATCCAGTTCTACTTGAATGATTTAGTAGGGAATGATTTCAACACACTGTTCAAAGGGTTATCTGTCATTCAAGATAAATATAAGAATGTTTCATGCTTTGCTATGGGTGCCCCTGGTTCCTTTCACGGCCGGCTTTTCCCTCAGAACTCCATGCATCTCATCCATTCCTCTTATGGTGTACAATGGCTCTCTAAGGTACCCAAAATGACAAGTGAAGGAGGGTTATCACCACCAAACAAAGGGAAGATCTACATATCGAAAACCAGTCCTCCTGCAGTGTGGAAGGCATACCTTTCTCAGTTTCAGGAAGACTTCCTTTCATTCTTGAGATGTCGATCTCCCGAGCTGGTACCTGATGGCCGCATGGTCTTGATAATTCATGGAAGGAAATCCGCAGATCCCACAACCAGAGAGAGCTGCTACACATGGGAAGTTCTAGCTGATGCCATCTCCTACCAGGTTTCACAGGGACTGATTGATGAAGAGAAACTAAACTCCTTCAATGTACCATACTATATTCCCTCCCAGGAAGAAGTTCGGGATTTAGTGAACAAGGAGGGTTCTTTCTTAACTGAGTTTGTAGACACAATTGAAGTTGAACTTGAAGGTATATGGACGGGACCGGAAAATGGAGCTAAAAACTTGAGATCCTTCACAGAGCCCATGATTTCTCACCAGTTTGGAGAAGAGGTAATGGACAAGCTATATGATAAGGTAAAAGATATTCTAGTAGAGGATTGCAAGCAGGAAAAACAATCAACCAGGGGAGTTAGTATTGTTCTTGAATTGAAAAAGAAAGAATCCCACCTCAGCTAAATGTATCATCATTCAGGAAACATTGAAGAAAACAATGTAGCAAAAGAATCAAGGTGATGAACATATGTAAAGAGTATTATTA TTAATTAATTTGCTAAGGAATCGAGAAGAAAAAAAAAAAAAAAGGATCCGGTACCTCTAGATCAG (SEQ ID NO: 9)

Example 5: Isolation of 5 'upstream region of N-methyltransferase mRNA by 5'-RACE method A commercially available set (5'-Full RACE Core Set: TAKARA) was used for isolation of 5' upstream region. . The following primers were designed based on the sequence information obtained from 3′-RACE results.
BCSn-RT: ACCCTCCTTGTTCACTAA (18mer) (SEQ ID NO: 4)
BCSn-AR: ACCTTAGAGAGCCATTGTA (19mer) (SEQ ID NO: 5)
BCSn-B: GCAGTGTGGAAGGCATACC (19mer) (SEQ ID NO: 6)
BCSn-CR: AAGAGGAATGGATGAGATGC (20mer) (SEQ ID NO: 7)
BCSn-D: GGACTGATTGATGAAGAGAA (20mer) (SEQ ID NO: 8)
First strand cDNA synthesis was performed using primer BCSn-RT (SEQ ID NO: 4), and after degradation of hybrid RNA and cyclization of single-stranded cDNA by ligation reaction, primers BCSn-AR (SEQ ID NO: 5) and BCSn PCR reaction was performed using -B (SEQ ID NO: 6). The reaction conditions were 94 ° C./3 minutes, 30 cycles of 94 ° C./30 seconds, 55 ° C./30 seconds, and 72 ° C./1 minute to obtain a reaction product. Further, using this as a template, primers BCSn-CR (SEQ ID NO: 7) and BCSn-D (SEQ ID NO: 8) were reacted at 94 ° C./3 minutes, followed by 94 ° C./30 seconds, 58 ° C./30 seconds, and 72 A cycle consisting of 30 ° C./30 seconds was set to 30 cycles to obtain a reaction product. The reaction product bands were separated by acrylamide electrophoresis, the DNA was collected from the gel, subcloned into the pT7blue vector, and a sample was prepared using a commercially available sequence kit (ThermoSequenase Cycle Sequencing Kit: Shimadzu Corporation). The base sequence of DNA was determined using the -2000L (S) system. The obtained base sequence is shown below.
ACACAAAGCAAGTTACATGCTTCAGAACAGAGAAAAGAAAATTATCAATGGAGGTGAAGGAAATGCTCTTCATGAACAAAGGAGATGGAGAAAATAGCTATGTTAAGACTTCAGGATACACGCAAAAAGTGGCAGCTGTAACCCAGCCAGTAGTTTACAGGGCAGCTCAATCTCTCTTCACAGGGAGGAATTCTTGCTCATACCAAGTCCTAAACGTGGCTGACTTGGGGTGTTCTTCAGGTCCAAACACTTTCACTGTGATGTCAACAGTGATAGAGAGCACAAGGGACAAATGCAGTGAATTGAATGGCAGATGCCAGAAATCCAGTTCTACTTGAATGATTTAGTAGGGAATGATTTCAACACACTGTTCAAAGGGTTATCTGTCATTCAAGATAAATATAAGAATGTTTCATGCTTTGCTATGGTGCCCCTGGTTCCTTTCACGGCCGGCTTTTCCCTCAGAACTCCATGCATCTCATCCATTCCTCTT (SEQ ID NO: 10)

Example 6: Preparation of N-methyltransferase gene BTS1 Using pT7blue vector (3 '/ pT7Blue sample) into which the cacao-derived N-methyltransferase fragment obtained in Example 3 was inserted as a template, BCSn-F and LCS1-ER were used. A PCR reaction was performed using the primer as a primer to prepare a fragment with an EcoRI site added to the 3 ′ end. As the reaction conditions, after the reaction at 95 ° C./1 minute, a cycle consisting of 94 ° C./1 minute, 53 ° C./1 minute, and 72 ° C./2 minutes was defined as 30 cycles. The reaction solution composition is shown below (Table 6).
BCSn-F: GGTTATCTGTCATTCAAG (18mer) (SEQ ID NO: 11)
LCS1-ER: GGAATTCCTAGAGGTACCG (19mer) (SEQ ID NO: 12)

In addition, a PCR reaction was performed by using the pT7blue vector (5 ′ / pT7Blue sample) into which the cacao-derived N-methyltransferase fragment obtained in Example 5 was inserted as a template and using BCSn-N1 and BCSn-CR as primers. A fragment having an NcoI site added to the terminal side was prepared. As the reaction conditions, after the reaction at 95 ° C./1 minute, a cycle comprising 94 ° C./1 minute, 53 ° C./1 minute, and 72 ° C./2 minutes was defined as 30 cycles. The reaction solution composition is shown below (Table 7).
BCSn-N1: GCCATGGAGGTGAAGGAAA (19mer) (SEQ ID NO: 13)
BCSn-CR: AAGAGGAATGGATGAGATGC (20mer) (SEQ ID NO: 14)

The sample after the reaction was separated by agarose gel electrophoresis, and the target fragment was recovered. A fragment containing the fragment obtained in Example 3 was cleaved with the restriction enzyme EcoRI, and a fragment containing the fragment obtained in Example 5 was cleaved with the restriction enzyme NcoI and ligated to the corresponding restriction enzyme site of the plasmid pET23d (Novagen). . The obtained plasmid was introduced into E. coli DH5α to obtain a plasmid into which the two fragments were inserted. Analysis of the base sequence confirmed that the base sequence shown below (hereinafter sometimes referred to as “BTS1”) was inserted into pET23d. A restriction enzyme map of this plasmid pET-BCS1 is shown in FIG.
ACACAAAGCAAGTTACATGCTTCAGAACAGAGAAAAGAAAATTATCAATGGAGGTGAAGGAAATGCTCTTCATGAACAAAGGAGATGGAGAAAATAGCTATGTTAAGACTTCAGGATACACGCAAAAAGTGGCAGCTGTAACCCAGCCAGTAGTTTACAGGGCAGCTCAATCTCTCTTCACAGGGAGGAATTCTTGCTCATACCAAGTCCTAAACGTGGCTGACTTGGGGTGTTCTTCAGGTCCAAACACTTTCACTGTGATGTCAACAGTGATAGAGAGCACAAGGGACAAATGCAGTGAATTGAACTGGCAGATGCCAGAAATCCAGTTCTACTTGAATGATTTAGTAGGGAATGATTTCAACACACTGTTCAAAGGGTTATCTGTCATTCAAGATAAATATAAGAATGTTTCATGCTTTGCTATGGGTGCCCCTGGTTCCTTTCACGGCCGGCTTTTCCCTCAGAACTCCATGCATCTCATCCATTCCTCTTATGGTGTACAATGGCTCTCTAAGGTACCCAAAATGACAAGTGAAGGAGGGTTATCACCACCAAACAAAGGGAAGATCTACATATCGAAAACCAGTCCTCCTGCAGTGTGGAAGGCATACCTTTCTCAGTTTCAGGAAGACTTCCTTTCATTCTTGAGATGTCGATCTCCCGAGCTGGTACCTGATGGCCGCATGGTCTTGATAATTCATGGAAGGAAATCCGCAGATCCCACAACCAGAGAGAGCTGCTACACATGGGAAGTTCTAGCTGATGCCATCTCCTACCAGGTTTCACAGGGACTGATTGATGAAGAGAAACTAAACTCCTTCAAtGTACCATACTATATTCCCTCCCAGGAAGAAGTTCGGGATTTAGTGAACAAGGAGGGTTCTTTCTTAACTGAGTTTGTAGACACAATTGAAGTTGAACTTGAAGGTATATGGACGGGACCGGAAAATGGAGCTAAAAACTTGAGATCCTTCACAGAGCCCATGATTTCTCACCA GTTTGGAGAAGAGGTAATGGACAAGCTATATGATAAGGTAAAAGATATTCTAGTAGAGGATTGCAAGCAGGAAAAACAATCAACCAGGGGAGTTAGTATTGTTCTTGAATTGAAAAAGAAAGAATCCCACCTCAGCTAAATGTATCATCATGAAAAAAATGTAGCAAAGTAAATTAAGGTGATGAATTTG

Example 7: Expression of N-methyltransferase in E. coli The isolated BTS1 gene was incorporated into an expression vector pET23d (Novagen), and this plasmid (pET-BCS1) was transformed into E. coli BL21 (DE3). After culturing the obtained Escherichia coli at 37 ° C. for 2 hours, isopropylthiogalactoside (IPTG) was added to a final concentration of 0.3 mmol / L, and further cultured at 30 ° C. for 4.5 hours. After completion of the culture, the cells were collected and mixed with 10 mmol / L-Tris-hydrochloric acid buffer (pH 7.5), 0.1 mol / L sodium chloride, and 1 mmol / L-EDTA-Na ₂ per 100 mL of the culture. The ultrasonic crushing was performed intermittently for 1 minute in 0.45 mL of the liquid. This was centrifuged at 14,000 rpm for 10 minutes, and the resulting supernatant was used as an enzyme solution. When the activity was measured by the following method using this enzyme solution, N-methyltransferase (theobromine synthase) activity was observed.

  The plasmid pET-BCS1 was introduced into Escherichia coli K12, and on September 11, 2003, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Takuba, Ibaraki, Japan 305-8565) It is deposited domestically in 1st, 1-Chome, Ichi-Chi, 1st Central 6). The accession number is FERM P-19522.

The reaction solution used in the N- methyltransferase (theobromine synthase) activity measurements, 100 mmol / L-Tris-HCl buffer (pH8.5), 0.2mmol / L- MgCl 2, 1mmol / L-7- methylxanthine, and The enzyme solution 56 μL was added to 46 μmol / L [methyl- ¹⁴ C] S-adenosylmethionine (1.85 kBq), and the volume of the reaction solution was 100 μL. The reaction was performed at 27 ° C. for 30 minutes, and the resulting reaction product was extracted by adding 1 mL of chloroform. The chloroform layer was concentrated and developed with butanol-acetic acid-water (4: 1: 1) using a cellulose thin layer plate, followed by autoradiography to identify the reaction product. When the expected reaction product was 7-methylxanthosine, the reaction solution was lyophilized, dissolved in 50% methanol, similarly separated by thin layer chromatography, and then identified by autoradiography. As a control, one obtained by removing 7-methylxanthine from the reaction solution was used. As a result of the activity measurement, it was found that 105.9 pmol of theobromine was produced.

Table 8 shows the substrate specificity of the N-methyltransferase enzyme obtained using the enzyme solution. The numerical values in the table are relative activities (%) when the activity against 7-methylxanthine is defined as 100. Further, “nd” in Table 2 means that it was below the detection limit. The measurement method was the same as described above except that the substrate shown in Table 2 was used. For comparison, the substrate specificity of caffeine synthase TCS1 derived from tea ( Camellia sinensis ) leaves was also described.

  The present invention can be applied to the production of a theobromine accumulating N-methyltransferase that can be used as an industrial, food, or medical enzyme.

   The description of “Artificial Sequence” is described in the numerical heading <223> of the following sequence listing. Specifically, the base sequence represented by SEQ ID NO: 3 is primer TCS-SAM1, the base sequence represented by SEQ ID NO: 4 is primer BCSn-RT, and the base sequence represented by SEQ ID NO: 5. Is a primer BCSn-AR, the base sequence represented by SEQ ID NO: 6 is primer BCSn-B, the base sequence represented by SEQ ID NO: 7 is primer BCSn-CR, and is represented by SEQ ID NO: 8. The base sequence represented by SEQ ID NO: 11 is the primer BCSn-F, the base sequence represented by SEQ ID NO: 12 is the primer LCS1-ER, The base sequence represented by No. 13 is primer BCSn-N1, and the base sequence represented by SEQ ID No. 14 is primer BCSn-CR.

It is explanatory drawing which shows the structure of the plasmid which introduce | transduced cacao origin N-methyltransferase (BTS1).

Claims (12)

(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or (b) an amino acid wherein one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 A modified polypeptide comprising a sequence and having N-methyltransferase activity.   2. The polypeptide of claim 1, which is derived from cocoa.   A polynucleotide encoding the polypeptide according to claim 1 or 2. (I) a polynucleotide comprising a base consisting of Nos. 48 to 1139 in the base sequence represented by SEQ ID NO: 1,
(Ii) including a base sequence in which one or several bases are deleted, substituted, or added in the bases of Nos. 48 to 1139 in the base sequence represented by SEQ ID NO: 1, and N-methyl Hybridizes under stringent conditions with a polynucleotide encoding a polypeptide having transferase activity, or (iii) a polynucleotide consisting of nucleotides 48 to 1139 in the nucleotide sequence represented by SEQ ID NO: 1; A polynucleotide encoding a polypeptide having N-methyltransferase activity.   An expression vector comprising the polynucleotide according to claim 3 or 4 and a structure for expressing a polypeptide having N-methyltransferase activity encoded by the polynucleotide in a microorganism and / or plant cell.   A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide according to claim 3 or 4 or a partial sequence thereof, and a polypeptide having N-methyltransferase activity in a microorganism and / or plant cell An expression vector having a configuration for suppressing the expression of.   A microorganism transformed with the expression vector according to claim 5 or 6.   A method for producing a polypeptide, comprising: culturing the microorganism according to claim 7; and collecting the polypeptide according to claim 1 or 2 from a culture obtained by the culture.   A plant cell, plant tissue, or plant transformed with the expression vector according to claim 5 or 6 or infected with the expression vector.   A method for producing a plant secondary metabolite using the polypeptide according to claim 1 or 2, or the plant cell, plant tissue, or plant according to claim 9.   The method according to claim 10, wherein the plant secondary metabolite is one or more compounds selected from xanthosine, 7-methylxanthosine, 7-methylxanthine, theobromine, theophylline, and caffeine.   The plant belongs to the genus Camellia, the plant belonging to the genus Coffee, the plant belonging to the genus Genus, the plant belonging to the genus Genus, the plant belonging to the genus Genus, the plant belonging to the Neae genus, the plant belonging to the Aegiri genus, the plant belonging to the genus Porinia The method according to claim 10, wherein the plant belongs to the genus Cacao.

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