JP2005130833A - 種子特異的プロモーターおよびその利用 - Google Patents
種子特異的プロモーターおよびその利用 Download PDFInfo
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- JP2005130833A JP2005130833A JP2003373815A JP2003373815A JP2005130833A JP 2005130833 A JP2005130833 A JP 2005130833A JP 2003373815 A JP2003373815 A JP 2003373815A JP 2003373815 A JP2003373815 A JP 2003373815A JP 2005130833 A JP2005130833 A JP 2005130833A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8234—Seed-specific, e.g. embryo, endosperm
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Abstract
【解決手段】 複数のイネ種子発現遺伝子のプロモーターを単離し、GUSレポーター遺伝子の上流に各プロモーターを挿入したバイナリーベクターをそれぞれ作製し、アグロバクテリウム法によりイネを形質転換した。GUS発現量を指標として、各プロモーターによる発現部位、種子成熟過程での発現、さらに種子での発現強度を検討したところ、種子の特定部位に特異的発現活性を有し、恒常的プロモーターや既知の種子特異的プロモーターと比較して高い活性をもつプロモーターを見出した。
【選択図】 なし
Description
(1)下記(a)から(c)のいずれかに記載の、種子においてプロモーター活性を有するDNA
(a)配列番号:1から配列番号:7のいずれかに記載された塩基配列からなるDNA
(b)配列番号:1から配列番号:7のいずれかに記載された塩基配列に1または複数個の塩基が付加、欠失、置換、または挿入された配列からなるDNA
(c)配列番号:1から配列番号:7のいずれかに記載された配列からなるDNAとストリンジェントな条件でハイブリダイズするDNA、
(2)上記(1)に記載されたDNAの下流に任意の遺伝子が機能的に結合されているDNA
(3)上記(1)または(2)に記載されたDNAを含むベクター、
(4)上記(2)に記載されたDNAを保持した形質転換植物細胞、
(5)上記(3)に記載されたベクターが導入された形質転換植物細胞、
(6)上記(4)または(5)に記載された細胞を保持した形質転換植物体
(7)上記(6)に記載された植物体の繁殖材料
(8)種子である、上記(7)に記載された繁殖材料
(9)下記(a)および(b)の工程を含む、任意の遺伝子を植物細胞の種子において発現させる方法
(a)上記(2)に記載されたDNAまたは上記(3)に記載されたベクターを植物細胞に導入する工程
(b)該植物細胞から植物体を再生させる工程
を提供するものである。
(A)胚乳特異的プロモーターDNAグループ(各DNAの塩基配列を配列番号:1〜4に記載)
(B)胚またはアリューロン組織特異的プロモーターDNAグループ(各DNAの塩基配列を配列番号:5および6に記載)
(C)種子全体で発現するプロモーターDNAグループ(各DNAの塩基配列を配列番号:7に記載)
[実施例1] プロモーター-GUSキメラ遺伝子の構築およびトランスジェニック植物の単離
調節因子と推定されるものを調べることではなく、種子で発現される複数の遺伝子の発現パターンおよびプロモーター活性を特徴付けることとした。サイズが0.8〜2.4kbの範囲にわたる15種のプロモーターを、ゲノムDNAまたはゲノムクローンをテンプレートとして用いるPCRによって単離した。
種子貯蔵タンパク質プロモーターによって導かれるGUSレポーター遺伝子発現の部位を明らかにするために、トランスジェニックイネの種子を組織化学染色によって検査した。組織化学的分析のために、開花後(DAF)17日の段階にある成熟中の種子をカミソリ刃で長軸方向に切片化した上で、0.5mM X-Gluc(5-ブロモ-4-クロロ-3-インドイルグルクロニド)および20%メタノールを含む50mMリン酸ナトリウム緩衝液(pH 7.0)中にて37℃でインキュベートした。染色反応のための至適インキュベーション時間は、GUS活性の程度に応じて30分から一晩までさまざまであった。
検討したプロモーターのほとんどが、GUS遺伝子の胚乳特異的または胚特異的な発現を示した。しかし、GUS活性は、10kDaプロラミン、PPDKおよびAGPアーゼのプロモーター(図3)、さらにはAlaATプロモーター(Kikuchiら、Plant Mol. Biol. 39, 149-159(1999))を有するトランスジェニックイネの栄養組織中でも検出された。これらのトランスジェニックイネでは、胚乳または種子全体に加えて、葉、葉鞘および茎における維管束の師部でもGUS活性が検出された(図3-a〜c)。これらのトランスジェニックイネでは根の内皮にもGUS活性が検出された。しかし、AGPアーゼプロモーターによって生じた発現パターンはPPDKおよび10kDaプロラミンのプロモーターによるものとは幾分異なり、特に前者のプロモーターは頂端分裂組織におけるGUSの高発現を生じさせたが、一方、後者の2つのプロモーターは根組織の均一な染色をもたらした。さらに、根でのGUS活性も異なり、AGPアーゼプロモーターはPPDKプロモーターおよび10kDaプロラミンプロモーターよりも強力であった。
なお、3'転写終結領域の単離に用いたプライマー対を配列番号:23および24に示す。
発生過程にある種子における導入遺伝子発現の分布を、7、12および17 DAFに採取した種子の縦断切片の組織染色によって検討した。具体的には、開花後(DAF)7、12および17日の段階にある成熟中の種子をカミソリ刃で長軸方向に切片化した上で、0.5mM X-Gluc(5-ブロモ-4-クロロ-3-インドイルグルクロニド)および20%メタノールを含む50mMリン酸ナトリウム緩衝液(pH 7.0)中にて37℃でインキュベートした。染色反応のための至適インキュベーション時間は、GUS活性の程度に応じて30分から一晩までさまざまであった。
種々のプロモーターの強度を評価するために、GUS活性の蛍光分析アッセイをJefferson(1987)に従って行った。17 DAFの成熟中の種子をGUS抽出バッファー(50mM NaPO4、pH 7.0、10mM 2-メルカプトエタノール、10mM Na2-EDTA、0.1%SDS、0.1%Triton X-100)中でホモジネート化した。遠心処理の後に、上清の10μlを、1mM 4-メチルウンベリフェリルβ-D-グルクロニド(MUG)を含むアッセイ用バッファー90μlと混合した。37℃で1時間インキュベートした後に、0.2M Na2CO3 900μlを添加することによって反応を停止させた。蛍光光度計の値を4-メチルウンベリフェロン(4MU)希釈系列の値と比較した。タンパク質含量は、Bio-Rad Protein Assayキットを用い、血清アルブミンを標準物質として用いて評価した。各トランスジェニック植物から種子を3個ずつアッセイした。
Claims (9)
- 下記(a)から(c)のいずれかに記載の、種子においてプロモーター活性を有するDNA。
(a)配列番号:1から配列番号:7のいずれかに記載された塩基配列からなるDNA
(b)配列番号:1から配列番号:7のいずれかに記載された塩基配列に1または複数個の塩基が付加、欠失、置換、または挿入された配列からなるDNA
(c)配列番号:1から配列番号:7のいずれかに記載された配列からなるDNAとストリンジェントな条件でハイブリダイズするDNA - 請求項1に記載されたDNAの下流に任意の遺伝子が機能的に結合されているDNA。
- 請求項1または2に記載されたDNAを含むベクター。
- 請求項2に記載されたDNAを保持した形質転換植物細胞。
- 請求項3に記載されたベクターが導入された形質転換植物細胞。
- 請求項4または請求項5に記載された細胞を保持した形質転換植物体。
- 請求項6に記載された植物体の繁殖材料。
- 種子である、請求項7に記載された繁殖材料。
- 下記(a)および(b)の工程を含む、任意の遺伝子を植物細胞の種子において発現させる方法
(a)請求項2に記載されたDNAまたは請求項3に記載されたベクターを植物細胞に導入する工程
(b)該植物細胞から植物体を再生させる工程。
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CA2494570A CA2494570C (en) | 2003-10-31 | 2004-10-29 | Seed-specific gene promoters and uses thereof |
US10/978,798 US7192774B2 (en) | 2003-10-31 | 2004-11-01 | Seed-specific promoter from the rice glutelin GluB-1 gene and uses thereof |
US11/414,882 US7619135B2 (en) | 2003-10-31 | 2006-05-01 | Seed-specific promoter from the rice glutelin GluB-4 gene and uses thereof |
US11/644,759 US7595384B2 (en) | 2003-10-31 | 2006-12-22 | Seed-specific gene promoter from the rice 10 KDa prolaminin gene and uses thereof |
US11/762,586 US7700835B2 (en) | 2003-10-31 | 2007-06-13 | AGPase promoter from rice and uses thereof |
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US7550578B2 (en) * | 2001-09-26 | 2009-06-23 | Syngenta Participations Ag | Rice promoters for regulation of plant expression |
JP4019147B2 (ja) * | 2003-10-31 | 2007-12-12 | 独立行政法人農業生物資源研究所 | 種子特異的プロモーターおよびその利用 |
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US8440891B2 (en) | 2009-09-22 | 2013-05-14 | Board of Trustees of the University of Akransas, N.A. | Rice cultivar CL 142-AR |
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