JP2005514069A - Dアミノ酸を用いる選択的植物成長 - Google Patents
Dアミノ酸を用いる選択的植物成長 Download PDFInfo
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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Abstract
Description
ける高度に効率的な形質転換方法となっている(Hieiら, (1994) The Plant Journal 6, 271-282)。
繁殖または増殖することを含んでもよい。
該ポリペプチドの蓄積を上記植物の細胞質ゾルにおいて起こさせるかまたは可能にし、そして;
上記植物をD-アミノ酸基質を用いて処理することを含むものであってもよい。
方法
シロイヌナズナ(Arabidopsis)形質転換
大腸菌(E. coli)遺伝子dsdA(D-セリンデヒドラターゼ(D-セリンデアミナーゼ)[EC:4.3.1.18]NCBI受託番号J01603)を、PCRによりプライマー5'-AATGGATCCTCATCTAAGCGCAAAGAGACGTACTATGGおよび5'-ATTGGATCCATGCTGCGTTGAAACGTTATTAACGGを用いて増幅した。PCR産物をpT-easy(Promega)中にサブクローニングしてDYEnamics配列決定キット(Amersham Pharmacia biotech)を用いて配列決定した。データベース配列を用いたアラインメント分析によりdsdAの全長クローニングが成功したことが確認された。次いでクローンを、アグロバクテリア・ツメファシエンス(Agrobacterium tumefaciens)の攻撃力を解除したTi-プラスミドであるバイナリーベクターpPCV702KmのCaMV 35S発現カセットのBamH1部位中にライゲートして、ベクターpPCV702:dsdAを作製した。エンドヌクレアーゼによるこのベクターの制限分析(restriction analysis)によって挿入方向を確認した。
非形質転換植物およびD-セリンアンモニアリアーゼ形質転換植物から得たシロイヌナズナ(A. thaliana)種子を、70%エタノールおよび0.1%Tween 80を用いて10分間表面殺菌し、そして95%エタノール中で簡単にリンス洗いした。それぞれのプレート上に10個の種子をまいて、その後、プレートをガス透過性テープを用いてシールした。成長培地は半強度MS(Murashige, T. & Skoog, F. Physiol Plant 15, 310-313 (1962))(0.5%w/vスクロースおよび0.8%w/v寒天による)で、窒素を排除したものかまたは3mM硝酸塩として含むもののいずれかであった。オートクレーブ処理の後、培地に濾過殺菌したD-セリンを加えた。植物を発芽後14日間、16時間光周期で24℃にて成長させた。植物をプレートから抜き取り、0.5mM CaCl2中で3回洗浄し、次いで40℃にて48時間乾燥した後に、乾燥重量を測定した。乾燥植物のN含量を元素分析計(PerkinElmer 2400 CHN)を用いて定量した。生物量および窒素含量はプレート基準で測定した(1プレート当たり10植物体があった)。6つの独立トランスジェニック系統をD-セリンで成長する能力について評価し、系統間に実質的差異は認められなかったが、ノーザンブロット分析によるdsdA転写物レベルは異なっていた。
野生型植物成長に対するD-アミノ酸の効果
異なるN源を用いる無菌寒天培養における野生型シロイヌナズナ植物の成長を観察した。その結果を図1に示す。D-アミノ酸培地で成長させた植物については、少ししかまたは全く成長が観察されなかった。
シロイヌナズナ(A. thaliana)を、CaMV 35Sプロモーターの制御下にありD-セリンアンモニアリアーゼ[EC:4.3.1.18]をコードする大腸菌(E. coli)遺伝子dsdAを用いて形質転換した。D-セリンアンモニアリアーゼはD-セリンを植物が容易に利用する産物であるアンモニウム、ピルビン酸および水に転化し、形質転換された植物はD-セリンを唯一のN源として成長することができる。
大腸菌(E. coli)D-グルタミン酸ラセマーゼを発現するシロイヌナズナ植物の構築を、上記と本質的に同じ方法で行った。遺伝子murI、NCBI受託番号AAC76949はラセマーゼEC: 5.1.1.3をコードする。該遺伝子をクローニングするためのプライマーを、データベース配列に基づいてオープンリーディングフレームにフランキングするように設計した。
酵母菌(Rhodotorula gracilis)D-アミノ酸オキシダーゼの2つの変異体を発現するシロイヌナズナ(A. thaliana)植物の構築を上記のように行った。
ポプラおよびタバコを、シロイヌナズナの形質転換について上に記載したのと同じベクターおよび形質転換プロトコルを用いて形質転換した。
Claims (19)
- D-アミノ酸代謝活性を有するポリペプチドをコードするヌクレオチド配列を含む単離された核酸であって、該配列が植物特異的調節エレメントと機能しうる形で連結された上記核酸。
- D-アミノ酸代謝活性がオキシダーゼ活性、ラセマーゼ活性、カルボキシラーゼ活性、トランスアミナーゼ活性およびデヒドラターゼ活性からなる群から選択される、請求項1に記載の核酸。
- ポリペプチドが表1または表2に示された、請求項2に記載の核酸。
- 上記D-アミノ酸がD-ser、D-ala、D-glu、D-arg、D-lys、D-his、D-asp、D-asn、またはD-glnからなる群から選択されることを特徴とする、請求項1〜3のいずれか1項に記載の核酸。
- 異種ポリペプチドをコードするヌクレオチド配列をさらに含む、請求項1〜4のいずれか1項に記載の核酸。
- D-アミノ酸代謝活性を有する上記ポリペプチドが、植物細胞の細胞内区画における上記酵素の蓄積をもたらすトランジットペプチドを含む請求項1〜5のいずれか1項に記載の核酸。
- 請求項1〜6のいずれか1項に記載の核酸を含む発現ベクター。
- 請求項7に記載の発現ベクターを含む植物細胞。
- 請求項8に記載の植物細胞を含む植物。
- 単子葉植物、双子葉植物、裸子植物、藻類、シダまたはコケである、請求項9に記載の植物。
- 植物細胞を請求項7に記載の発現ベクターを用いて形質転換し;そしてD-アミノ酸を含む培地上で上記植物を上記細胞から再生することを含む、トランスジェニック植物を生産する方法。
- 培地が単一窒素源を含み、上記窒素源がD-アミノ酸からなる、請求項11に記載の方法。
- 請求項9または請求項10に記載の植物に対して選択的に肥料となる、D-アミノ酸を含む肥料組成物。
- 除草剤活性を有しかつ1以上のD-アミノ酸を含む組成物。
- 請求項13に記載の組成物を用いて植物を処理することを含む、植物成長を制御する方法。
- 植物中にD-アミノ酸基質を酸化するポリペプチドを発現させ、そして上記植物を上記D-アミノ酸基質を用いて処理することを含む、植物のストレス耐性を刺激する方法。
- D-アミノ酸基質を酸化するポリペプチドであるオキシダーゼを発現するトランスジェニック植物の成長を阻害する方法であって、上記植物の細胞質ゾル中に上記ポリペプチドを蓄積させ、そして上記D-アミノ酸基質を用いて植物を処理することを含む上記方法。
- D-アミノ酸基質がD-ileまたはD-asnである、請求項17に記載の方法。
- 形質転換した植物細胞を選択する方法における、請求項1〜6のいずれか1項に記載の核酸の使用。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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GBGB0201043.7A GB0201043D0 (en) | 2002-01-17 | 2002-01-17 | Plants methods and means |
PCT/EP2003/000222 WO2003060133A2 (en) | 2002-01-17 | 2003-01-13 | Selective plant growth using d-amino acids |
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JP2005514069A true JP2005514069A (ja) | 2005-05-19 |
JP4377241B2 JP4377241B2 (ja) | 2009-12-02 |
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JP2003560217A Expired - Fee Related JP4377241B2 (ja) | 2002-01-17 | 2003-01-13 | Dアミノ酸を用いる選択的植物成長 |
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US (2) | US7105349B2 (ja) |
EP (1) | EP1468096B1 (ja) |
JP (1) | JP4377241B2 (ja) |
CN (1) | CN1620506B (ja) |
AT (1) | ATE324457T1 (ja) |
AU (1) | AU2003235629B2 (ja) |
BR (1) | BR0306978A (ja) |
CA (1) | CA2471517C (ja) |
DE (1) | DE60304823T2 (ja) |
DK (1) | DK1468096T3 (ja) |
ES (1) | ES2261938T3 (ja) |
GB (1) | GB0201043D0 (ja) |
IL (2) | IL162644A0 (ja) |
MX (1) | MXPA04006606A (ja) |
NZ (1) | NZ534469A (ja) |
PT (1) | PT1468096E (ja) |
WO (1) | WO2003060133A2 (ja) |
ZA (1) | ZA200406486B (ja) |
Cited By (3)
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JP2009529863A (ja) * | 2006-03-17 | 2009-08-27 | ビーエーエスエフ プラント サイエンス ゲーエムベーハー | ダイズに対するd−アミノ酸選択 |
JP2012197249A (ja) * | 2011-03-22 | 2012-10-18 | Ajinomoto Co Inc | 農園芸用高温ストレス耐性付与剤およびそれを用いた高温ストレス耐性付与法 |
WO2022244846A1 (ja) * | 2021-05-19 | 2022-11-24 | Kagami株式会社 | 細胞増殖の調整のための組成物及び細胞増殖の調整方法 |
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RU2320722C2 (ru) | 2002-02-26 | 2008-03-27 | Зингента Лимитед | Способ избирательного получения растений с мужской или женской стерильностью |
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JP2009529863A (ja) * | 2006-03-17 | 2009-08-27 | ビーエーエスエフ プラント サイエンス ゲーエムベーハー | ダイズに対するd−アミノ酸選択 |
JP2012197249A (ja) * | 2011-03-22 | 2012-10-18 | Ajinomoto Co Inc | 農園芸用高温ストレス耐性付与剤およびそれを用いた高温ストレス耐性付与法 |
WO2022244846A1 (ja) * | 2021-05-19 | 2022-11-24 | Kagami株式会社 | 細胞増殖の調整のための組成物及び細胞増殖の調整方法 |
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CN1620506B (zh) | 2010-05-26 |
US7741540B2 (en) | 2010-06-22 |
GB0201043D0 (en) | 2002-03-06 |
AU2003235629A1 (en) | 2003-07-30 |
WO2003060133A3 (en) | 2003-12-24 |
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US20050076409A1 (en) | 2005-04-07 |
ZA200406486B (en) | 2005-10-26 |
PT1468096E (pt) | 2006-07-31 |
EP1468096B1 (en) | 2006-04-26 |
MXPA04006606A (es) | 2005-03-31 |
WO2003060133A2 (en) | 2003-07-24 |
CN1620506A (zh) | 2005-05-25 |
US20070016973A1 (en) | 2007-01-18 |
EP1468096A2 (en) | 2004-10-20 |
ES2261938T3 (es) | 2006-11-16 |
IL162644A0 (en) | 2005-11-20 |
BR0306978A (pt) | 2004-11-09 |
NZ534469A (en) | 2005-08-26 |
CA2471517A1 (en) | 2003-07-24 |
CA2471517C (en) | 2011-04-05 |
ATE324457T1 (de) | 2006-05-15 |
US7105349B2 (en) | 2006-09-12 |
DE60304823D1 (de) | 2006-06-01 |
DE60304823T2 (de) | 2007-01-25 |
IL162644A (en) | 2010-06-30 |
DK1468096T3 (da) | 2006-07-24 |
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