JP2021509023A - 修飾dhs遺伝子を有する植物 - Google Patents
修飾dhs遺伝子を有する植物 Download PDFInfo
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
Description
表1:
ヒトDHSの活動領域。
表2:
DHSの機能活性に重要であることが実証されているヒトDHS中のアミノ酸残基、およびアルファルファ(M.sativa)中の対応する残基。
369アミノ酸のヒトDHSタンパク質における、前述の変異、置換または欠失、およびそれらのNAD+結合、スペルミジン結合、eIF5A(Lys)結合、およびデオキシハイプシン合成酵素活性を含むDHS活性に対する影響。
1つ以上のgRNAは、アルファルファのゲノム内の所望の部位とアニールし、1つ以上のCmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質との相互作用を可能にするように設計される。これらのgRNAを、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(gRNAカセット)。Cmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質をコードする1つ以上の遺伝子を、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(CRISPRヌクレアーゼカセット)。gRNAカセットおよびCRISPRヌクレアーゼカセットを、それぞれ植物形質転換に好適なベクターにクローニングし、その後、このベクターを、アグロバクテリウム細胞に形質転換する。これらの細胞を、形質転換に好適なアルファルファ組織と接触させる。このアグロバクテリウム細胞とのインキュベーションの後、アルファルファ細胞を、アグロバクテリウム細胞に対する選択を有するインタクトな植物の再生に好適な組織培養培地で培養する。アルファルファ植物を、CRISPRヌクレアーゼカセットおよびgRNAカセットを含むベクターを保有するアグロバクテリウム細胞と接触させた細胞から再生する。アルファルファ植物の再生後、植物組織を収穫し、組織からDNAを抽出する。T7エンドヌクレアーゼI(T7EI)アッセイおよび/または配列決定アッセイを、必要に応じて実施し、所望のゲノム位置でDNA配列に変化が生じたかどうかを決定する。
1つ以上のgRNAは、Oryza sativaのゲノム内の所望の部位とアニールし、1つ以上のCmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質との相互作用を可能にするように設計される。これらのgRNAを、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(gRNAカセット)。Cmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質をコードする1つ以上の遺伝子を、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(CRISPRヌクレアーゼカセット)。gRNAカセットおよびCRISPRヌクレアーゼカセットを、それぞれ植物形質転換に好適なベクターにクローニングし、その後、このベクターを、アグロバクテリウム細胞に形質転換する。これらの細胞を、アグロバクテリウム細胞に対する選択を有し、形質転換に好適なOryza sativa組織と接触させる。このアグロバクテリウム細胞とのインキュベーションの後、Oryza sativa細胞を、インタクトな植物の再生に好適な組織培養培地で培養する。Oryza sativa植物を、CRISPRヌクレアーゼカセットおよびgRNAカセットを含むベクターを保有するアグロバクテリウム細胞と接触させた細胞から再生する。Oryza sativa植物の再生後、植物組織を収穫し、組織からDNAを抽出する。T7EIアッセイおよび/または配列決定アッセイを、必要に応じて実施し、所望のゲノム位置でDNA配列に変化が生じたかどうかを決定する。
1つ以上のgRNAは、Brassica napusのゲノム内の所望の部位とアニールし、1つ以上のCmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質との相互作用を可能にするように設計される。これらのgRNAを、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(gRNAカセット)。Cmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質をコードする1つ以上の遺伝子を、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(CRISPRヌクレアーゼカセット)。gRNAカセットおよびCRISPRヌクレアーゼカセットを、それぞれ植物形質転換に好適なベクターにクローニングし、その後、このベクターを、アグロバクテリウム細胞に形質転換する。これらの細胞を、形質転換に好適なBrassica napus組織と接触させる。このアグロバクテリウム細胞とのインキュベーションの後、Brassica napus細胞を、アグロバクテリウム細胞に対する選択を有するインタクトな植物の再生に好適な組織培養培地で培養する。Brassica napus植物を、CRISPRヌクレアーゼカセットおよびgRNAカセットを含むベクターを保有するアグロバクテリウム細胞と接触させた細胞から再生する。Brassica napus植物の再生後、植物組織を収穫し、組織からDNAを抽出する。T7EIアッセイおよび/または配列決定アッセイを、必要に応じて実施し、所望のゲノム位置でDNA配列に変化が生じたかどうかを決定する。
第1のgRNAは、目的の植物のゲノム内の第1の所望の部位とアニールし、1つ以上のCmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質との相互作用を可能にするように設計される。第2のgRNAは、目的の植物のゲノム内の第2の所望の部位とアニールし、1つ以上のCRISPRヌクレアーゼタンパク質との相互作用を可能にするように設計される。
これらのgRNAを、それぞれ植物細胞で動作可能なプロモーターに動作可能に連結し、その後、植物形質転換に好適なベクターにクローニングする。Cmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質をコードする1つ以上の遺伝子を、植物細胞で動作可能なプロモーターに動作可能に連結されるように、ベクターにクローニングする(「CRISPRヌクレアーゼカセット」)。CRISPRヌクレアーゼカセットおよびgRNAカセットを、単一の植物形質転換ベクターにクローニングし、その後、アグロバクテリウム細胞に形質転換する。これらの細胞を、形質転換に好適な植物組織と接触させる。このアグロバクテリウム細胞とのインキュベーションの後、植物細胞を、アグロバクテリウム細胞に対する選択を有するインタクトな植物の再生に好適な組織培養培地で培養する。あるいは、CRISPRヌクレアーゼカセットおよびgRNAカセットを含むベクターを、植物細胞の照射に好適な金またはチタンビーズ上にコーティングする。細胞に照射し、その後、インタクトな植物の再生に好適な組織培養培地に移す。gRNA−CRISPRヌクレアーゼ複合体は、所望のゲノム遺伝子座で二本鎖切断を引き起こし、一部の場合、DNA修復機構は、gRNA配列の近くまたはその中に位置したいくつかの天然DNA配列が欠失されるように、DNAを修復する。植物は、CRISPRヌクレアーゼカセットおよびgRNAカセットを含むベクターを有するアグロバクテリウム細胞と接触させた細胞から、またはこのベクターでコーティングされたビーズで照射された細胞から再生される。植物の再生後、植物組織を収穫し、組織からDNAを抽出する。T7EIアッセイおよび/または配列決定アッセイを、必要に応じて実施し、所望のゲノム位置または複数の位置でDNA配列に変化が生じたかどうかを決定する。
gRNAは、目的の植物のゲノム内の所望の部位とアニールし、1つ以上のCmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質との相互作用を可能にするように設計される。gRNAを、植物細胞内で動作可能なプロモーターに動作可能に連結し、その後、植物の形質転換に適したベクターにクローニングする。Cmslまたは他のCRISPR二本鎖ヌクレアーゼタンパク質をコードする1つ以上の遺伝子を、ベクターにクローニングし、それらが、宿主ゲノム内の標的DNAに相同であるが、相同性指向修復によって生じる1つ以上の標的変異を引き起こす特異的塩基変化を含有する配列から構成される高発現の一本鎖または二本鎖ドナーDNAオリゴヌクレオチドとともに、植物細胞内で動作可能なプロモーター(CRISPRヌクレアーゼカセット)に動作可能に連結されるようにする[Miki et al.(2018)Nature Comm.9:1967−1975]。CRISPRヌクレアーゼカセット、gRNAカセット、および高発現オリゴヌクレオチドを、単一の植物形質転換ベクターにクローニングし、その後、アグロバクテリウム細胞に形質転換する。これらの細胞を、形質転換に好適な植物組織と接触させる。上記ドナーDNAオリゴヌクレオチドは、植物ゲノム内の所望の部位に挿入されるDNA分子を含み、上流および下流隣接領域に隣接する。上流隣接領域は、gRNAの標的となるゲノム遺伝子座の上流のゲノムDNA領域と相同であり、下流隣接領域は、gRNAの標的となるゲノム遺伝子座の下流のゲノムDNA領域と相同である。上流および下流隣接領域は、DNAの植物ゲノムの所望の部位への組み換えを媒介する。アグロバクテリウム細胞とのインキュベーションおよびドナーDNAの導入後、植物細胞を、アグロバクテリウム細胞に対する選択を有するインタクトな植物の再生に好適な組織培養培地で培養する。植物は、CRISPRヌクレアーゼカセット、gRNAカセット、およびドナーDNAオリゴヌクレオチドを含むベクターを有するアグロバクテリウム細胞と接触させた細胞から再生される。植物の再生後、植物組織を収集し、組織からDNAを抽出する。T7EIアッセイおよび/または配列決定アッセイは、所望の塩基変化が、所望のゲノムのある位置または複数の位置で生じたかどうかを決定するために、必要に応じて実施される。
Brassica napus(キャノーラ)は、重要な油料種子の作物であり、双子葉植物である。B.napusの複二倍体ゲノムは、2つのコピーのDHS遺伝子を有し、B.rapa(AA)およびB.oleracea(CC)のゲノムからそれぞれ1つが寄与される。B.napusのLOC106419026 DHS遺伝子(DHS1)を、CRISPR−Cas9システムを使用して編集し、老化を遅延させることができるかどうかを決定した。この実施例のための方法は、B.napusにおける以前の遺伝子編集研究から適合されたもので、それらの全体が本明細書に援用される[Yang et al.,Scientific Reports 7:7489,2017]。
Claims (66)
- 遅延老化を有する植物の生産方法であって、前記植物中のデオキシハイプシン合成酵素(DHS)をコードする遺伝子の少なくとも1つのコピーに、少なくとも1つのヌクレオチドの欠失、挿入または置換を誘導することを含み、前記ヌクレオチドの欠失、挿入または置換が、前記植物中の前記遺伝子によってコードされるDHSの活性を減少させる、方法。
- 前記ヌクレオチドの欠失、挿入または置換が、DHS活性に必要な少なくとも1つのアミノ酸のコード領域において生じる、請求項1に記載の方法。
- 前記DHS活性が、前記植物中の真核生物翻訳開始因子5A(eIF−5A)のハイプシン化である、請求項2に記載の方法。
- 前記ヌクレオチドの欠失、挿入または置換が、前記植物中の前記遺伝子によってコードされる前記DHSの活性を減少させる、請求項1に記載の方法。
- 前記遅延老化が、前記植物における種子収量を増加させる、請求項1〜4のいずれか1項に記載の方法。
- 前記遅延老化が、葉および根バイオマスを増加させる、請求項1〜4のいずれか1項に記載の方法。
- 前記遅延老化が、干ばつまたは栄養ストレス中に植物の生存を増強する、請求項1〜4のいずれか1項に記載の方法。
- 前記遅延老化が、前記植物の病害抵抗性を増加させる、請求項1〜4のいずれかに1項に記載の方法。
- 前記遅延老化が、前記植物の葉、茎、種子および果実が保存され、使用に好適なままでありうる期間を増加させる、請求項1〜4のいずれか1項に記載の方法。
- 前記植物が、一倍体、二倍体、四倍体、または倍数体である、請求項1〜9のいずれか1項に記載の方法。
- 前記植物中のDHSをコードする遺伝子の少なくとも2つのコピーに、少なくとも1つのヌクレオチドの欠失、挿入または置換を誘導することを含む、請求項1〜10のいずれか1項に記載の方法。
- 前記植物が、アルファルファである、請求項1〜11のいずれか1項に記載の方法。
- 前記アルファルファが、Medicago sativaである、請求項12に記載の方法。
- 前記植物が、トマト植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記トマト植物が、Solanum lycopersicumである、請求項14に記載の方法。
- 前記植物におけるが、コムギである、請求項1〜11のいずれか1項に記載の方法。
- 前記コムギが、Triticum aestivumである、請求項16に記載の方法。
- 前記植物が、バナナ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記バナナ植物が、Musa acuminateである、請求項18に記載の方法。
- 前記植物が、キャノーラ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記キャノーラ植物が、Brassica napusである、請求項20に記載の方法。
- 前記植物が、ワタ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ワタ植物が、Gossypium hirsutumである、請求項22に記載の方法。
- 前記植物が、ヒヨコマメ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ヒヨコマメ植物が、Cicer arietinumである、請求項24に記載の方法。
- 前記植物が、ダイズ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ダイズ植物が、Glycine maxである、請求項26に記載の方法。
- 前記植物が、インゲンマメ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記マメ植物が、Phaseolus vulgarisである、請求項28に記載の方法。
- 前記植物が、コットンウッド植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記コットンウッド植物が、Populus deltoidesである、請求項30に記載の方法。
- 前記植物が、ビート植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ビート植物が、Beta vulgaris subsp.vulgarisである、請求項32に記載の方法。
- 前記植物が、ジャガイモ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ジャガイモ植物が、Solanum tuberosumである、請求項34に記載の方法。
- 前記植物が、イネ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記イネ植物が、Oryza sativa Japonica Groupである、請求項36に記載の方法。
- 前記植物が、モロコシ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記モロコシ植物が、Sorghum bicolorである、請求項38に記載の方法。
- 前記植物が、トウモロコシ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記トウモロコシ植物が、Zea maysである、請求項40に記載の方法。
- 前記植物が、Camelina sativaである、請求項1〜11のいずれか1項に記載の方法。
- 前記植物が、Brassica rapaである、請求項1〜11のいずれか1項に記載の方法。
- 前記植物が、ピーナッツ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ピーナッツ植物が、Arachis hypogaeaである、請求項44に記載の方法。
- 前記植物が、ブドウ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記ブドウ植物が、Vitis viniferaまたはVitis labruscaである、請求項46に記載の方法。
- 前記植物が、カカオである、請求項1〜11のいずれか1項に記載の方法。
- 前記カカオ植物が、Theobroma cacaoである、請求項48に記載の方法。
- 前記植物が、チャ植物である、請求項1〜11のいずれか1項に記載の方法。
- 前記チャ植物が、Camellia sinensisである、請求項50に記載の方法。
- 前記植物が、コーヒーである、請求項1〜11のいずれか1項に記載の方法。
- 前記コーヒー植物が、Coffea canephoraである、請求項52に記載の方法。
- 前記植物が、レタスである、請求項1〜11のいずれか1項に記載の方法。
- 前記レタス植物が、Lactuca sativaである、請求項54に記載の方法。
- 前記植物が、キャッサバである、請求項1〜11のいずれか1項に記載の方法。
- 前記キャッサバ植物が、Manihot esculentaである、請求項56に記載の方法。
- 前記植物が、ランである、請求項1〜11のいずれか1項に記載の方法。
- 前記ラン植物が、Phalaenopsis equestrisである、請求項58に記載の方法。
- 前記植物が、バラである、請求項1〜11のいずれか1項に記載の方法。
- 前記バラ植物が、Rosa chinensisである、請求項60に記載の方法。
- 遺伝子の少なくとも1つのコピーへの前記欠失、挿入、または置換が、配列番号2のK334、K292、K343、E328、A329、V330、S331、W332、G333、K144、D318からなる群から選択される少なくとも1つのアミノ酸、または関連植物種における対応するアミノ酸のコード領域にある、請求項1〜61のいずれか1項に記載の方法。
- 前記老化が、加齢性老化である、請求項1〜62のいずれか1項に記載の方法。
- 前記老化が、環境ストレス誘導性老化である、請求項1〜63のいずれか1項に記載の方法。
- 請求項1〜64のいずれか1項に記載の方法によって生産される植物。
- 請求項65に記載の植物の子孫。
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EP (1) | EP3731626A4 (ja) |
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CA3087064A1 (en) * | 2017-12-28 | 2019-07-04 | Agribody Technologies, Inc. | Plants with modified dhs genes |
CN111206035B (zh) * | 2020-02-17 | 2021-12-28 | 河南科技学院 | 调节目的植物叶片衰老进程的基因及方法及其在棉花作物上的应用 |
WO2024011167A2 (en) * | 2022-07-06 | 2024-01-11 | Agribody Technologies, Inc. | Plants with modified deoxyhypusine synthase genes |
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US20050155109A1 (en) * | 2003-11-17 | 2005-07-14 | Anawah Inc. | Tomato having reduced deoxyhypusine synthase activity caused by non-transgenic alterations in a deoxyhypusine synthase gene |
JP2007524384A (ja) * | 2003-06-20 | 2007-08-30 | セネスコ テクノロジーズ,インコーポレイティド | eIF−5Aアイソフォーム:老化誘導eIF−5A;損傷誘導eIF−5A;成長eIF−5A;及びDHS |
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US7262338B2 (en) * | 1998-11-13 | 2007-08-28 | Performance Plants, Inc. | Stress tolerance and delayed senescence in plants |
US20060294623A1 (en) * | 1999-07-06 | 2006-12-28 | Thompson John E | Isolated eIF-5A and polynucleotides encoding same |
US7663028B2 (en) * | 1999-07-06 | 2010-02-16 | Senesco Technologies, Inc. | Method of increasing seed yield and growth eIF-5A |
US6878860B1 (en) * | 1999-07-06 | 2005-04-12 | Senesco, Inc. | DNA encoding a plant deoxyhypusine synthase, a plant eukaryotic initiation factor 5A, transgenic plants and a method for controlling senescence programmed and cell death in plants |
EP2019142A3 (en) * | 2004-12-03 | 2009-04-01 | Senesco Technologies, Inc. | Isoforms of elF-5A and methods of using same |
EP2003953A4 (en) * | 2006-03-28 | 2009-11-11 | Cornell Res Foundation Inc | USE OF NAP-GEN TO MANIPULATE LEAFENESCENCE IN PLANTS |
CA3087064A1 (en) * | 2017-12-28 | 2019-07-04 | Agribody Technologies, Inc. | Plants with modified dhs genes |
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2018
- 2018-12-28 CA CA3087064A patent/CA3087064A1/en active Pending
- 2018-12-28 EP EP18896661.8A patent/EP3731626A4/en active Pending
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JP2007524384A (ja) * | 2003-06-20 | 2007-08-30 | セネスコ テクノロジーズ,インコーポレイティド | eIF−5Aアイソフォーム:老化誘導eIF−5A;損傷誘導eIF−5A;成長eIF−5A;及びDHS |
US20050155109A1 (en) * | 2003-11-17 | 2005-07-14 | Anawah Inc. | Tomato having reduced deoxyhypusine synthase activity caused by non-transgenic alterations in a deoxyhypusine synthase gene |
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EP3731626A1 (en) | 2020-11-04 |
EP3731626A4 (en) | 2021-10-06 |
BR112020013326A2 (pt) | 2020-12-01 |
US20210259177A1 (en) | 2021-08-26 |
US20190203220A1 (en) | 2019-07-04 |
AR114072A1 (es) | 2020-07-15 |
CA3087064A1 (en) | 2019-07-04 |
WO2019133871A1 (en) | 2019-07-04 |
AU2018395410A1 (en) | 2020-07-09 |
US10973187B2 (en) | 2021-04-13 |
MX2020006869A (es) | 2020-11-11 |
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