JP2006149293A - New anthocyanidin glucosyl transferase gene - Google Patents
New anthocyanidin glucosyl transferase gene Download PDFInfo
- Publication number
- JP2006149293A JP2006149293A JP2004345322A JP2004345322A JP2006149293A JP 2006149293 A JP2006149293 A JP 2006149293A JP 2004345322 A JP2004345322 A JP 2004345322A JP 2004345322 A JP2004345322 A JP 2004345322A JP 2006149293 A JP2006149293 A JP 2006149293A
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- gene
- protein
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- anthocyanidin
- transferring
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Abstract
Description
本発明は、グルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質をコードする遺伝子及びその利用に関するものである。 The present invention relates to a gene encoding a protein having an activity to transfer a glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and / or an activity to transfer a glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside, and use thereof. .
植物色素の中で最も多く見られるものがアントシアニンであり、アントシアニンは淡青色から濃赤色まで多くの色を呈する。産業的にも植物の色は最も重要な形質の一つであり、花色の多様化や果実の発色の良さ、着色の安定化・均一化などに見られるように花卉類、果樹類、野菜類において大きな経済的要因となっている。直接、目で確認できる形質であるアントシアニンによる発色は多くの遺伝学、生化学、分子生物学的研究の対象となり、今日ではアントシアニンを含むフラボノイドを含めて、フラボノイド生合成系に関わる遺伝子がペチュニア、キンギョソウ、アラビドプシス等の花卉類やリンゴ、ブドウなどの果実類、ナス、シソ等の野菜類からクローニングされている。また、アントシアニン発現の仕組みが天然物化学及び生理学的解析によって解明されつつある。 The most common plant pigment is anthocyanin, and anthocyanins exhibit many colors from light blue to deep red. Industrially, plant color is one of the most important traits. As seen in the diversification of flower color, good color development of fruits, stabilization and uniformity of coloration, etc., flowers, fruit trees, vegetables Has become a major economic factor. Color development by anthocyanins, which are directly visible identities, has been the subject of many genetics, biochemistry, and molecular biological studies, and today, including flavonoids containing anthocyanins, genes related to flavonoid biosynthesis systems are petunia, It has been cloned from florets such as Snapdragon and Arabidopsis, fruits such as apples and grapes, and vegetables such as eggplant and perilla. The mechanism of anthocyanin expression is being elucidated by natural product chemistry and physiological analysis.
これまでに５００種近くのアントシアニンが様々な植物種から報告されている。アントシアニジンはアントシアニンの骨格を成すアグリコンであるが、そのままでは植物体内に存在せず、必ず配糖体化された形（アントシアニン）で存在する。配糖体化されることによりアントシアニジンは無毒なアントシアニンになって安定化し、また、水溶性になるので細胞の液胞内に溶けるようになる。アントシアニンは液胞内でフラボン/フラボノールなどの補助色素や金属イオンと分子内会合や分子間会合、金属イオンとの配位結合によって複雑な色素高分子を形成し各植物固有の色を呈する。それ故、アントシアニジンの配糖体化はアントシアニンを形成する最初の反応として重要であり、その後の発色にとって必須の化学反応である。 To date, nearly 500 anthocyanins have been reported from various plant species. Anthocyanidin is an aglycone that forms the skeleton of anthocyanin, but it does not exist in the plant as it is, but it always exists in a glycosylated form (anthocyanin). As a result of glycosylation, anthocyanidins become non-toxic anthocyanins, stabilize, and become water-soluble so that they can dissolve in the vacuole of cells. Anthocyanins form complex pigment polymers in the vacuole by co-bonding with auxiliary dyes such as flavones / flavonols, metal ions, intramolecular associations, intermolecular associations, and metal ions, and exhibit a unique color of each plant. Therefore, glycosylation of anthocyanidins is important as the first reaction to form anthocyanins and is an essential chemical reaction for subsequent color development.
アントシアニンの水酸基に結合する糖残基はＣ環の3位、Ａ環の5位、7位、Ｂ環の3'位、5’位などに見られるが、これらの糖残基が結合する順序は様々な植物種による報告によって種を越えた植物共通の反応経路であると考えられている。アントシアニジンの水酸基の配糖体化はＣ環の3位から始まることは多くの植物で知られており、このことから配糖体化の最初の反応過程として一般に認識されている。バラにおいては3位と5位の水酸基に糖残基が結合し、3位から5位の順序で配糖体化が起こると考えられているが、バラの配糖体化を行う糖転移酵素は酵素学的にも分子生物学的にも未だ見つけられていない。 The sugar residues that bind to the hydroxyl group of anthocyanins are found at the 3rd position of the C ring, the 5th and 7th positions of the A ring, the 3 'position and the 5' position of the B ring, etc. Is considered to be a common reaction pathway across plants, as reported by various plant species. It is known in many plants that glycosylation of the hydroxyl group of anthocyanidin begins at the 3-position of the C ring, and this is generally recognized as the first reaction process of glycosylation. In roses, sugar residues are thought to bind to the hydroxyl groups at the 3rd and 5th positions, and glycosylation occurs in the order from the 3rd to the 5th position. Has yet to be found both enzymatically and molecularly.
アントシアニジンの3位水酸基へ糖残基を転移させる反応を触媒する酵素（糖転移酵素(配糖体化酵素)）の遺伝子はトウモロコシからトランスポゾンを用いて始めて単離された（Fedoroff et al.,(1984) Proc. Natl. Acad. Sci. USA 81: 3825-3829）。その後、この遺伝子をプローブとして他の植物種から、同種の糖転移酵素の遺伝子が単離されている（Wise et al., (1990) Plant Mol. Biol. 14: 277-279, Ford et al., (1998) J. Biol. Chem. 273: 9224-9233, Yamazaki et al., (2002) Plant Mol. Biol. 48: 401-411）。また、アントシアニンの5位水酸基にグルコース残基を転移させる反応を触媒する酵素の遺伝子は赤シソからディファレンシャル・ディスプレイ法を用いて始めて単離された（特許文献１, Yamazaki et al., (1999) J. Biol. Chem. 274: 7405-7411）。また、この遺伝子を用いてペチュニアからも見つけられた（Yamazaki et al., (2002) Plant Mol. Biol. 48: 401-411）。他のフラボノイドの糖転移酵素がクローン化された例も幾つか報告がある。フラボノールの3位水酸基へガラクトース残基を転移させる反応を触媒する酵素の遺伝子（Mato et al., (1998) Plant Cell Physiol. 39: 1145-1155, Miller et al., (1999) J. Biol. Chem. 274: 34011-34019）、フラボンの7位水酸基にグルコース残基を転移させる反応を触媒する酵素の遺伝子（Hirotani et al., (2000) Planta 210: 1006-1013）、そして、フラボノールの4'位または7位にグルコース残基を転移させる反応を触媒するベタニジン5位糖転移酵素の遺伝子（Vogt et al., (1997) Planta 203: 349-361, Vogt, (1999) Plant J. 19: 509-519）が挙げられる。また、B環の3’位にグルコース残基を転移させる反応を触媒する酵素の遺伝子がリンドウ（特許文献２）から見出されている。アントシアニジンの異なる二カ所の水酸基へ逐次的にグルコース残基を転移させる反応を触媒する酵素の遺伝子としては、3位から5位の水酸基へ逐次的に糖残基を転移させる反応を触媒する酵素の遺伝子がリンドウから単離されている（特許文献３）。バラでは糖残基を転移させる反応を触媒する酵素は今まで知られておらず、酵素の活性が測定されたことも、酵素が精製されたことも、遺伝子がクローン化されたこともない。その大きな原因としてバラの花弁にはタンニン類や他の二次代謝物質が多く存在しており、他の植物で成功した方法を用いても単離が困難であったことがあげられる。また、現在までに約7,000種以上のバラが作出されているが、その赤色のバラ花弁にはアントシアニンであるシアニジンの3位と5位がグルコース残基により配糖体化されたシアニジン3,5-ジグルコシドが普遍的に含まれており、逆に3位のみが配糖体化されたシアニジン3-グルコシドのみを有するバラは報告されていない。
The gene for an enzyme that catalyzes the transfer of a sugar residue to the hydroxyl group at the 3-position of anthocyanidin (glycosyltransferase (glycosylase)) was first isolated from maize using a transposon (Fedoroff et al., ( 1984) Proc. Natl. Acad. Sci. USA 81: 3825-3829). Subsequently, the same glycosyltransferase gene has been isolated from other plant species using this gene as a probe (Wise et al., (1990) Plant Mol. Biol. 14: 277-279, Ford et al. (1998) J. Biol. Chem. 273: 9224-9233, Yamazaki et al., (2002) Plant Mol. Biol. 48: 401-411). In addition, a gene for an enzyme that catalyzes a reaction of transferring a glucose residue to the 5-position hydroxyl group of anthocyanin was isolated from red perilla using a differential display method (
本発明は、グルコシル基を転移させる活性を有するタンパク質をコードする遺伝子、好ましくは、既知の順序とは異なる順序でアントシアニジンの5位及び3位の水酸基にグルコシル基を転移させる活性を有するタンパク質をコードする遺伝子を得ることを課題とした。本発明で得られたグルコシル基転移活性を有するタンパク質をコードする遺伝子あるいは同様の遺伝子を植物に導入して、発現させることにより、蓄積するフラボノイド化合物の種類を改変して、花色や果実などの植物体の発色変換をすることが可能になる。また、バラにおいては本遺伝子を用いたRNAi法、遺伝子破壊法等による遺伝子発現制御と、これまでに報告のあるアントシアニンにおける既知の修飾酵素（糖転移酵素（配糖体化酵素）、アシル化酵素）を遺伝子導入することで、本来バラには存在し得なかったアントシアニンをバラ花弁中で生産させ、これまでにない花色のバラを作出することができる。 The present invention encodes a gene encoding a protein having an activity to transfer a glucosyl group, preferably a protein having an activity to transfer a glucosyl group to the 5- and 3-position hydroxyl groups of anthocyanidin in an order different from the known order. The problem was to obtain the gene to be used. By introducing a gene encoding a protein having a glucosyl group transfer activity obtained in the present invention or a similar gene into a plant and expressing it, the kind of the accumulated flavonoid compound is modified, and plants such as flower color and fruit It becomes possible to change the color of the body. In rose, gene expression control by RNAi method, gene disruption method, etc. using this gene and known modification enzymes (glycosylase (glycosylase), acylase) in anthocyanins reported so far ) Can be used to produce anthocyanins that could not be present in roses in rose petals, and to produce unprecedented flower-colored roses.
本発明者は、アントシアニジンの5位の水酸基へグルコシル基を転移させた後、3位の水酸基へグルコシル基を転移させる反応を触媒する酵素をバラの花弁から見出し、その遺伝子をクローニングした。糖転移酵素に共通して存在する塩基配列に基づき合成した縮重プライマーを用いたRT-PCRを行いcDNA断片の増幅し、その塩基配列を決定した。得られた塩基配列情報を基に遺伝子のタンパク質コード領域をRT-PCRによってクローン化した。得られたクローンの機能解析は大腸菌の組換えタンパク質合成系を利用して得られたタンパク質を用い、その酵素活性を解析した。本発明は、以上の知見に基づき完成されたものである。 The present inventor found an enzyme that catalyzes the reaction of transferring a glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and then transferred the glucosyl group to the hydroxyl group at the 3-position, and cloned the gene thereof. RT-PCR was carried out using degenerate primers synthesized based on the nucleotide sequence that is common to glycosyltransferases to amplify the cDNA fragment, and the nucleotide sequence was determined. Based on the obtained base sequence information, the protein coding region of the gene was cloned by RT-PCR. For the functional analysis of the obtained clone, the enzyme activity was analyzed using a protein obtained by using a recombinant protein synthesis system of Escherichia coli. The present invention has been completed based on the above findings.
即ち、本発明は、以下の〔１〕〜〔１６〕を提供するものである。 That is, the present invention provides the following [1] to [16].
〔１〕本発明の第一は、グルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質をコードする遺伝子である。 [1] The first of the present invention is a gene encoding a protein having an activity to transfer a glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and / or an activity to transfer the glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside. is there.
〔２〕本発明の第二は、以下の(a)〜(d)のタンパク質をコードする〔１〕記載の遺伝子である。 [2] A second aspect of the present invention is the gene according to [1], which encodes the following proteins (a) to (d):
(a)配列番号２に記載のアミノ酸配列を有するタンパク質、
(b)配列番号２に記載のアミノ酸配列において一個又は複数個のアミノ酸が付加、欠失及び／又は他のアミノ酸により置換されているアミノ酸配列を有するタンパク質、
(c)配列番号２に記載のアミノ酸配列に対して20%以上の相同性を示すアミノ酸配列を有するタンパク質、
(d)配列番号２に記載のアミノ酸配列に対して70%以上の相同性を示すアミノ酸配列を有するタンパク質、
〔３〕本発明の第三は、配列番号１に記載の塩基配列で表される核酸、又は配列番号２に記載のアミノ酸配列をコードする核酸の一部又は全部に対して、ストリンジェントな条件下でハイブリダイズする遺伝子であって、グルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質をコードする遺伝子である。
(a) a protein having the amino acid sequence of SEQ ID NO: 2,
(b) a protein having an amino acid sequence in which one or more amino acids are added, deleted and / or substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 2;
(c) a protein having an amino acid sequence showing 20% or more homology to the amino acid sequence set forth in SEQ ID NO: 2;
(d) a protein having an amino acid sequence showing 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 2;
[3] The third aspect of the present invention is a stringent condition for the nucleic acid represented by the nucleotide sequence set forth in SEQ ID NO: 1 or a part or all of the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 2. A gene that encodes a protein that has the activity of transferring the glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and / or the activity of transferring the glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside. is there.
〔４〕本発明の第四は、グルコシル基をアントシアニジンの5位の水酸基へ転移させる活性とグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性の両者を有するタンパク質をコードする〔１〕〜〔３〕のいずれか記載の遺伝子である。 [4] The fourth of the present invention encodes a protein having both an activity of transferring a glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and an activity of transferring the glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside [1] ] It is a gene in any one of [3].
〔５〕本発明の第五は、〔１〕〜〔３〕のいずれかに記載の遺伝子を含んでいるベクターである。 [5] A fifth aspect of the present invention is a vector containing the gene according to any one of [1] to [3].
〔６〕本発明の第六は、〔４〕に記載の遺伝子を含んでいるベクターである。 [6] A sixth aspect of the present invention is a vector containing the gene according to [4].
〔７〕本発明の第七は、〔５〕又は〔６〕に記載のベクターにより形質転換された宿主細胞である。 [7] A seventh aspect of the present invention is a host cell transformed with the vector according to [5] or [6].
〔８〕本発明の第八は、〔１〕〜〔４〕のいずれか1項に記載の遺伝子によってコードされるタンパク質である。 [8] An eighth aspect of the present invention is a protein encoded by the gene according to any one of [1] to [4].
〔９〕本発明の第九は、〔７〕に記載の宿主細胞を培養し、又は生育させ、その後、該宿主細胞からグルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質を採取することを特徴とする該タンパク質の製造方法である。 [9] A ninth aspect of the present invention is the activity and / or glucosyl group for culturing or growing the host cell according to [7], and then transferring the glucosyl group from the host cell to the hydroxyl group at the 5-position of anthocyanidin. A method for producing a protein, comprising collecting a protein having an activity of transferring an anthocyanidin 5-glucoside to a hydroxyl group at the 3-position.
〔１０〕本発明の第十は、〔１〕〜〔４〕のいずれかに記載の遺伝子を用いた無細胞タンパク質合成系による該タンパク質の製造方法である。 [10] A tenth aspect of the present invention is a method for producing the protein by a cell-free protein synthesis system using the gene according to any one of [1] to [4].
〔１１〕本発明の第十一は、〔１〕〜〔４〕のいずれかに記載の遺伝子、又は〔５〕又は〔６〕に記載のベクターが導入され、形質転換された植物である。 [11] An eleventh aspect of the present invention is a plant transformed by introducing the gene according to any one of [1] to [4] or the vector according to [5] or [6].
〔１２〕本発明の第十二は、〔１１〕に記載の植物と同じ性質を有する該植物の子孫である。 [12] A twelfth aspect of the present invention is a progeny of the plant having the same properties as the plant according to [11].
〔１３〕本発明の第十三は、〔１１〕に記載の植物又は〔１２〕に記載の該植物の子孫の組織である。 [13] A thirteenth aspect of the present invention is the plant according to [11] or the offspring tissue of the plant according to [12].
〔１４〕本発明の第十四は、〔１１〕に記載の植物又は〔１２〕に記載の該植物の子孫の切り花である。 [14] A fourteenth aspect of the present invention is a cut flower of the plant according to [11] or the progeny of the plant according to [12].
〔１５〕本発明の第十五は、〔４〕に記載の遺伝子、又は〔６〕に記載のベクターを植物又は植物細胞に導入し、該遺伝子を発現させることによる、アントシアニジンの5位の水酸基へグルコシル基を転移させた後、3位の水酸基へグルコシル基を転移させる方法である。 [15] A fifteenth aspect of the present invention is the hydroxyl group at the 5-position of anthocyanidin produced by introducing the gene according to [4] or the vector according to [6] into a plant or plant cell and expressing the gene. This is a method of transferring the glucosyl group to the hydroxyl group at the 3-position after transferring the glucosyl group.
〔１６〕本発明の第十六は、〔１〕〜〔４〕のいずれかに記載の遺伝子、又は〔５〕又は〔６〕に記載のベクターを植物又は植物細胞に導入し、該遺伝子を発現させることにより、植物体の花色を調節する方法である。 [16] The sixteenth aspect of the present invention is the introduction of the gene according to any one of [1] to [4] or the vector according to [5] or [6] into a plant or plant cell, It is a method of adjusting the flower color of a plant body by making it express.
〔１７〕本発明の第十七は、〔１〕〜〔４〕のいずれかに記載の遺伝子を持つ植物において、該遺伝子の発現を抑制することにより、植物体の花色を調節する方法である。 [17] A seventeenth aspect of the present invention is a method for regulating flower color of a plant body by suppressing expression of the gene in a plant having the gene according to any one of [1] to [4]. .
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
（１）遺伝子
本発明の遺伝子は、グルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質をコードするものである。
(1) Gene The gene of the present invention encodes a protein having an activity of transferring a glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and / or an activity of transferring the glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside. is there.
本発明の遺伝子としては、例えば、以下の（Ａ）〜（Ｄ）の遺伝子を例示できる。 Examples of the gene of the present invention include the following genes (A) to (D).
（Ａ）配列番号２に記載のアミノ酸配列を有し、前記二つの酵素活性のいずれか一方又は両方を有するタンパク質をコードする遺伝子
ここで「配列番号２に記載のアミノ酸配列を有する」とは、配列番号２に記載のアミノ酸配列のみからなるタンパク質だけでなく、このようなタンパク質のN末端又はC末端側に複数のアミノ酸が付加したタンパク質をも含む趣旨である。付加するアミノ酸の個数は、上記グルコシル基転移活性を失わせない範囲であれば特に限定されないが、通常は400個以内であり、好ましくは50個以内である。
(A) A gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2 and having either one or both of the two enzyme activities. Here, “having the amino acid sequence set forth in SEQ ID NO: 2” This includes not only a protein consisting only of the amino acid sequence shown in SEQ ID NO: 2, but also a protein having a plurality of amino acids added to the N-terminal or C-terminal side of such a protein. The number of amino acids to be added is not particularly limited as long as the above glucosyl group transfer activity is not lost, but is usually 400 or less, preferably 50 or less.
（Ｂ）配列番号２に記載のアミノ酸配列において一個又は複数個のアミノ酸が付加、欠失及び／又は他のアミノ酸により置換されているアミノ酸配列を有し、かつ前記二つの酵素活性のいずれか一方又は両方を有するタンパク質をコードする遺伝子
このような付加、欠失、置換されているアミノ酸配列を有するタンパク質は、天然のものでもよく、また、人工的なものであってもよい。付加、欠失、置換するアミノ酸の個数は上記グルコシル基転移活性を失わせない範囲であれば特に限定されないが、通常は20個以内であり、好ましくは5個以内である。
(B) the amino acid sequence of SEQ ID NO: 2 has an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted with another amino acid, and one of the two enzyme activities Alternatively, a gene encoding a protein having both. A protein having such an added, deleted, or substituted amino acid sequence may be natural or artificial. The number of amino acids to be added, deleted, or substituted is not particularly limited as long as the above glucosyl group transfer activity is not lost, but is usually 20 or less, preferably 5 or less.
（Ｃ）配列番号２に記載のアミノ酸配列に対して一定以上の相同性を示すアミノ酸配列を有し、かつ前記二つの酵素活性のいずれか一方又は両方を有するタンパク質をコードする遺伝子
ここで「一定以上の相同性」とは、通常は20％以上の相同性、好ましくは50％以上の相同性、より好ましくは60％以上の相同性、最も好ましくは70％以上の相同性を意味する。
(C) a gene encoding a protein having an amino acid sequence having a certain homology to the amino acid sequence of SEQ ID NO: 2 and having either one or both of the two enzyme activities The term “homology above” usually means 20% or more homology, preferably 50% or more homology, more preferably 60% or more homology, and most preferably 70% or more homology.
（Ｄ）配列番号１に記載の塩基配列で表される核酸、又は配列番号２に記載のアミノ酸配列をコードする核酸の一部又は全部に対して、ストリンジェントな条件下でハイブリダイズする遺伝子であって、かつ前記二つの酵素活性のいずれか一方又は両方を有するタンパク質をコードする遺伝子
ここで「核酸の一部」とは、例えば、コンセンサス配列領域の6個以上のアミノ酸配列をコードする部分などを意味する。
(D) a gene that hybridizes under stringent conditions to part or all of the nucleic acid represented by the nucleotide sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2 And a gene encoding a protein having one or both of the two enzyme activities. Here, “part of nucleic acid” means, for example, a portion encoding 6 or more amino acid sequences of a consensus sequence region, etc. Means.
「ストリンジェントな条件」とは、特異的なハイブリダイゼーションのみが起き、非特異的なハイブリダイゼーションが起きないような条件をいい、例えば、温度が50℃、塩濃度が5×SSC（又はこれと同等の塩濃度）といった条件である。なお、適切なハイブリダイゼーション温度は、核酸の塩基配列やその核酸の長さによって異なり、例えば、アミノ酸6個をコードする18塩基からなるDNAフラグメントをプローブとした場合には50℃以下の温度が好ましい。 “Stringent conditions” refers to conditions in which only specific hybridization occurs and non-specific hybridization does not occur. For example, the temperature is 50 ° C. and the salt concentration is 5 × SSC (or this). Equivalent salt concentration). The appropriate hybridization temperature varies depending on the base sequence of the nucleic acid and the length of the nucleic acid. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable. .
この様なハイブリダイゼーションによって選択される遺伝子としては、天然の遺伝子、例えば、植物由来の遺伝子、特にバラ由来の遺伝子が挙げられる。また、ハイブリダイゼーションによって選択される遺伝子はcDNAであってもよく、ゲノムDNAであっても良い。 Examples of genes selected by such hybridization include natural genes, for example, plant-derived genes, particularly rose-derived genes. Further, the gene selected by hybridization may be cDNA or genomic DNA.
上記遺伝子のうち、天然に存在するものは、後述する実施例に示すように、cDNAライブラリーのスクリーニング等により得ることができる。また、天然には存在しない遺伝子も部位特異的変異誘発法やPCR法などを利用することにより得ることができる。 Among the above genes, naturally occurring ones can be obtained by screening a cDNA library or the like, as shown in the examples described later. In addition, genes that do not exist in nature can also be obtained by utilizing site-directed mutagenesis or PCR.
（２）ベクター
本発明のベクターは、既知の発現ベクターに（１）の遺伝子を挿入することにより作製できる。
(2) Vector The vector of the present invention can be prepared by inserting the gene of (1) into a known expression vector.
ここで使用する既知の発現ベクターは、適当なプロモーター、ターミネーター、複製起点等を含有するものであれば特に限定されない。プロモーターは、例えば、細菌中で発現させるのであれば、trcプロモーター、tacプロモーター、lacプロモーター等が使用でき、酵母中で発現させるのであれば、グリセルアルデヒド３リン酸デヒドロゲナーゼプロモーター、PH05プロモーター等が使用でき、糸状菌中で発現させるのであれば、アミラーゼプロモーター、trpCプロモーター等が使用でき、動物細胞中で発現させるのであれば、SV40アーリープロモーター、SV40レートプロモーター等が使用できる。 The known expression vector used here is not particularly limited as long as it contains an appropriate promoter, terminator, origin of replication and the like. For example, if the promoter is expressed in bacteria, the trc promoter, tac promoter, lac promoter, etc. can be used. If the promoter is expressed in yeast, the glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. are used. If it is expressed in filamentous fungi, an amylase promoter, trpC promoter or the like can be used. If it is expressed in animal cells, an SV40 early promoter, SV40 rate promoter or the like can be used.
（３）形質転換された宿主細胞
本発明における形質転換された宿主細胞とは、（２）のベクターにより形質転換された宿主細胞である。
(3) Transformed host cell The transformed host cell in the present invention is a host cell transformed with the vector of (2).
宿主細胞は、原核生物、真核生物のいずれでもよい。原核生物としては、例えば、大腸菌（Escherichia coli）、バチルス・スブチリス（Bacillus subtilis）等を用いることができる。真核生物としては、酵母、糸状菌などのほか、動物及び植物の培養細胞を使用することができる。酵母としては、サッカロミセス・セレビシエ（Saccharomyces cerevisiae）等を例示でき、糸状菌としては、アスペルギルス・オリゼ（Aspergillus oryzae）、アスペルギルス・ニガー（Aspergillus niger）等を例示でき、動物細胞としては、マウス、ハムスター、サル、ヒト、カイコ等の培養細胞を例示できる。 The host cell may be either prokaryotic or eukaryotic. Prokaryotes, such as E. coli (Escherichia coli), it is possible to use Bacillus subtilis (Bacillus subtilis) and the like. Examples of eukaryotes include yeast and filamentous fungi, as well as animal and plant cultured cells. As yeast, it can be exemplified Saccharomyces cerevisiae (Saccharomyces cerevisiae) or the like, as the filamentous fungi, Aspergillus oryzae (Aspergillus oryzae), can be exemplified Aspergillus niger (Aspergillus niger). Examples of animal cells, a mouse, hamster, Examples include cultured cells such as monkeys, humans, and silkworms.
（２）のベクターにより形質転換する方法は特に限定されず、常法に従って行うことができる。 The method of transforming with the vector of (2) is not particularly limited, and can be performed according to a conventional method.
（４）タンパク質
上記（１）の遺伝子によってコードされるタンパク質も本発明に含まれる。このタンパク質は、例えば、後述する（５）の方法によって製造することができる。
(4) Protein A protein encoded by the gene of (1) is also included in the present invention. This protein can be produced, for example, by the method (5) described later.
（５）タンパク質の製造方法
本発明のタンパク質の製造方法は、（３）の宿主細胞を培養し、又は生育させ、その後、該宿主細胞からグルコシル基をアントシアニジンの5位の水酸基へ転移させる活性及び／又はグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性を有するタンパク質を採取すること、もしくは無細胞タンパク質合成系などによるタンパク質の製造を特徴とするものである。
(5) Protein production method The protein production method of the present invention comprises culturing or growing the host cell of (3), and then transferring the glucosyl group from the host cell to the hydroxyl group at the 5-position of anthocyanidin and It is characterized by collecting a protein having an activity of transferring a glucosyl group to the hydroxyl group at the 3-position of anthocyanidin 5-glucoside, or producing a protein by a cell-free protein synthesis system or the like.
宿主細胞の培養又は生育は、その宿主細胞の種類に応じた方法に従って行うことができる。また、無細胞タンパク質合成系によるタンパク質の製造は常法に従って行うことができる。 The culture or growth of the host cell can be performed according to a method according to the type of the host cell. Moreover, protein production by a cell-free protein synthesis system can be carried out according to a conventional method.
タンパク質の採取は常法に従って行うことができ、例えば、培養物等から濾過、遠心分離、細胞の破砕、ゲル濾過、イオン交換クロマトグラフィー等により回収、精製することにより、目的のタンパク質を得ることができる。 The protein can be collected according to a conventional method. For example, the target protein can be obtained by collecting and purifying from a culture etc. by filtration, centrifugation, cell disruption, gel filtration, ion exchange chromatography, etc. it can.
（６）植物
本発明の植物は、上記（１）の遺伝子又は（２）のベクターが導入され形質転換されたものである。
(6) Plant The plant of the present invention has been transformed by introducing the gene (1) or the vector (2).
遺伝子等の導入対象とする植物は特に限定されず、例えば、バラ、キク、シネラリア、キンギョソウ、シクラメン、ラン、トルコギキョウ、フリージア、ガーベラ、グラジオラス、カスミソウ、カランコエ、ユリ、ペラルゴニウム、ゼラニウム、ペチュニア、チューリップ、イネ、オオムギ、コムギ、ナタネ、ポテト、トマト、ポプラ、バナナ、ユーカリ、サツマイモ、ダイズ、アルファルファ、ルーピン、トウモロコシ、カリフラワー、ロベリア、リンゴ、ブドウ、モモ、カキ、スモモ、柑橘類、などを挙げることができる。 Plants to be introduced into genes and the like are not particularly limited. Rice, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, soybean, alfalfa, lupine, corn, cauliflower, lobelia, apple, grape, peach, oyster, plum, citrus, etc. .
（７）植物の子孫
上記（６）の植物の子孫も本発明に含まれる。
(7) Progeny of plant The progeny of the plant of the above (6) is also included in the present invention.
（８）植物等の組織
上記（６）の植物又は（７）の植物の子孫の組織も本発明に含まれる。
(8) Tissues of plants and the like Tissues of the plants of (6) or (7) are also included in the present invention.
（９）植物等の切り花
上記（６）の植物又は（７）の植物の子孫の切り花も本発明に含まれる。
(9) Cut flowers such as plants The cut flowers of the plant of (6) or the progeny of the plant of (7) are also included in the present invention.
（１０）グルコシル基の転移方法
本発明のグルコシル基の転移方法は、上記（１）の遺伝子のうちグルコシル基をアントシアニジンの5位の水酸基へ転移させる活性とグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性の両者を有するタンパク質をコードするもの、又は上記（２）のベクターのうちグルコシル基をアントシアニジンの5位の水酸基へ転移させる活性とグルコシル基をアントシアニジン5-グルコシドの3位の水酸基へ転移させる活性の両者を有するタンパク質をコードする遺伝子を含んでいるものを、植物又は植物細胞に導入し、該遺伝子を発現させることによる、アントシアニジンの5位の水酸基へグルコシル基を転移させた後、3位の水酸基へグルコシル基を転移させるものである。
(10) Glucosyl group transfer method The glucosyl group transfer method of the present invention comprises the activity of transferring the glucosyl group to the 5-position hydroxyl group of anthocyanidin in the gene of (1) above and the 3rd-position of anthocyanidin 5-glucoside. One encoding a protein having both the activity of transferring to the hydroxyl group of the above or the activity of transferring the glucosyl group to the hydroxyl group at the 5-position of anthocyanidin and the glucosyl group at the 3-position of anthocyanidin 5-glucoside in the vector of (2) above A glucosyl group was transferred to the hydroxyl group at the 5-position of anthocyanidin by introducing a gene encoding a protein having both activities to transfer to a hydroxyl group into a plant or plant cell and expressing the gene. Thereafter, the glucosyl group is transferred to the hydroxyl group at the 3-position.
（１１）花色の調節方法
本発明の花色の調節方法は、上記（１）の遺伝子又は（２）のベクターを植物又は植物細胞に導入し、該遺伝子を発現させることにより、あるいは、上記（１）の遺伝子を持つ植物において、該遺伝子の発現を抑制することにより、植物体の花や果実の色を調節するものである。遺伝子導入の対象とする植物は特に限定されず、例えば、上記（６）で例示した植物を使用できる。また、（１）の遺伝子の抑制対象とする植物は（１）の遺伝子を持つ植物であれば特に限定されない。
(11) Method for regulating flower color The method for regulating flower color of the present invention comprises introducing the gene of (1) or the vector of (2) into a plant or plant cell and expressing the gene, or (1 ), The color of the flowers and fruits of the plant body is regulated by suppressing the expression of the gene. The plant which is the target of gene transfer is not particularly limited, and for example, the plant exemplified in (6) above can be used. Moreover, if the plant made into the suppression target of the gene of (1) is a plant which has the gene of (1), it will not be specifically limited.
（１）の遺伝子の導入及び発現は常法に従って行うことができる。また、（１）の遺伝子の発現抑制も常法（例えば、アンチセンス法やコサプレッション法やRNAi法）に従って行うことができる。 The introduction and expression of the gene of (1) can be performed according to a conventional method. Moreover, the gene expression suppression of (1) can also be performed according to a conventional method (for example, an antisense method, a cosuppression method, or an RNAi method).
本発明により得られた遺伝子の発現産物を用いることにより、アントシアニジンの5位の水酸基へグルコシル基を転移させた後、3位の水酸基へグルコシル基を転移させることができる。これにより、花や果実などアントシアニンの蓄積により発色している植物組織の改変を行うことが可能になる。 By using the gene expression product obtained by the present invention, the glucosyl group can be transferred to the hydroxyl group at the 3-position after transferring the glucosyl group to the hydroxyl group at the 5-position of anthocyanidin. This makes it possible to modify plant tissues that are colored by the accumulation of anthocyanins such as flowers and fruits.
以下実施例に従って、本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
〔実施例１〕 酵素反応の基質及び標準化合物の調製
（１）植物材料の調製
バラ‘クリムソングローリー’（Rosa hybrida cv. Crimson Glory）（広島バラ園）の苗を、10号ポリポットに移植してガラス温室にて育苗した。その後、アントシアニン抽出に用いた‘クリムソングローリー’の開花花弁は35℃で通風乾燥した後、アントシアニン抽出に用いるまで真空デシケーター内で保存した。
[Example 1] Preparation of enzyme reaction substrate and standard compound (1) Preparation of plant material A seedling of Rosa hybrida cv. Crimson Glory (Hiroshima Rose Garden) was transplanted to No. 10 polypot. Raised seedlings in a glass greenhouse. Thereafter, the flower petals of “Crimson lorry” used for anthocyanin extraction were dried by ventilation at 35 ° C. and then stored in a vacuum desiccator until used for anthocyanin extraction.
（２）基質および反応産物の決定
酵素反応基質および反応産物の同定は、薄層クロマトグラフィー(TLC)、高速液体クロマトグラフィー(HPLC)分析による標準物質とのコクロマトグラフィーおよび分光学的手法により行った。シアニジン、シアニジン3-グルコシド、シアニジン3,5-グルコシドは市販品（フナコシ（株）製）を用いた。
(2) Determination of substrates and reaction products Identification of enzyme reaction substrates and reaction products is performed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis with co-chromatography and spectroscopic methods. It was. As for cyanidin, cyanidin 3-glucoside, and cyanidin 3,5-glucoside, commercially available products (manufactured by Funakoshi Co., Ltd.) were used.
（３）シアニジン5-グルコシドの単離
バラ乾燥花弁（後述のHPLC（520nm）分析の結果、粗抽出液でシアニジン3,5-ジグルコシドを95％以上含む：生重量100ｇ）を１Ｌの0.05％塩酸水溶液にて一晩常温で放置し粗抽出液を得た後、石油エーテル、クロロホルム及び酢酸エチルにより分画操作を行った。アンバーライトXAD-7（オルガノ（株）製）を充填したガラスカラム（30 mm i.d.×600 mm）を0.01％塩酸水溶液にて平衡化したのち２倍に希釈した水層画分をカラムに通し、色素を吸着させた。未吸着物質を洗浄した後、0.01％塩酸を含む80％メタノール水溶液にて色素を溶出した。溶出液は乾燥固化させた。
(3) Isolation of cyanidin 5-glucoside 1 L of 0.05% hydrochloric acid with dried rose petals (as a result of HPLC (520 nm) described later, the crude extract contains 95% or more of cyanidin 3,5-diglucoside: raw weight 100 g) The crude extract was obtained by standing overnight at room temperature in an aqueous solution, and then fractionated with petroleum ether, chloroform and ethyl acetate. A glass column (30 mm id × 600 mm) packed with Amberlite XAD-7 (manufactured by Organo Corp.) was equilibrated with 0.01% hydrochloric acid aqueous solution and then diluted twice, and the aqueous layer fraction was passed through the column. The dye was adsorbed. After washing the unadsorbed material, the dye was eluted with 80% aqueous methanol solution containing 0.01% hydrochloric acid. The eluate was dried and solidified.
乾燥固化させた色素粉末を0.05％塩酸水溶液に再溶解させ、そこに12％塩酸水溶液を等量加え80℃で20分間加熱し酸加水分解を行った。加熱後、その溶液に等量のイソアミルアルコールを加え激しく攪拌後遠心し、イソアミルアルコール層を取り除いた。これを３回行った後、酢酸エチルを等量加え攪拌後、遠心し水層を分画した。 The dried and solidified pigment powder was redissolved in 0.05% aqueous hydrochloric acid solution, and an equal amount of 12% aqueous hydrochloric acid solution was added thereto, followed by heating at 80 ° C. for 20 minutes for acid hydrolysis. After heating, an equal amount of isoamyl alcohol was added to the solution, and the mixture was vigorously stirred and centrifuged to remove the isoamyl alcohol layer. After performing this three times, an equal amount of ethyl acetate was added and stirred, and then centrifuged to separate the aqueous layer.
上述の方法で得られた水層から２回のペーパークロマトグラフィー（ワットマン３MM）を行うことでシアニジン5-グルコシドを単離した。有隅の方法（有隅 山口大学農学部学術報告 第18号1967年）を一部改変し、展開溶媒 2メチル-1-プロパノール：塩酸：水＝5：2：4の上清を用い1回目のクロマトを行い3種類（シアニジン3-グルコシド、シアニジン5-グルコシド、シアニジン3,5-ジグルコシド）の色素画分を得た。展開後風乾させ、320nmのUV下で強い蛍光を示す画分（シアニジン5-グルコシド）を２回目のペーパークロマトグラフィーに用いた。展開溶媒 塩酸：酢酸：水＝15：3：82を用い展開精製した。１種類の色素画分を得、溶出後、HPLC分析により単一に精製されたことを確認し、その後の酵素反応の基質とした。また、単一でない場合はHPLCによる分取を行った。 Cyanidin 5-glucoside was isolated from the aqueous layer obtained by the above-mentioned method by performing paper chromatography (Whatman 3MM) twice. First modification using Arisumi's method (Arisumi Yamaguchi University Faculty of Agriculture, Scientific Report No.18, 1967) and using supernatant of developing solvent 2methyl-1-propanol: hydrochloric acid: water = 5: 2: 4 Chromatography was performed to obtain three types of pigment fractions (cyanidin 3-glucoside, cyanidin 5-glucoside, cyanidin 3,5-diglucoside). After the development, it was air-dried, and a fraction (cyanidine 5-glucoside) showing strong fluorescence under UV at 320 nm was used for the second paper chromatography. Developing solvent Purified with hydrochloric acid: acetic acid: water = 15: 3: 82. One type of dye fraction was obtained, and after elution, it was confirmed that it was purified by HPLC analysis, and used as a substrate for the subsequent enzyme reaction. Moreover, when it was not single, fractionation by HPLC was performed.
HPLCによる分析および分取には逆層カラム Wakosil-II5C18ARカラム（4.6 mm i.d.×250 mm）を用い、カラム温度35℃で行った。流速１ml/minにて0.5％TFA、5％アセトニトリルを含む水溶液（Ａ）に対して5％から25％の0.5％TFAを含むアセトニトリル溶液（Ｂ）による直線的濃度勾配で20分間の溶出時間でPDAを用いて検出した。 For the analysis and fractionation by HPLC, a reverse layer column Wakosil-II5C18AR column (4.6 mm i.d. × 250 mm) was used at a column temperature of 35 ° C. Elution time of 20 minutes with a linear concentration gradient from 5% to 25% acetonitrile solution (B) containing 0.5% TFA to an aqueous solution (A) containing 0.5% TFA and 5% acetonitrile at a flow rate of 1 ml / min. Detection was performed using PDA.
〔実施例２〕 バラ花弁でのアントシアニンの5位及び3位にグルコシル基を転移する酵素活性の測定
0.1Mトリス-塩酸緩衝液（pH8.0）、0.5mMシアニジンまたはシアニジン５-グルコシドまたはシアニジン３-グルコシド、1mM UDP-グルコース及び粗酵素液30μlを含む反応液 50μlを30℃で反応させた。1M塩酸水溶液10μlを加えて反応を停止させ、クロロホルム：メタノール＝2：1を等量加え攪拌後遠心し、上清を凍結乾燥機にて乾燥させHPLC溶媒 A/B=95:5を10μl加え再溶解後遠心し上清を分析サンプルとした。
[Example 2] Measurement of enzyme activity to transfer glucosyl group to 5- and 3-positions of anthocyanins in rose petals
50 μl of a reaction solution containing 0.1 M Tris-HCl buffer (pH 8.0), 0.5 mM cyanidin or cyanidin 5-glucoside or cyanidin 3-glucoside, 1 mM UDP-glucose and 30 μl of the crude enzyme solution was reacted at 30 ° C. Stop the reaction by adding 10 μl of 1M aqueous hydrochloric acid solution, stir after adding an equal volume of chloroform: methanol = 2: 1, centrifuge, dry the supernatant with a freeze dryer, and add 10 μl of HPLC solvent A / B = 95: 5 After redissolving, the sample was centrifuged and the supernatant was used as an analysis sample.
酵素反応産物のHPLC分析は、上述の方法で行った。本条件による各化合物の溶出時間は以下のとおりである。シアニジン 3,5-ジグルコシド：6.12 分、シアニジン3-グルコシド：9.34 分、シアニジン5-グルコシド：9.72 分、シアニジン：14.17 分。 The HPLC analysis of the enzyme reaction product was performed by the method described above. The elution time of each compound under these conditions is as follows. Cyanidin 3,5-diglucoside: 6.12 min, cyanidin 3-glucoside: 9.34 min, cyanidin 5-glucoside: 9.72 min, cyanidin: 14.17 min.
〔実施例３〕 アントシアニジン5位グルコシル基転移酵素及びアントシアニジン5-グルコシドの3位グルコシル基転移酵素の活性の検出
バラ‘クリムソングローリー’（Rosa hybrida cv. Crimson Glory）‘ローテローゼ（cv. Rote Roze）‘カールレッド’(cv. Carl Red)の花弁をそれぞれ用い、シアニジン、シアニジン5-グルコシドおよびシアニジン 3-グルコシドを基質に用いて活性測定を行った。以下の実験は０〜４℃で行った。
Example 3 anthocyanidin 5-position glucosyl sulfotransferase 3-position glucosyl sulfotransferase activity of and anthocyanidin 5-glucosides detection Rose 'Crimson Glory' (Rosa hybrida cv. Crimson Glory ) ' Roteroze (cv. Rote Roze)' Activity was measured using cyanidin, cyanidin 5-glucoside, and cyanidin 3-glucoside as substrates, respectively, using petals of cv. Carl Red. The following experiment was performed at 0-4 degreeC.
（１）粗酵素液の調製
バラの凍結花弁１０gをポリビニルポリピロリドン（PVPP）５gと共に液体窒素存在下で乳鉢及び乳棒で磨砕した。100mlの緩衝液A（100mMリン酸カリウム緩衝液（pH7.0）、14mM 2-メルカプトエタノール、1%ポリビニルピロリドンK-30（PVP）、100mM 塩化ナトリウム、5mM EDTA、5mM アスコルビン酸ナトリウム、10%グリセロール、0.1% Triton X-100）を加え、攪拌溶出した。抽出懸濁液を四重のガーゼにて濾過後12,000 rpmで20分間遠心した。
(1) Preparation of crude enzyme solution 10 g of frozen rose petals were ground with a mortar and pestle in the presence of liquid nitrogen together with 5 g of polyvinylpolypyrrolidone (PVPP). 100 ml Buffer A (100 mM potassium phosphate buffer (pH 7.0), 14 mM 2-mercaptoethanol, 1% polyvinylpyrrolidone K-30 (PVP), 100 mM sodium chloride, 5 mM EDTA, 5 mM sodium ascorbate, 10% glycerol 0.1% Triton X-100) was added and the mixture was eluted with stirring. The extracted suspension was filtered through quadruple gauze and then centrifuged at 12,000 rpm for 20 minutes.
（２）硫安分画
濾液（約100ｍl）を10％飽和硫安で40分間塩析を行った後、不溶物を12,000 rpmで20分間遠心することにより除去した。70％飽和硫安で再び塩析を行い、12,000 rpmで20分間遠心することにより沈殿を得た。沈殿を最少量の緩衝液B（50mMトリス塩酸（pH 8.0）、14mM 2-メルカプトエタノール）に再溶解した後、緩衝液Bで平衡化したSephadex G-25 Fine（30 mm i.d.×500 mm；Amersham Bioscience）カラムを用いて脱塩した。タンパク質画分（約20ml）を回収して以下のクロマトグラフィーに供した。
(2) Ammonium sulfate fraction The filtrate (about 100 ml) was salted out with 10% saturated ammonium sulfate for 40 minutes, and then insoluble matters were removed by centrifugation at 12,000 rpm for 20 minutes. Salting out was performed again with 70% saturated ammonium sulfate, and a precipitate was obtained by centrifuging at 12,000 rpm for 20 minutes. The precipitate was redissolved in a minimum amount of buffer B (50 mM Tris-HCl (pH 8.0), 14 mM 2-mercaptoethanol), and then equilibrated with buffer B, Sephadex G-25 Fine (30 mm id × 500 mm; Amersham Desalted using a Bioscience) column. The protein fraction (about 20 ml) was collected and subjected to the following chromatography.
（３）オープンカラムよる陰イオン交換樹脂の吸脱着
陰イオン交換樹脂であるDE52(ワットマン製)をカラム（30 mm i.d.×50 mm）に充填して、緩衝液Bで平衡化した。カラムへ２倍希釈した酵素溶液を添加し、緩衝液Bで洗浄後、塩化ナトリウム0.3Ｍを含む緩衝液Bにより溶出し、粗酵素液とした。
(3) Adsorption / desorption of anion exchange resin by an open column DE52 (manufactured by Whatman), an anion exchange resin, was packed in a column (30 mm id × 50 mm) and equilibrated with buffer B. The enzyme solution diluted 2-fold was added to the column, washed with buffer B, and then eluted with buffer B containing 0.3 M sodium chloride to obtain a crude enzyme solution.
〔実施例４〕 粗酵素液の反応特性
上述の粗酵素液を用い酵素活性測定を行った。HPLC分析は、実施例１および２に記載の方法に従って行った。その結果、シアニジンを基質とした場合、その反応産物はシアニジン5-グルコシド、シアニジン3,5-ジグルコシドおよび極微量のシアニジン3-グルコシドであり、シアニジン3-グルコシドを基質とした場合、シアニジン3,5-ジグルコシドは得られなかった。さらに、シアニジン5-グルコシドを基質とした時シアニジン3,5-ジグルコシドが得られた。基質別の反応結果を図1に示す。つまり、これまで考えられていたアントシアニジンの3位への配糖体化、その後の5位への配糖体化という生合成(Tanaka et al., (1998) Plant Cell Physiol. 39: 1119-1126、Forkmann, (2003) in: Encyclopedia of rose science Vol.1. 256-262, Elsevier academic press, Oxford).とは逆の、最初に5位の配糖体化、そして3位の配糖体化がバラ粗酵素液で検出された。さらにバラ花弁に含まれるペラルゴニジン3,5-ジグルコシドのアグリコンであるペラルゴニジンを基質とした場合もシアニジンと同様の配糖体化反応が起こり、ペラルゴニジン3-グルコシドを基質とした場合はシアニジン3-グルコシド同様、反応産物は得られなかった。
Example 4 Reaction Characteristics of Crude Enzyme Solution Enzyme activity was measured using the above-mentioned crude enzyme solution. HPLC analysis was performed according to the method described in Examples 1 and 2. As a result, when cyanidin was used as a substrate, the reaction products were cyanidin 5-glucoside, cyanidin 3,5-diglucoside and a trace amount of cyanidin 3-glucoside. When cyanidin 3-glucoside was used as a substrate, cyanidin 3,5 -Diglucoside was not obtained. Furthermore, cyanidin 3,5-diglucoside was obtained when cyanidin 5-glucoside was used as a substrate. The reaction results for each substrate are shown in FIG. In other words, the biosynthesis of anthocyanidins that had been considered so far was glycosylated at position 3 and then glycosylated at position 5 (Tanaka et al., (1998) Plant Cell Physiol. 39: 1119-1126. , Forkmann, (2003) in: Encyclopedia of rose science Vol.1 256-262, Elsevier academic press, Oxford). First, glycosylation at position 5 and glycosylation at position 3. Was detected in the crude enzyme solution. Furthermore, pelargonidin 3,5-diglucoside aglycon contained in rose petals also has a glycosylation reaction similar to cyanidin when pelargonidin is used as a substrate, and when pelargonidin 3-glucoside is used as a substrate, it is similar to cyanidin 3-glucoside. No reaction product was obtained.
これらの結果から、アントシアニジンの5位の水酸基に対するグルコシルトランスフェラーゼ活性を有する酵素およびアントシアニジン5-グルコシドの3位の水酸基に対するグルコシルトランスフェラーゼ活性を有する酵素がバラ花弁中には存在することが強く示唆された。なお、これらの知見はこれまで一切報告されていない。 These results strongly suggested that an enzyme having glucosyltransferase activity for the hydroxyl group at the 5-position of anthocyanidin and an enzyme having glucosyltransferase activity for the hydroxyl group at the 3-position of anthocyanidin 5-glucoside are present in the rose petals. These findings have never been reported so far.
〔実施例５〕 バラフラボノイド糖転移酵素遺伝子の遺伝子断片の増幅
（１）RNAの調製
バラ品種、クリムソングローリーの花弁から改変CTAB法（Chang et al., 1993, 向井・山本 植物細胞工学シリーズ7 pp.57-62）により全RNAを調製した。poly(A)+RNAはこの全RNA（250μg）からOligotex-dT30 super（Takara）を用い、製造者の推奨する方法にて調製した。
[Example 5] Amplification of gene fragment of rose flavonoid glycosyltransferase gene (1) RNA preparation Modified CTAB method (Chang et al., 1993, Mukai / Yamamoto Plant
（２）3’RACE
調製した全RNAを99℃で２分間処理し、氷冷後、First strand cDNA synthesis kit (Pharmacia)を用い、製造者の示した方法によりcDNAを合成した。合成したcDNAを鋳型として、植物二次代謝産物配糖化酵素（Plant Secondary Product Glycosyltransferase）に高度に保存されている領域PSPG-box（Huges and Huges (1994) DNA Seq., 5: 41-49）のアミノ酸配列を基に設計した縮重プライマーGTSPFd［WCICAYTGYGGITGGAAYTC：配列番号３］及びcDNA合成に用いたNot Id(T)18に存在する配列を基に設計したNotIR1［AACTGGAAGAATTCGCGGC：配列番号４］をプライマーとし、PCR反応を行った。PCR反応液は、2μl cDNA，１x Expand HF バッファー（BOEHRINGER MANNHEIM）, 0.2mM dNTPs、プライマー各1.6pmol/μｌ、Expand High Fidelity PCR system 酵素ミックス1.75 unitからなる総体積50μlに調製した。反応は、94℃で2分反応させた後、94℃・30秒、48℃・30秒、72℃・30秒の反応を40サイクル行い、最後に72℃で１分間処理した。得られた反応産物を0.8%アガロースゲル電気泳動し、予想されるサイズのDNA断片を回収した。回収したDNA断片を鋳型に、GTSPFd及びNot Id(T)18に存在する配列を基にNotIR1の3’側に設計したNotR2(GAACGCGGCCGCAGGAAT：配列番号５)をプライマーとしPCR反応を行った。PCR反応液は、2μl cDNA，１ｘ Expand HF バッファー（BOEHRINGER MANNHEIM）, 0.2mM dNTPs、プライマー各1.6pmol/μｌ、Expand High Fidelity PCR system 酵素ミックス1.75 unitからなる総体積50μｌに調製した。反応は、94℃で2分反応させた後、94℃・30秒、48℃・30秒、72℃・30秒の反応を40サイクル行い、最後に72℃で１分間処理した。得られた反応産物を0.8%アガロースゲル電気泳動し、予想されるサイズのDNA断片を回収した。回収したDNA産物をpGEM Easy Tベクター（Promega）にクローニングした。このようにして得られたプラスミドのいくつかのクローンをABIＴＭ BigDyeＴＭTerminator cycle sequencing reaction ver.3.1（Applied Biosystem）を用いて反応し、DNA Sequencer model 3100を用いて、全塩基配列を決定した後、BLASTX 検索を行い、糖転移酵素遺伝子に相同性の認められるクローン RhGT fra.42を見出した。
(2) 3'RACE
The prepared total RNA was treated at 99 ° C. for 2 minutes, cooled on ice, and then cDNA was synthesized using First strand cDNA synthesis kit (Pharmacia) by the method indicated by the manufacturer. Using the synthesized cDNA as a template, the region PSPG-box (Huges and Huges (1994) DNA Seq., 5: 41-49) highly conserved in plant secondary metabolite glycosyltransferases (Plant Secondary Product Glycosyltransferase) The degenerate primer GTSPFd [WCICAYTGYGGITGGAAYTC: SEQ ID NO: 3] designed based on the amino acid sequence and NotIR1 [AACTGGAAGAATTCGCGGC: SEQ ID NO: 4] designed based on the sequence present in Not Id (T) 18 used for cDNA synthesis were used as primers. A PCR reaction was performed. The PCR reaction solution was prepared in a total volume of 50 μl consisting of 2 μl cDNA, 1 × Expand HF buffer (BOEHRINGER MANNHEIM), 0.2 mM dNTPs, each primer 1.6 pmol / μl, and Expand High Fidelity PCR system enzyme mix 1.75 unit. The reaction was carried out at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 48 ° C for 30 seconds, 72 ° C for 30 seconds, and finally treated at 72 ° C for 1 minute. The obtained reaction product was subjected to 0.8% agarose gel electrophoresis, and a DNA fragment having the expected size was recovered. Using the recovered DNA fragment as a template, PCR reaction was performed using NotR2 (GAACGCGGCCGCAGGAAT: SEQ ID NO: 5) designed on the 3 ′ side of NotIR1 based on the sequences present in GTSPFd and Not Id (T) 18 as primers. The PCR reaction solution was prepared in a total volume of 50 μl consisting of 2 μl cDNA, 1 × Expand HF buffer (BOEHRINGER MANNHEIM), 0.2 mM dNTPs, each primer 1.6 pmol / μl, Expand High Fidelity PCR system enzyme mix 1.75 unit. The reaction was carried out at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 48 ° C for 30 seconds, 72 ° C for 30 seconds, and finally treated at 72 ° C for 1 minute. The obtained reaction product was subjected to 0.8% agarose gel electrophoresis, and a DNA fragment having the expected size was recovered. The recovered DNA product was cloned into pGEM Easy T vector (Promega). After several clones of the plasmids thus obtained were reacted using ABI ™ BigDye ™ Terminator cycle sequencing reaction ver.3.1 (Applied Biosystem) and the entire nucleotide sequence was determined using DNA Sequencer model 3100 A BLASTX search was carried out, and a clone RhGT fra.42 was found that showed homology to the glycosyltransferase gene.
（３）5’RACE
5’RACEに用いた5’末端を有するｍRNAに対するcDNAはGeneRacer kit (Invitrogen) を用い、製造者の示した方法に従って合成した。合成したcDNAを鋳型にGeneRacer kitのGeneRacer 5’primer (CGACTGGAGCACGAGGACACTGA：配列番号６)及び RhGT Fra.42の内部配列に設定したプライマー42R1(TTATGCGGGCCCAACATGCC：配列番号７)を用い1st-PCRを行った。PCR反応液は、1μl cDNA、１ｘ LA PCR Buffer II（Takara），0.2mM dNTPs、 3μl GeneRacer 5’primer (10μM)、2μl 42R1(5μM)、2mM MgCl2、Takara LA Taq 2.5 unitからなる総体積50μlに調製した。反応は、94℃で2分反応させた後、94℃・30秒、58℃・30秒、72℃・２分の反応を40サイクル行い、最後に72℃で２分間処理した。この反応液を0.8％アガロース電気泳動にかけ、増幅産物のバンドを切り出し、Qiagen Gel Extraction kit（Qiagen）により増幅産物を回収した。回収した増幅産物を鋳型にGeneRacer kitのGeneRacer 5’Nested primer 及びRhGT Fra.42の内部配列に設定したプライマー42R2（CCACAGCTGAGCCAACTTGG：配列番号８）を用いNeated-PCRを行った。PCR反応液は、1μl cDNA，１ｘ LA PCR Buffer II（Takara），0.2mM dNTPs、1μl GeneRacer 5’primer (10μM)、2μl 42R1(5μM)、2mM MgCl2、Takara LA Taq 2.5 unitからなる総体積50μlに調製した。反応は、94℃で2分反応させた後、94℃・30秒、58℃・30秒、72℃・２分の反応を40サイクル行い、最後に72℃で２分間処理した。この反応液を0.8％アガロース電気泳動にかけ、増幅産物のバンドを切り出し、Qiagen Gel Extraction kit（Qiagen）により増幅産物を回収した。得られたPCR産物をpGEM Easy Tベクター（Promega）にクローニングした。このようにして得られたプラスミドのいくつかのクローンをABIＴＭ BigDyeＴＭTerminator cycle sequencing reaction ver.3.1（Applied Biosystem）を用いて反応し、DNA Sequencer model 3100を用いて、全塩基配列を決定した後、DNASISを用い、コードされるアミノ酸配列を推定した。
(3) 5'RACE
CDNA for mRNA having 5 ′ end used in 5 ′ RACE was synthesized using GeneRacer kit (Invitrogen) according to the method indicated by the manufacturer. 1st-PCR was performed using GeneRacer kit GeneRacer 5'primer (CGACTGGAGCACGAGGACACTGA: SEQ ID NO: 6) and primer 42R1 (TTATGCGGGCCCAACATGCC: SEQ ID NO: 7) set to the internal sequence of RhGT Fra.42 using the synthesized cDNA as a template. PCR reaction solution is 1 μl cDNA, 1 × LA PCR Buffer II (Takara), 0.2 mM dNTPs, 3 μl GeneRacer 5'primer (10 μM), 2 μl 42R1 (5 μM), 2 mM MgCl 2 , Takara LA Taq 2.5 unit total volume 50 μl Prepared. The reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 2 minutes, and finally treated at 72 ° C for 2 minutes. This reaction solution was subjected to 0.8% agarose electrophoresis, the amplification product band was cut out, and the amplification product was recovered using a Qiagen Gel Extraction kit (Qiagen). Using the recovered amplified product as a template, Neated-PCR was performed using GeneRacer 5′Nested primer of GeneRacer kit and primer 42R2 (CCACAGCTGAGCCAACTTGG: SEQ ID NO: 8) set to the internal sequence of RhGT Fra.42. The PCR reaction solution is 1 μl cDNA, 1 × LA PCR Buffer II (Takara), 0.2 mM dNTPs, 1 μl GeneRacer 5'primer (10 μM), 2 μl 42R1 (5 μM), 2 mM MgCl 2 , Takara LA Taq 2.5 unit total volume 50 μl Prepared. The reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 2 minutes, and finally treated at 72 ° C for 2 minutes. This reaction solution was subjected to 0.8% agarose electrophoresis, the amplification product band was cut out, and the amplification product was recovered using a Qiagen Gel Extraction kit (Qiagen). The resulting PCR product was cloned into pGEM Easy T vector (Promega). After several clones of the plasmids thus obtained were reacted using ABI ™ BigDye ™ Terminator cycle sequencing reaction ver.3.1 (Applied Biosystem) and the entire nucleotide sequence was determined using DNA Sequencer model 3100 The encoded amino acid sequence was estimated using DNASIS.
〔実施例６〕RhGT Fra.42の全タンパク質コード領域のクローニング及び大腸菌における組換えタンパク質の発現
（１）RhGT Fra.42の全タンパク質コード領域のクローニング
実施例５によって得られたクローンの塩基配列から予想されるタンパク質コード領域をPCRにより増幅後、大腸菌発現用pETベクターにクローニングした。全RNAに対するcDNAをFirst strand cDNA synthesis kit（Pharmacia）を用い、製造者の推奨する方法にてNot I dTプライマーを用い逆転写反応を行い合成した。大腸菌発現用pET30Ek/LIC（Novagen）にクローニングするための配列と実施例５で得られた塩基配列から予想されるタンパク質の開始コドンを含むプライマーRhGTFd（GACGACGACAAGATGGGTGGTGATGCTATAGTTTG：配列番号９）及び終始コドンを含むプライマーRhGTRv（GAGGAGAGCCCGGTTCATTTTTGCTTCCACAGCTGAGCC：配列番号１０）を製造者の推奨する方法に従って合成した。PCR反応液は2μl cDNA，１x Expand HF バッファー（BOEHRINGER MANNHEIM）, 0.2mM dNTPs、プライマー各1.6pmol/μｌ、Expand High Fidelity PCR system 酵素ミックス1.75 unitからなる総体積50μｌに調製した。反応は、94℃で2分反応させた後、94℃・30秒、55℃・30秒、72℃・２分の反応を40サイクル行い、最後に72℃で２分間処理した。得られた反応産物を0.8%アガロースゲル電気泳動し、増幅産物をQIAquick Gel Extraction Kit（Qiagen）を用いてゲルからDNAを精製した。得られたDNAをpET30Ek/LIC（Novagen）に製造者の推奨する方法に従って連結した後、大腸菌JM109に形質転換して幾つかのクローンをT7 プロモーター、T7ターミネータープライマーを用い、BigDye Terminator Ver. 3.1を用いたCycle Sequencing Reactionを行った後、ABI3100 Genetic Analyserを用いて塩基配列の決定を行った。塩基配列の決定を行ったpETクローンのうちpETRhGT1は1,422塩基対（この塩基配列を配列番号１に示す。）からなり、473アミノ酸残基（このアミノ酸配列を配列番号２に示す。）からなるタンパク質をコードしていた。このpETRhGT1を用いて機能解析を行った。
[Example 6] Cloning of the entire protein coding region of RhGT Fra.42 and expression of recombinant protein in E. coli (1) Cloning of the entire protein coding region of RhGT Fra.42 From the base sequence of the clone obtained in Example 5 The expected protein coding region was amplified by PCR and then cloned into an E. coli expression pET vector. CDNA for total RNA was synthesized by reverse transcription reaction using Not I dT primer using First strand cDNA synthesis kit (Pharmacia) and the method recommended by the manufacturer. Primer RhGTFd (GACGACGACAAGATGGGTGGTGATGCTATAGTTTG: SEQ ID NO: 9) containing a sequence for cloning into pET30Ek / LIC (Novagen) for expression in E. coli and a protein predicted from the base sequence obtained in Example 5 and a termination codon RhGTRv (GAGGAGAGCCCGGTTCATTTTTGCTTCCACAGCTGAGCC: SEQ ID NO: 10) was synthesized according to the method recommended by the manufacturer. The PCR reaction solution was prepared in a total volume of 50 μl consisting of 2 μl cDNA, 1 × Expand HF buffer (BOEHRINGER MANNHEIM), 0.2 mM dNTPs, each primer 1.6 pmol / μl, and Expand High Fidelity PCR system enzyme mix 1.75 units. The reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, and finally treated at 72 ° C for 2 minutes. The obtained reaction product was subjected to 0.8% agarose gel electrophoresis, and the amplified product was purified from the gel using QIAquick Gel Extraction Kit (Qiagen). After ligating the obtained DNA to pET30Ek / LIC (Novagen) according to the method recommended by the manufacturer, E. coli JM109 was transformed and several clones were used with T7 promoter and T7 terminator primer, and BigDye Terminator Ver. After performing the used Cycle Sequencing Reaction, the base sequence was determined using ABI3100 Genetic Analyzer. Among the pET clones for which the base sequence was determined, pETRhGT1 consists of 1,422 base pairs (this base sequence is shown in SEQ ID NO: 1), and a protein consisting of 473 amino acid residues (this amino acid sequence is shown in SEQ ID NO: 2). Was coded. Functional analysis was performed using this pETRhGT1.
（２）組換えタンパク質の発現
pETRhGT1を大腸菌BL21(DE3)（Novagen）に形質転換した。形質転換株はカナマイシン30μg/mlを含むLB培地3mlで37℃で一晩振盪培養した。この培養液500μlをカナマイシン30μg/mlを含むLB培地に加え、600nmにおける吸光度が0.4に達するまで振盪培養した後、イソプロピル-β-D-チオガラクトシド（IPTG）を最終濃度0.4mMになるように添加し、22℃で12時間振盪培養した後、冷却遠心（8000rpm, 4℃, 20分間）して菌体を回収した。
(2) Recombinant protein expression
pETRhGT1 was transformed into E. coli BL21 (DE3) (Novagen). The transformant was cultured overnight at 37 ° C. in 3 ml of LB medium containing 30 μg / ml of kanamycin. Add 500 μl of this culture to LB medium containing 30 μg / ml of kanamycin, shake culture until the absorbance at 600 nm reaches 0.4, and then add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM. After culturing at 22 ° C. for 12 hours, the cells were recovered by centrifuging (8000 rpm, 4 ° C., 20 minutes).
〔実施例７〕 pETRhGT1 cDNAがコードするタンパク質の酵素活性測定
（１）組換えタンパク質の抽出
組換えタンパク質の抽出には、菌体のペレットを、１mlの溶解緩衝液（pH8.0）（100mMトリス塩酸、300mM塩化ナトリウム、14mM 2-メルカプトエタノール0.1% Protease and Phosphatase Inhibitor Cocktails (SIGMA社製)）に再懸濁し、超音波破砕機により破壊し、冷却遠心機で分離した。上清を組換えタンパク質液とし、酵素活性測定に用いた。
[Example 7] Measurement of enzyme activity of the protein encoded by pETRhGT1 cDNA (1) Extraction of recombinant protein For extraction of recombinant protein, 1 ml of lysis buffer (pH 8.0) (100 mM Tris) was used. Resuspended in hydrochloric acid, 300 mM sodium chloride, 14 mM 2-mercaptoethanol 0.1% Protease and Phosphatase Inhibitor Cocktails (manufactured by SIGMA)), disrupted with an ultrasonic crusher, and separated with a cooling centrifuge. The supernatant was used as a recombinant protein solution and used for enzyme activity measurement.
（２）組換えタンパク質の酵素活性測定と酵素反応産物の同定
組換えpETRhGT1タンパク質を用い酵素反応を行った。組換えタンパク質液30μlを用い〔実施例２〕と同様の方法で行った。HPLCによる分析には逆層カラム Wakosil-II5C18ARカラム（4.6 mm i.d.×250 mm）を用い、カラム温度35℃で行った。流速１ml/minにて0.5％TFA、5％アセトニトリルを含む水溶液に対して5％から20％の0.5％TFAを含むアセトニトリル溶液による直線的濃度勾配で20分間の溶出時間でPDAを用いて検出した。基質別の反応結果を図2に示す。標準物質とのコクロマトグラフィー分析により、反応産物をそれぞれシアニジン3，5-ジグルコシド及びシアニジン5-グルコシドと同定した。さらにシアニジン3-グルコシドを基質とした場合、その反応産物は得られなかった。本条件による各化合物の溶出時間は以下のとおりである。シアニジン 3,5-ジグルコシド：6.31 分、シアニジン3-グルコシド：9.85 分、シアニジン5-グルコシド：10.68 分、シアニジン：16.34 分。以上の結果より、pETRhGT1クローンの組換え酵素はUDP-glucose: cyanidin 5-O-glucosyltransferase及びUDP-glucose: cyanidin 5-O-glucoside 3-O-glucosyltransferase活性の両方を持つことがあきらかになった。従って、RhGT1遺伝子はアントシアニジンの5位及び3位の水酸基に逐次グリコシル基を転移する活性がある、UDP-glucose:anthocyanidin 5,3-O-glucosyltransferaseをコードしていることが確認された。
(2) Enzyme activity measurement of recombinant protein and identification of enzyme reaction product Enzymatic reaction was performed using recombinant pETRhGT1 protein. 30 μl of the recombinant protein solution was used in the same manner as in [Example 2]. For the analysis by HPLC, a reverse layer column Wakosil-II5C18AR column (4.6 mm id × 250 mm) was used, and the column temperature was 35 ° C. Detection was performed with PDA at an elution time of 20 minutes in a linear concentration gradient with an acetonitrile solution containing 5% to 20% 0.5% TFA against an aqueous solution containing 0.5% TFA and 5% acetonitrile at a flow rate of 1 ml / min. . The reaction results for each substrate are shown in FIG. The reaction products were identified as cyanidin 3,5-diglucoside and cyanidin 5-glucoside, respectively, by co-chromatographic analysis with standard substances. Furthermore, when cyanidin 3-glucoside was used as a substrate, the reaction product was not obtained. The elution time of each compound under these conditions is as follows. Cyanidin 3,5-diglucoside: 6.31 min, cyanidin 3-glucoside: 9.85 min, cyanidin 5-glucoside: 10.68 min, cyanidin: 16.34 min. From the above results, it was revealed that the recombinant enzyme of the pETRhGT1 clone has both UDP-glucose: cyanidin 5- O- glucosyltransferase and UDP-glucose: cyanidin 5- O- glucoside 3- O- glucosyltransferase activities. Therefore, it was confirmed that the RhGT1 gene encodes UDP-glucose: anthocyanidin 5,3- O- glucosyltransferase, which has an activity of sequentially transferring a glycosyl group to the hydroxyl groups at the 5- and 3-positions of anthocyanidins.
Claims (17)
(a)配列番号２に記載のアミノ酸配列を有するタンパク質、
(b)配列番号２に記載のアミノ酸配列において一個又は複数個のアミノ酸が付加、欠失及び／又は他のアミノ酸により置換されているアミノ酸配列を有するタンパク質、
(c)配列番号２に記載のアミノ酸配列に対して20%以上の相同性を示すアミノ酸配列を有するタンパク質、
(d)配列番号２に記載のアミノ酸配列に対して70%以上の相同性を示すアミノ酸配列を有するタンパク質。 The gene according to claim 1, which encodes the following proteins (a) to (d):
(a) a protein having the amino acid sequence of SEQ ID NO: 2,
(b) a protein having an amino acid sequence in which one or more amino acids are added, deleted and / or substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 2;
(c) a protein having an amino acid sequence showing 20% or more homology to the amino acid sequence set forth in SEQ ID NO: 2;
(d) a protein having an amino acid sequence showing 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 2.
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| WO2012096307A1 (en) | 2011-01-14 | 2012-07-19 | サントリーホールディングス株式会社 | Novel glycosyltransferase gene and use thereof |
| WO2013108794A1 (en) | 2012-01-17 | 2013-07-25 | サントリーホールディングス株式会社 | Novel glycosyltransferase gene and use thereof |
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| CN114807160B (en) * | 2022-03-10 | 2023-11-14 | 上海师范大学 | Gene RcGT for regulating and controlling petal color of China rose, protein, recombinant vector, recombinant transformant, application and method |
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| JP2008048612A (en) * | 2006-08-22 | 2008-03-06 | Bio Taxol:Kk | Glycosyltransferase gene |
| JP2009213413A (en) * | 2008-03-11 | 2009-09-24 | Iwate Prefecture | 3-deoxyanthocyanidin glycosyltransferase gene and use thereof |
| WO2012096307A1 (en) | 2011-01-14 | 2012-07-19 | サントリーホールディングス株式会社 | Novel glycosyltransferase gene and use thereof |
| US9365836B2 (en) | 2011-01-14 | 2016-06-14 | Suntory Holdings Limited | Glycosyltransferase gene and use thereof |
| WO2013108794A1 (en) | 2012-01-17 | 2013-07-25 | サントリーホールディングス株式会社 | Novel glycosyltransferase gene and use thereof |
| KR20140116394A (en) | 2012-01-17 | 2014-10-02 | 산토리 홀딩스 가부시키가이샤 | Novel glycosyltransferase gene and use thereof |
| US10059956B2 (en) | 2012-01-17 | 2018-08-28 | Suntory Holdings Limited | Glycosyltransferase gene and use thereof |
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