JP2004105075A - Coffee-derived caffeine synthase and gene encoding the enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently obtain caffeine synthase useful as an enzyme for foods and medical application for (1) industrial use, to modify the caffeine biosynthetic metabolism of plant tissue or plant cell and to efficiently obtain a caffeine metabolism-based compound in (2) a caffeine production plant and to modify the caffeine biosynthetic metabolism of plant tissue or plant cell and to modify the formation ratio of the compound group of the caffeine metabolism system in (3) the caffeine production plant by incorporating the whole or a part of a DNA encoding caffeine synthase in a sense or an antisense form into a bacterium or a plant cell. <P>SOLUTION: Caffeine synthase is produced using a bacterium, a plant body or a cultured cell transformed by a vector containing a DNA or RNA encoding a polypeptide having an amino acid sequence described by sequence number 1 and enzyme activity of caffeine synthase or the amount of expression of caffeine synthase is controlled by antisense RNA. In the method, a caffeine content in a plant is varied and caffeine synthase is produced. <P>COPYRIGHT: (C)2004,JPO

Description

反応条件は３０℃１０分、５０℃４０分、９５℃５分後氷冷とした。
【００５７】
得られた生成物を鋳型とし、以下の反応溶液中で、９５℃／１分間反応させた後、９４℃／１分間、５５℃／１分間、７２℃／１．５分間を３０サイクルでＰＣＲを行なった。Ｐｒｉｍｅｒ１には配列番号５の配列を用いた。

【００５８】
得られた生成物を鋳型とし、以下の反応液中で、９４℃／１分間、５５℃／１分間、７２℃／４０秒間を３０サイクルでＰＣＲを行ない、ＰＣＲ増幅産物を得た。Ｐｒｉｍｅｒ１には配列番号６の配列を用いた。

【００５９】
得られた反応生成物を０．８％　アガロースゲルを用いてＴＡＥ中で電気泳動し、得られた目的生成物のバンドを切り取り、ＧＥＮＥ　ＣＬＥＡＮ（フナコシ）を用いてゲルからＤＮＡを回収した。回収したＤＮＡをｐＴ７ｂｌｕｅベクター（Ｎｏｖａｇｅｎ）にライゲーションした後、大腸菌ＤＨ５αにトランスフォーメーションした。Ｘ−ｇａｌを用いてカラーセレクションを行った後に、アンピシリンを含むＬＢ培地で液体培養を行い、アルカリ−ＳＤＳ法によりプラスミドを抽出した。インサートの有無はアガロース電気泳動によって確認した。
【００６０】
得られた生成物の塩基配列を決定するため、単離したプラスミドを用いて下記の反応液中でプライマーエクステンションを行った。反応条件は９６℃／１分間反応させた後、９６℃／０．２分間、５０℃／０．１分間、６０℃／４分間を２５サイクル行った。反応液に対してエタノール沈殿を行い、得られたＤＮＡ　をＴｅｍｐｌａｔｅ　ｓｕｐｐｒｅｓｓｉｏｎ　ｒｅａｇｅｎｔに溶解し、ＡＢＩ−３１０ジェネティックアナライザーを用いて分析した。
【００６１】
プライマーエクステンション反応液
プラスミドＤＮＡ　（２０ｎｇ）　２μｌ
Ｐｒｅｍｉｘ　　　　　　　　　　４μｌ
Ｐｒｉｍｅｒ　　　　　　　　　　１μｌ
Ｈ２Ｏ　　　　　　　　　　　　　３μｌ
【００６２】
（２）　５’ＲＡＣＥ法による５’上流域の単離
５’上流域の単離には、５’−Ｆｕｌｌ　ＲＡＣＥ　Ｃｏｒｅ　Ｓｅｔ（ＴＡＫＡＲＡ）を用いた。３’−ＲＡＣＥ法で得られた配列を元に配列番号７〜９のプライマーを設計し、下記の反応液中で１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡを合成した。反応中のＰｒｉｍｅｒには配列番号７の配列を用いた。
変性ＲＮＡ（約１５μｇ）　　　　　　　　　　　　　　１５μｌ
１０×ＲＴバッファー　　　　　　　　　　　　　　　　　３μｌ
ＲＮａｓｅ　ｉｎｈｉｂｉｔｏｒ　　　　　　　　　　　　　１μｌ
Ｐｒｉｍｅｒ（ＣＳ１０ｒ）　　　　　　　　　　　　　　　　　１０μｌ
ＡＭＶ　ＲＴ　ｅｎｚｙｍｅ　　　　　　　　　　　　　　　１μｌ
【００６３】
得られた３０μｌのＲＮＡ−ｃＤＮＡハイブリッドに、２５μｌ　　５×バッファー、４５μｌ　　Ｈ２Ｏ、２μｌ　　ＲＮａｓｅＨを加え、３０℃　１ｈｒで反応させ、ＲＮＡを分解した。得られた１本鎖ｃＤＮＡは、Ｔ７−ＲＮＡ　ｌｉｇａｓｅでセルフライゲーションまたはコンカテマー化した。これを鋳型にし、以下の反応溶液中で、９４℃／１分間、６２℃／１分間、７２℃／１．５分間を３０サイクルでＰＣＲを行い反応生成物を得た。Ｐｒｉｍｅｒ１、２は、配列番号８、９の配列を用いた。アクリルアミド電気泳動により反応生成物のバンドを分離し、ゲルからＤＮＡを回収し、ｐＧＥＭ−Ｔ　Ｅａｓｙベクター（Ｐｒｏｍｅｇａ）にサブクローニングした。

サブクローニングした配列の塩基配列は上述の方法で決定した。得られた塩基配列を配列表の配列番号１に示す。
【００６４】
［実施例３］　カフェインシンターゼの大腸菌での発現と活性測定
単離した新規カフェインシンターゼ遺伝子を発現ベクターｐＥＴ３２ａ（Ｎｏｖａｇｅｎ）に組み込み、このプラスミドを大腸菌ＢＬ２１（ＤＥ３）にトランスフォーメーションした。得られた大腸菌を３７℃で２時間培養した後に、ＩＰＴＧを最終濃度０．３ｍＭになるように加え、２５℃でさらに８時間培養を行った。培養終了後集菌し、２００μｌのＴＥＳ−Ｇ　ｂｕｆｆｅｒ（５０ｍＭ　Ｔｒｉｓ−塩酸緩衝液（ｐＨ８．５）、５ｍＭ　ＤＴＴ、５０ｍＭ　ＮａＣｌ、２ｍＭ　ＥＤＴＡ−Ｎａ２、２０％　ｇｌｙｃｅｒｏｌ）に懸濁し、−８０℃で凍結後、１分間断続的に超音波破砕を行った。これを１４，０００ｒｐｍ、１０分間の遠心分離を行い、得られた上清を酵素液とした。
【００６５】
カフェインシンターゼ活性測定のための反応液は、１００ｍＭ　Ｔｒｉｓ−塩酸緩衝液（ｐＨ８．５）、０．２ｍＭ　ＭｇＣｌ２、０．２ｍＭ　７−メチルキサンチン、４μＭ　［メチル−１４Ｃ］Ｓ−アデノシルメチオニン（０．９ｋＢｑ）に酵素液１０μｌを加えた物とし、反応液の体積は１００μｌとした。２７℃で１０分間の反応を行い、得られた１４Ｃ−カフェインを１ｍｌのクロロホルムを加えて抽出し、クロロホルム層の放射活性を測定した。対照としては、反応液から７−メチルキサンチンを除いたものを用いた。活性測定の結果、カフェインが生成したことが判明した。
【００６６】
組み換え酵素の基質特異性を表１に示す。７−メチルキサンチンに対する活性を１００としたときの相対活性（％）で示した。比較のため、チャ（Ｃａｍｅｌｌｉａ　ｓｉｎｅｎｓｉｓ）葉由来カフェインシンターゼの基質特異性も記載した。
【００６７】
【表１】

【００６８】
［実施例４］　　アンチセンス法によるカフェイン生合成の抑制
アンチセンスＮ−メチルトランスフェラーゼ遺伝子を有する組換えベクターを以下の方法により構築した。
実施例２で得られたコーヒー由来新規カフェインシンターゼ遺伝子を、ｐＢＩベクター（ｃｌｏｎｔｅｃｈ社製）にＣａＭＶ３５Ｓプロモーターの下流に該遺伝子がアンチセンス方向に挿入される様に連結した。このようにして、アンチセンスＮ−メチルトランスフェラーゼ遺伝子が挿入された所望の組換えベクターを得た。そしてこれを以下の形質転換に用いた。
【００６９】
コーヒーの組織培養によるカフェイン生合成の従来法については、これまでにもＰｌａｎｔａ，　１０８，　３３９　（１９７２），　Ｐｌａｎｔ　Ｃｅｌｌ　Ｒｅｐｏｒｔｓ，　２，　１０９　（１９８３）などの多くの報告がある。これらの従来法に従ってコーヒーの茎頂ないし幼葉からカルスを誘導した。得られたカルスにパーティクルガン法で上記で構築された組換えベクターを導入した。もしくは、該カルスのプロトプラストを調製して、このプロトプラストにエレクトロポレーション法により組換えベクターを導入した。導入後、マーカー耐性を示す細胞を選抜した。選択された細胞は明条件下で培養し、形質転換細胞に対して実施例３に記載の方法により酵素活性を測定した。その結果、アンチセンスＮ−メチルトランスフェラーゼＤＮＡを導入した細胞におけるカフェインの生産は、アンチセンスＮ−メチルトランスフェラーゼＤＮＡを導入していない通常細胞と比較して有意に減少していることが判明した。
【００７０】
さらに、形質転換したコーヒー培養細胞を再分化させ、幼植物体を得た。再分化の方法はＺ．Ｐｆｌａｎｚｅｎｐｈｙｓｉｏｌ．　Ｂｄ．，　８１，　３９５　（１９７７）、Ｐｌａｎｔ　Ｃｅｌｌ，　Ｔｉｓｓｕｅ　ａｎｄＯｒｇａｎ　Ｃｕｌｔｕｒｅ，　８，　２４３　（１９８７）を含む文献に記載の従来法に従って行った。幼植物体の葉を用いて実施例３に記載の方法により酵素活性を測定した。その結果、アンチセンスＮ−メチルトランスフェラーゼＤＮＡを導入した植物体のカフェイン生産は、アンチセンスＮ−メチルトランスフェラーゼＤＮＡを導入していない植物体と比較して有意に減少していることが判明した。
【００７１】
【発明の効果】
本発明によれば、工業用、食品用または医療用酵素として利用できるカフェインシンターゼを効率よく生産する事が可能になる。
本発明によれば、カフェイン産生植物、植物組織または植物細胞のカフェイン生合成代謝を改変してカフェイン代謝系の化合物を効率よく生産する事が可能になる。
本発明によれば、カフェイン産生植物、植物組織または植物細胞のカフェイン生合成代謝を改変してカフェイン代謝系の化合物群の生成比を改変することが可能になる。
【００７２】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to caffeine and caffeine or a precursor thereof using caffeine synthase, DNA or RNA encoding the enzyme, microorganisms and plants transformed with the DNA, and the plant or its cultured cells or tissues. And a method for changing the amounts of caffeine and theobromine or a precursor thereof with a nucleic acid construct containing the RNA.
[0002]
[Prior art]
[0003]
[Non-Patent Document 1] Phytochemistry, $ 31, $ 2575- (1992)
[0004]
[Non-Patent Document 2] Biochem. J. , $ 146, $ 87- (1975)
[0005]
[Non-Patent Document 3] Phytochemistry, $ 37, $ 1577- (1994)
[0006]
[Non-Patent Document 4] Physiol. Plant. , $ 98, $ 629- (1996)
[0007]
[Patent Document 1] Japanese Patent Application No. 11-146358
[0008]
[Patent Document 2] Japanese Patent Application No. 2000-151718
[0009]
[Patent Document 3] Japanese Patent Application No. 2000-275063)
[0010]
[Patent Document 4] Japanese Patent Application No. 2001-209072
Caffeine is used for tea (Camellia sinensis) Such as Camellia, Camellia plant, coffee (Coffea arabicaPurine alkaloids contained in Rubiaceae coffee genus plants such as ii) and used as pharmaceutical raw materials and food additives. At present, caffeine is produced by extraction from caffeine-producing plants including the above plant species, or by organic synthesis. Also, in luxury items such as tea and coffee, in order to reduce or enhance their irritation, the content of caffeine and its intermediates is reduced or increased using classical breeding techniques and the like. ing.
[0011]
14C-tracer experiments have shown that caffeine is biosynthesized from xanthosine via theobromine by a three-step N-methylation. $ [Phytochemistry, $ 31, $ 2575- (1992)]. This enzymatic activity to catalyze methylation was first reported in a study using a crude extract of tea leaves in 1975 [Biochem. J. , $ 146, $ 87- (1975)]. Attempts have been made to purify methyltransferase in coffee [Phytochemistry, # 37, # 1577- (1994)], but the purification ratio was extremely low. In Cha, there have been reports of partial purification of methyltransferase [Physiol. Plant. , 98, 629- (1996)], and the enzyme protein was not isolated. Under such circumstances, the amino acid sequence of caffeine synthase, that is, an N-methyltransferase that catalyzes a two-step methylation reaction of 7-methylxanthine-theobromine-caffeine, which is the final reaction of caffeine biosynthesis, and its amino acid sequence For the DNA encodingCamellia sinensisKato et al. Have been the first in the world to isolate and purify and report (Japanese Patent Application Nos. 11-146358, 2000-151718, and 2000-275063).
[0012]
The same group has isolated and reported the presence of an enzyme that catalyzes only one-step methylation of 7-methylxanthine to theobromine in coffee (Japanese Patent Application No. 2001-209072). However, in coffee, the existence of an N-methyltransferase that catalyzes a two-step methylation reaction of 7-methylxanthine-theobromine-caffeine, which is the final reaction of caffeine biosynthesis, has not been known so far. Was.
[0013]
[Problems to be solved by the invention]
The present invention aims to achieve the following object by incorporating all or a part of DNA or RNA encoding caffeine synthase into a microorganism or a plant cell in a sense or antisense form.
(1) Efficiently produce caffeine synthase that can be used as an industrial, food or medical enzyme.
(2) Caffeine-producing plants, plant tissues or plant cells are modified to alter the caffeine biosynthetic metabolism to efficiently produce caffeine metabolic compounds.
(3) Caffeine-producing plants, plant tissues or plant cells are modified to alter the caffeine biosynthesis metabolism to alter the ratio of the generation of caffeine metabolic compounds.
[0014]
[Means for Solving the Problems]
The present inventors have determined that the previously isolated tea (Camellia sinensis), A DNA probe is prepared based on the DNA sequence of caffeine synthase, and coffee (Coffea arabicaAs a result of screening the cDNA library of (1), the target DNA was isolated from coffee, but these were theobromine synthases using only 7-methylxanthine as a substrate. Therefore, as a result of diligent research, the present inventors have focused on different conserved sequences of theobromine synthase which the present inventors have isolated, and have isolated a completely new homolog gene. This DNA was inserted into a vector and then introduced into Escherichia coli to express a large amount of a protein derived from the DNA. Examination of the enzymatic properties of this protein showed that it recognized 1-methylxanthine, 3-methylxanthine, paraxanthine, and theobromine as substrates in addition to 7-methylxanthine, and the DNA was caffeine biosynthesis. It was identified as a gene encoding a novel caffeine synthase that catalyzes major methylation of the pathway. The present inventors have completed the present invention based on the above findings.
[0015]
That is, the present invention is as follows.
[1] The following nucleotide sequence:
(A) N-methyltransferase which is a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase A coding nucleotide sequence,
(B) a mutant nucleotide obtained by performing nucleotide substitution, deletion or insertion in the nucleotide sequence (a) as long as the polypeptide encoded by the nucleotide sequence (a) can maintain the enzyme activity; Array
A DNA molecule having any of the following.
[2] The DNA molecule according to claim 1, wherein the nucleotide sequence (a) and the mutant nucleotide sequence (b) are capable of hybridizing under stringent conditions.
[3] The DNA molecule according to claim 1 or 2, wherein the nucleotide sequence (a) comprises the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.
[4] The following nucleotide sequence:
(A) N-methyltransferase which is a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase A coding nucleotide sequence,
(B) a mutant nucleotide obtained by performing nucleotide substitution, deletion or insertion in the nucleotide sequence (a) as long as the polypeptide encoded by the nucleotide sequence (a) can maintain the enzyme activity; An RNA molecule having any of the sequences.
[5] The RNA molecule according to claim 4, wherein the nucleotide sequence (a) and the mutant nucleotide sequence (b) can hybridize under stringent conditions.
[6] The RNA molecule according to claim 4 or 5, wherein the sequence (a) comprises the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.
[7] {characterized by having the DNA molecule according to any one of the above [1] to [3] and a configuration for expressing the N-methyltransferase encoded by the DNA molecule in a plant cell. Expression vector.
[8] A transformed cell obtained by transforming a host cell with the expression vector according to [7].
[9] The transformed cell according to [8], wherein the host cell is a microbial cell.
[10] A method for producing an N-methyltransferase having the enzyme activity by culturing the transformed cell according to [8] or [9].
[11] A nucleotide sequence complementary to all or a part of the nucleotide sequence of the DNA molecule according to any one of [1] to [3], which is expressed by being introduced into a plant cell having the enzyme activity. A DNA molecule capable of inhibiting the enzyme activity of the plant cell when performed.
[12] A nucleotide sequence complementary to all or a part of the nucleotide sequence of the RNA molecule according to any one of the above [4] to [6], which is introduced into a plant cell having the enzyme activity and expressed. An RNA molecule capable of inhibiting the enzyme activity of the plant cell when performed.
[13] A vector comprising the DNA molecule or RNA molecule according to any one of [1] to [6] and [11] to [12].
[14] A N-methyltransferase having an enzymatic activity of 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase can be expressed in at least one cell of a microorganism and a plant, or The vector according to the above [13], which has a function of inhibiting the expression of the N-methyltransferase.
[15] A microorganism transformed with the vector according to [13] or [14].
[16] A plant cell, plant tissue or plant transformed with the vector according to [13] or [14].
[17] The plant cell, plant tissue or plant according to [16], wherein the vector according to [13] or [14] has been introduced by infection.
[18] A method for producing a secondary metabolite of a plant using the plant cell, plant tissue or plant according to the above [16] or [17].
[19] A method for modifying the composition of a plant secondary metabolite using the plant cell, plant tissue or plant according to the above [16] or [17].
[20] A method for producing a plant secondary metabolite by culturing the plant cell or plant tissue or cultivating a plant according to the above [16] or [17].
[21] A method of culturing the plant cell or plant tissue or cultivating a plant according to the above [16] or [17] to modify the composition of a plant secondary metabolite.
[22] At least one plant secondary metabolite selected from the group consisting of xanthosine, 7-methylxanthosine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, theobromine, theophylline, paraxanthine, and caffeine The method according to any one of the above [18] to [21], which is one or more compounds.
[23] The transformed plant is a plant belonging to the genus Camellia, the plant belonging to the genus Coffee (Coffea), the plant belonging to the genus Colla (Cola), the plant belonging to the genus Molex (Ilex), the plant belonging to the genus Neeea, or the plant belonging to the genus Firiana. The method according to any one of the above [18] to [21], which is a plant of the genus Paulinia or a plant of the genus Theobroma.
[24] N-methyltransferase having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase,
(A) the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or
(B) having, in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, a mutated amino acid sequence obtained by substituting, inserting, or deleting an amino acid within the amino acid sequence within a range that does not impair the enzyme activity. N-methyltransferase characterized by the following.
[25] The above-mentioned [24], wherein the nucleotide sequence encoding the amino acid sequence (a) and the nucleotide sequence encoding the mutant amino acid sequence (b) can hybridize under stringent conditions. N-methyltransferase.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The N-methyltransferase according to the present invention is a polypeptide derived from coffee having enzymatic activities simultaneously as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase.
[0017]
The N-methyltransferase has the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid within a range that does not impair the desired N-methyltransferase activity. And those having a mutated amino acid sequence obtained by insertion or deletion. That is, a polypeptide having the desired amino acid sequence of SEQ ID NO: 1 having the above-mentioned N-methyltransferase activity and a mutant sequence thereof are collectively referred to as N-methyltransferase.
[0018]
The polypeptide having the above-mentioned mutant amino acid sequence has a function substantially equivalent to that of coffee N-methyltransferase, and is stringent at a site related to the amino acid sequence of SEQ ID NO: 1 and the enzyme activity. Mention may be made of polypeptides encoded by nucleotide sequences that hybridize under conditions.
[0019]
The nucleotide sequence encoding the N-methyltransferase according to the present invention, that is, the N-methyltransferase gene includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and specific examples thereof include SEQ ID NO: 1 and the RNA sequence of SEQ ID NO: 2. Nucleotide sequences that hybridize to these N-methyltransferase genes under stringent conditions and encode the polypeptide having the N-methyltransferase activity are also included in the N-methyltransferase encoding gene of the present invention. .
[0020]
As the mutant amino acid sequence maintaining the desired N-methyltransferase activity, a mutant nucleotide sequence encoding this mutant amino acid sequence and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 are hybridized under stringent conditions. What can be soyed can be suitably used practically.
[0021]
In addition, as the mutant N-methyltransferase gene, a gene that can hybridize with a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 under stringent conditions can be suitably used practically. Specific examples thereof include a DNA molecule capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and an RNA molecule capable of hybridizing to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions. Can be.
[0022]
Hybridization under stringent conditions can be performed, for example, by Molecular Cloning: Cold Spring Harbor Laboratory Press, Current Protocols in Molecular Biology; a method described in the commercially available system by the method described in the University of Wiley; Amersham). Specifically, hybridization can be performed by the following operation.
[0023]
The membrane onto which the DNA or RNA molecule to be tested has been transferred is hybridized with the labeled probe in a protocol-specific hybridization buffer according to the product protocol. The composition of the hybridization buffer consisted of 0.1% by weight of SDS, 5% by weight of dextran sulfate, 1/20 volume of the blocking reagent attached to the kit, and 2 to 7 × SSC. As the blocking reagent, for example, 100 × Denhardt's solution, 2% (weight / volume) Bovine serum serum albumin 2% (weight / volume) Ficoll ™ 400, 2% (weight / volume) polyvinylpyrrolidone prepared at a 5-fold concentration. Diluted 1/20. 20 × SSC is a 3M sodium chloride, 0.3M citric acid solution, and SSC is more preferably used at a concentration of 3 to 6 × SSC, more preferably 4 to 5 × SSC.
[0024]
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, washing is performed with a washing buffer. The temperature for washing is preferably room temperature, more preferably the temperature for hybridization. The composition of the washing buffer is 6 × SSC + 0.1% by weight SDS solution, more preferably 4 × SSC + 0.1% by weight SDS solution, further preferably 2 × SSC + 0.1% by weight SDS solution, further preferably 1 × SSC + 0.1%. % SDS solution, most preferably 0.1 × SSC + 0.1% SDS solution. The membrane is washed with such a washing buffer, and the DNA or RNA molecule hybridized with the probe can be identified using the label used for the probe.
[0025]
The mutation may be caused in nature or artificially caused by site mutation in the nucleotide sequence.
[0026]
The DNA molecule having an N-methyltransferase gene of the present invention can be obtained, for example, by a PCR technique using an oligonucleotide that specifically hybridizes to a DNA molecule encoding N-methyltransferase as a primer (PCR experiment protocol for plants (cell engineering). Separate volume, Plant Cell Engineering Series 2) Shujunsha (1995)) can be used to separate cells from cells producing the N-methyltransferase of the present invention.
[0027]
Specifically, by isolating the full-length sequence of the target cDNA by, for example, binding a linker to cDNA synthesized from mRNA and performing PCR between the linker and DNA encoding the amino acid sequence constituting N-methyltransferase. Can be.
[0028]
A DNA molecule encoding N-methyltransferase obtained by such a hybridization technique or a PCR technique, at least at a site used for isolation, can be combined with the N-methyltransferase gene of SEQ ID NO: 1 under stringent conditions. Nucleotides that hybridize below.
[0029]
As an organism used for isolating a DNA molecule or an RNA molecule having a nucleotide sequence (gene) encoding the N-methyltransferase according to the present invention, any organism that produces caffeine or a precursor thereof can be used. However, among them, Camellia plants of the family Camellia, such as tea, plants of the genus Coffea, such as coffee, and plants of the genus Coleoptera, Cola, such as Chinese lantern can be exemplified.
[0030]
Next, a method for constructing an expression vector containing caffeine synthase, a method for producing a transformed plant, and a method for suppressing expression of the caffeine synthase enzyme by the RNAi method will be described.
[0031]
In order to express the caffeine synthase gene or its antisense RNA in this report in plant cells, (i) a promoter that can be transcribed in plant cells, and (ii) a sense or antisense direction downstream of the promoter. An expression cassette comprising all or a part of the linked caffeine synthase gene DNA, and (iii) a terminator sequence containing a polyadenylation site necessary for stabilizing a transcript linked downstream of the gene DNA as necessary. Is introduced into plant cells.
[0032]
Here, as a template of the antisense RNA, a nucleotide sequence obtained by linking all or a part of a DNA molecule having an N-methyltransferase gene or a DNA molecule that hybridizes with them under stringent conditions in the antisense direction, or N A nucleotide sequence in which all or a part of an RNA molecule having a methyltransferase gene or an RNA molecule that hybridizes with them under stringent conditions is linked in the antisense direction, and a host that 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 a cell.
[0033]
Here, a part of the N-methyltransferase gene is defined as an inhibitory mRNA obtained based on a sequence having complementarity with the part, that is, an antisense RNA is formed in the host cell. Is a portion that binds to mRNA for expressing N-methyltransferase in and inhibits N-methyltransferase expression in host cells. This part is a part necessary for the formation of such antisense mRNA, and includes, for example, a part having a length of at least 14 bases.
[0034]
Examples of the DNA molecule for forming an antisense mRNA include a DNA molecule having complementarity with all or a part of the nucleotide sequence of SEQ ID NO: 1, and examples of the antisense RNA molecule include SEQ ID NO: : RNA molecules having complementarity with all or part of the nucleotide sequence of SEQ ID NO: 2. A DNA or RNA molecule having complementarity with all or part of a mutant sequence capable of hybridizing with these nucleotide sequences under stringent conditions can also be used for such a purpose.
[0035]
These DNA or RNA molecules for inhibition may not necessarily encode the N-methyltransferase of the present invention.
[0036]
The expression cassette may contain a promoter for constitutively or inducibly expressing the inserted DNA. Examples of the promoter for constant expression include a cauliflower mosaic virus 35S promoter and a rice actin promoter. In addition, as a promoter for inducible expression, it is known that expression is caused by external factors such as infection or invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, anaerobic conditions, and spraying of a specific compound. Promoters and the like. Examples of such a promoter include a promoter of a rice chitinase gene expressed by infection or invasion of a filamentous fungus, a bacterium, and a virus, a promoter of a PR protein gene of tobacco, and a promoter of a rice “lip19” gene induced by low temperature. And the promoter of the Arabidopsis "HSP18.2" gene induced by high temperature, the promoter of the rice "rab" gene induced by drought, the promoter of the maize alcohol dehydrogenase gene induced by anaerobic conditions, and the like. The rice chitinase gene promoter and the tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and the rice “rab” gene promoter is induced by spraying the plant hormone abscisic acid.
[0037]
Alternatively, as a promoter for expressing the DNA inserted in the expression cassette, a method of isolating and using a promoter of the caffeine synthase gene can also be mentioned. One example of a specific method for isolating a promoter is to select a genomic DNA fragment using a caffeine synthase gene, for example, a hybridization technique using all or part of the DNA of the caffeine synthase gene as a probe. And a method for specifying the upstream DNA.
[0038]
To prepare for the introduction of the recombinant DNA molecule into plants, a number of cloning vectors are available that contain a replication signal for E. coli and a marker gene for selecting transformed bacterial cells. Examples of such vectors include pBR322, pUC, M13mp, and the like. The target 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 cut and ligated to another DNA. The sequence of each plasmid DNA can be cloned into the same plasmid or another plasmid.
[0039]
Various techniques can be used to introduce the expression cassette into a plant host cell. These techniques include Agrobacterium tumefaciens (Agrobacterium tumefaciens) Or Agrobacterium perizogenes (Agrobacterium rhizogenes), Transformation of plant cells with T-DNA, direct introduction into protoplasts (injection method, electroporation method, etc.), particle gun method, and other possibilities.
[0040]
For direct introduction into protoplasts, no special vectors are required. For example, a simple plasmid such as a pUC derivative can be used. Depending on the method of introducing the target gene into plant cells, 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 sequence at the right end, usually both ends of the T-DNA region of the Ti and Ri plasmids is used as a region adjacent to the gene to be introduced. Must be connected to.
[0041]
When Agrobacterium is used for transformation, the expression cassette to be introduced must be cloned into a special plasmid, ie, 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 the T-DNA, it is incorporated into a Ti or Ri plasmid of Agrobacterium by homologous recombination. Agrobacterium used as a host must contain a vir region. Usually, a Ti or Ri plasmid contains a vir region, and the T-DNA can be transferred to a plant cell by its function.
[0042]
On the other hand, since a binary vector can be replicated and maintained in Agrobacterium, when incorporated into Agrobacterium by a helper plasmid or an electroporation method, the host vir region acts on the binary vector. Can be transferred to plant cells.
[0043]
The intermediate vector or binary vector containing the expression cassette thus obtained, and microorganisms such as Escherichia coli and Agrobacterium containing the expression cassette are also objects of the present invention.
[0044]
The transformed plant cells can be converted into plants by undergoing a regeneration process. The method of regeneration differs depending on the type of plant cell. For example, in rice, Fujimura et al. [Plant Tissue Culture Lett. , 2, 74- (1995)], for corn, the method of Shellito et al. [Bio / Technology, 7, 581- (1989)], and for Arabidopsis, the method of Akayama et al. [Plant Cell Rep. , 12,
7- (1992)].
[0045]
Plants produced by these methods have altered expression levels of the caffeine synthase protein of the present invention compared to wild-type plants that produce caffeine and caffeine or its precursor, Changes in the amount of caffeine and theobromine metabolic compounds produced by the alteration of the plant, and changes in the production ratio of caffeine and theobromine metabolic compounds resulting from the alteration of the metabolism of the host plant. The transgenic plants thus obtained are the subject of the present invention. In the present invention, the term "plant" refers to all or a part of organs (eg, leaves, stems, roots, flowers, fruits, seeds, etc.) or cultured cells of a biological individual classified as a plant.
[0046]
Examples of plants that produce caffeine using the above-mentioned SAM as a methyl group donor include camellia family camellias such as tea (CamelliaGenus plants, coffee and other Rubiaceae coffee (CoffeaGenus plant, kakaonoki (Theobroma) Genus plants, bonito etc.ColaA) Caffeine-producing plants such as genus plants.
[0047]
Examples of microorganisms for introducing a DNA encoding caffeine synthase according to the present invention to express a large amount of caffeine synthase protein include bacteria such as Escherichia coli and Bacillus subtilis, and viruses such as baculovirus.
[0048]
Further, for the purpose of improving the productivity of a specific compound or modifying the production ratio of a specific compound group by modifying the metabolism of a host cell, a DNA encoding the caffeine synthase according to the present invention may be in a sense or antisense form. Any plant that produces caffeine or a precursor thereof can be used as a plant to be incorporated in, but among them, camellia family camellias such as tea (CamelliaGenus plants, coffee and other Rubiaceae coffee (CoffeaGenus plant, kakaonoki (Theobroma) Genus plants, bonito etc.ColaAnd genus plants.
[0049]
Plants produced by these methods have altered expression levels of the N-methyltransferase of the present invention compared to wild-type plants that produce caffeine or its precursor, Changes in the amount of caffeine metabolic compounds produced and changes in the production ratio of caffeine metabolic compounds due to alterations in the metabolism of the host plant occur. The transgenic plants thus obtained are the subject of the present invention.
[0050]
In recent years, from studies of plant post-translational gene silencing, it is possible to suppress the expression of a target gene by utilizing the defense mechanism inherent in plants against foreign nucleic acids such as viruses. (Cell, 95, 177-187 (1998), Chemistry and Biology, 37, 532- (1999), Protein Nucleic Acid Enzyme, 44, 1396- (1999)). According to this, when a DNA virus, an RNA virus, or the like enters a plant, the plant transcribes the aberrant RNA from these templates, and specifically transcribes the double-stranded RNA with the transcript of the sequence originally possessed by the plant. Form. When this double-stranded RNA is degraded by RNase, it becomes possible to suppress the expression of the target gene (Cell, 96, $ 303- (1999)). One of the important features of this method is that it is not always necessary to transform a target plant into a sequence whose expression is to be suppressed. Further, a further feature of the present method is that if a target nucleic acid is introduced into a part of a plant (lower leaf or the like) by infection or the like, the effect spreads to the whole plant. Specific methods for suppressing expression include double-stranded RNA containing the whole or a part of the N-methyltransferase gene or a partial sequence of the N-methyltransferase gene or a sequence that hybridizes therewith under stringent conditions; Agrobacterium having single-stranded DNA is transmitted to lower leaves of plants. Here, a part of the N-methyltransferase gene is defined as an inhibitory mRNA obtained on the basis of a sequence having complementarity with the part, that is, when an aberrant RNA is formed in a host cell, Is a portion that binds to mRNA for expressing N-methyltransferase in and inhibits N-methyltransferase expression in host cells. This portion is a portion necessary for forming such an average mRNA, and includes, for example, a portion having a length of at least 14 bases.
[0051]
Plants that have been subjected to these methods have altered expression levels of the N-methyltransferase protein of the present invention compared to wild-type plants that produce caffeine or theobromine or a precursor thereof, and Changes in the amount of caffeine metabolic compounds caused by the alteration of the compound, and changes in the production ratio of the caffeine metabolic compounds caused by alteration in the metabolism of the host plant. The plants thus obtained are the subject of the present invention. The plant in the present invention also includes specific tissues or cells of the plant, such as leaves, flowers, fruits, and seeds.
[0052]
Cultivating plant cells or plant tissues transformed with or infected with the vector of the above-described configuration, or cultivating similarly transformed plants, secondary to caffeine and theobromine synthesis system Production of metabolites and 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 7-methylxanthine, paraxanthine, theobromine and caffeine.
[0053]
As a source of a plant cell, plant tissue or plant used for transformation, for example, the transformed plant is a plant of the genus Camellia, a plant of the genus Coffee (Coffea), a plant of the genus Collan (Cola), or a plant of Ilex (Ilex) ) Genus plants, Neea genus plants, Firmiana genus plants, Paulinia genus plants, or Kakaonoki (Theobroma) genus plants. Among them, preferred are Camellia plants of the family Camellia, such as tea, plants of the genus Coffea, such as coffee, and plants of the genus Coleana, such as Chinese lantern.
[0054]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples.
[Example 1] Preparation of RNA from coffee
(1) Total RNA isolation
0.5g of coffee obtained from University of Tsukuba Agriculture and Forestry Technology Center (Coffea arabica) Was crushed using a pestle and a mortar in the presence of liquid nitrogen. After sublimation of liquid nitrogen, it is dissolved in 5 ml of 2% CTAB, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4 M NaCl, and 5% β-mercaptoethanol, and left at 65 ° C. for 10 minutes. An equal volume of a chloroform solution was added and stirred, and the total volume was reduced to about 10 ml. Centrifuge at 15,000 rpm for 10 minutes, remove the aqueous layer, add 2.5 ml of 10M lithium chloride solution, and leave at -20 ° C for at least 2 hours. Centrifugation was performed at 4 ° C., 15,000 rpm for 10 minutes, the supernatant was removed, the precipitate was dissolved in 1 ml of TE, and centrifuged again. The supernatant was recovered, and 1 ml of a TE-saturated phenol solution was added thereto, mixed by inversion, and centrifuged at 15,000 rpm for 10 minutes. Further, phenol / chloroform was added to the supernatant, and the mixture was stirred and centrifuged at 15,000 rpm for 10 minutes. The aqueous layer is collected, an equal volume of a chloroform solution is added, and the mixture is stirred. The mixture is centrifuged at 15,000 rpm for 10 minutes, the aqueous layer is collected, a 1/4 volume of a 10 M lithium chloride solution is added, and the mixture is left at −20 ° C. for at least 2 hours. After centrifugation at 15,000 rpm for 10 minutes, 70% ethanol was added to the obtained precipitate, followed by centrifugation again. The precipitate was dried up with a vacuum pump and dissolved in 200 μl of water to obtain a total RNA fraction.
[0055]
(2) Isolation of mRNA
After subjecting the total RNA (2 mg) obtained by the above method to heat treatment at 65 ° C. for 5 minutes, an equal volume of a 2 × concentration A solution (10 mM Tris-HCl buffer (pH 7.5), 1 mM Na 2 -EDTA) , 0.1% SDS, 0.5M NaCl). 0.1 g of oligo (dT) -Cellulose @ Type7 (Pharmacia) in 2 ml of solution B (10 mM Tris-HCl buffer (pH 7.5), 1 mM Na2-EDTA, 0.1% SDS, 0.1 M NaCl). After swelling, the suspension was poured into a blue chip filled with glass wool at the tip, washed with 2.5 ml of 0.1 N NaOH, and then equilibrated by flowing 5 ml of solution A. After applying total RNA to the column and flowing 3 ml of solution A and 4 ml of solution B, the mRNA was added to 3 ml of solution C (10 mM Tris-HCl buffer (pH 7.5), 1 mM Na2-EDTA, 0.05 mM). % @ SDS). The eluate was concentrated by ethanol precipitation, dried up, dissolved in water, and stored at -80 ° C.
[0056]
Example 2 Isolation of Caffeine Synthase cDNA from Coffee
(1) 3′-RACE isolation by 3′-RACE method
To date, the present inventors have isolated a plurality of N-methyltransferases from coffee, but they are polypeptides having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase, and paraxanthine N3 methyltransferase. N-methyltransferase could not be isolated from coffee. Therefore, the present inventors newly designed primers of SEQ ID NOS: 5 to 6 based on CTS1 (SEQ ID NO: 3) and CTS2 (SEQ ID NO: 4), which are coffee-derived N-methyltransferases isolated so far. , A novel caffeine synthase cDNA partial sequence was isolated. For 3'-RACE, 3'-Full \ RACE \ CORE \ Set (TAKARA) was used to synthesize 1st \ strand \ cDNA in the following reaction solution.

The reaction conditions were 30 ° C for 10 minutes, 50 ° C for 40 minutes, and 95 ° C for 5 minutes, followed by ice cooling.
[0057]
Using the obtained product as a template and reacting in the following reaction solution at 95 ° C for 1 minute, PCR was performed at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1.5 minutes for 30 cycles. Was performed. The sequence of SEQ ID NO: 5 was used for Primer 1.

[0058]
Using the obtained product as a template, PCR was carried out in the following reaction solution at 30 cycles of 94 ° C./1 minute, 55 ° C./1 minute, and 72 ° C./40 seconds to obtain a PCR amplification product. The sequence of SEQ ID NO: 6 was used for Primer 1.

[0059]
The obtained reaction product was subjected to electrophoresis in TAE using 0.8% @ agarose gel, the band of the obtained target product was cut out, and DNA was recovered from the gel using GENE @ CLEAN (Funakoshi). The collected DNA was ligated to a pT7blue vector (Novagen), and then transformed into Escherichia coli DH5α. After performing color selection using X-gal, liquid culture was performed in an LB medium containing ampicillin, and a plasmid was extracted by an alkali-SDS method. The presence or absence of the insert was confirmed by agarose electrophoresis.
[0060]
To determine the nucleotide sequence of the obtained product, primer extension was performed in the following reaction solution using the isolated plasmid. The reaction was performed at 96 ° C for 1 minute, followed by 25 cycles of 96 ° C for 0.2 minutes, 50 ° C for 0.1 minutes, and 60 ° C for 4 minutes. The reaction solution was subjected to ethanol precipitation, and the obtained DNA was dissolved in Template \ suppression \ reagent and analyzed using an ABI-310 Genetic Analyzer.
[0061]
Primer extension reaction solution
Plasmid DNA (20 ng) 2 μl
Premix 4μl
Primer 1 µl
H2O 3μl
[0062]
(2) Isolation of 5 'upstream region by' 5 'RACE method
For isolation of the 5 'upstream region, 5'-Full \ RACE \ Core \ Set (TAKARA) was used. Primers of SEQ ID NOS: 7 to 9 were designed based on the sequence obtained by the 3'-RACE method, and 1st strand cDNA was synthesized in the following reaction solution. The sequence of SEQ ID NO: 7 was used as a Primer during the reaction.
Denatured RNA (about 15 μg) μ15 μl
10 × RT buffer 3μl
RNase inhibitor 1μl
Primer (CS10r) 10 μl
AMV RT enzyme 1μl
[0063]
To 30 μl of the obtained RNA-cDNA hybrid, 25 μl 5 × buffer, 45 μl H2O, and 2 μl RNaseH were added and reacted at 30 ° C. for 1 hr to degrade RNA. The obtained single-stranded cDNA was subjected to self-ligation or concatemerization with T7-RNA ligase. Using this as a template, PCR was performed in the following reaction solution for 30 cycles of 94 ° C./1 minute, 62 ° C./1 minute, and 72 ° C./1.5 minutes to obtain a reaction product. The primers 1 and 2 used the sequences of SEQ ID NOs: 8 and 9. The band of the reaction product was separated by acrylamide electrophoresis, DNA was recovered from the gel, and subcloned into pGEM-T @ Easy vector (Promega).

The nucleotide sequence of the subcloned sequence was determined by the method described above. The obtained base sequence is shown as SEQ ID NO: 1 in the sequence listing.
[0064]
[Example 3] Expression of caffeine synthase in Escherichia coli and activity measurement
The isolated novel caffeine synthase gene was incorporated into an expression vector pET32a (Novagen), and this plasmid was transformed into Escherichia coli BL21 (DE3). After culturing the obtained Escherichia coli at 37 ° C for 2 hours, IPTG was added to a final concentration of 0.3 mM, and culturing was further performed at 25 ° C for 8 hours. After completion of the culture, the cells are collected, suspended in 200 μl of TES-G buffer (50 mM Tris-HCl buffer (pH 8.5), 5 mM DTT, 50 mM NaCl, 2 mM EDTA-Na2, 20% glycocerol), and frozen at −80 ° C. Thereafter, ultrasonic crushing was performed intermittently for 1 minute. This was centrifuged at 14,000 rpm for 10 minutes, and the obtained supernatant was used as an enzyme solution.
[0065]
The reaction solution for measuring caffeine synthase activity was 100 mM Tris-HCl buffer (pH 8.5), 0.2 mM MgCl2, 0.2 mM 7-methylxanthine, 4 μM {Methyl-14C] S-adenosylmethionine (0 9.9 kBq) and 10 μl of the enzyme solution, and the volume of the reaction solution was 100 μl. The reaction was carried out at 27 ° C. for 10 minutes, and the obtained 14C-caffeine was extracted by adding 1 ml of chloroform, and the radioactivity of the chloroform layer was measured. As a control, a reaction solution from which 7-methylxanthine was removed was used. As a result of activity measurement, it was found that caffeine was generated.
[0066]
Table 1 shows the substrate specificity of the recombinant enzyme. The relative activity (%) is shown with the activity for 7-methylxanthine as 100. For comparison, Cha (Camellia sinensis) The substrate specificity of leaf-derived caffeine synthase is also described.
[0067]
[Table 1]

[0068]
[Example 4] Inhibition of caffeine biosynthesis by antisense method
A recombinant vector having an antisense N-methyltransferase gene was constructed by the following method.
The coffee-derived novel caffeine synthase gene obtained in Example 2 was ligated to a pBI vector (manufactured by clonetech) so that the gene was inserted in the antisense direction downstream of the CaMV35S promoter. Thus, a desired recombinant vector into which the antisense N-methyltransferase gene was inserted was obtained. This was used for the following transformation.
[0069]
There are many reports on conventional methods of caffeine biosynthesis by coffee tissue culture, such as Planta, {108, {339} (1972), {Plant Cell} Reports, {2, {109} (1983). Callus was induced from the shoot apex or young leaf of coffee according to these conventional methods. The recombinant vector constructed above was introduced into the obtained calli by a particle gun method. Alternatively, a protoplast of the callus was prepared, and a recombinant vector was introduced into the protoplast by electroporation. After the introduction, cells exhibiting marker resistance were selected. The selected cells were cultured under light conditions, and the enzymatic activity of the transformed cells was measured by the method described in Example 3. As a result, it was found that the production of caffeine in cells into which antisense N-methyltransferase DNA had been introduced was significantly reduced as compared to normal cells into which antisense N-methyltransferase DNA had not been introduced.
[0070]
Furthermore, the transformed coffee culture cells were redifferentiated to obtain seedlings. The method of regeneration is described in Z. Pflanzenphysiol. {Bd. , {81, {395} (1977), Plant Cell, Tissue and Organ Culture, 8, 8, 243} (1987). Enzyme activity was measured by the method described in Example 3 using the leaves of the young plant. As a result, it was found that the production of caffeine in the plant into which the antisense N-methyltransferase DNA had been introduced was significantly reduced as compared to the plant into which the antisense N-methyltransferase DNA had not been introduced.
[0071]
【The invention's effect】
According to the present invention, it becomes possible to efficiently produce caffeine synthase that can be used as an industrial, food or medical enzyme.
ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to modify the caffeine biosynthesis metabolism of a caffeine producing plant, a plant tissue, or a plant cell, and to efficiently produce a caffeine metabolic compound.
ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to modify caffeine biosynthesis metabolism of a caffeine producing plant, a plant tissue, or a plant cell, and to modify the production ratio of the compound group of a caffeine metabolism system.
[0072]
[Sequence list]

Claims (25)

The following nucleotide sequence:
(A) N-methyltransferase which is a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase A coding nucleotide sequence,
(B) a mutant nucleotide obtained by performing nucleotide substitution, deletion or insertion in the nucleotide sequence (a) as long as the polypeptide encoded by the nucleotide sequence (a) can maintain the enzyme activity; A DNA molecule having any of the sequences. The DNA molecule according to claim 1, wherein the nucleotide sequence (a) and the mutant nucleotide sequence (b) are capable of hybridizing under stringent conditions. 3. The DNA molecule according to claim 1, wherein the nucleotide sequence (a) comprises the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. The following nucleotide sequence:
(A) N-methyltransferase which is a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase A coding nucleotide sequence,
(B) a mutant nucleotide obtained by performing nucleotide substitution, deletion or insertion in the nucleotide sequence (a) as long as the polypeptide encoded by the nucleotide sequence (a) can maintain the enzyme activity; An RNA molecule having any of the sequences. The RNA molecule according to claim 4, wherein the nucleotide sequence (a) and the mutant nucleotide sequence (b) can hybridize under stringent conditions. The RNA molecule according to claim 4 or 5, wherein the sequence (a) comprises the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. An expression vector, comprising: the DNA molecule according to any one of claims 1 to 3; and a configuration for expressing the N-methyltransferase encoded by the DNA molecule in a plant cell. A transformed cell, which is obtained by transforming a host cell with the expression vector according to claim 7. The transformed cell according to claim 8, wherein the host cell is a microbial cell. A method for producing an N-methyltransferase having the enzymatic activity by culturing the transformed cell according to claim 8 or 9. A nucleotide sequence complementary to all or a part of the nucleotide sequence of the DNA molecule according to any one of claims 1 to 3, wherein the plant is expressed when introduced into a plant cell having the enzyme activity. A DNA molecule capable of inhibiting the enzyme activity of a cell. A nucleotide sequence complementary to all or a part of the nucleotide sequence of the RNA molecule according to any one of claims 4 to 6, which is introduced into a plant cell having the enzymatic activity and is expressed in the plant. An RNA molecule characterized by being capable of inhibiting the enzyme activity of a cell. A vector comprising the DNA molecule or the RNA molecule according to any one of claims 1 to 6 and 11 to 12. N-methyltransferase having the enzymatic activity of 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase can be expressed in at least one cell of a microorganism and a plant, 14. The vector according to claim 13, which has a function of inhibiting the expression of methyltransferase. A microorganism transformed with the vector according to claim 13. A plant cell, plant tissue or plant transformed with the vector according to claim 13. The plant cell, plant tissue or plant according to claim 16, wherein the vector according to claim 13 or 14 has been introduced by infection. A method for producing a secondary plant metabolite using the plant cell, plant tissue or plant according to claim 16. A method for modifying the composition of a plant secondary metabolite using the plant cell, plant tissue or plant according to claim 16 or 17. A method for producing a plant secondary metabolite by culturing the plant cell or plant tissue according to claim 16 or 17, or cultivating a plant. A method for culturing the plant cell or plant tissue according to claim 16 or 17 or cultivating a plant to modify the composition of a plant secondary metabolite. The plant secondary metabolite is at least one or more selected from the group consisting of xanthosine, 7-methylxanthosine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, theobromine, theophylline, paraxanthine, and caffeine. The method according to any one of claims 18 to 21, which is a compound. The transformed plant is a plant of the genus Camellia, a plant of the genus Coffee (Coffea), a plant of the genus Collan (Cola), a plant of the genus Ilex, a plant of the genus Neee, a plant of the genus Aerugiana (Firmiana), 22. The method according to any one of claims 18 to 21, which is a plant of the genus Paululinia or a plant of the genus Theobroma. An N-methyltransferase having enzymatic activity as 7-methylxanthine N3 methyltransferase, theobromine N1 methyltransferase and paraxanthine N3 methyltransferase,
(A) substitution or insertion of an amino acid into the amino acid sequence of SEQ ID NO: 1 in the sequence listing or (b) the amino acid sequence of SEQ ID NO: 1 in the sequence listing within a range that does not impair the enzyme activity. Or an N-methyltransferase having a mutant amino acid sequence obtained by deletion. The N-methyltransferase according to claim 24, wherein the nucleotide sequence encoding the amino acid sequence (a) and the nucleotide sequence encoding the mutant amino acid sequence (b) can hybridize under stringent conditions. .