JP4505627B2 - Promoter with green tissue specific expression activity - Google Patents
Promoter with green tissue specific expression activity Download PDFInfo
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- JP4505627B2 JP4505627B2 JP2003416951A JP2003416951A JP4505627B2 JP 4505627 B2 JP4505627 B2 JP 4505627B2 JP 2003416951 A JP2003416951 A JP 2003416951A JP 2003416951 A JP2003416951 A JP 2003416951A JP 4505627 B2 JP4505627 B2 JP 4505627B2
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Description
本発明は、緑色組織特異的発現活性を有するプロモーター及びその使用に関するものである。 The present invention relates to a promoter having a green tissue-specific expression activity and use thereof.
植物細胞内で機能可能なプロモーターの下流に発現させたい目的タンパク質をコードする遺伝子を連結させた遺伝子を植物細胞に導入し、得られた植物細胞を通常の植物組織培養技術により再生させる方法によって、所望の形質を有する改良植物体を作出することが行われている。植物で一般に用いられるプロモーターとしては、例えば、カリフラワーモザイクウイルス(CaMV)35Sプロモーター、アグロバクテリウムTiプラスミド由来ノパリン合成酵素遺伝子のプロモーター（Pnos)、トウモロコシ由来ユビキチンプロモーター、イネ由来のアクチンプロモーターなどが知られている。これらのプロモーターは、植物細胞内で目的とするタンパク質を構成的（constitutive)に発現させることが知られており、汎用されている。 By introducing a gene in which a gene encoding a target protein to be expressed downstream of a promoter that can function in the plant cell is introduced into the plant cell, and regenerating the obtained plant cell by a normal plant tissue culture technique, An improved plant having a desired character has been produced. Examples of promoters commonly used in plants include cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium Ti plasmid-derived nopaline synthase gene promoter (Pnos), maize-derived ubiquitin promoter, rice-derived actin promoter, and the like. ing. These promoters are known to constitutively express a target protein in plant cells, and are widely used.
しかしながら、植物を改良する場合、例えば、目的タンパク質を局所的に発現させることにより効果的な改良が行えることがあり、このような改良により新しいタイプの高機能性植物を開発する一環として、組織特異的な発現をもたらす植物プロモーターの探索が望まれている。 However, when plants are improved, effective improvements may be achieved by, for example, local expression of the target protein, and as a part of developing new types of highly functional plants by such improvements, tissue specific It is desired to search for plant promoters that bring about specific expression.
これまでに、植物において目的タンパク質を組織特異的に発現させることのできるプロモーターとしては、イネを例にすると、葯特異的プロモーターRA8（特許文献１）、葯又は花粉特異的プロモーターCatA（特許文献２）、雄ずい特異的プロモーターT72, T23, T42, T155, E1（特許文献３）、花器特異的プロモーターRPC213（特許文献４）、葉肉細胞特異的プロモーターrbcS（非特許文献１）、胚乳特異的プロモーターGluB-1、GluA-2（非特許文献２、３）、カルス特異的プロモーターPRO3（特許文献５）等が報告されている。 So far, as a promoter capable of tissue-specific expression of a target protein in a plant, rice is taken as an example, a rice cake-specific promoter RA8 (Patent Document 1), a rice cake or pollen-specific promoter CatA (Patent Document 2) ), Stamen-specific promoters T72, T23, T42, T155, E1 (patent document 3), flower organ-specific promoter RPC213 (patent document 4), mesophyll cell-specific promoter rbcS (non-patent document 1), endosperm-specific promoter GluB-1, GluA-2 (Non-patent Documents 2 and 3), callus-specific promoter PRO3 (Patent Document 5) and the like have been reported.
葉や茎などの緑色組織に存在する葉緑体は植物固有のオルガネラの一分化形態であり、植物が有する最も重要な機能である光合成の場である。植物が有する光合成機能を様々な環境条件で最適化することは、食料源供給の安定化、植物の良好な生長と分化を目指す上で重要である。また、大気中のCO2削減には、植物の持つ光合成能力の強化がもっとも直接的な手法である。このためには光合成の場である葉緑体の機能を改変し、CO2固定能を高めた植物を創製する技術が次世代技術として注目されている。さらに、葉緑体は、従来の大腸菌や枯草菌等の微生物に代わる、産業上有用な酵素等の異種タンパク質発現系としても着目されている。従って、植物における有用物質の生産、病害抵抗性の付与、環境耐性・高光合成機能植物の作出、植物の生長制御等のために、葉緑体が存在する葉や茎などの緑色組織に特異的発現をもたらすプロモーターの新規開発・実用化が強く望まれている。
従って、本発明の目的は、緑色組織に特異的発現をもたらすプロモーター活性を有するDNA、該DNAを含有する遺伝子を緑色組織で特異的に発現させることを可能にした組換えプラスミド、該組換えベクターを導入した形質転換植物体を提供することを目的とする。 Accordingly, an object of the present invention is to provide DNA having promoter activity that causes specific expression in green tissue, a recombinant plasmid that enables specific expression of a gene containing the DNA in green tissue, and the recombinant vector It aims at providing the transformed plant body which introduce | transduced.
本発明者らは、上記課題を解決すべく、イネゲノム配列情報、イネESTの出現頻度情報、イネ完全長cDNA配列情報等（http://www.dna.affrc.go.jp/）を参考にしつつ鋭意研究を重ねた結果、種々の組織に特異的な発現パターンを有すると期待されるイネ遺伝子を約100種選定し、選定された各遺伝子の５’上流域をポリメラーゼ連鎖反応（PCR）で増幅した断片の中から、緑色組織において特異的に目的タンパク質を発現させるプロモーター活性を有するDNA断片を得ることに成功し、本発明を完成させるに至った。 In order to solve the above problems, the present inventors refer to rice genome sequence information, rice EST appearance frequency information, rice full-length cDNA sequence information, etc. (http://www.dna.affrc.go.jp/). As a result of extensive research, we selected about 100 rice genes that are expected to have expression patterns specific to various tissues, and polymerase chain reaction (PCR) was performed on the 5 'upstream region of each selected gene. From the amplified fragments, the present inventors have succeeded in obtaining a DNA fragment having a promoter activity that specifically expresses a target protein in a green tissue, thereby completing the present invention.
即ち、本発明は以下の発明を包含する。
(1) 以下の（ａ）、（ｂ）又は（ｃ）に示す、プロモーターとして機能しうるDNA。
（ａ）配列表の配列番号１に示す塩基配列からなるDNA
（ｂ）配列表の配列番号１に示す塩基配列において、１若しくは数個の塩基が欠失、置換若しくは付加された塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNA
（ｃ）配列表の配列番号１に示す塩基配列の一部の塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNA
(2) (1)に記載のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNA。
(3) (1)又は(2)に記載のDNAを含有する組換えベクター。
(4) (1)又は(2)に記載のDNAと目的タンパク質をコードする遺伝子を含有する組換えベクター。
(5) (3)又は(4)に記載の組換えベクターを導入した形質転換植物体。
That is, the present invention includes the following inventions.
(1) DNA which can function as a promoter, as shown in the following (a), (b) or (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(B) a DNA comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and having a promoter activity that brings about specific expression in green tissue
(C) DNA consisting of a part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and having promoter activity that brings about specific expression in green tissue
(2) A DNA having a promoter activity that hybridizes with a DNA comprising a base sequence complementary to the DNA according to (1) under stringent conditions and brings about expression specific to a green tissue.
(3) A recombinant vector containing the DNA according to (1) or (2).
(4) A recombinant vector containing the DNA according to (1) or (2) and a gene encoding a target protein.
(5) A transformed plant into which the recombinant vector according to (3) or (4) has been introduced.
本発明によれば、緑色組織に特異的な発現をもたらすプロモーター活性を有するDNAが提供される。このプロモーター活性を有するDNAを利用すれば、緑葉における有用物質の生産性向上、耐病性関連遺伝子の発現による耐病性付与、植物の生長制御、光合成関連遺伝子の改変や高発現による光合成能の向上などが可能となる。具体的には、例えば、ベータカロチン、アントシアニンなどの色素成分、インターフェロン、ビタミン類などの医薬成分、ショ糖などの光合成産物の緑葉での生産性向上、あるいは、生長抑制遺伝子導入によって茎伸長を阻害し、穀粒が増えても倒伏しにくい、収穫作業を容易にするなどの効果を得ることができる。 According to the present invention, DNA having promoter activity that provides specific expression in green tissue is provided. Utilizing DNA with this promoter activity improves productivity of useful substances in green leaves, imparts disease resistance by expressing disease resistance-related genes, controls plant growth, improves photosynthetic ability by altering and synthesizing photosynthetic genes Is possible. Specifically, for example, pigment components such as beta-carotene and anthocyanins, pharmaceutical components such as interferons and vitamins, photosynthetic products such as sucrose are improved in productivity in green leaves, or stem growth is inhibited by introducing a growth-suppressing gene However, even if the number of grains increases, it is difficult to fall down, and effects such as facilitating harvesting can be obtained.
１．プロモーター及びその単離
本発明に係るプロモーターとして機能しうるDNA（以下、「プロモーター」という）は、配列番号１に示す塩基配列からなるDNAである。
1. Promoter and Isolation thereof DNA capable of functioning as a promoter according to the present invention (hereinafter referred to as “promoter”) is a DNA comprising the base sequence shown in SEQ ID NO: 1.
本発明のDNAは、目的タンパク質をコードする遺伝子（以下、「目的遺伝子」という）の翻訳開始点の５’側に挿入することにより、該目的遺伝子の緑色組織における発現を誘導し、又は該目的遺伝子を緑色組織において高レベルで発現させることができる。 The DNA of the present invention induces the expression of the target gene in green tissue by inserting it on the 5 ′ side of the translation start point of the gene encoding the target protein (hereinafter referred to as “target gene”), or Genes can be expressed at high levels in green tissue.
また、本発明のDNAには、配列番号１に示す塩基配列において１以上の塩基が置換、欠失、付加又は挿入された塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNAも含まれる。ここで、置換、欠失、付加又は挿入されてもよい塩基の数は特に限定されないが、好ましくは１個〜数個である。例えば、配列番号１に示す塩基配列の１〜１０個、好ましくは１〜５個の塩基が欠失してもよく、配列番号１に示す塩基配列に１〜１０個、好ましくは１〜５個の塩基が付加してもよく、あるいは、配列番号１に示す塩基配列の１〜１０個、好ましくは１〜５個の塩基が他の塩基に置換してもよい。 The DNA of the present invention comprises a nucleotide sequence in which one or more bases are substituted, deleted, added or inserted in the nucleotide sequence shown in SEQ ID NO: 1, and has a promoter activity that brings about specific expression in green tissue. Also included is the DNA it has. Here, the number of bases that may be substituted, deleted, added or inserted is not particularly limited, but is preferably 1 to several. For example, 1 to 10, preferably 1 to 5 bases of the nucleotide sequence shown in SEQ ID NO: 1 may be deleted, and 1 to 10, preferably 1 to 5 nucleotide sequences shown in SEQ ID NO: 1. May be added, or 1 to 10, preferably 1 to 5 bases of the base sequence shown in SEQ ID NO: 1 may be substituted with other bases.
さらに、本発明のDNAには、配列番号１に示す塩基配列の一部の塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNAも含まれる。 Furthermore, the DNA of the present invention includes a DNA consisting of a part of the base sequence shown in SEQ ID NO: 1 and having promoter activity that brings about specific expression in green tissue.
配列番号１に示す塩基配列からなるDNAにおけるプロモーター活性に必須な部分は、該DNAの様々な欠失体、例えば、５’上流側から様々な長さに欠損させたDNA断片をベータグルクロニダーゼ（GUS）遺伝子等のリポーター遺伝子を融合させたプラスミドを宿主に導入し、プロモーター活性を測定することによって特定でき、そのような活性部分の特定のための手法は、当業者には公知である。 The essential part of the promoter activity in the DNA consisting of the base sequence shown in SEQ ID NO: 1 is a variety of deletions of the DNA, for example, DNA fragments deficient in various lengths from the 5 ′ upstream side to beta-glucuronidase (GUS ) It can be identified by introducing a plasmid fused with a reporter gene such as a gene into a host and measuring the promoter activity. Techniques for identifying such an active moiety are known to those skilled in the art.
このような変異体DNAは、緑色組織に特異的な発現をもたらすプロモーター活性（以下、「緑色組織特異的プロモーター活性」という）を有していればよく、その活性の大きさは特に限定されないが、配列番号１に示す塩基配列からなるDNAの緑色組織特異的プロモーター活性を実質的に保持することが好ましい。「配列番号１に示す塩基配列からなるDNAの緑色組織特異的プロモーター活性を実質的に保持する」とは、該プロモーター活性を利用した実際の使用態様において、配列番号１に示す塩基配列からなるDNAと、同一の条件でほぼ同様の利用が可能な程度の活性が維持されていることをいう。 Such a mutant DNA may have a promoter activity (hereinafter referred to as “green tissue-specific promoter activity”) that causes expression specific to green tissue, and the magnitude of the activity is not particularly limited. It is preferable that the DNA consisting of the base sequence shown in SEQ ID NO: 1 substantially retains the green tissue-specific promoter activity. “Substantially retains the green tissue-specific promoter activity of the DNA consisting of the base sequence shown in SEQ ID NO: 1” means that the DNA consisting of the base sequence shown in SEQ ID NO: 1 in the actual usage mode utilizing the promoter activity. And that the activity is maintained to the extent that the same use is possible under the same conditions.
本発明において「緑色組織特異的プロモーター活性」とは、緑色組織において、同じ植物体の他の組織又は器官の少なくとも１種におけるよりも目的遺伝子を優先的かつ高度に発現させる活性をいう。 In the present invention, “green tissue-specific promoter activity” refers to an activity that preferentially and highly expresses a target gene in green tissue over at least one other tissue or organ of the same plant.
また、上記「緑色組織」とは、葉緑体が存在する組織であれば特に限定はないが、主として葉（イネ科植物では葉身と葉鞘、また多くの双子葉植物では葉身と葉柄からなる）、および茎（イネ科植物では稈と称する）を指す。また、イネ等の植物に見られる葉耳・葉舌（これらは葉身と葉鞘の境界に存在）、葉枕（葉の付け根にあって、内部の圧力を調節して葉を動かすことを可能にした肥厚組織。オジギソウ等のマメ科植物他に存在）などの特殊組織もこれに含まれる。ただし本発明では、葉緑体をわずかしか含まない、あるいは葉緑体が存在しないような維管束組織（茎・稈に存在、および葉身内を走る葉脈）は原則として含まない。 In addition, the “green tissue” is not particularly limited as long as it is a tissue in which chloroplasts are present, but mainly leaves (from grasses and leaf sheaths in gramineous plants, and from leaf blades and petiole in many dicotyledons). ) And stem (referred to as moth in gramineous plants). In addition, leaf ears and tongues found in plants such as rice (these are at the boundary between leaf blades and leaf sheaths) and leaf pillows (at the base of the leaf, it is possible to move the leaves by adjusting the internal pressure) This includes special tissues such as thickened tissues, which are present in leguminous plants such as sea urchins. However, in the present invention, in principle, vascular tissue (existing in stems and folds and leaf veins running in leaf blades) containing little or no chloroplast is not included.
上記のような変異体DNAを取得するための遺伝子変異導入は、Kunkel法又は Gapped duplex法等の公知手法又はこれに準ずる方法によって行うことができ、例えば部位特異的突然変異誘発法を利用した変異導入用キット（例えばMutant-K(TAKARA社製）やMutant-G(TAKARA社製）)、TAKARA社のLA PCR in vitro Mutagenesis シリーズキットを利用することができる。 The introduction of gene mutation for obtaining mutant DNA as described above can be carried out by a known technique such as the Kunkel method or the Gapped duplex method or a method analogous thereto, for example, mutation using site-directed mutagenesis. Kits for introduction (for example, Mutant-K (manufactured by TAKARA) or Mutant-G (manufactured by TAKARA)) and LA PCR in vitro Mutagenesis series kits by TAKARA can be used.
さらに、当業者であれば、配列番号１に示す塩基配列の全部又は一部からなるDNAを用いて、配列番号１に示す塩基配列からなるDNAと同様の機能、すなわち、緑色組織特異的プロモーター活性を有する他の塩基配列からなるDNAを種々の生物から新たに取得し、利用することも容易である。このような他の塩基配列からなるDNAの取得は、例えば、配列番号１に示す塩基配列の全部又は一部からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズさせるハイブリダイゼーション、該塩基配列の一部をプライマーとして用いるPCR等によって行うことができる。ここで、ストリンジェントな条件とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。例えば、相同性が高い核酸、すなわち配列番号１に示す塩基配列と90％以上、好ましくは95%以上の相同性を有する塩基配列からなるDNAの相補鎖がハイブリダイズし、それより相同性が低い核酸の相補鎖がハイブリダイズしない条件が挙げられる。より具体的には、ナトリウム濃度が10〜300mM、好ましくは15〜75mMであり、温度が25℃〜70℃、好ましくは42℃〜55℃での条件をいう。 Furthermore, those skilled in the art can use DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1 to perform the same function as DNA consisting of the base sequence shown in SEQ ID NO: 1, ie, green tissue-specific promoter activity. It is also easy to newly obtain and use DNA consisting of other base sequences having For obtaining such a DNA consisting of another base sequence, for example, it is hybridized under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1. It can be performed by hybridization, PCR using a part of the base sequence as a primer, or the like. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a highly homologous nucleic acid, that is, a complementary strand of DNA consisting of a nucleotide sequence having a homology of 90% or more, preferably 95% or more, with the nucleotide sequence shown in SEQ ID NO: 1 hybridizes and has a lower homology. A condition in which the complementary strand of the nucleic acid does not hybridize is mentioned. More specifically, the sodium concentration is 10 to 300 mM, preferably 15 to 75 mM, and the temperature is 25 ° C to 70 ° C, preferably 42 ° C to 55 ° C.
上記のように取得した変異体DNAやハイブリダイゼーションにより得られるホモログがプロモーターとしての活性を有するか否かは、種々のレポーター遺伝子、例えばベータグルクロニダーゼ（GUS）、ルシフェラーゼ（LUC）、Green fluorescent protein（GFP）、
クロラムフェニコールアセチルトランスフェラーゼ（CAT）、ベータガラクトシダーゼ（GAL）、ノパリン合成酵素（NOS）、オクトピン合成酵素（OCS）等の遺伝子を上記プロモーターの下流域に連結したベクターを作製し、該ベクターを用いて従来から周知慣用されている種々の形質転換法（後述）により植物細胞のゲノムに挿入した後、該レポーター遺伝子の発現を測定することにより確認できる。
Whether the mutant DNA obtained as described above or the homolog obtained by hybridization has activity as a promoter depends on various reporter genes such as beta glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP ),
A vector in which genes such as chloramphenicol acetyltransferase (CAT), beta galactosidase (GAL), nopaline synthase (NOS), octopine synthase (OCS) and the like are linked to the downstream region of the above promoter is prepared, and the vector is used. It can be confirmed by measuring the expression of the reporter gene after insertion into the genome of a plant cell by various transformation methods (to be described later) that have been well known and conventionally used.
例えば、レポーター遺伝子がGUSの場合には、宿主細胞内でのプロモーター活性は、(ｉ)ヒストケミカルなGUS染色による方法（EMBO J. 6, 3901-3907 (1987）)により、及び／又は(ii)蛍光基質を用いるCastle＆Morrisの方法（Plant Molecular Biology Manual, B5, 1-16 (1994); S.B.Gelvin & R.A.Schilperoort, Kluwer Academic Publishers）に従ってGUS活性を測定し、さらにBradfordの方法（Anal. Biochem. 72, 248-254（1976)）に従ってタンパク質量を測定して、GUS活性をタンパク量当たりに換算する（nmole 4-MU/min/mg proteinとして算出する）ことにより、それぞれ確認することができる。 For example, when the reporter gene is GUS, the promoter activity in the host cell is determined by (i) a method by histochemical GUS staining (EMBO J. 6, 3901-3907 (1987)) and / or (ii) ) GUS activity was measured according to the Castle & Morris method (Plant Molecular Biology Manual, B5, 1-16 (1994); SBGelvin & RASchilperoort, Kluwer Academic Publishers) using a fluorescent substrate, and Bradford's method (Anal. Biochem. 72). , 248-254 (1976)), and the GUS activity can be confirmed by converting the GUS activity per protein amount (calculated as nmole 4-MU / min / mg protein).
本発明のプロモーターは、イネ（日本晴）の第４番染色体ゲノム上に存在し、配列番号３に示すアミノ酸配列を有するプロトクロロフィリドレダクターゼ前駆体（Protochlorophyllide reductase precursor、別名：Protochlorophyllide oxidoreductase、省略名称：POR）をコードするイネ遺伝子〔DDBJ（http://www.ddbj.nig.ac.jp）:アクセッション番号AK065236〕の塩基配列（配列番号２）に基づき、該遺伝子（以下、本明細書において「イネPOR遺伝子」という）の上流領域を単離することによって取得できる。プロモーター領域を単離する方法としては、特に限定されないが、例えば、インバースPCR、ゲノムDNAライブラリーから単離する方法等を例示することができる。 The promoter of the present invention is a protochlorophyllide reductase precursor (also known as: Protochlorophyllide oxidoreductase, abbreviated name: POR) present on the chromosome 4 genome of rice (Nipponbare) and having the amino acid sequence shown in SEQ ID NO: 3. ) -Based rice gene [DDBJ (http://www.ddbj.nig.ac.jp): Accession No. AK065236] based on the base sequence (SEQ ID No. 2), the gene (hereinafter referred to as “ It can be obtained by isolating the upstream region of the “rice POR gene”. The method for isolating the promoter region is not particularly limited, and examples thereof include inverse PCR, a method for isolating from a genomic DNA library, and the like.
インバースPCRによる場合は、イネPOR遺伝子の塩基配列情報に基づいて一対のプライマーを合成し、これら一対のプライマーと所定の制限酵素で処理した後にセルフライゲーションさせたゲノムDNA断片とを用いてPCRを行うことによって、イネPOR遺伝子の上流領域を増幅することができる。その後、イネPOR遺伝子の上流領域をクローニングし塩基配列を決定することによって、イネPOR遺伝子のプロモーター領域の単離、及び塩基配列（配列番号１）の決定を行うことができる。 In the case of inverse PCR, a pair of primers is synthesized based on the nucleotide sequence information of the rice POR gene, and PCR is performed using these paired primers and a genomic DNA fragment that has been treated with a predetermined restriction enzyme and then self-ligated. Thus, the upstream region of the rice POR gene can be amplified. Thereafter, by cloning the upstream region of the rice POR gene and determining the base sequence, the promoter region of the rice POR gene can be isolated and the base sequence (SEQ ID NO: 1) can be determined.
また、ゲノムDNAライブラリーから単離する場合には、イネPOR遺伝子を含むcDNAをプローブとして、定法に従って調製したゲノムDNAライブラリーからイネPOR遺伝子を含むゲノムDNAをスクリーニングする。その後、スクリーニングしたゲノムDNAの塩基配列を決定することによってイネPOR遺伝子の上流領域に存在するプロモーター領域を特定することができ、さらに、該プロモーター領域のみをPCR等によって増幅してクローニングすることによって単離することができる。 In addition, when isolating from a genomic DNA library, genomic DNA containing a rice POR gene is screened from a genomic DNA library prepared according to a conventional method using cDNA containing the rice POR gene as a probe. Thereafter, by determining the base sequence of the screened genomic DNA, the promoter region existing in the upstream region of the rice POR gene can be identified, and further, only the promoter region is amplified and cloned by PCR or the like. Can be separated.
いったん本発明のプロモーターの塩基配列が確定されると、その後は化学合成によって、あるいはその塩基配列の一部からなるDNAをプライマーとして合成し、イネの全DNAを鋳型として用いて、該プライマーを用いるPCRによって容易に得ることができる。 Once the base sequence of the promoter of the present invention has been determined, the subsequent synthesis is carried out by chemical synthesis or by using a DNA consisting of a part of the base sequence as a primer, and using the rice total DNA as a template. It can be easily obtained by PCR.
さらに、単離したプロモーター領域（配列番号１）の一部を用いてプロモーター活性を測定することによって、単離したプロモーター領域（配列番号１）において、プロモーター活性に寄与している領域を特定することができる。プロモーター領域の一部は、該プロモーター領域の一部をPCRによって増幅する方法、プロモーター領域を所定の制限酵素で処理して断片化する方法等を適宜使用して得ることができる。 Further, by measuring the promoter activity using a part of the isolated promoter region (SEQ ID NO: 1), the region contributing to the promoter activity in the isolated promoter region (SEQ ID NO: 1) is specified. Can do. A part of the promoter region can be obtained by appropriately using a method in which a part of the promoter region is amplified by PCR, a method in which the promoter region is treated with a predetermined restriction enzyme, and fragmented.
得られたプロモーター領域の一部は、発現量を定量できる遺伝子の上流に組み込み、該遺伝子の発現量を定量することによってプロモーター活性を測定することができる。すなわち、得られたプロモーター領域の一部及び所定の遺伝子を組み込んでなる組換えベクターを構築し、該組換えベクターを用いて形質転換した細胞における該遺伝子の発現量を定量することによって、得られたプロモーター領域の一部におけるプロモーター活性を測定することができる。 A part of the obtained promoter region can be incorporated upstream of a gene whose expression level can be quantified, and the promoter activity can be measured by quantifying the expression level of the gene. That is, it can be obtained by constructing a recombinant vector incorporating a part of the obtained promoter region and a predetermined gene, and quantifying the expression level of the gene in cells transformed with the recombinant vector. Promoter activity in a part of the promoter region can be measured.
２. 組換えベクター
本発明の組換えベクターは、上記１．のプロモーターに目的遺伝子を連結した遺伝子を適当なベクターに導入することにより構築することができる。ここで、ベクターとしては、アグロバクテリウムを介して植物に目的遺伝子を導入することができる、pBI系、pPZP系（Hajdukiewicz P, Svab Z, Maliga P.: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation., Plant Mol Biol., 25: 989-94, 1994）、pCAMBIA系（http://www.cambia.org/main/r_et_camvec.htm）、pSMA系のベクターなどが好適に用いられる。特にpBI系のバイナリーベクター又は中間ベクター系が好適に用いられ、例えば、pBI121、pBI101、pBI101.2、pBI101.3等が挙げられる。バイナリーベクターとは大腸菌（Escherichia coli）及びアグロバクテリウムにおいて複製可能なシャトルベクターで、バイナリーベクターを保持するアグロバクテリムを植物に感染させると、ベクター上にあるLB配列とRB配列より成るボーダー配列で囲まれた部分のDNAを植物核DNAに組み込むことが可能である（EMBO Journal, 10(3), 697-704 (1991)）。一方、pUC系のベクターは、植物に遺伝子を直接導入することができ、例えば、pUC18、pUC19、pUC9等が挙げられる。また、カリフラワーモザイクウイルス（CaMV)、インゲンマメモザイクウイルス（BGMV)、タバコモザイクウイルス（TMV）等の植物ウイルスベクターも用いることができる。
2. Recombinant vector The recombinant vector of the present invention comprises the above-mentioned 1. The gene can be constructed by introducing a gene in which the target gene is linked to the above promoter into an appropriate vector. Here, as a vector, a target gene can be introduced into a plant via Agrobacterium, pBI system, pPZP system (Hajdukiewicz P, Svab Z, Maliga P .: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation., Plant Mol Biol., 25: 989-94, 1994), pCAMBIA (http://www.cambia.org/main/r_et_camvec.htm), pSMA vectors, and the like are preferably used. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A binary vector is a shuttle vector that can replicate in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium that holds the binary vector, it is a border sequence consisting of LB and RB sequences on the vector. It is possible to incorporate the enclosed DNA into plant nuclear DNA (EMBO Journal, 10 (3), 697-704 (1991)). On the other hand, a pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) can also be used.
ベクターに目的遺伝子を挿入するには、まず、精製されたDNAを適当な制限酵素で切断し、適当なベクター DNAの制限酵素部位又はマルチクローニングサイトに挿入してベクターに連結する方法などが採用される。 In order to insert a target gene into a vector, first, a method in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or a multicloning site, and then ligated to the vector is employed. The
目的遺伝子としては、対象となる植物における内因性遺伝子、または外来遺伝子であって、その遺伝子産物の発現が緑色組織において所望される任意の遺伝子をいう。かかる遺伝子としては、有用物質（医薬、色素、芳香成分など）生産遺伝子、植物生長制御（促進/抑制）遺伝子、糖代謝関連遺伝子、耐病虫害性〔昆虫食害抵抗性、カビ（菌類）及び細菌病抵抗性、ウイルス（病）抵抗性など〕遺伝子、環境ストレス（低温、高温、乾燥、光障害、紫外線）抵抗性関連遺伝子等が挙げられるが、これらに限定はされない。 The target gene refers to any gene that is an endogenous gene in a target plant or a foreign gene, and the expression of the gene product is desired in green tissue. Such genes include useful substance (pharmaceuticals, pigments, aromatic components, etc.) production genes, plant growth control (promotion / suppression) genes, sugar metabolism-related genes, insect pest resistance [insect feeding resistance, fungi (fungi) and bacterial diseases Resistance, virus (disease) resistance, etc.] genes, environmental stress (low temperature, high temperature, dryness, light damage, ultraviolet light) resistance related genes and the like, but are not limited thereto.
上記の目的遺伝子は、その遺伝子の機能が発揮されるようにベクターに組み込まれることが必要である。そこで、ベクターには、目的遺伝子の上流、内部、あるいは下流に、本発明のプロモーター、エンハンサー、イントロン、ポリA付加シグナル、5'-UTR配列、選抜マーカー遺伝子などを連結することができる。 The target gene described above needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the promoter, enhancer, intron, poly A addition signal, 5′-UTR sequence, selectable marker gene and the like of the present invention can be linked to the vector upstream, inside, or downstream of the target gene.
エンハンサーとしては、例えば、目的遺伝子の発現効率を高めるために用いられ、CaMV35Sプロモーター内の上流側の配列を含むエンハンサー領域などが挙げられる。 Examples of the enhancer include an enhancer region that is used to increase the expression efficiency of the target gene and includes an upstream sequence in the CaMV35S promoter.
ターミネーターとしては、前記プロモーターにより転写された遺伝子の転写を終結できる配列であればよく、例えば、ノパリン合成酵素遺伝子のターミネーター、オクトピン合成酵素遺伝子のターミネーター、CaMV 35Ｓ RNA遺伝子のターミネーター等が挙げられる。 The terminator may be any sequence that can terminate transcription of the gene transcribed by the promoter, and examples thereof include nopaline synthase gene terminator, octopine synthase gene terminator, CaMV 35S RNA gene terminator, and the like.
選抜マーカー遺伝子としては、例えば、ハイグロマイシン耐性遺伝子、カナマイシン耐性遺伝子、ビアラホス耐性遺伝子、ブラストサイジンS耐性遺伝子、アセト乳酸合成酵素（Acetolactate synthase）遺伝子などが挙げられる。 Examples of the selection marker gene include a hygromycin resistance gene, a kanamycin resistance gene, a bialaphos resistance gene, a blasticidin S resistance gene, an acetolactate synthase gene, and the like.
また、選抜マーカー遺伝子は、上記のように目的遺伝子とともに同一のプラスミドに連結させて組換えベクターを調製してもよいが、あるいは、選抜マーカー遺伝子をプラスミドに連結して得られる組換えベクターと、目的遺伝子をプラスミドに連結して得られる組換えベクターとを別々に調製してもよい。別々に調製した場合は、各ベクターを宿主にコトランスフェクト（共導入）する。 Alternatively, the selection marker gene may be prepared by ligating the same gene together with the target gene as described above to prepare a recombinant vector, or alternatively, a recombinant vector obtained by linking the selection marker gene to a plasmid, A recombinant vector obtained by ligating the target gene to a plasmid may be prepared separately. When prepared separately, each vector is co-transfected (co-introduced) into the host.
３．形質転換植物体
上記２．で調製した組換えベクターを用いて、対象植物を形質転換し、形質転換植物体を調製することができる。
3. Transformed plant body 2. Using the recombinant vector prepared in Step 1, a target plant can be transformed to prepare a transformed plant body.
形質転換植物体を調製する際には、既に報告され、確立されている種々の方法を適宜利用することができ、その好ましい例として、アグロバクテリウム法、ＰＥＧ−リン酸カルシウム法、エレクトロポレーション法、リポソーム法、パーティクルガン法、マイクロインジェクション法等が挙げられる。アグロバクテリウム法を用いる場合は、プロトプラストを用いる場合、組織片を用いる場合、プロトプラストを用いる場合、組織片を用いる場合、及び植物体そのものを用いる場合（in planta法）がある。プロトプラストを用いる場合は、Tiプラスミドをもつアグロバクテリウムと共存培養する方法、スフェロプラスト化したアグロバクテリウムと融合する方法（スフェロプラスト法）、組織片を用いる場合は、対象植物の無菌培養葉片（リーフディスク）に感染させる方法やカルスに感染させる等により行うことができる。また、種子あるいは植物体を用いるin planta法を適用する場合、すなわち植物ホルモン添加の組織培養を介さない系では、吸水種子、幼植物（幼苗）、鉢植え植物などへのアグロバクテリウムの直接処理等にて実施可能である。 In preparing a transformed plant body, various methods already reported and established can be used as appropriate. Preferred examples thereof include the Agrobacterium method, the PEG-calcium phosphate method, the electroporation method, Examples include the liposome method, particle gun method, and microinjection method. When using the Agrobacterium method, there are cases where protoplasts are used, tissue pieces are used, protoplasts are used, tissue pieces are used, and plants themselves are used (in planta method). When using protoplasts, co-culture with Agrobacterium with Ti plasmid, fusing with spheroplasted Agrobacterium (spheroplast method), and when using tissue fragments, aseptic culture of the target plant It can be performed by infecting leaf pieces (leaf discs) or infecting callus. In addition, when the in planta method using seeds or plants is applied, that is, in systems that do not involve tissue culture with the addition of plant hormones, direct treatment of Agrobacterium to water-absorbing seeds, seedlings (plant seedlings), potted plants, etc. Can be implemented.
遺伝子が植物体に組み込まれたか否かの確認は、PCR法、サザンハイブリダイゼーション法、ノーザンハイブリダイゼーション法、ウェスタンブロッティング法等により行うことができる。例えば、形質転換植物体からDNAを調製し、DNA特異的プライマーを設計してPCRを行う。PCRを行った後は、増幅産物についてアガロースゲル電気泳動、ポリアクリルアミドゲル電気泳動又はキャピラリー電気泳動等を行い、臭化エチジウム、SYBR Green液等により染色し、そして増幅産物を1本のバンドとして検出することにより、形質転換されたことを確認することができる。また、予め蛍光色素等により標識したプライマーを用いてPCRを行い、増幅産物を検出することもできる。さらに、マイクロプレート等の固相に増幅産物を結合させ、蛍光又は酵素反応等により増幅産物を確認する方法でもよい。 Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. After PCR, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band. By doing so, it can confirm that it transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, the amplification product may be bound to a solid phase such as a microplate, and the amplification product may be confirmed by fluorescence or enzymatic reaction.
本発明において形質転換に用いられる植物としては、イネ、ムギ、トウモロコシ、ネギ、ユリ、ラン等の単子葉植物、ダイズ、ナタネ、トマト、バレイショ、キク、バラ、カーネーション、ペチュニア、カスミソウ、シクラメン等の双子葉植物などの植物が挙げられ、特に限定はされない。好ましくは、本発明のDNAが単離されたイネ科の植物、例えば、イネ、オオムギ、コムギ、トウモロコシ、サトウキビ、シバ、ソルガム、アワ、及びヒエなどの植物が挙げられる。 Plants used for transformation in the present invention include monocotyledonous plants such as rice, wheat, corn, leek, lily, orchid, soybean, rapeseed, tomato, potato, chrysanthemum, rose, carnation, petunia, gypsophila, cyclamen and the like. Plants such as dicotyledonous plants can be mentioned, and are not particularly limited. Preferably, plants of the family Gramineae from which the DNA of the present invention is isolated, for example, plants such as rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, and millet.
本発明において、形質転換の対象とする植物材料としては、例えば、根、茎、葉、種子、胚、胚珠、子房、茎頂（植物の芽の先端の生長点）、葯、花粉等の植物組織やその切片、未分化のカルス、それを酵素処置して細胞壁を除いたプロプラスト等の植物培養細胞が挙げられる。またin planta法適用の場合、吸水種子や植物体全体を利用し得る。 In the present invention, examples of plant materials to be transformed include roots, stems, leaves, seeds, embryos, ovules, ovaries, shoot tips (growth points at the tips of plant buds), buds, pollen and the like. Examples thereof include plant tissue cells and slices thereof, undifferentiated callus, and plant cultured cells such as proplasts obtained by removing the cell wall by enzyme treatment. In the case of applying the in planta method, water-absorbing seeds or the entire plant body can be used.
また、本発明において形質転換植物体とは、植物体全体、植物器官（例えば根、茎、葉、花弁、種子、種子、実等）植物組織(例えば表皮、師部、柔組織、木部、維管束等)、植物培養細胞のいずれをも意味するものである。 In the present invention, the transformed plant body means the whole plant body, plant organ (eg, root, stem, leaf, petal, seed, seed, fruit, etc.) plant tissue (eg, epidermis, phloem, soft tissue, xylem, Vascular bundles, etc.) and plant cultured cells.
植物培養細胞を対象とする場合において、得られた形質転換細胞から形質転換体を再生させるためには既知の組織培養法により器官又は個体を再生させればよい。このような操作は、植物細胞から植物体への再生方法として一般的に知られている方法により、当業者であれば容易に行うことができる。植物細胞から植物体への再生については、例えば、以下のように行うことができる。 When plant cultured cells are targeted, in order to regenerate a transformant from the obtained transformed cells, an organ or an individual may be regenerated by a known tissue culture method. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. Regeneration from plant cells to plants can be performed, for example, as follows.
まず、形質転換の対象とする植物材料して植物組織又はプロトプラストを用いた場合、これらを無機要素、ビタミン、炭素源、エネルギー源としての糖類、植物生長調節物質（オーキシン、サイトカイニン等の植物ホルモン）等を加えて滅菌したカルス形成用培地中で培養し、不定形に増殖する脱分化したカルスを形成させる（以下「カルス誘導」という）。このように形成されたカルスをオーキシン等の植物生長調節物質を含む新しい培地に移しかえて更に増殖（継代培養）させる。 First, when plant materials or protoplasts are used as plant materials to be transformed, these are inorganic elements, vitamins, carbon sources, sugars as energy sources, plant growth regulators (plant hormones such as auxin and cytokinin) And so on, and cultured in a sterilized callus formation medium to form dedifferentiated callus that grows irregularly (hereinafter referred to as “callus induction”). The callus thus formed is transferred to a new medium containing a plant growth regulator such as auxin, and further grown (subcultured).
カルス誘導は寒天等の固型培地で行い、継代培養は例えば液体培養で行うと、それぞれの培養を効率良くかつ大量に行うことができる。次に、上記の継代培養により増殖したカルスを適当な条件下で培養することにより器官の再分化を誘導し（以下、「再分化誘導」という）、最終的に完全な植物体を再生させる。再分化誘導は、培地におけるオーキシンやサイトカイニン等の植物生長調節物質、炭素源等の各種成分の種類や量、光、温度等を適切に設定することにより行うことができる。かかる再分化誘導により、不定胚、不定根、不定芽、不定茎葉等が形成され、更に完全な植物体へと育成させる。あるいは、完全な植物体になる前の状態（例えばカプセル化された人工種子、乾燥胚、凍結乾燥細胞及び組織等）で貯蔵等を行ってもよい。 When callus induction is performed in a solid medium such as agar and subculture is performed in, for example, liquid culture, each culture can be performed efficiently and in large quantities. Next, callus grown by the above subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as “redifferentiation induction”), and finally regenerates a complete plant body. . Redifferentiation induction can be performed by appropriately setting the types and amounts of various components such as plant growth regulators such as auxin and cytokinin, carbon sources, and the like, light, temperature and the like in the medium. By such redifferentiation induction, somatic embryos, adventitious roots, adventitious shoots, adventitious foliage, etc. are formed and further grown into complete plants. Alternatively, storage or the like may be performed in a state before becoming a complete plant body (for example, encapsulated artificial seeds, dried embryos, freeze-dried cells and tissues).
本発明の形質転換植物体は、形質転換を施した再分化当代である「T1世代」のほか、その植物の種子から得られた後代である「T2世代」、薬剤選抜あるいはサザン法等による解析によりトランスジェニックであることが判明した「T2世代」植物の花を自家受粉して得られる次世代（T3世代）などの後代植物をも含む。 The transformed plant of the present invention is not only the “T1 generation” that is the regenerated generation that has undergone transformation, but also the “T2 generation” that is a progeny obtained from the seeds of the plant, analysis by drug selection or Southern method, etc. It includes progeny plants such as the next generation (T3 generation) obtained by self-pollination of "T2 generation" plants that have been found to be transgenic.
以下、実施例によって本発明を更に具体的に説明するが、これらの実施例は本発明を限定するものでない。
（実施例１） イネPOR遺伝子プロモーター配列の単離及び形質転換用ベクターへの導入
（１）プロモーターの単離
配列番号２に示す塩基配列を有するイネPOR遺伝子（DDBJ（http://www.ddbj.nig.ac.jp）:アクセッション番号AK065236）の5’上流域に位置するプロモーター配列の単離をゲノミックPCR法にて行った。イネ（品種：日本晴）幼植物体の緑葉よりDNeasy Plant Mini Kit（キアゲン社）を使って抽出したゲノムDNAを鋳型とし、DDBJ（http://www.ddbj.nig.ac.jp：アクセッション番号AL732349）に登録されているイネゲノム配列情報から設計されたプライマー#26-5［5’-ATGCAAGCTTAACGATAAGGACGAGTGTGCATGC-3’（下線部はHindIII制限酵素認識部位）：配列番号４]と#26-3 [5’-AGAGCCATGGGTGTGAGTGTTTGAGTTTGG-3’（下線部は、NcoI制限酵素認識部位）：配列番号５]をプライマーとして用い、PCRを行った。プライマーは、POR遺伝子のmRNAの情報から予測される翻訳開始コドンATGの5’側2塩基上流までに位置する1979塩基対［配列番号１において7位（1位〜6位はHindIII認識配列）の塩基から1985位（うち1985位にはNcoI認識配列ccATGgの一部）までの塩基に対応］の配列を増幅するように設計した。なお、この1979塩基対の配列中には開始コドンより106bp上流にTATA配列が存在する。また、プライマーは増幅断片のクローニングを容易にするため、増幅断片の5’末端（#26-5）及び3’末端（#26-3）に、それぞれ唯一のHindIII及びNcoI制限酵素認識部位を導入した。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.
Example 1 Isolation of rice POR gene promoter sequence and introduction into transformation vector (1) Isolation of promoter
Genomic isolation of a promoter sequence located 5 'upstream of the rice POR gene (DDBJ (http://www.ddbj.nig.ac.jp): accession number AK065236) having the base sequence shown in SEQ ID NO: 2 PCR was performed. DDBJ (http://www.ddbj.nig.ac.jp: Accession number) Using genomic DNA extracted from green leaves of rice (variety: Nipponbare) seedlings using DNeasy Plant Mini Kit (Qiagen) as a template Primer # 26-5 [5'-ATGC AAGCTT AACGATAAGGACGAGTGTGCATGC-3 '(underlined part is HindIII restriction enzyme recognition site): SEQ ID NO: 4] and # 26-3 [designed from AL732349) PCR was performed using 5′-AGAG CCATGG GTGTGAGTGTTTGAGTTTGG-3 ′ (underlined is the NcoI restriction enzyme recognition site): SEQ ID NO: 5] as a primer. The primer is a 1979 base pair [position 7 in SEQ ID NO: 1 (positions 1 to 6 are HindIII recognition sequences)] located 2 bases upstream of the translation initiation codon ATG predicted from the mRNA information of the POR gene. It was designed to amplify the sequence from the base to the position of 1985 (of which 1985 corresponds to a part of the NcoI recognition sequence ccATGg). In this 1979 base pair sequence, there is a TATA sequence 106 bp upstream from the start codon. The primers also introduce unique HindIII and NcoI restriction enzyme recognition sites at the 5 'end (# 26-5) and 3' end (# 26-3), respectively, to facilitate cloning of the amplified fragment. did.
PCR後、増幅断片（約2．0 kb）をEx Taqポリメラーゼ（Takara）を用いてdATP存在下で72℃、5分間処理して末端にAを付加し、Promega社のpGEM-T easy vector systemを用いてpGEM-T easyベクターにクローニングした。 After PCR, the amplified fragment (about 2.0 kb) was treated with Ex Taq polymerase (Takara) in the presence of dATP at 72 ° C for 5 minutes to add A to the end, and Promega pGEM-T easy vector system Was used to clone into pGEM-T easy vector.
クローニング後、インサートの塩基配列を解読し、上記の登録イネゲノム配列情報と同一であることを確認した。配列番号１にその塩基配列を示す。なお、配列番号１の塩基配列において1位〜6位までの塩基配列、1985位〜1990位までの塩基配列がクローニングに用いた制限酵素部位であり、1915位以降の塩基配列が転写領域である。
以上のイネ遺伝子プロモーター配列のクローニング手順を図１に示す。
After cloning, the base sequence of the insert was decoded and confirmed to be the same as the above registered rice genome sequence information. SEQ ID NO: 1 shows the base sequence. In the base sequence of SEQ ID NO: 1, the base sequence from the 1st to the 6th position, the base sequence from the 1985th to the 1990th position are the restriction enzyme sites used for cloning, and the base sequence from the 1915th position is the transcription region. .
The cloning procedure of the rice gene promoter sequence described above is shown in FIG.
（２）植物形質転換用バイナリーTiプラスミドベクターの構築
上記のプロモーター配列をイネに導入するために、植物形質転換用バイナリーTiプラスミドベクターpSMAHdN627-M2GUSを構築した（図２）。図２に示すように、本バイナリーベクターには、T-DNA上に植物用選抜マーカー遺伝子として、アグロバクテリウムTiプラスミド由来Nos（ノパリン合成酵素遺伝子）プロモーター：：大腸菌由来HPT（ハイグロマシン耐性）遺伝子のコード領域：：Tiプラスミド由来TiaaM（トリプトファンモノオキシゲナーゼ遺伝子）ターミネーターを配置し、形質転換された植物体がハイグロマイシンB耐性を示すようにした。また、その隣接部位にベータグルクロニダーゼ（GUS）遺伝子のコード領域：：Tiプラスミド由来Nosターミネーターから構成されるキメラ遺伝子を配置し、GUS遺伝子の5’上流側にプロモーター配列を挿入できるようにマルチクローニング部位を設けた。また、left border（LB）配列とright border（RB）の配列の外側には、微生物細胞で機能し得るスペクチノマイシン耐性（SpR）遺伝子、大腸菌で機能するpBR322由来（ColE1型）複製開始領域、アグロバクテリウム細胞内においてプラスミドが安定に保持されるための配列Sta、及び複製開始領域Repを設けた。
(2) Construction of Binary Ti Plasmid Vector for Plant Transformation In order to introduce the above promoter sequence into rice, a binary Ti plasmid vector pSMAHdN627-M2GUS for plant transformation was constructed (FIG. 2). As shown in FIG. 2, this binary vector has an Agrobacterium Ti plasmid-derived Nos (nopaline synthase gene) promoter :: E. coli-derived HPT (hygromachine resistance) gene as a plant selection marker gene on T-DNA. The coding region of :: Ti plasmid-derived TiaaM (tryptophan monooxygenase gene) terminator was placed so that the transformed plant body was resistant to hygromycin B. In addition, a chimeric gene composed of a beta glucuronidase (GUS) gene coding region :: Ti plasmid-derived Nos terminator is placed in the adjacent site, and a multicloning site is inserted so that a promoter sequence can be inserted 5 'upstream of the GUS gene. Was provided. Also, outside of the left border (LB) and right border (RB) sequences, a spectinomycin resistance (Sp R ) gene that can function in microbial cells, a pBR322-derived (ColE1 type) replication initiation region that functions in E. coli The sequence Sta for stably maintaining the plasmid in Agrobacterium cells and the replication initiation region Rep were provided.
（３）バイナリーベクターへのプロモーター配列の導入
pGEM-T easyベクターをHindIII及びNcoIで二重消化することにより、イネPOR遺伝子プロモーター断片を切り出し、（２）で構築したpSMAHdN627-M2GUSベクターの対応する部位にクローニングした。得られたプラスミドをpSMAHdN627-PRPGUSと命名し、バイオラッド社のE. coliパルサーを用いたエレクトロポレーション法（0.2 cmキュベット、パルス条件：2.4kV/cm、25μF、200Ω）により、アグロバクテリウムEHA105系統に導入した。
(3) Introduction of promoter sequence into binary vector
The rice POR gene promoter fragment was excised by double digestion of pGEM-T easy vector with HindIII and NcoI, and cloned into the corresponding site of the pSMAHdN627-M2GUS vector constructed in (2). The obtained plasmid was named pSMAHdN627-PRPGUS, and Agrobacterium EHA105 was obtained by electroporation (0.2 cm cuvette, pulse conditions: 2.4 kV / cm, 25 μF, 200Ω) using E. coli pulsars manufactured by Biorad. It was introduced into the system.
（実施例２） イネ形質転換
イネの形質転換は超迅速形質転換法（WO 01/06844 A1 (2001)参照）により行った。イネ（品種：日本晴）種子を、70 ％エタノール、続いて次亜塩素酸ナトリウムで殺菌し、滅菌蒸留水ですすいで水を切った後、胚が上向きになるようN6D寒天培地〔N6 salts 及びvitamins (Chu C.C., C.S.Wang, C.C.Sun, C. Hsu, K.C. Yin, C.Y. Chu, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica, 18, 659-668 (1975))、30 g/L ショ糖、 0.3 g/L カザミノ酸、2.8 g/L プロリン、2 mg/L 2,4-D, 2 g/L ゲルライト、pH5.7〕に置床し、30℃かつ明条件下で5日間培養した。
(Example 2) Rice transformation Rice was transformed by an ultra-rapid transformation method (see WO 01/06844 A1 (2001)). Rice (variety: Nipponbare) seeds are sterilized with 70% ethanol followed by sodium hypochlorite, rinsed with sterile distilled water, drained, and N6D agar medium [N6 salts and vitamins so that the embryos face up (Chu CC, CSWang, CCSun, C. Hsu, KC Yin, CY Chu, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica, 18, 659-668 (1975)), 30 g / L sucrose, 0.3 g / L casamino acid, 2.8 g / L proline, 2 mg / L 2,4-D, 2 g / L gellite, pH 5.7) at 30 ° C under bright conditions For 5 days.
一方、イネPOR遺伝子プロモーター断片を挿入したプラスミドpSMAHdN627-PRPGUSを保持するアグロバクテリウムEHA105系統を、25 mg/Lクロラムフェニコール、25 mg/Lリファンピシン、及び100 mg/Lスペクチノマイシンを含むLB寒天培地にて28 ℃で3日間培養した。続いて増殖した菌体をミクロスパーテルで少量かきとり、20 mg/Lアセトシリンゴンを含むAAM液体培地 〔Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J., 6, 271-282 (1994)〕に懸濁した。このアグロバクテリウム懸濁液に、N6D培地で5日間培養したイネ発芽種子を浸し、菌液をよく切ったあと、20 mg/Lアセトシリンゴンを含むAAM寒天培地に置床し、25℃の暗条件下で3日間培養した（共存培養）。共存培養後のイネ発芽種子を滅菌水、続いて500 mg/Lカルベニシリンを含む滅菌水で洗浄し、滅菌ろ紙上で余分な水分を切った後にシュート基部を四分割し、500 mg/Lカルベニシリン及び30 mg/LハイグロマイシンBを含むN6D寒天培地に置床し、30℃の明条件下で14日間培養した。その後、成長してきたシュート基部を、組換え体再分化及び選択培地〔植物体再分化培地：MS salts及びvitamins (Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant, 15, 473-497 (1962))、 30 g/l ショ糖、30 g/l ソルビトール、2 g/l カザミノ酸、0.02 mg/l NAA、2 mg/l カイネチン、2 g/l ゲルライト、pH 5.8〕に、300 mg/Lカルベニシリン + 30 mg/LハイグロマイシンBを添加したもの； Toki, S., 1997, Rapid and Efficient Agrobacterium-mediated transfomation of rice, Plant Mol Biol. Rep., 15: 16-21参照〕に移し、30℃かつ明条件下で14日間培養し、さらにこの操作をもう一度繰り返し、ハイグロマイシン耐性を有する植物体を再分化させた。このようにして得られた再分化個体をMSホルモンフリー寒天培地〔MS salts 及びvitamins（Murashige, T.ら、前掲）、30g/l ショ糖、4 g/l ゲルライト、pH5.8〕に移植し、30℃、明条件下で1〜2週間生育させ、シュート（地上部）の伸長、及び発根とその伸長を促した。シャーレ（直径9 cm）内で形質転換体のシュート及び根の長さが約10 cmあるいはそれ以上の長さに到達した際、培養土に移植し、遺伝子組換え体育成用グロースチャンバーにて明期14時間（30℃）- 暗期10時間（25℃）のサイクルでさらに生育させた。 On the other hand, Agrobacterium EHA105 strain carrying plasmid pSMAHdN627-PRPGUS inserted with rice POR gene promoter fragment was transformed into LB containing 25 mg / L chloramphenicol, 25 mg / L rifampicin, and 100 mg / L spectinomycin. The cells were cultured on an agar medium at 28 ° C. for 3 days. Subsequently, the grown cells are scraped in a small amount with microspatel, and AAM liquid medium containing 20 mg / L acetosyringone [Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J., 6, 271-282 (1994)]. Rice seeds cultured for 5 days in N6D medium were immersed in this Agrobacterium suspension, and the bacterial solution was thoroughly cut off, then placed on an AAM agar medium containing 20 mg / L acetosyringone and darkened at 25 ° C. Cultivation was performed under conditions for 3 days (co-culture). Wash the rice germinated seeds after co-cultivation with sterilized water, followed by sterilized water containing 500 mg / L carbenicillin, drain excess water on a sterilized filter paper, divide the shoot base into four parts, and add 500 mg / L carbenicillin and It was placed on an N6D agar medium containing 30 mg / L hygromycin B and cultured under light conditions at 30 ° C. for 14 days. Thereafter, the grown shoot base was transformed into a recombinant redifferentiation and selective medium [plant regeneration medium: MS salts and vitamins (Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue. cultures, Physiol. Plant, 15, 473-497 (1962)), 30 g / l sucrose, 30 g / l sorbitol, 2 g / l casamino acid, 0.02 mg / l NAA, 2 mg / l kinetin, 2 g / l Gelrite, pH 5.8] with 300 mg / L carbenicillin + 30 mg / L hygromycin B added; Toki, S., 1997, Rapid and Efficient Agrobacterium-mediated transformation of rice, Plant Mol Biol. Rep. , 15: 16-21], and cultured for 14 days at 30 ° C. and in light conditions, and this operation was repeated once more to redifferentiate plants having hygromycin resistance. The redifferentiated individuals thus obtained were transplanted to MS hormone-free agar medium [MS salts and vitamins (Murashige, T. et al., Supra), 30 g / l sucrose, 4 g / l gellite, pH 5.8]. It was grown for 1 to 2 weeks at 30 ° C. under light conditions to promote the elongation of the shoot (aboveground part) and the rooting and elongation. When the length of the shoot and root of the transformant reaches about 10 cm or more in the petri dish (9 cm in diameter), it is transplanted to the culture soil and clarified in the growth chamber for the genetically modified organism. Further growth was carried out in a cycle of 14 hours (30 ° C.)-dark 10 hours (25 ° C.).
（実施例３） 植物組織切片の作製とGUS染色による導入遺伝子の発現の観察
イネに導入したPOR遺伝子プロモーターの活性を観察するため、GUS酵素活性の組織化学的染色を実施した。実験に用いた植物組織材料のうち、カルス、花器官、根についてはイネ個体あるいはカルス（培養細胞）から切り取った材料をそのまま反応液に浸漬した。葉身については展開葉を5〜10 mmの幅で切り取り５％の寒天に包埋し、マイクロスライサー（堂阪イーエム、DTK1000）を用いて80μmの厚さの切片を作製した〔植物細胞工学 第4巻281-285頁(1992)〕。稈基部については、根を切り取った稈（かん：イネ科植物の茎を表す用語）の根元から5-10 mmの組織を切り取り、また茎頂については前出の稈基部の直上部分を10 mmほど切り取り、葉身と同様の方法で80μmの厚さの切片を作製した。こうして作製した植物材料をGUS活性測定用の反応液〔50 mMリン酸ナトリウムバッファー(pH 7.0)、1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc)、5%(v/v) メタノール、10μg/mlシクロヘキシミド、1 mMジチオスレイトール〕に浸漬し、37℃で穏やかに振盪しながら24時間置いた。その後、100％エタノールで反応液を置換して反応を停止し、さらに24時間静置することにより緑色組織の細胞より葉緑素を除去した。次にエタノールを純水で置換した後、実体顕微鏡あるいは光学顕微鏡で染色パターンを観察した。その結果、葉身、稈（茎）基部、茎頂周辺組織（幼葉部分）において、GUS遺伝子の発現を示す青色の呈色が特に強く検出された。その他の組織・器官では、カルス、花器官（穎・葯）でGUS発現が認められたが、根、維管束、冠根原基、茎頂分裂組織、節では発現が認められなかった（図３）。
(Example 3) Preparation of plant tissue section and observation of transgene expression by GUS staining In order to observe the activity of the POR gene promoter introduced into rice, histochemical staining of GUS enzyme activity was performed. Of the plant tissue materials used in the experiment, callus, flower organs, and roots were directly immersed in the reaction solution after being cut from rice individuals or callus (cultured cells). For leaf blades, the developed leaves were cut to a width of 5-10 mm, embedded in 5% agar, and 80 μm-thick sections were prepared using a microslicer (Dosaka EM, DTK1000). 4 281-285 (1992)]. For the heel base, cut 5-10 mm of tissue from the root of the cut potato (cane: a term representing the stem of the grass family), and for the shoot apex, the portion immediately above the heel base is 10 mm. A section having a thickness of 80 μm was prepared in the same manner as the blade. The plant material thus prepared was used as a reaction solution for measuring GUS activity (50 mM sodium phosphate buffer (pH 7.0), 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 5% (v / v) methanol, 10 μg / ml cycloheximide, 1 mM dithiothreitol] and placed at 37 ° C. with gentle shaking for 24 hours. Thereafter, the reaction solution was replaced with 100% ethanol to stop the reaction, and the mixture was further allowed to stand for 24 hours to remove chlorophyll from the cells of the green tissue. Next, after replacing ethanol with pure water, the staining pattern was observed with a stereomicroscope or an optical microscope. As a result, the blue color indicating the expression of the GUS gene was detected particularly strongly in the leaf blades, the heel (stem) base, and the tissue around the shoot apex (young leaf part). In other tissues / organs, GUS expression was observed in callus and flower organs (穎 / 葯), but not in roots, vascular bundles, coronal root primordia, shoot apical meristems, and nodes (Fig. 3). ).
Claims (4)
（ａ）配列表の配列番号１に示す塩基配列からなるDNA
（ｂ）配列表の配列番号１に示す塩基配列において、１若しくは数個の塩基が欠失、置換若しくは付加された塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNA
（ｃ）配列表の配列番号１に示す塩基配列に対して95％以上の相同性を有する塩基配列からなり、かつ緑色組織に特異的な発現をもたらすプロモーター活性を有するDNA DNA which can function as a promoter, shown in the following (a), (b) or (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(B) a DNA comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and having a promoter activity that brings about specific expression in green tissue
(C) DNA comprising a nucleotide sequence having a homology of 95% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and having a promoter activity that brings about specific expression in green tissue
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| WO2000058454A1 (en) * | 1999-03-26 | 2000-10-05 | Japan As Represented By Director General Of National Institute Of Agrobiological Resources, Ministry Of Agriculture, Forestry And Fisheries | Promoter sequences expressed in anthers and pollens |
| WO2000071704A1 (en) * | 1999-05-21 | 2000-11-30 | National Institute Of Agrobiological Sciences | Promoter having specific activity to pistil tissue |
| WO2002077248A1 (en) * | 2001-03-27 | 2002-10-03 | Nationalinstitute Of Agrobiological Sciences | Vegetative growth-specific promoter and genetically modified plants obtained thereby |
| WO2002077247A1 (en) * | 2001-03-27 | 2002-10-03 | National Institute Of Agrobiological Sciences | Rice-origin high expression polypeptide chain elongation factor promoter and method of using the same |
| WO2003004649A1 (en) * | 2001-07-06 | 2003-01-16 | Consejo Superior De Investigaciones Cientificas | Method of interfering with a virus infection in plants |
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| WO2000071704A1 (en) * | 1999-05-21 | 2000-11-30 | National Institute Of Agrobiological Sciences | Promoter having specific activity to pistil tissue |
| WO2002077248A1 (en) * | 2001-03-27 | 2002-10-03 | Nationalinstitute Of Agrobiological Sciences | Vegetative growth-specific promoter and genetically modified plants obtained thereby |
| WO2002077247A1 (en) * | 2001-03-27 | 2002-10-03 | National Institute Of Agrobiological Sciences | Rice-origin high expression polypeptide chain elongation factor promoter and method of using the same |
| WO2003004649A1 (en) * | 2001-07-06 | 2003-01-16 | Consejo Superior De Investigaciones Cientificas | Method of interfering with a virus infection in plants |
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