JP6463724B2 - 構成ダイズプロモーター - Google Patents
構成ダイズプロモーター Download PDFInfo
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- JP6463724B2 JP6463724B2 JP2016501197A JP2016501197A JP6463724B2 JP 6463724 B2 JP6463724 B2 JP 6463724B2 JP 2016501197 A JP2016501197 A JP 2016501197A JP 2016501197 A JP2016501197 A JP 2016501197A JP 6463724 B2 JP6463724 B2 JP 6463724B2
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Description
本出願は、2013年3月15日出願の米国仮特許出願第61/790,907号明細書(その内容はその全体が参照により本明細書に組み込まれる)の利益を主張する。
配列リストのコピーは、4.14キロバイトのサイズを有する2014年3月10日作成のファイル名「2912939−20179WO01_Sequence_Listing.txt」のASCII形式配列リストとしてEFS−Webを介して電子的に送付され、本明細書と同時に提出されている。このASCII形式ドキュメントに含まれる配列リストは、本明細書の一部であり、その全体が参照により本明細書に組み込まれる。
本発明に係るヌクレオチド配列は、配列番号1および2に示されるプロモーター配列およびその変異体を含む。「プロモーター」とは、下流コード配列の転写を指令するように機能する核酸配列であることが意図される。プロモーターは、一般的には、転写開始(キャップ)部位の5’側にある約10〜30塩基対のコンセンサス5’−TATAAT−3’(TATAボックス)と相同なDNA配列を含み、これによりRNAポリメラーゼにRNA合成を開始するように指令可能である。プロモーターは、一般的にはTATAボックスの上流または5’側に、上流プロモーターエレメントとして参照される転写開始速度に影響を及ぼす他の認識配列をさらに含みうる。これらは、CAATボックスを含み、これは、多くの場合、TATAボックスの5’側に約30〜70塩基対で見いだされ、カノニカル形5’−CCAAT−3’に対する相同性を有する(Breathnach and Chambon(1981)Ann.Rev.Biochem.50:349−383)。植物では、CAATボックスは、トリプレットG(またはT)NGに対称的にフランキングするアデニン残基を有する領域であるAGGAボックスとして知られる配列と置き換えられることがある(Messing et al.(1983),in Genetic Engineering of Plants,T.Kosuge,C.Meredith and A.Hollaender(eds.),Plenum Press,New York,pp.211−227)。これらのエレメントは、他の転写および翻訳調節核酸配列(「制御配列」とも称される)と共に、対象のDNA配列の発現に必要である。本明細書に記載されていない調節エレメント、たとえば、エンハンサーおよびコード領域の組織発現または時間発現に関与するエレメントを単離および同定する方法は、当技術分野で周知である。たとえば、米国特許第5,635,618号明細書、同第6,218,140号明細書、同第6,303,370号明細書、同第6,310,197号明細書、および同第6,355,864号明細書を参照されたい。
本発明に係る方法は、植物および植物細胞において本発明に係るプロモーター配列の制御下で異種ヌクレオチド配列を発現させることに関する。トランスジェニック植物は、病原体または虫類に対する防衛機構の改変、1種以上の除草剤に対する耐性の増加、ストレスを引き起こす環境条件に耐える能力の増加、デンプンを産生する能力の修飾、デンプン産生レベルの修飾、油の含有率および/または組成の修飾、窒素を利用、分配、および/または貯蔵する能力の修飾などを含めて、表現型の変化を有しうるが、これらに限定されるものではない。これらの結果は、植物において、異種遺伝子の発現を介してまたは内因性産物の発現の増加により、達成可能である。他の選択肢として、結果は、植物において、1種以上の内因性産物、特定的には、酵素、トランスポーター、もしくは補因子の発現を低減することにより、または養分吸収に影響を及ぼすことにより、達成可能である。
本明細書に開示されたダイズプロモーターに機能可能に結合された異種ヌクレオチド配列は、標的遺伝子のアンチセンスヌクレオチド配列でありうる。「アンチセンスヌクレオチド配列」とは、ヌクレオチド配列の5’側から3’側への通常の方向とは逆の方向の配列であることが意図される。植物細胞におけるアンチセンスDNA配列の発現は、標的遺伝子の正常発現を妨害する。アンチセンスヌクレオチド配列は、標的遺伝子の転写により産生された内因性メッセンジャーRNA(mRNA)に相補的なかつそれにハイブリダイズ可能なRNA転写物をコードする。このようにして、標的遺伝子によりコードされた天然タンパク質の産生が阻害され、所望の表現型反応が達成される。配列が対応するmRNAにハイブリダイズして発現を妨害するかぎり、アンチセンス配列の修飾を行いうる。対応するアンチセンス配列に対して約70%、80%、85%、90%、または95%の配列同一性を有するアンチセンス構築物を使用しうる。さらに、アンチセンスヌクレオチドの一部分を用いて標的遺伝子の発現を撹乱しうる。一般的には、少なくとも50隣接ヌクレオチド、100隣接ヌクレオチド、200隣接ヌクレオチド、またはそれ以上の配列を使用しうる。したがって、植物において、本明細書に開示されたプロモーター配列をアンチセンスDNA配列に機能可能に結合して、天然タンパク質の発現を低減または阻害しうる。
当技術分野で公知のいくつかの技術の1つにより、植物細胞の形質転換を達成することが可能である。「植物」とは、全植物体、植物器官(たとえば、葉、茎、根など)、種子、植物細胞、繁殖体、胚、およびその後代であることが意図される。植物細胞は、分化または未分化でありうる(たとえば、カルス、懸濁培養細胞、プロトプラスト、葉細胞、根細胞、篩部細胞、花粉)。「トランスジェニック植物」または「形質転換植物」または「安定に形質転換された」植物もしくは細胞もしくは組織とは、外因性の核酸配列またはDNA断片が植物細胞に取り込まれたまたは一体化された植物を意味する。「安定な形質転換」とは、植物に導入されたヌクレオチド構築物が植物のゲノムに一体化されてその後代に遺伝可能であることが意図される。
本発明に係る方法は、植物中にヌクレオチド構築物を導入することを含む。「導入」とは、構築物が植物の細胞の内部へのアクセスを取得するように、植物にヌクレオチド構築物を提示することが意図される。本発明に係る方法は、植物にヌクレオチド構築物を導入する特定の方法を使用する必要はないが、ただし、ヌクレオチド構築物が植物の少なくとも1つの細胞の内部へのアクセスを取得する。植物中にヌクレオチド構築物を導入する方法は、限定されるものではないが、安定形質転換法、一過性形質転換法、およびウイルス媒介法を含めて、当技術分野で公知である。
本発明は、限定されるものではないが、単子葉植物および双子葉植物を含めて、任意の植物種の形質転換に使用されうる。対象の植物の例としては、トウモロコシ(メイズ)、ソルガム、コムギ、ヒマワリ、トマト、アブラナ、コショウ、ジャガイモ、ワタ、コメ、ダイズ、テンサイ、サトウキビ、タバコ、オオムギ、およびセイヨウアブラナ、アブラナ属(Brassica)の種、アルファルファ、ライムギ、雑穀、サフラワー、ラッカセイ、サツマイモ、キャッサバ、コーヒー、ココヤシ、パイナップル、柑橘類、ココア、茶、バナナ、アボカド、イチジク、グアバ、マンゴー、オリーブ、パパイヤ、カシュー、マカダミア、アーモンド、エンバク、野菜、観賞植物、および針葉樹が挙げられるが、これらに限定されるものではない。
異種外来DNAを植物細胞内に導入した後、種々の方法により、たとえば、一体化DNAに関連する核酸またはタンパク質および代謝物の解析により、植物ゲノムにおける異種DNAによる形質転換または一体化が確認される。
植物においてプロモーター活性を評価するための多くの方法が利用可能である。調節制御下での対象の遺伝子の発現時のプロモーター機能は、転写または翻訳の段階で試験されうる。転写の段階では、DNA−RNAハイブリダイゼーションアッセイ(すなわち、ノーザンブロット解析)、競合逆転写酵素PCR、およびRNアーゼ保護アッセイにより、RNAレベルを試験しうる。翻訳の段階では、合成タンパク質に特異的な機能アッセイを用いて(たとえば、酵素活性によりまたはタンパク質の免疫アッセイにより)、プロモーター活性を決定しうる。たとえば、レポーター遺伝子活性、たとえば、β−グルクロニダーゼ活性、ルシフェラーゼ活性、またはGFP蛍光を、形質転換後の種々の時間で、モニターしうる。レポーター遺伝子活性は、酵素活性により、レポーター遺伝子によりコードされた酵素に対する基質を用いて細胞または組織を染色することにより、または適切な波長の光の下で直接可視化することにより、モニターしうる(たとえば、Wang et al.(2000)Plant Science 156:201−211を参照されたい)。タンパク質上に存在する1つ以上のエピトープに結合する抗体を用いた標準的手順により、トランスジェニック植物でウェスタンブロットを行って、TripPro5プロモーターに機能可能に結合された対象の遺伝子によりコードされたタンパク質の存在を確認しうる(Sambrook and Russell,2001,前掲)。全長プロモーター配列、プロモーター配列の欠失および突然変異をアッセイし、それらの発現レベルを比較しうる。たとえば、米国特許第6,072,050号明細書、およびSambrook et al.(1989)Molecular Cloning:A Laboratory Manual(2d ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York)(参照により本明細書に組み込まれる)を参照されたい。
実施例1.ダイズに由来する構成プロモーターの同定
公共のダイズトランスクリプトームデータベースを用いて、異なる組織(葉、莢、花、根など)で高度に発現される遺伝子を同定した。これらの遺伝子のプロモーター領域(最初のATGの上流)をダイズゲノムDNA(Jack)からPCR増幅し、ルシフェラーゼ遺伝子コード領域およびPinIIターミネーターに結合した。これらのプロモーター含有ベクターをアグロバクテリウム(Agrobacterium)に導入して形質転換させた。形質転換アグロバクテリウム(Agrobacterium)を用いて、幼少ダイズまたはインゲンマメ葉片を浸潤させた。16時間の光の下、25℃で2日間インキュベートした後、タンパク質抽出のために葉片をPBS緩衝液中でホモジナイズした。次いで、Promega STEADY−GLO(登録商標)ルシフェラーゼアッセイシステムを用いて、ルシフェラーゼ活性に関して可溶性タンパク質をアッセイした。各ベクターに対して浸潤したダイズ葉片の3つの独立したセットの平均のルシフェラーゼ活性を図1に示す。Pbdc6およびPbdc7は、アラビドプシスに由来するPubi3と同等の活性を呈した。Pbdc6(配列番号1)は、γ液胞膜内在性タンパク質をコードするGlyma03g34310から取得した。Pbdc7(配列番号2)は、形質膜内在性タンパク質をコードするGlyma23g42220から取得した。
プロモーターPbdc6およびPbdc7をそれぞれ有するDNA配列をpSZ8133中にクローニングし、これらのプロモーターをgrg23Ace5に結合した。得られたバイナリーベクターpSZ8806およびpSZ8807をアグロバクテリウム(Agrobacterium)LBA4404中に導入して形質転換させ、これを用いてトランスジェニックダイズ植物を生成した。4×グリホセートスプレーを用いて、各ベクターの約150のトランスジェニックイベントをアッセイした。スプレーの1週間後、4×グリホセートに対する耐性をスコア付けした(表2、0は耐性がないことを意味し、4は最も強い耐性を表す)。UBQ3を対照として使用した。この場合も、Pbdc6およびPbdc7は、Pubi3Atと同等の強度を示した。
Claims (20)
- 異種核酸に機能可能に結合されているヌクレオチド配列を含んでなる核酸分子を含んでなる発現カセットであって、前記ヌクレオチド配列が
(a)配列番号2に示されるヌクレオチド配列、および
(b)前記配列番号2に示される配列に対して少なくとも99%の配列同一性を有するヌクレオチド配列であって、前記配列が植物細胞において前記異種核酸の転写を開始する、ヌクレオチド配列、
からなる群から選択される、発現カセット。 - 請求項1に記載の発現カセットを含んでなるベクター。
- 植物細胞であって、そのゲノム中に請求項1に記載の発現カセットが安定に取り込まれており、前記ヌクレオチド配列が対象の異種核酸に機能可能に結合されている、細胞。
- 前記植物細胞が双子葉植物に由来する、請求項3に記載の植物細胞。
- 前記双子葉植物がダイズである、請求項4に記載の植物細胞。
- 植物であって、そのゲノム中に請求項1に記載の発現カセットが安定に取り込まれていおり、前記ヌクレオチド配列が対象の異種核酸に機能可能に結合されている、植物。
- 前記植物が双子葉植物である、請求項6に記載の植物。
- 前記双子葉植物がダイズである、請求項7に記載の植物。
- 請求項1に記載の発現カセットを含むトランスジェニック種子。
- 前記対象の異種核酸が、除草剤、塩、病原体、または有害生物に対する耐性を付与する遺伝子産物をコードする、請求項6に記載の植物。
- 植物において対象の異種核酸を発現させる方法であって、
対象の異種核酸に機能可能に結合されたプロモーターを含む発現カセットを植物細胞中に導入することであって、前記プロモーターが、
(a)配列番号2に示されるヌクレオチド配列、および、
(b)前記配列番号2に示される配列に対して少なくとも99%の配列同一性を有するヌクレオチド配列であって、前記ヌクレオチド配列が植物細胞において対象の異種核酸の転写を開始する、ヌクレオチド配列、
からなる群から選択されるヌクレオチド配列を含む、導入することと、
前記植物細胞から形質転換植物を再生することであって、前記植物のゲノム中に前記発現カセットが安定に取り込まれている、再生することと、
を含む、方法。 - 前記植物が単子葉植物である、請求項11に記載の方法。
- 前記植物が双子葉植物である、請求項11に記載の方法。
- 前記単子葉植物がメイズである、請求項12に記載の方法。
- 前記異種核酸が、除草剤耐性または有害生物に対する耐性を付与する遺伝子産物をコードする、請求項11に記載の方法。
- 植物細胞において対象の異種核酸を発現させる方法であって、
対象の異種核酸に機能可能に結合されたプロモーターを含む発現カセットを植物細胞中に導入することを含み、前記プロモーターが、
(a)配列番号2に示されるヌクレオチド配列、および
(b)前記配列番号2に示される配列に対して少なくとも99%の配列同一性を有するヌクレオチド配列であって、前記配列が植物細胞において対象の異種核酸の転写を開始する、ヌクレオチド配列、
からなる群から選択されるヌクレオチド配列を含む、方法。 - 前記植物細胞が単子葉植物細胞である、請求項16に記載の方法。
- 前記植物細胞が双子葉植物細胞である、請求項16に記載の方法。
- 前記単子葉植物がメイズである、請求項17に記載の方法。
- 前記異種核酸が、除草剤耐性または有害生物に対する耐性を付与する遺伝子産物をコードする、請求項16に記載の方法。
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2014
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- 2014-03-11 EP EP14719208.2A patent/EP2970405B1/en active Active
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WO2014150449A2 (en) | 2014-09-25 |
US20150376643A1 (en) | 2015-12-31 |
JP2016514963A (ja) | 2016-05-26 |
BR112015022495A2 (pt) | 2017-10-24 |
EP2970405A2 (en) | 2016-01-20 |
CN105102474B (zh) | 2019-11-12 |
EP2970405B1 (en) | 2018-09-12 |
BR112015022495A8 (pt) | 2022-06-28 |
CA2903226C (en) | 2022-03-15 |
WO2014150449A3 (en) | 2014-11-13 |
CN105102474A (zh) | 2015-11-25 |
MX359956B (es) | 2018-10-16 |
CA2903226A1 (en) | 2014-09-25 |
RU2714724C2 (ru) | 2020-02-19 |
AU2014237167A1 (en) | 2015-10-01 |
US9944938B2 (en) | 2018-04-17 |
RU2015144014A (ru) | 2017-04-21 |
AU2014237167B2 (en) | 2018-07-12 |
ES2702289T3 (es) | 2019-02-28 |
UA118845C2 (uk) | 2019-03-25 |
MX2015012171A (es) | 2015-11-30 |
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