JPH0965886A

JPH0965886A - Dna of gene relating to photosynthesis of plant and plant introduced with the same

Info

Publication number: JPH0965886A
Application number: JP8157060A
Authority: JP
Inventors: Nobuyuki Imaizumi; 信之今泉; Ritsu Osugi; 立大杉; Muneaki Samejima; 宗明鮫島
Original assignee: JAPAN TURF GLASS KK
Current assignee: JAPAN TURF GLASS KK
Priority date: 1995-06-22
Filing date: 1996-06-18
Publication date: 1997-03-11

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA of a gene relating to photosynthesis of a plant useful for developing new plant species, tailoring culture cells for metabolite production, etc., by coding an arrangement in which a part of a specific amino acid arrangement of phosphoenol pyruvic carboxykinase is varied. SOLUTION: This new DNA of phosphoenol pyruvic carboxykinase has a base arrangement coding the amino acid arrangement expressed by the formula, etc., or the amino acid arrangement accompanied by substitutions, insertions, omissions, etc. The new DNA is useful for giving a plant low temperature resistance in C4 photosynthesis, enhancing photosynthesizing function, developing new plant species, tailoring culture cells for producing metabolite, etc. The gene DNA is obtained by extracting the total RNA from leaves of a plant belonging to Spartina-anglica, isolating mRNA from the total RNA, preparing a cDNA library using the same, selecting a positive clone by an immunoscreening method using an antibody and recovering the DNA.

Description

Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】本発明は、Sparti na anglica
及びノシバ（Zo ysia japonic a)に由来する植物の光合成
に関わるホスホエノールピルビン酸カルボキシキナーゼ
（以下ＰＣＫという）遺伝子のＤＮＡ、及びそれを導入
した植物に関する。本発明のＰＣＫ遺伝子のＤＮＡは、
遺伝子組換えを利用した植物の新品種の開発や、代謝産
物を生産させるための培養細胞の改変に利用し得る。TECHNICAL FIELD The present invention relates to Sparti na anglica.
And Zoysia (Zo ysia japonic a) phosphoenolpyruvate carboxykinase kinase involved in photosynthesis of plants derived from the (hereinafter referred PCK) gene DNA, and relates to a plant introduced with it. The DNA of the PCK gene of the present invention is
It can be used for the development of new plant varieties utilizing gene recombination and the modification of cultured cells for producing metabolites.

【０００２】[0002]

【従来の技術及び発明が解決しようとする課題】植物
は、一般に光合成のＣＯ₂ 固定経路の違いによって、Ｃ
₃ 光合成を行うＣ₃ 植物（イネ、ムギ、ベントグラスな
ど）とＣ₄ 光合成を行うＣ₄ 植物（ノシバ、トウモロコ
シ、サトウキビなど）に分けることができる。Ｃ₄ 光合
成はＣ₃ 光合成にＣＯ₂ 濃縮機構を付加したもので、Ｃ
₃ 光合成の１．５倍から２倍のＣＯ₂ 固定能力をもつ。2. Description of the Related Art Plants are generally C-induced by differences in CO ₂ fixation pathways of photosynthesis.
C ₃ plants performing ₃ photosynthesis (rice, wheat, bentgrass and the like) can be divided into C ₄ plants to be and the C ₄ photosynthesis (Zoysia japonica, corn, sugarcane, etc.). C ₄ photosynthesis is a combination of C ₃ photosynthesis and a CO ₂ concentration mechanism.
_{3 It has} a CO ₂ fixing capacity 1.5 to 2 times that of photosynthesis.

【０００３】これまで、イネ、ムギなどに高い光合成能
力を持たせるために、雌雄交雑による品種改良が行われ
てきた。近年、これに代わる育種技術として外来遺伝子
導入による形質転換技術が開発された。この形質転換技
術を用いて、Ｃ₃ 光合成にＣＯ₂ 濃縮経路を付加し、Ｃ
₄ 光合成のような高い光合成を行わせることができれ
ば、イネ、ムギ、牧草などの収量増大が期待できる。Hitherto, in order to give rice, wheat and the like a high photosynthetic ability, breeding has been carried out by male and female crossing. In recent years, a transformation technique by introducing a foreign gene has been developed as an alternative breeding technique. This transformation technique was used to add a CO ₂ enrichment pathway to C ₃ photosynthesis
_If high photosynthesis such as ₄ photosynthesis can be performed, it is expected to increase the yield of rice, wheat, grass and the like.

【０００４】また、Ｃ₄ 植物であるノシバ、コウライシ
バなどは、冬期に休眠状態となり、緑色を失ってしま
う。ゴルフ場や競馬場などでは、冬期の緑色を維持する
ためにＣ₃ 植物である寒地型シバ草（イタリアンライグ
ラス、トールフェスクなど）を混植している。このよう
に、Ｃ₄ 植物は、一般に低温ストレスに弱く、１０℃以
下の低温での生育は困難である。このことはＣ₄ 光合成
系にその一因があることを示しており、Ｃ₄ 光合成系で
働く酵素の中で、律速となっている酵素に低温耐性能力
を付加できれば、Ｃ₄ 光合成を低温に強くできることが
期待される。[0004] In addition, C ₄ plants such as Noshiba and Koishibushi become dormant in winter and lose their green color. In such as golf courses and racetracks, have been planting C ₃ plant at a cold region lawn grass the (Italian ryegrass, tall fescue, etc.) in order to maintain the winter of green. Thus, C ₄ plants are generally vulnerable to low temperature stress and difficult to grow at low temperatures of 10 ° C. or lower. This indicates that the C ₄ photosynthesis system has one of the causes. Among the enzymes that work in the C ₄ photosynthesis system, if the low temperature resistance ability can be added to the rate-limiting enzyme, the C ₄ photosynthesis can be performed at a low temperature. Expected to be strong.

【０００５】[0005]

【課題を解決するための手段】ホスホエノールピルビン
酸カルボキシキナーゼ（ＰＣＫ）はＣ₄ 光合成系の脱炭
酸酵素であり、Ｃ₄ 植物の維管束鞘細胞でＣ₃ 光合成系
にＣＯ₂ を供給するための重要な酵素である。本発明者
らは、Ｃ₄ 植物のＰＣＫ遺伝子のｃＤＮＡを利用して、
Ｃ₃ 植物で活発な発現を示すプロモーターと結合させ、
これをイネや他のＣ₃ 植物に遺伝子導入してやることに
より、Ｃ₄ 植物と同様の発現を行わせれば、ＣＯ₂ 固定
効率に優れたＣ₄ 光合成機能を植物に付与することがで
き、その結果イネなどＣ₃ 植物の光合成能力を１．５〜
２倍に上昇させることができるのはないかと考えた。ま
た、Ｃ₄ 植物の内で最も北限に自生しているSpartina a
nglicaを用いて、ノシバとの比較により、Ｃ₄ 光合成の
低温における律速酵素はＰＣＫであることを見い出し、
またSpartina anglicaのＰＣＫは低温耐性であることを
明らかにできたので、Spartin a anglicaの低温耐性ＰＣ
Ｋ遺伝子を単離し、維管束鞘細胞で特異的に発現するｒ
ｂｃＳプロモーターの発現制御のもとで、Ｃ₄ 植物へこ
の遺伝子を導入してやれば、ノシバなどの冬期の緑化に
貢献できると考え、Ｃ₄ 植物のノシバとSpartin a angli
caから、ＰＣＫ遺伝子を単離した。[Means for Solving the Problems] Phosphoenolpyruvate carboxykinase (PCK) is a decarboxylase of the C ₄ photosynthesis system, and is used to supply CO ₂ to the C ₃ photosynthesis system in vascular sheath cells of C ₄ plants. Is an important enzyme. The present inventors have utilized the cDNA of the PCK gene of C ₄ plants to
Ligated with a promoter showing active expression in C ₃ plants,
If this gene is introduced into rice or other C ₃ plants to achieve the same expression as in C ₄ plants, it is possible to impart C ₄ photosynthetic function excellent in CO ₂ fixation efficiency to the plants, and as a result, The photosynthetic ability of C ₃ plants such as rice is 1.5-
I wondered if it could be doubled. In addition, Spartina a, which is the northernmost of the C ₄ plants
By using nglica and comparing with Noshiba , it was found that the temperature-limiting enzyme of C ₄ photosynthesis at low temperature was PCK,
Moreover, since it was clarified that the PCK of Spartina anglica is cold-resistant, the PCK of Spartin a anglica is cold-resistant
K gene isolated and specifically expressed in vascular sheath cells
Under expression control of bcS promoter, do it by introducing this gene into C ₄ plants, believed to contribute to winter greening such as Zoysia, C ₄ plants of lawn grass and Spartin a angli
The PCK gene was isolated from ca.

【０００６】すなわち、本発明のｃＤＮＡは、後記配列
表の配列番号１で示されるアミノ酸配列をコードする塩
基配列を有するSparti na anglicaのＰＣＫ遺伝子のＤＮ
Ａ、特に配列番号１で示される塩基配列を有するＤＮＡ
である。また、本発明のＤＮＡは、配列番号２で示され
るアミノ酸配列をコードする塩基配列を有するノシバの
ＰＣＫ遺伝子のＤＮＡ、特に配列番号２で示される塩基
配列を有するＤＮＡである。That is, the cDNA of the present invention is DN of the PCK gene of Sparti na anglica which has a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing below.
A, especially a DNA having the nucleotide sequence shown in SEQ ID NO: 1
It is. Further, the DNA of the present invention is a DNA of the PCK gene of Noshiba having a base sequence encoding the amino acid sequence shown by SEQ ID NO: 2, particularly a DNA having the base sequence shown by SEQ ID NO: 2.

【０００７】本発明のＤＮＡは、機能を実質的に損なわ
ず実質的に同一の活性を有する限り、配列番号１及び２
に示される塩基配列又はアミノ酸配列の一部が、置換、
挿入、欠失等を伴う塩基配列又はアミノ酸配列を有する
誘導体であってもよく、これらの誘導体のいずれもが本
発明のｃＤＮＡに包含される。As long as the DNA of the present invention has substantially the same activity without substantially impairing its function, it has SEQ ID NOS: 1 and 2.
A part of the nucleotide sequence or amino acid sequence shown in
It may be a derivative having a base sequence or amino acid sequence accompanied by insertion, deletion, etc., and any of these derivatives is included in the cDNA of the present invention.

【０００８】以下、本発明を詳細に説明する。ＰＣＫ遺
伝子は次のように単離する。まず、Spartina anglicaの
葉身からＲＮＡを抽出し、ｃＤＮＡを合成する。このｃ
ＤＮＡをタンパク質発現ファージベクターに接続し、ｃ
ＤＮＡライブラリーを作成する。ＰＣＫ抗体を用いてイ
ムノスクリーニング法により、ｃＤＮＡライブラリーの
中からＰＣＫのｃＤＮＡを含むクローンを単離する。Hereinafter, the present invention will be described in detail. The PCK gene is isolated as follows. First, RNA is extracted from the leaf blades of Spartina anglica to synthesize cDNA. This c
DNA is connected to a protein expression phage vector, c
Create a DNA library. A clone containing PCK cDNA is isolated from the cDNA library by the immunoscreening method using the PCK antibody.

【０００９】また、ノシバの葉身からＲＮＡを抽出し、
ｃＤＮＡを合成する。このｃＤＮＡをファージベクター
に接続し、ｃＤＮＡライブラリーを作成する。次にSp ar
tinaanglicaのＰＣＫｃＤＮＡの一部のＤＮＡ断片を
プローブとして、上記ｃＤＮＡライブラリーの中からＰ
ＣＫのｃＤＮＡを含むクローンを単離する。RNA is extracted from the leaf blades of Noshiba,
Synthesize cDNA. This cDNA is connected to a phage vector to prepare a cDNA library. Then Sp ar
Using a partial DNA fragment of tinaanglica PCK cDNA as a probe, P
A clone containing the CK cDNA is isolated.

【００１０】Spartina an glicaのＰＣＫ遺伝子のｃＤＮ
Ａ、及びノシバのＰＣＫ遺伝子のｃＤＮＡの塩基配列
を、それぞれ配列表の配列番号１及び２に示す。Sp arti
na angli ca及びノシバのＰＣＫ遺伝子のｃＤＮＡについ
て、配列番号１の塩基番号６５〜６７及び配列番号２の
塩基番号８３〜８５の各ＡＴＧは、翻訳開始点を示し、
配列番号１の塩基番号２１８７〜２１９２のＡＡＴＡＡ
Ａは、ポリＡシグナルを示す。また、実施例２に記載の
方法により前記本発明のＰＣＫ遺伝子のＤＮＡを含む組
換えベクターで植物を形質転換して、植物体を得ること
ができる。The cDNA of the PCK gene of Spartina an glica
The nucleotide sequences of the cDNAs of A and PCK genes of Noshiba are shown in SEQ ID NOS: 1 and 2 of the Sequence Listing, respectively. Sp arti
Regarding the cDNAs of the PCK genes of na angli ca and Noshiba, each ATG of nucleotide numbers 65 to 67 of SEQ ID NO: 1 and nucleotide numbers 83 to 85 of SEQ ID NO: 2 represents a translation initiation point,
AATAAA of base numbers 2187 to 2192 of SEQ ID NO: 1
A indicates poly A signal. In addition, a plant can be obtained by transforming a plant with the recombinant vector containing the DNA of the PCK gene of the present invention by the method described in Example 2.

【００１１】以下、実施例によって本発明をさらに詳し
く説明するが、本発明はこれらの実施例に限定されるも
のではない。Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

【００１２】[0012]

【Example】

実施例１ＰＣＫ遺伝子は以下のようにクローニングした。まず、
Spartina anglicaの緑葉よりグアニジンチオシアネート
法により全ＲＮＡを抽出した。この全ＲＮＡよりオリゴ
ｄＴセルロースカラムを用いて、polyＡ⁺ −ＲＮＡを単
離した。polyＡ⁺ −ＲＮＡは、全ＲＮＡの約１％の収量
であった。polyＡ⁺ −ＲＮＡよりオリゴｄＴヌクレオチ
ドをプライマーとして、市販のｃＤＮＡ合成キット（フ
ァルマシア社製）を使用し、ｃＤＮＡを合成した。合成
したｃＤＮＡを、ファージベクターであるλｇｔ１１
（Stratagene社製）のＥｃｏＲＩ部位に接続し、Strata
gene社製のパッケイジングキットを用いてｃＤＮＡライ
ブラリーを作成した。Example 1 The PCK gene was cloned as follows. First,
Total RNA was extracted from the green leaves of Spartina anglica by the guanidine thiocyanate method. From this total RNA, polyA ⁺ -RNA was isolated using an oligo dT cellulose column. The polyA ⁺ -RNA was in a yield of about 1% of total RNA. cDNA was synthesized from polyA ⁺ -RNA using a commercially available cDNA synthesis kit (Pharmacia) with oligo dT nucleotide as a primer. The synthesized cDNA was used as a phage vector λgt11.
Connect to the EcoRI site (from Stratagene) to create Strata
A cDNA library was prepared using a packaging kit manufactured by gene.

【００１３】Panicum maximum var.trichoglume(グリー
ンパニック）により精製したＰＣＫタンパク質を抗原と
して作成したＰＣＫ抗体を用いて、イムノスクリーニン
グ法により、上記ｃＤＮＡライブラリーの中からポジテ
ィブクローンを得た。この塩基配列を、ＡＢＩ社製のシ
ークエンサーで決定した。この塩基配列は、微生物（大
腸菌等）から得られた既知のＰＣＫ遺伝子のｃＤＮＡと
比較的高いホモロジー（約５０％以上）を示し、またＰ
ＣＫの活性中心であるＡＴＰ結合部位は８０％以上の非
常に高いホモロジーを示すことから、Spart ina anglica
のＰＣＫ遺伝子のｃＤＮＡであると断定した。A positive clone was obtained from the above cDNA library by an immunoscreening method using a PCK antibody prepared by using the PCK protein purified by Panicum maximum var. Trichoglume (Green Panic) as an antigen. This base sequence was determined by a sequencer manufactured by ABI. This nucleotide sequence shows a relatively high homology (about 50% or more) with the cDNA of a known PCK gene obtained from a microorganism (E. coli etc.), and P
Since the ATP binding site, which is the active center of CK, shows a very high homology of 80% or more, Spart ina anglica
It was determined to be the cDNA of the PCK gene of.

【００１４】また、ノシバの緑葉より、同様にしてｃＤ
ＮＡライブラリーを作成した。先に単離したSpartina a
nglicaのＰＣＫ遺伝子のｃＤＮＡの一部をプローブとし
て、プラークハイブリダイゼーション法により、このｃ
ＤＮＡライブラリーの中からポジティブクローンを得
た。この塩基配列を、ＡＢＩ社製のシークエンサーで決
定した。この塩基配列は、Spartina ang licaのＰＣＫ遺
伝子のｃＤＮＡと約９０％の高いホモロジーを示すこと
から、ノシバのＰＣＫ遺伝子のｃＤＮＡであると判断し
た。In addition, from the green leaves of Noshiba, cD
An NA library was created. Spartina a isolated earlier
By using a part of cDNA of PCK gene of nglica as a probe, this c
A positive clone was obtained from the DNA library. This base sequence was determined by a sequencer manufactured by ABI. Since this nucleotide sequence shows a high homology of about 90% with the cDNA of the PCK gene of Spartina ang lica , it was determined to be the cDNA of the PCK gene of Noshiba.

【００１５】実施例２ノシバの完熟種子由来又は生長点由来の再分化能を有す
る液体培養系から単離したプロトプラストに、プラスミ
ドｐＲＢＣＰＣＫ〔ＰＶＣ１９のＨｉｎｄIII，Ｅｃｏ
ＲＩ部位にノシバrbc Ｓプロモーター（遺伝子発現の誘
導方法：特開平６−７０７８１号参照）＋実施例１で得
たSpartina anglicaのＰＣＫ遺伝子＋ＮＯＳターミネー
ターを挿入したもの〕をＰＥＧ法により導入した。ま
た、抗生物質ハイグロマイシン抵抗性を付与するプラス
ミドｐｌＪ（ＣａＭＶ３５Ｓプロモーター＋カタラーゼ
イントロン＋ハイグロマイシンホスホトランスフェラー
ゼ遺伝子＋ＮＯＳターミネーター）を選抜マーカーとし
て導入した。Example 2 A plasmid pRBCPCK [HindIII, Eco of PVC19] was added to protoplasts isolated from a liquid culture system having a redifferentiation ability derived from mature seeds or growing points of Noshiba.
The Noshiba rbc S promoter (method of inducing gene expression: see JP-A-6-70781) + Spartina anglica PCK gene + NOS terminator obtained in Example 1) was introduced into the RI site by the PEG method. In addition, a plasmid plJ (CaMV35S promoter + catalase intron + hygromycin phosphotransferase gene + NOS terminator), which confers resistance to the antibiotic hygromycin, was introduced as a selectable marker.

【００１６】〈プロトプラストへの遺伝子導入〉４５℃
で５分間加温したノシバのプロトプラストに、上記プラ
スミドＤＮＡ（５０μg/ml）及び導入バッファー（０．
４M マンニトール、１５mM塩化マグネシウム、０．１％
ＭＥＳ）を加え、プロトプラスト密度を２×１０⁸/mlに
調整した後、ポリエチレングリコール処理を行った。即
ち最終濃度が２０％となるようにポリエチレングリコー
ル液を加え、しばらく静置の後、塩化ナトリウム、塩化
カルシウム及び塩化カリウムを含む希釈液を加え、遠心
洗浄を行った（ゾイシア属植物形質転換体及びその作出
方法：特願平６−３３２４２１号参照）。<Gene transfer into protoplasts> 45 ° C.
The above plasmid DNA (50 μg / ml) and transfer buffer (0.
4M mannitol, 15mM magnesium chloride, 0.1%
MES) was added to adjust the protoplast density to 2 × 10 ⁸ / ml, followed by polyethylene glycol treatment. That is, a polyethylene glycol solution was added so that the final concentration would be 20%, and after standing for a while, a diluent containing sodium chloride, calcium chloride and potassium chloride was added, and centrifugal washing was performed (Zoisia plant transformants and The production method: Japanese Patent Application No. 6-332421).

【００１７】〈導入処理したプロトプラストの培養とＰ
ＣＫ遺伝子が導入された細胞集塊の選抜及び植物体の再
生方法〉プロトプラストをＫ８ｐ培地を基本とした液体
培地で培養した。導入１４日後、培養培地中にハイグロ
マイシン４００μg/mlを添加し抗生物質抵抗性遺伝子の
導入された細胞集塊の選抜を開始した。選抜培養２８日
後に選抜された抗生物質抵抗性の細胞集塊をハイグロマ
イシン４００μg/mlの培地に移植した。１カ月間培養し
て増殖させた後、再分化培地にカルスを移植し、不定胚
を再生させ、それより植物体を再生させた。<Cultivation of introduced protoplasts and P
Method of selecting cell clumps into which CK gene has been introduced and regenerating plant> Protoplasts were cultured in a liquid medium based on K8p medium. 14 days after the introduction, 400 μg / ml of hygromycin was added to the culture medium to start selection of cell clumps into which the antibiotic resistance gene had been introduced. After 28 days from the selective culture, the antibiotic-resistant cell clumps selected were transplanted to a hygromycin 400 μg / ml medium. After culturing for one month and allowing it to grow, callus was transplanted to a regeneration medium to regenerate somatic embryos and thereby regenerate plants.

【００１８】〈ＰＣＫ遺伝子の植物体内における確認〉
得られた抗生物質抵抗性植物体から常法によりＤＮＡを
単離し、抗生物質抵抗性遺伝子と同時に導入したＰＣＫ
遺伝子の存在をＰＣＲ法により確認した。<Confirmation of PCK gene in plant>
PCK in which DNA was isolated from the obtained antibiotic-resistant plant by a conventional method and introduced at the same time as the antibiotic-resistant gene
The presence of the gene was confirmed by the PCR method.

【００１９】[0019]

【発明の効果】ＰＣＫ遺伝子は、Ｃ₄ 光合成系の重要な
酵素の遺伝子であり、Ｃ₃ 光合成又はＣ₄ 光合成の改良
に利用できる。すなわち、ＰＣＫはＣ₄ 光合成系の一員
であり、ＰＣＫ遺伝子の利用により、Ｃ₄ 光合成のよう
な炭酸固定効率に優れた光合成機能をＣ₃ 植物に付与で
きる可能性がある。特に、Spartina anglicaのＰＣＫ遺
伝子は、他のＣ₄ 植物のＰＣＫにない低温に強い特性を
持っていることから、本遺伝子をＣ₄ 植物に導入するこ
とにより、低温耐性能力を付与し、冬期の芝草などの緑
葉の維持に貢献できる。INDUSTRIAL APPLICABILITY The PCK gene is a gene for an important enzyme in the C ₄ photosynthesis system and can be used for improving C ₃ photosynthesis or C ₄ photosynthesis. That is, PCK is a member of the C ₄ photosynthesis system, and the use of the PCK gene may possibly impart a photosynthetic function such as C ₄ photosynthesis, which is excellent in carbon dioxide fixation efficiency, to C ₃ plants. In particular, since the PCK gene of Spartina anglica has a strong characteristic at low temperature that PCK of other C ₄ plants does not have, by introducing this gene into C ₄ plants, low temperature tolerance ability is imparted, and It can contribute to the maintenance of green leaves such as turfgrass.

【００２０】[0020]

[Sequence list]

配列番号：１配列の長さ：２２１８配列の型：核酸配列の種類：ｃＤＮＡ起源生物名：Spartina anglica 配列の特徴特徴を表す記号：polyA signal 存在位置：２１８７…２１９２特徴を表す記号：５’ＵＴＲ存在位置：１…６４／partial 特徴を表す記号：３’ＵＴＲ存在位置：２０４２…２２１８特徴を表す記号：ＣＤＳ存在位置：６５…２０４１ GTTCCATTCC GGGACGCCAC CCAAAGCACG CCGTTCTTTT TTCATCGAAA TCAGCTCGAG 60 CGAG ATG GCG ACC CCG GAC GCA TTG GCG CGG ATC GAG ACT AAC GGG AAG 109 Met Ala Thr Pro Asp Ala Leu Ala Arg Ile Glu Thr Asn Gly Lys 1 5 10 15 AAG AGT CAC GAG GAC GCG GTG TGG GAC GAC GAC ATC TCG GCG CCG GTG 157 Lys Ser His Glu Asp Ala Val Trp Asp Asp Asp Ile Ser Ala Pro Val 20 25 30 CGC GCG CAG AAC ATC GAC GAG CTG CAC TCC CTG CAA AAG AAG AGG TCG 205 Arg Ala Gln Asn Ile Asp Glu Leu His Ser Leu Gln Lys Lys Arg Ser 35 40 45 GCG CCC ACC ACG CCC ATC AAG GAC GGC GCC GCC GCC GCC TTC GCC GCC 253 Ala Pro Thr Thr Pro Ile Lys Asp Gly Ala Ala Ala Ala Phe Ala Ala 50 55 60 GCA CTC ACC GAG GAG CAG CGC CAG AAG CAG CAG CTC CAG TCC ATT AGC 301 Ala Leu Thr Glu Glu Gln Arg Gln Lys Gln Gln Leu Gln Ser Ile Ser 65 70 75 TCG TCG TTG GCA TCT CTG ACA CGT GAG ACC GGC CCC AAG GTC CTC AGG 349 Ser Ser Leu Ala Ser Leu Thr Arg Glu Thr Gly Pro Lys Val Leu Arg 80 85 90 95 GGC GAT CCG GCC AGG AAG AGC GAG GCC TCC GTG AAG AGC ACA CCG GCG 397 Gly Asp Pro Ala Arg Lys Ser Glu Ala Ser Val Lys Ser Thr Pro Ala 100 105 110 CCG AGC CCG CAG GTG CCG GCG CCG AAA CCG GCC GCC CCC ACA ATC GCC 445 Pro Ser Pro Gln Val Pro Ala Pro Lys Pro Ala Ala Pro Thr Ile Ala 115 120 125 GTC AGC GAC AGC TCC CTC AAG TTC ACA CAT GTG CTC TAC AAC CTC TCC 493 Val Ser Asp Ser Ser Leu Lys Phe Thr His Val Leu Tyr Asn Leu Ser 130 135 140 CCT GGC GAC CTG TAC GAG CAT GCG GTC AAG TGC AAT ATG GCT TCC TTC 541 Pro Gly Asp Leu Tyr Glu His Ala Val Lys Cys Asn Met Ala Ser Phe 145 150 155 ATC ACG TCC ACT GGC GCG CTG GCG ACG CTG TCC GGC GCC AAG ACC GGG 589 Ile Thr Ser Thr Gly Ala Leu Ala Thr Leu Ser Gly Ala Lys Thr Gly 160 165 170 175 CGC TCG CCC AGG GAC AGG CGC GTG GTC AAG GAC GCG GCC GCG GCG AAG 637 Arg Ser Pro Arg Asp Arg Arg Val Val Lys Asp Ala Ala Ala Ala Lys 180 185 190 GAC CTG TGG TGG GGC AAG GGA TCT CCG AAC ATC GAG ATA GAC GAG CAC 685 Asp Leu Trp Trp Gly Lys Gly Ser Pro Asn Ile Glu Ile Asp Glu His 195 200 205 ACG TTC CTC ACC AAC AGG GAG AGG GCC GTC GAC TAC CTC AAC TCC CTC 733 Thr Phe Leu Thr Asn Arg Glu Arg Ala Val Asp Tyr Leu Asn Ser Leu 210 215 220 GAC AAG GTG TTC GTG AAC GAC CAG TTC CTG AAC TGG GAC CCG GAG AAC 781 Asp Lys Val Phe Val Asn Asp Gln Phe Leu Asn Trp Asp Pro Glu Asn 225 230 235 CGC ATC AAG GTC CGC ATC ATC TCT GCC AGG TCG TAC CAC TCC CTC TTC 829 Arg Ile Lys Val Arg Ile Ile Ser Ala Arg Ser Tyr His Ser Leu Phe 240 245 250 255 ATG CAC AAC ATG TGC ATC CGC CCC ACG GCC GAG GAG CTG AGG AAC TTT 877 Met His Asn Met Cys Ile Arg Pro Thr Ala Glu Glu Leu Arg Asn Phe 260 265 270 GGC ACG CCG GAC TTC ACC ATT TAC AAC GCC GGG ATG TTC CCG TGC AAC 925 Gly Thr Pro Asp Phe Thr Ile Tyr Asn Ala Gly Met Phe Pro Cys Asn 275 280 285 CGT TAC ACG AAC TAC ATG ACA TCA TCG ACG AGC ATA AAC ATC AAT CTC 973 Arg Tyr Thr Asn Tyr Met Thr Ser Ser Thr Ser Ile Asn Ile Asn Leu 290 295 300 GCT AGG AGG GAG ATG GTC ATC CTG GGC ACC CAG TAC GCC GGG GAG ATG 1021 Ala Arg Arg Glu Met Val Ile Leu Gly Thr Gln Tyr Ala Gly Glu Met 305 310 315 AAG AAG GGG CTC TTC GCC GTC ATG CAC TAC CTC ATG CCC AAA CGA CAA 1069 Lys Lys Gly Leu Phe Ala Val Met His Tyr Leu Met Pro Lys Arg Gln 320 325 330 335 ATC CTC TCC CTC CAC TCC GGA TGC AAC ATG GGC AAA CAC GGT GAC GTC 1117 Ile Leu Ser Leu His Ser Gly Cys Asn Met Gly Lys His Gly Asp Val 340 345 350 GCC CTA TTC TTT GGG CTC TCA GGT ACC GGG AAG ACA ACG CTG TCA ACA 1165 Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser Thr 355 360 365 GAC CAC AAC AGG CTG TTG ATC GGC GAT GAT GAG CAC TGT TGG AGT GAC 1213 Asp His Asn Arg Leu Leu Ile Gly Asp Asp Glu His Cys Trp Ser Asp 370 375 380 AAC GGC GTC TCC AAC ATC GAA GGT GGG TGC TAC GCC AAG TGT ATA GAC 1261 Asn Gly Val Ser Asn Ile Glu Gly Gly Cys Tyr Ala Lys Cys Ile Asp 385 390 395 CTC GCT CAG GAG AAG GAG CCT GAC ATC TGG AAC GCC ATC AAG TTT GGA 1309 Leu Ala Gln Glu Lys Glu Pro Asp Ile Trp Asn Ala Ile Lys Phe Gly 400 405 410 415 ACT GTG TTG GAG AAC GTT GTG TTT GAT GAG CGT ACT CGT GAG GTT GAC 1357 Thr Val Leu Glu Asn Val Val Phe Asp Glu Arg Thr Arg Glu Val Asp 420 425 430 TAC ACC GAT AAA TCT GTC ACC GAG AAC ACT CGT GCT GCG TAC CCG ATC 1405 Tyr Thr Asp Lys Ser Val Thr Glu Asn Thr Arg Ala Ala Tyr Pro Ile 435 440 445 GAG TAC ATC CCC AAC GCA AAG ATA CCG TGC GTC GGA CCG CAC CCG AAG 1453 Glu Tyr Ile Pro Asn Ala Lys Ile Pro Cys Val Gly Pro His Pro Lys 450 455 460 AAC GTC ATC CTC CTG GCC TGC GAC GCG TTC GGT GTG CTC CCG CCT GTC 1501 Asn Val Ile Leu Leu Ala Cys Asp Ala Phe Gly Val Leu Pro Pro Val 465 470 475 AGC AAG CTC AAC CTT GCA CAG ACC ATG TAC CAT TTC ATC AGC GGC TAC 1549 Ser Lys Leu Asn Leu Ala Gln Thr Met Tyr His Phe Ile Ser Gly Tyr 480 485 490 495 ACT GCT CTG GTT GCC GGC ACG GAA GAA GGC ATC AAG GAG CCA CAG GCC 1597 Thr Ala Leu Val Ala Gly Thr Glu Glu Gly Ile Lys Glu Pro Gln Ala 500 505 510 ACG TTC TCC GCC TGC TTC GGT GCG GCA TTC ATC ATG CTC CAC CCG ACC 1645 Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Ile Met Leu His Pro Thr 515 520 525 AAG TAC GCC GCC ATG CTC GCC GAG AAG ATG CAG AAG TAT GGC GCC ACC 1693 Lys Tyr Ala Ala Met Leu Ala Glu Lys Met Gln Lys Tyr Gly Ala Thr 530 535 540 GGG TGG CTT GTG AAC ACC GGC TGG TCC GGA GGG AGG TAC GGC GTT GGC 1741 Gly Trp Leu Val Asn Thr Gly Trp Ser Gly Gly Arg Tyr Gly Val Gly 545 550 555 AAG AGG ATC AAG CTG CCG TAC ACC AGG AAG ATC ATC GAC GCC ATC CAC 1789 Lys Arg Ile Lys Leu Pro Tyr Thr Arg Lys Ile Ile Asp Ala Ile His 560 565 570 575 TCC GGC GAG CTC CTC ACC GCC AAC TAC AAG AAA ACC GAG GTC TTC GGC 1837 Ser Gly Glu Leu Leu Thr Ala Asn Tyr Lys Lys Thr Glu Val Phe Gly 580 585 590 CTG GAG ATC CCC ACC GAG ATC AAC GGC GTG CCG TCC GAA ATC CTC GAC 1885 Leu Glu Ile Pro Thr Glu Ile Asn Gly Val Pro Ser Glu Ile Leu Asp 595 600 605 CCT ATC AAC ACA TGG ACG GAC AAG GCC GCG TAC AAG GAG GCG CTC ATG 1933 Pro Ile Asn Thr Trp Thr Asp Lys Ala Ala Tyr Lys Glu Ala Leu Met 610 615 620 AAG CTG GCC GGG TTG TTC AAG AAC AAC TTC GAG GTG TTC GCA AGC TAC 1981 Lys Leu Ala Gly Leu Phe Lys Asn Asn Phe Glu Val Phe Ala Ser Tyr 625 630 635 AAG ATC GGG GAC GAC AAC AGC CTG ACC GAC GAG ATC CTC GCC GCA GGC 2029 Lys Ile Gly Asp Asp Asn Ser Leu Thr Asp Glu Ile Leu Ala Ala Gly 640 645 650 655 CCC AAC TTC TGATCTGAAG TGGTGTTGCA CTATATCAGT AGCTGTTGCA 2078 Pro Asn Phe 658 GCGGCGTACA TAGCTTGTGC TATGTTAGAT TACATCTTGT CAGCTTGTTC GACTGGGATT 2138 GCGCTTGGCT GCTTGCAGCA TTGTCCATGT ACTCTCTCCA CTTTCCGAAA TAAAAAATGA 2198 TTTCGAAACT AAAAAAAAAA 2218 SEQ ID NO: 1 Sequence length: 2218 Sequence type: Nucleic acid Sequence type: cDNA Origin organism name: Spartina anglica Sequence characteristics: Symbol representing the characteristic: polyA signal Location: 2187 ... 2192 Symbol representing the characteristic: 5'UTR Location: 1 ... 64 / partial Characteristic symbol: 3'UTR Location: 2042 ... 2218 Characteristic symbol: CDS Location: 65 ... 2041 GTTCCATTCC GGGACGCCAC CCAAAGCACG CCGTTCTTTT TTCATCGAAA TCAGCTCGAG 60 CGAG ATG GCG ACC CCG GAC GCA TTG GCG CGG ATC GAG ACT AAC GGG AAG 109 Met Ala Thr Pro Asp Ala Leu Ala Arg Ile Glu Thr Asn Gly Lys 1 5 10 15 AAG AGT CAC GAG GAC GCG GTG TGG GAC GAC GAC ATC TCG GCG CCG GTG 157 Lys Ser His Glu Asp Ala Val Trp Asp Asp Asp Ile Ser Ala Pro Val 20 25 30 CGC GCG CAG AAC ATC GAC GAG CTG CAC TCC CTG CAA AAG AAG AGG TCG 205 Arg Ala Gln Asn Ile Asp Glu Leu His Ser Leu Gln Lys Lys Arg Ser 35 40 45 GCG CCC ACC ACG CCC ATC AAG GAC GGC GCC GC C GCC GCC TTC GCC GCC 253 Ala Pro Thr Thr Pro Ile Lys Asp Gly Ala Ala Ala Ala Phe Ala Ala 50 55 60 GCA CTC ACC GAG GAG CAG CGC CAG AAG CAG CAG CTC CAG TCC ATT AGC 301 Ala Leu Thr Glu Glu Gln Arg Gln Lys Gln Gln Leu Gln Ser Ile Ser 65 70 75 TCG TCG TTG GCA TCT CTG ACA CGT GAG ACC GGC CCC AAG GTC CTC AGG 349 Ser Ser Leu Ala Ser Leu Thr Arg Glu Thr Gly Pro Lys Val Leu Arg 80 85 90 95 GGC GAT CCG GCC AGG AAG AGC GAG GCC TCC GTG AAG AGC ACA CCG GCG 397 Gly Asp Pro Ala Arg Lys Ser Glu Ala Ser Val Lys Ser Thr Pro Ala 100 105 110 CCG AGC CCG CAG GTG CCG GCG CCG AAA CCG GCC GCC CCC ACA ATC GCC 445 Pro Ser Pro Gln Val Pro Ala Pro Lys Pro Ala Ala Pro Thr Ile Ala 115 120 125 GTC AGC GAC AGC TCC CTC AAG TTC ACA CAT GTG CTC TAC AAC CTC TCC 493 Val Ser Asp Ser Ser Leu Lys Phe Thr His Val Leu Tyr Asn Leu Ser 130 135 140 CCT GGC GAC CTG TAC GAG CAT GCG GTC AAG TGC AAT ATG GCT TCC TTC 541 Pro Gly Asp Leu Tyr Glu His Ala Val Lys Cys Asn Met Ala Ser Phe 145 150 155 ATC ACG TCC ACT GGC GCG CTG GCG ACG CTG TCC GGC GCC AAG ACC GGG 589 Ile Thr Ser Thr Gly Ala Leu Ala Thr Leu Ser Gly Ala Lys Thr Gly 160 165 170 175 CGC TCG CCC AGG GAC AGG CGC GTG GTC AAG GAC GCG GCC GCG GCG AAG 637 Arg Ser Pro Arg Asp Arg Arg Val Val Lys Asp Ala Ala Ala Ala Lys 180 185 190 GAC CTG TGG TGG GGC AAG GGA TCT CCG AAC ATC GAG ATA GAC GAG CAC 685 Asp Leu Trp Trp Gly Lys Gly Ser Pro Asn Ile Glu Ile Asp Glu His 195 200 205 ACG TTC CTC ACC AAC AGG GAG AGG GCC GTC GAC TAC CTC AAC TCC CTC 733 Thr Phe Leu Thr Asn Arg Glu Arg Ala Val Asp Tyr Leu Asn Ser Leu 210 215 220 GAC AAG GTG TTC GTG AAC GAC CAG TTC CTG AAC TGG GAC CCG GAG AAC 781 Asp Lys Val Phe Val Asn Asp Gln Phe Leu Asn Trp Asp Pro Glu Asn 225 230 235 CGC ATC AAG GTC CGC ATC ATC TCT GCC AGG TCG TAC CAC TCC CTC TTC 829 Arg Ile Lys Val Arg Ile Ile Ser Ala Arg Ser Tyr His Ser Leu Phe 240 245 250 255 ATG CAC AAC ATG TGC ATC CGC CCC ACG GCC GAG GAG CTG AGG AAC TTT 877 Met His Asn Met Cys Ile Arg Pro Thr Ala Glu Glu Leu Arg Asn Phe 260 265 270 GGC ACG CCG GAC TTC ACC ATT TAC AAC GCC GGG ATG TTC CCG TGC AAC 925 Gly Thr Pro Asp Phe Thr Ile Tyr Asn Ala Gly Met Phe Pro Cys Asn 275 280 285 CGT TAC ACG AAC TAC ATG ACA TCA TCG ACG AGC ATA AAC ATC AAT CTC 973 Arg Tyr Thr Asn Tyr Met Thr Ser Ser Thr Ser Ile Asn Ile Asn Leu 290 295 300 GCT AGG AGG GAG ATG GTC ATC CTG GGC ACC CAG TAC GCC GGG GAG ATG 1021 Ala Arg Arg Glu Met Val Ile Leu Gly Thr Gln Tyr Ala Gly Glu Met 305 310 315 AAG AAG GGG CTC TTC GCC GTC ATG CAC TAC CTC ATG CCC AAA CGA CAA 1069 Lys Lys Gly Leu Phe Ala Val Met His Tyr Leu Met Pro Lys Arg Gln 320 325 330 335 ATC CTC TCC CTC CAC TCC GGA TGC AAC ATG GGC AAA CAC GGT GAC GTC 1117 Ile Leu Ser Leu His Ser Gly Cys Asn Met Gly Lys His Gly Asp Val 340 345 350 GCC CTA TTC TTT GGG CTC TCA GGT ACC GGG AAG ACA ACG CTG TCA ACA 1165 Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser Thr 355 360 365 GAC CAC AAC AGG CTG TTG ATC GGC GAT GAT GAG CAC TGT TGG AGT GAC 1213 Asp His Asn Arg Leu Leu Ile Gly Asp Asp Glu His Cys Trp Ser Asp 370 375 380 AAC GG C GTC TCC AAC ATC GAA GGT GGG TGC TAC GCC AAG TGT ATA GAC 1261 Asn Gly Val Ser Asn Ile Glu Gly Gly Cys Tyr Ala Lys Cys Ile Asp 385 390 395 CTC GCT CAG GAG AAG GAG CCT GAC ATC TGG AAC GCC ATC AAG TTT GGA 1309 Leu Ala Gln Glu Lys Glu Pro Asp Ile Trp Asn Ala Ile Lys Phe Gly 400 405 410 415 ACT GTG TTG GAG AAC GTT GTG TTT GAT GAG CGT ACT CGT GAG GTT GAC 1357 Thr Val Leu Glu Asn Val Val Phe Asp Glu Arg Thr Arg Glu Val Asp 420 425 430 TAC ACC GAT AAA TCT GTC ACC GAG AAC ACT CGT GCT GCG TAC CCG ATC 1405 Tyr Thr Asp Lys Ser Val Thr Glu Asn Thr Arg Ala Ala Tyr Pro Ile 435 440 445 GAG TAC ATC CCC AAC GCA AAG ATA CCG TGC GTC GGA CCG CAC CCG AAG 1453 Glu Tyr Ile Pro Asn Ala Lys Ile Pro Cys Val Gly Pro His Pro Lys 450 455 460 AAC GTC ATC CTC CTG GCC TGC GAC GCG TTC GGT GTG CTC CCG CCT GTC 1501 Asn Val Ile Leu Leu Ala Cys Asp Ala Phe Gly Val Leu Pro Pro Val 465 470 475 AGC AAG CTC AAC CTT GCA CAG ACC ATG TAC CAT TTC ATC AGC GGC TAC 1549 Ser Lys Leu Asn Leu Ala Gln Thr Met Tyr His Phe Ile Ser Gly Ty r 480 485 490 495 ACT GCT CTG GTT GCC GGC ACG GAA GAA GGC ATC AAG GAG CCA CAG GCC 1597 Thr Ala Leu Val Ala Gly Thr Glu Glu Gly Ile Lys Glu Pro Gln Ala 500 505 510 ACG TTC TCC GCC TGC TTC GGT GCG GCA TTC ATC ATG CTC CAC CCG ACC 1645 Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Ile Met Leu His Pro Thr 515 520 525 AAG TAC GCC GCC ATG CTC GCC GAG AAG ATG CAG AAG TAT GGC GCC ACC 1693 Lys Tyr Ala Ala Met Leu Ala Glu Lys Met Gln Lys Tyr Gly Ala Thr 530 535 540 GGG TGG CTT GTG AAC ACC GGC TGG TCC GGA GGG AGG TAC GGC GTT GGC 1741 Gly Trp Leu Val Asn Thr Gly Trp Ser Gly Gly Arg Tyr Gly Val Gly 545 550 555 AAG AGG ATC AAG CTG CCG TAC ACC AGG AAG ATC ATC GAC GCC ATC CAC 1789 Lys Arg Ile Lys Leu Pro Tyr Thr Arg Lys Ile Ile Asp Ala Ile His 560 565 570 575 TCC GGC GAG CTC CTC ACC GCC AAC TAC AAG AAA ACC GAG GTC TTC GGC 1837 Ser Gly Glu Leu Leu Thr Ala Asn Tyr Lys Lys Thr Glu Val Phe Gly 580 585 590 CTG GAG ATC CCC ACC GAG ATC AAC GGC GTG CCG TCC GAA ATC CTC GAC 1885 Leu Glu Ile Pro Thr Glu Ile Asn Gly Val Pro Ser Glu Ile Leu Asp 595 600 605 CCT ATC AAC ACA TGG ACG GAC AAG GCC GCG TAC AAG GAG GCG CTC ATG 1933 Pro Ile Asn Thr Trp Thr Asp Lys Ala Ala Tyr Lys Glu Ala Leu Met 610 615 620 AAG CTG GCC GGG TTG TTC AAG AAC AAC TTC GAG GTG TTC GCA AGC TAC 1981 Lys Leu Ala Gly Leu Phe Lys Asn Asn Phe Glu Val Phe Ala Ser Tyr 625 630 635 AAG ATC GGG GAC GAC AAC AGC CTG ACC GAC GAG ATC CTC GCC GCA GGC 2029 Lys Gly Asp Asp Asn Ser Leu Thr Asp Glu Ile Leu Ala Ala Gly 640 645 650 655 CCC AAC TTC TGATCTGAAG TGGTGTTGCA CTATATCAGT AGCTTC 2 ACT

【００２１】配列番号：２配列の長さ：２２０９配列の型：核酸配列の種類：ｃＤＮＡ生物名：Zoysia japonica 配列の特徴特徴を表す記号：５’ＵＴＲ存在位置：１…８２／partial 特徴を表す記号：３’ＵＴＲ存在位置：２０４５…２２０９／partial 特徴を表す記号：ＣＤＳ存在位置：８３…２０４４ GCGTACACCG CGACACAGAC AGAGCACGCC GTTCTCCTCG TACGCGGATC AACGACCACC 60 GTGCCGCACG CGCGCCGGCG AG ATG GCG ACC CCG AAC GGC CTG GCC CAG ATC 112 Met Ala Thr Pro Asn Gly Leu Ala Gln Ile 1 5 10 GAG ACG AAC GGG AGG AAG AAG CAC GAG AAC GTG GTG TGC CAC GAC GAC 160 Glu Thr Asn Gly Arg Lys Lys His Glu Asn Val Val Cys His Asp Asp 15 20 25 AGC GCT GCG CCA GTG CGC GCG CAG ACC ATC GAC GAG CTG CAC TCC CTG 208 Ser Ala Ala Pro Val Arg Ala Gln Thr Ile Asp Glu Leu His Ser Leu 30 35 40 CAG CGG AAG CGT TCG GCG CCC AGC ACG CCC AAG GAC AGC GCC GCC TCC 256 Gln Arg Lys Arg Ser Ala Pro Ser Thr Pro Lys Asp Ser Ala Ala Ser 45 50 55 GCC TTC GCC GCT GCG CTC ACC GAG GAG CAG CGC GAG GAG CAG CAG CTG 304 Ala Phe Ala Ala Ala Leu Thr Glu Glu Gln Arg Glu Glu Gln Gln Leu 60 65 70 CAG TCC ATC AGC GCG TCA CTG GCG TCT CTG ACG CGT GAG ACC GGC CCC 352 Gln Ser Ile Ser Ala Ser Leu Ala Ser Leu Thr Arg Glu Thr Gly Pro 75 80 85 90 AAG GTC GTC AGG GGA GAT CCG GCC AGG AAG GGT GAG GCG TCC GCG AAG 400 Lys Val Val Arg Gly Asp Pro Ala Arg Lys Gly Glu Ala Ser Ala Lys 95 100 105 AGC ACG CCG GCG CAC CAC CAC CAG CAC CCC GCC GCC CCC GCC GTC GCC 448 Ser Thr Pro Ala His His His Gln His Pro Ala Ala Pro Ala Val Ala 110 115 120 GTC AGC GAC AGC TCC CTC AAG TCC ACC CAT GTC CTC TCC AAC CTC TCG 496 Val Ser Asp Ser Ser Leu Lys Ser Thr His Val Leu Ser Asn Leu Ser 125 130 135 CCT GCC GAG CTG TAC GAG CAG GCG ATC AAG TAC GAG AAG GGT TCG TTC 544 Pro Ala Glu Leu Tyr Glu Gln Ala Ile Lys Tyr Glu Lys Gly Ser Phe 140 145 150 ATC ACG TCC ACG GGT GCG CTG GCG ACG CTG TCC GGC GCG AAG ACC GGC 592 Ile Thr Ser Thr Gly Ala Leu Ala Thr Leu Ser Gly Ala Lys Thr Gly 155 160 165 170 CGC GCG CCC AGG GAC AAG CGC GTG GTC AAG GAC GAG GCC GCG GCG CGG 640 Arg Ala Pro Arg Asp Lys Arg Val Val Lys Asp Glu Ala Ala Ala Arg 175 180 185 GAG CTG TGG TGG GGC AAG GGG TCG CCG AAC ATC GAG ATG GAC GAG CAC 688 Glu Leu Trp Trp Gly Lys Gly Ser Pro Asn Ile Glu Met Asp Glu His 190 195 200 ACG TTC CTC ATC AAC AGG GAG AGG GCC GTC GAC TAC CTC AAC TCC CTC 736 Thr Phe Leu Ile Asn Arg Glu Arg Ala Val Asp Tyr Leu Asn Ser Leu 205 210 215 GAC AAG GTG TTC GTG AAC GAC CAG TTC CTG AAC TGG GAC CCG GAG AAC 784 Asp Lys Val Phe Val Asn Asp Gln Phe Leu Asn Trp Asp Pro Glu Asn 220 225 230 CGC ATC AAG GTC CGC ATC ATC TCC GCC AGG GCG TAC CAC TCC CTC TTC 832 Arg Ile Lys Val Arg Ile Ile Ser Ala Arg Ala Tyr His Ser Leu Phe 235 240 245 250 ATG CAC AAC ATG TGC ATC CGC CCG ACG GAT GAG GAG CTG GAG AAC TTC 880 Met His Asn Met Cys Ile Arg Pro Thr Asp Glu Glu Leu Glu Asn Phe 255 260 265 GGC ACG CCG GAC TTC ACC ATT TAC AAC GCC GGG CAG TTC CCT TGT AAC 928 Gly Thr Pro Asp Phe Thr Ile Tyr Asn Ala Gly Gln Phe Pro Cys Asn 270 275 280 CGA TAC ACG CAC TAC ATG ACG TCG TCG TCG AGC GTA GCC ATC AAC CTG 976 Arg Tyr Thr His Tyr Met Thr Ser Ser Ser Ser Val Ala Ile Asn Leu 285 290 295 GCT AGG AGG GAG ATG GTG ATC CTG GGC ACG CAG TAC GCC GGG GAG ATG 1024 Ala Arg Arg Glu Met Val Ile Leu Gly Thr Gln Tyr Ala Gly Glu Met 300 305 310 AAG AAG GGA CTC TTC GGC CTC ATG CAC TAC CTC ATG CCC AAA CGG CAG 1072 Lys Lys Gly Leu Phe Gly Leu Met His Tyr Leu Met Pro Lys Arg Gln 315 320 325 330 ATC CTC TCG CTG CAC TCC GGG TGC AAC ATG GGC AAG GGC GGT GAC GTC 1120 Ile Leu Ser Leu His Ser Gly Cys Asn Met Gly Lys Gly Gly Asp Val 335 340 345 GCC CTC TTC TTC GGG CTG TCA GGT ACT GGG AAG ACG ACG CTA TCG ACA 1168 Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser Thr 350 355 360 GAC CAC AAC AGG CTT CTG ATC GGC GAC GAC GAG CAC TGC TGG AGC GAC 1216 Asp His Asn Arg Leu Leu Ile Gly Asp Asp Glu His Cys Trp Ser Asp 365 370 375 AAT GGC GTC TCC AAC ATT GAG GGT GGG TGC TAC GCC AAG TGT ATA GAC 1264 Asn Gly Val Ser Asn Ile Glu Gly Gly Cys Tyr Ala Lys Cys Ile Asp 380 385 390 CTC TCT CGG GAG AAA GAG CCT GAC ATC TGG AAC GCC ATC AAG TTT GGA 1312 Leu Ser Arg Glu Lys Glu Pro Asp Ile Trp Asn Ala Ile Lys Phe Gly 395 400 405 410 ACC GTG TTG GAG AAC GTT GTG TTT GAC GAG CGC ACC CGT GAA GTC GAC 1360 Thr Val Leu Glu Asn Val Val Phe Asp Glu Arg Thr Arg Glu Val Asp 415 420 425 TAC ACC GAT AAA TCC GTG ACA GAG AAC ACT CGT GCT GCT TAC CCG ATA 1408 Tyr Thr Asp Lys Ser Val Thr Glu Asn Thr Arg Ala Ala Tyr Pro Ile 430 435 440 GAG TAC ATC CCC AAC GCG AAG ATA CCG TGC GTC GGG CCG CAC CCG AAA 1456 Glu Tyr Ile Pro Asn Ala Lys Ile Pro Cys Val Gly Pro His Pro Lys 445 450 455 AAC GTC ATT CTC CTG GCC TGC GAC GCG TTC GGC GTC CTC CCG CCT GTC 1504 Asn Val Ile Leu Leu Ala Cys Asp Ala Phe Gly Val Leu Pro Pro Val 460 465 470 AGC AAG CTC AAC CTT GCG CAG ACC ATG TAC CAT TTC ATC AGT GGC TAC 1552 Ser Lys Leu Asn Leu Ala Gln Thr Met Tyr His Phe Ile Ser Gly Tyr 475 480 485 490 ACT GCT CTT GTT GCC GGC ACG GAA GAA GGT ATC AAG GAG CCA CAG GCC 1600 Thr Ala Leu Val Ala Gly Thr Glu Glu Gly Ile Lys Glu Pro Gln Ala 495 500 505 ACG TTC TCC GCC TGC TTC GGC GCG GCT TTC ATC ATG CTC CAC CCG ACC 1648 Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Ile Met Leu His Pro Thr 510 515 520 AAG TAC GCC GCC ATG CTC GCC GAG AAG ATG CAG AAG TAC GGC GCC ACA １６
９６ＬｙｓＴｙｒＡｌａＡｌａＭｅｔＬｅｕＡｌａＧｌｕＬｙｓ
ＭｅｔＧｌｎＬｙｓＴｙｒＧｌｙＡｌａＴｈｒ５２５５３０
５３５ＧＧＧＴＧＧＣＴＴＧＴＧＡＡＣＡＣＴＧＧＣＴＧＧＴＣＣ
ＧＧＣＧＧＧＡＧＧＴＡＣＧＧＣＧＴＣＧＧＣ１７４４ＧｌｙＴｒｐＬｅｕＶａｌＡｓｎＴｈｒＧｌｙＴｒｐＳｅｒ
ＧｌｙＧｌｙＡｒｇＴｙｒＧｌｙＶａｌＧｌｙ５４０５４５
５５０ＡＡＧＡＧＧＡＴＴＡＡＧＣＴＧＣＣＧＴＡＣＡＣＣＡＧＧ
ＡＡＧＡＴＣＡＴＴＧＡＣＧＣＣＡＴＣＣＡＣ１７９２ＬｙｓＡｒｇＩｌｅＬｙｓＬｅｕＰｒｏＴｙｒＴｈｒＡｒｇ
ＬｙｓＩｌｅＩｌｅＡｓｐＡｌａＩｌｅＨｉｓ５５５５６０
５６５５７０ＴＣＣＧＧＣＧＡＧＣＴＴＣＴＣＡＧＣＧＣＣＡＧＣＴＡＣ
ＡＡＧＡＡＧＡＣＣＧＡＧＧＴＣＴＴＣＧＧＣ１８４０ＳｅｒＧｌｙＧｌｕＬｅｕＬｅｕＳｅｒＡｌａＳｅｒＴｙｒ
ＬｙｓＬｙｓＴｈｒＧｌｕＶａｌＰｈｅＧｌｙ５７５
５８０５８５ＣＴＧＧＡＧＡＴＣＣＣＣＡＣＴＣＡＧＡＴＣＧＡＣＧＧＣ
ＧＴＧＣＣＧＴＣＧＧＡＡＡＴＣＣＴＣＧＡＣ１８８８ＬｅｕＧｌｕＩｌｅＰｒｏＴｈｒＧｌｎＩｌｅＡｓｐＧｌｙ
ＶａｌＰｒｏＳｅｒＧｌｕＩｌｅＬｅｕＡｓｐ５９０５９５
６００ＣＣＧＡＴＣＡＡＣＡＣＧＴＧＧＧＡＧＧＡＣＡＡＧＧＣＣ
ＧＣＧＴＡＣＡＡＧＧＡＧＡＣＧＣＴＣＣＴＧ１９３６ＰｒｏＩｌｅＡｓｎＴｈｒＴｒｐＧｌｕＡｓｐＬｙｓＡｌａ
ＡｌａＴｙｒＬｙｓＧｌｕＴｈｒＬｅｕＬｅｕ６０５６１０
６１５ＡＡＧＣＴＧＧＣＣＧＧＣＣＴＧＴＴＣＡＡＧＡＡＣＡＡＣ
ＴＴＣＧＡＧＧＴＧＴＴＣＧＣＣＡＧＣＴＡＣ１９８４ＬｙｓＬｅｕＡｌａＧｌｙＬｅｕＰｈｅＬｙｓＡｓｎＡｓｎ
ＰｈｅＧｌｕＶａｌＰｈｅＡｌａＳｅｒＴｙｒ６２０６２５
６３０ＡＡＧＡＴＣＧＧＧＧＡＣＧＡＣＡＧＣＡＧＣＣＴＧＡＣＣ
ＧＡＣＧＡＧＡＴＣＣＴＣＧＣＣＧＣＡＧＧＣ２０３２ＬｙｓＩｌｅＧｌｙＡｓｐＡｓｐＳｅｒＳｅｒＬｅｕＴｈｒ
ＡｓｐＧｌｕＩｌｅＬｅｕＡｌａＡｌａＧｌｙ６３５６４０
６４５６５０ＣＣＣＡＧＣＴＴＣＴＧＡＴＣＴＴＧＡＡＧＡＡＧＡＡＧＧＣＧＡＣ
ＧＣＧＡＴＧＧＡＧＴＧＧＧＴＡＴＴＣ２０８１ＰｒｏＳｅｒＰｈｅ６５３ＡＧＡＡＴＡＡＡＣＧＧＴＧＧＴＧＴＴＴＧＴＣＴＡＴＣＡＣＧＡＴＧＡ
ＴＧＡＴＧＡＣＧＡＡＡＡＡＡＡＴＡＴＧＴＴＧＴＡＴＣＧ２１４１ＧＴＧＴＧＴＧＡＧＴＡＣＴＡＣＡＴＴＴＴＣＴＣＡＧＣＴＴＧＡＴＧＡ
ＣＣＴＧＴＧＴＧＣＧＣＧＣＴＴＧＧＣＴＧＴＴＴＧＣＡＧ２２０１ＣＡＴＴＧＴＣＣ
２２０９SEQ ID NO: 2 Sequence length: 2209 Sequence type: Nucleic acid Sequence type: cDNA Organism name: Zoysia japonica Sequence features Characteristic symbols: 5'UTR Location: 1 ... 82 / partial Features Symbol: 3'UTR Location: 2045 ... 2209 / partial Characteristic symbol: CDS Location: 83 ... 2044 GCGTACACCG CGACACAGAC AGAGCACGCC GTTCTCCTCG TACGCGGATC AACGACCACC 60 GTGCCGCACG CGCGCCGGCG AG ATG GCG ACC CCG AAC GGC CTG GTC CTG GCC CTG GCC ATG Asn Gly Leu Ala Gln Ile 1 5 10 GAG ACG AAC GGG AGG AAG AAG CAC GAG AAC GTG GTG TGC CAC GAC GAC 160 Glu Thr Asn Gly Arg Lys Lys His Glu Asn Val Val Cys His Asp Asp 15 20 25 AGC GCT GCG CCA GTG CGC GCG CAG ACC ATC GAC GAG CTG CAC TCC CTG 208 Ser Ala Ala Pro Val Arg Ala Gln Thr Ile Asp Glu Leu His Ser Leu 30 35 40 CAG CGG AAG CGT TCG GCG CCC AGC ACG CCC AAG GAC AGC GCC GCC TCC 256 Gln Arg Lys Arg Ser Ala Pro Ser Thr Pro Lys Asp Ser Ala Ala Ser 45 50 55 GCC TTC GCC GCT GCG CTC ACC GAG GAG CAG CGC GAG GAG CAG CAG CTG 304 Ala Phe Ala Ala Ala Leu Thr Glu Glu Gln Arg Glu Glu Gln Gln Leu 60 65 70 CAG TCC ATC AGC GCG TCA CTG GCG TCT CTG ACG CGT GAG ACC GGC CCC 352 Gln Ser Ile Ser Ala Ser Leu Ala Ser Leu Thr Arg Glu Thr Gly Pro 75 80 85 90 AAG GTC GTC AGG GGA GAT CCG GCC AGG AAG GGT GAG GCG TCC GCG AAG 400 Lys Val Val Arg Gly Asp Pro Ala Arg Lys Gly Glu Ala Ser Ala Lys 95 100 105 AGC ACG CCG GCG CAC CAC CAC CAG CAC CCC GCC GCC CCC GCC GTC GCC 448 Ser Thr Pro Ala His His Gln His Pro Ala Ala Pro Ala Val Ala 110 115 120 GTC AGC GAC AGC TCC CTC AAG TCC ACC CAT GTC CTC TCC AAC CTC TCG 496 Val Ser Asp Ser Ser Leu Lys Ser Thr His Val Leu Ser Asn Leu Ser 125 130 135 CCT GCC GAG CTG TAC GAG CAG GCG ATC AAG TAC GAG AAG GGT TCG TTC 544 Pro Ala Glu Leu Tyr Glu Gln Ala Ile Lys Tyr Glu Lys Gly Ser Phe 140 145 150 ATC ACG TCC ACG GGT GCG CTG GCG ACG CTG TCC GGC GCG AAG ACC GGC 592 Ile Thr Ser Thr Gly Ala Leu Ala Thr Leu Ser Gly Ala Lys Thr Gly 155 160 165 170 CGC GCG CCC AGG GAC AAG CGC GTG GTC AAG GAC GAG GCC GCG GCG CGG 640 Arg Ala Pro Arg Asp Lys Arg Val Val Lys Asp Glu Ala Ala Ala Arg 175 180 185 GAG CTG TGG TGG GGC AAG GGG TCG CCG AAC ATC GAG ATG GAC GAG CAC 688 Glu Leu Trp Trp Gly Lys Gly Ser Pro Asn Ile Glu Met Asp Glu His 190 195 200 ACG TTC CTC ATC AAC AGG GAG AGG GCC GTC GAC TAC CTC AAC TCC CTC 736 Thr Phe Leu Ile Asn Arg Glu Arg Ala Val Asp Tyr Leu Asn Ser Leu 205 210 215 GAC AAG GTG TTC GTG AAC GAC CAG TTC CTG AAC TGG GAC CCG GAG AAC 784 Asp Lys Val Phe Val Asn Asp Gln Phe Leu Asn Trp Asp Pro Glu Asn 220 225 230 CGC ATC AAG GTC CGC ATC ATC TCC GCC AGG GCG TAC CAC TCC CTC TTC 832 Arg Ile Lys Val Arg Ile Ile Ser Ala Arg Ala Tyr His Ser Leu Phe 235 240 245 250 ATG CAC AAC ATG TGC ATC CGC CCG ACG GAT GAG GAG CTG GAG AAC TTC 880 Met His Asn Met Cys Ile Arg Pro Thr Asp Glu Glu Leu Glu Asn Phe 255 260 265 GGC ACG CCG GAC TTC ACC ATT TAC AAC GCC GGG CAG TTC CCT TGT AAC 928 Gly Thr Pro Asp Phe Thr Ile Tyr Asn Ala Gly Gln Phe Pro Cys Asn 270 275 280 CGA TAC ACG CAC TAC ATG ACG TCG TCG TCG AGC GTA GCC ATC AAC CTG 976 Arg Tyr Thr His Tyr Met Thr Ser Ser Ser Val Ala Ile Asn Leu 285 290 295 GCT AGG AGG GAG ATG GTG ATC CTG GGC ACG CAG TAC GCC GGG GAG ATG 1024 Ala Arg Arg Glu Met Val Ile Leu Gly Thr Gln Tyr Ala Gly Glu Met 300 305 310 AAG AAG GGA CTC TTC GGC CTC ATG CAC TAC CTC ATG CCC AAA CGG CAG 1072 Lys Lys Gly Leu Phe Gly Leu Met His Tyr Leu Met Pro Lys Arg Gln 315 320 325 330 ATC CTC TCG CTG CAC TCC GGG TGC AAC ATG GGC AAG GGC GGT GAC GTC 1120 Ile Leu Ser Leu His Ser Gly Cys Asn Met Gly Lys Gly Gly Asp Val 335 340 345 GCC CTC TTC TTC GGG CTG TCA GGT ACT GGG AAG ACG ACG CTA TCG ACA 1168 Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser Thr 350 355 360 GAC CAC AAC AGG CTT CTG ATC GGC GAC GAC GAG CAC TGC TGG AGC GAC 1216 Asp His Asn Arg Leu Leu Ile Gly Asp Asp Glu His Cys Trp Ser Asp 365 370 375 AAT GGC GTC TCC AAC ATT GAG GGT GGG TGC TAC GCC AAG TGT ATA GAC 1264 Asn Gly Val Ser Asn Ile G lu Gly Gly Cys Tyr Ala Lys Cys Ile Asp 380 385 390 CTC TCT CGG GAG AAA GAG CCT GAC ATC TGG AAC GCC ATC AAG TTT GGA 1312 Leu Ser Arg Glu Lys Glu Pro Asp Ile Trp Asn Ala Ile Lys Phe Gly 395 400 405 410 ACC GTG TTG GAG AAC GTT GTG TTT GAC GAG CGC ACC CGT GAA GTC GAC 1360 Thr Val Leu Glu Asn Val Val Phe Asp Glu Arg Thr Arg Glu Val Asp 415 420 425 TAC ACC GAT AAA TCC GTG ACA GAG AAC ACT CGT GCT GCT TAC CCG ATA 1408 Tyr Thr Asp Lys Ser Val Thr Glu Asn Thr Arg Ala Ala Tyr Pro Ile 430 435 440 GAG TAC ATC CCC AAC GCG AAG ATA CCG TGC GTC GGG CCG CAC CCG AAA 1456 Glu Tyr Ile Pro Asn Ala Lys Ile Pro Cys Val Gly Pro His Pro Lys 445 450 455 AAC GTC ATT CTC CTG GCC TGC GAC GCG TTC GGC GTC CTC CCG CCT GTC 1504 Asn Val Ile Leu Leu Ala Cys Asp Ala Phe Gly Val Leu Pro Pro Val 460 465 470 AGC AAG CTC AAC CTT GCG CAG ACC ATG TAC CAT TTC ATC AGT GGC TAC 1552 Ser Lys Leu Asn Leu Ala Gln Thr Met Tyr His Phe Ile Ser Gly Tyr 475 480 485 490 ACT GCT CTT GTT GCC GGC ACG GAA GAA GGT ATC AAG GAG CCA CAG GCC 1600 Th r Ala Leu Val Ala Gly Thr Glu Glu Gly Ile Lys Glu Pro Gln Ala 495 500 505 ACG TTC TCC GCC TGC TTC GGC GCG GCT TTC ATC ATG CTC CAC CCG ACC 1648 Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Ile Met Leu His Pro Thr 510 515 520 AAG TAC GCC GCC ATG CTC GCC GAG AAG ATG CAG AAG TAC GGC GCC ACA 16
96 Lys Tyr Ala Ala Met Leu Ala Glu Lys
Met Gln Lys Tyr Gly Ala Thr 525 530
535 GGG TGG CTT GTG AAC ACT GGC TGG TCC
GGC GGG AGG TAC GGC GTC GGC 1744 Gly Trp Leu Val Asn Thr Gly Trp Ser
Gly Gly Arg Tyr Gly Val Gly 540 545
550 AAG AGG ATT AAG CTG CCG TAC ACC AGG
AAG ATC ATT GAC GCC ATC CAC 1792 Lys Arg Ile Lys Leu Pro Tyr Thr Arg
Lys Ile Ile Asp Ala Ile His 555 560
565 570 TCC GGC GAG CTT CTC AGC GCC AGC TAC
AAG AAG ACC GAG GTC TTC GGC 1840 Ser Gly Glu Leu Leu Ser Ala Ser Tyr
Lys Lys Thr Glu Val Phe Gly 575
580 585 CTG GAG ATC CCC ACT CAG ATC GAC GGC
GTG CCG TCG GAA ATC CTC GAC 1888 Leu Glu Ile Pro Thr Gln Ile Asp Gly
Val Pro Ser Glu Ile Leu Asp 590 595
600 CCG ATC AAC ACG TGG GAG GAC AAG GCC
GCG TAC AAG GAG ACG CTC CTG 1936 Pro Ile Asn Thr Trp Glu Asp Lys Ala
Ala Tyr Lys Glu Thr Leu Leu 605 610
615 AAG CTG GCC GGC CTG TTC AAG AAC AAC
TTC GAG GTG TTC GCC AGC TAC 1984 Lys Leu Ala Gly Leu Phe Lys Asn Asn
Phe Glu Val Phe Ala Ser Tyr 620 625
630 AAG ATC GGG GAC GAC AGC AGC CTG ACC
GAC GAG ATC CTC GCC GCA GGC 2032 Lys Ile Gly Asp Asp Ser Ser Leu Thr
Asp Glu Ile Leu Ala Ala Gly 635 640
645 650 CCC AGC TTC TGATCTTGAA GAAGAAGGCG AC
GCGATGGA GTGGGTATTC 2081 Pro Ser Phe 653 AGAATAAACG GTGGTGTTTG TCTATCACGA TGA
TGATGAC GAAAAAAATA TGTTTGTATCG 2141 GTGTGTGAGT ACTACATTTT CTCAGCTTGA TGA
CCTGTGT GCGCGCTTGGC TGTTTGCAG 2201 CATGTCC
2209

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.⁶ 識別記号庁内整理番号ＦＩ技術表示箇所 // Ｃ０７Ｋ 14/415 Ｃ１２Ｎ 5/00 Ｃ (Ｃ１２Ｎ 5/10 Ｃ１２Ｒ 1:91） (Ｃ１２Ｎ 9/12 Ｃ１２Ｒ 1:91） ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // C07K 14/415 C12N 5/00 C (C12N 5/10 C12R 1:91) (C12N 9 / 12 C12R 1:91)

Claims

[Claims]

1. A DNA of a phosphoenolpyruvate carboxykinase gene having a base sequence encoding an amino acid sequence represented by SEQ ID NO: 1 or 2 or a part of the amino acid sequence with substitution, insertion, deletion and the like.

2. The DNA according to claim 1, which has a base sequence represented by SEQ ID NO: 1 or 2 or a part of the base sequence with substitution, insertion, deletion and the like.

3. The DNA according to claim 2, which has a nucleotide sequence represented by nucleotide numbers 65 to 2038 of SEQ ID NO: 1 and which is derived from a plant belonging to Spartina anglica .

4. The DNA according to claim 2, which has a base sequence represented by base numbers 83 to 2041 of SEQ ID NO: 2 which is derived from a plant belonging to Noshiba ( Zoisia japonica ).

5. The DNA according to claim 1, which can impart low temperature tolerance of C ₄ photosynthesis to a plant.

6. A method for enhancing the photosynthetic ability of a plant by introducing the DNA according to any one of claims 1 to 4 into the plant.

7. The DN according to any one of claims 1 to 4.
A recombinant vector containing A.

8. A plant transformed with the recombinant vector of claim 7.

9. The plant according to claim 8, wherein the plant is Noshiba.