JP2020521512A - バナナの貯蔵寿命を延長するための組成物及び方法 - Google Patents
バナナの貯蔵寿命を延長するための組成物及び方法 Download PDFInfo
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Abstract
Description
米国特許出願公開第20130097732号明細書、
米国特許出願公開第20140075593号明細書、
Zhang,Y.ら、Efficient and transgene−free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.Nat Commun,2016.7:12617頁、
Woo,J.W.ら、DNA−free genome editing in plants with preassembled CRISPR−Cas9 ribonucleoproteins.Nat Biotechnol,2015.33(11):1162−4頁、
Svitashev,S.ら、Genome editing in maize directed by CRISPR−Cas9 ribonucleoprotein complexes.Nat Commun,2016.7:13274頁、
Luo,S.ら、Non−transgenic Plant Genome Editing Using Purified Sequence−Specific Nucleases.Mol Plant,2015.8(9):1425−7頁、
Hoffmann 2017 PlosOne 12(2):e0172630、
Chiangら、2016.SP1,2,3.Sci Rep.2016 Apr 15;6:24356。
(a)バナナ植物細胞を、バナナのエチレン生合成経路における成分をコードする核酸配列に向けられたDNA編集剤に供し、上記エチレン生合成経路をコードする核酸配列の中に機能喪失型変異を生じる工程と、
(b)上記植物細胞から植物を再生する工程と
を備える方法が提供される。
(a)本明細書に記載される植物を成長させる工程と、
(b)上記植物から果実を収穫する工程と
を備える方法が提供される。
(a)バナナ植物の1以上の細胞を請求項14から請求項22のいずれか一項に記載の核酸構築物で形質転換する工程と、
(b)上記エチレン生合成経路の上記タンパク質成分をコードするゲノム核酸配列の中に機能喪失型変異を生成する工程であって、この変異は上記タンパク質のレベルの低下又は活性の低下を生じる工程と、
(c)上記植物細胞から植物を再生する工程と
を備える方法が提供される。
(a)本明細書に記載される植物を成長させる工程と、
(b)上記植物から果実を収穫する工程と
を備える方法が提供される。
(a)バナナ植物細胞を、バナナのエチレン生合成経路における成分をコードする核酸配列に向けられたDNA編集剤に供し、上記エチレン生合成経路をコードする核酸配列の中に機能喪失型変異を生じる工程と、
(b)上記植物細胞から植物を再生する工程と
を備える方法が提供される。
二倍体マレーヤマバショウ(Musa acuminata)、野生のバナナ植物及び栽培品種の両方
Chinganバナナ
Lacatanバナナ
Lady Fingerバナナ(Sugarバナナ)
Pisang jari buaya(Crocodile fingersバナナ)
Senoritaバナナ(Monkoy、Arnibalバナナ、Cuarenta dias、Carinosa、Pisang Empat Puluh Hari、Pisang Lampung)[12]
Sinwobogiバナナ
三倍体マレーヤマバショウ、野生のバナナ植物及び栽培品種の両方
Cavendish亜族
「Dwarf Cavendish」
「Giant Cavendish」(「Williams」)
「Grand Nain」(「Chiquita」)
「Masak Hijau」
「Robusta」
「Red Dacca」
Dwarf Redバナナ
Gros Michelバナナ
East African Highlandバナナ(AAA−EA亜族)
四倍体マレーヤマバショウ、野生のバナナ及び栽培品種の両方
Bodles Altafortバナナ
Golden Beautyバナナ
マレーヤマバショウとリュウキュウバショウとの間の交雑種(Musa×paradisiaca)の四倍体栽培品種
Atanバナナ
Goldfingerバナナ
マレーヤマバショウとリュウキュウバショウとの間の交雑種の三倍体栽培品種。この群は、「純粋の」プランテン又はアフリカプランテン − その多様性の中心は中央アフリカ及び西アフリカである − から構成されるプランテン亜族を含み、中央アフリカ及び西アフリカでは、おそらく2000〜3000年前にアジアからの先祖のプランテンの導入のあと、非常に多くの栽培品種が栽培化された。
Maoli−Popo’ulu亜族 − 太平洋地域で栽培化された料理用バナナの亜族
Maquenoバナナ
Popouluバナナ
Mysore亜族 − 料理用及びデザートのバナナ[15]
Mysoreバナナ
Pisang Raja亜族
Pisang Rajaバナナ
プランテン亜族
Frenchプランテン
Green Frenchバナナ
Horn プランテン & Rhino Hornバナナ
Nendranバナナ
Pink Frenchバナナ
Tigerバナナ
Pome亜族
Pomeバナナ
Prata−anaバナナ(Dwarf Brazilianバナナ、Dwarf Prata)
Silk亜族
Latundanバナナ(Silkバナナ、Appleバナナ)
その他
Pisang Seribuバナナ
pluバナナ
マレーヤマバショウとリュウキュウバショウとの間の交雑種の四倍体栽培品種
Kalamagolバナナ
Pisang Awak(Ducasseバナナ)
マレーヤマバショウとリュウキュウバショウとの間の交雑種の二倍体栽培品種
Ney Poovanバナナ
マレーヤマバショウとリュウキュウバショウとの間の交雑種の三倍体栽培品種
Blue Javaバナナ(Ice Creamバナナ、Ney mannan、Ash プランテン、Pata hina、Dukuru、Vata)
Bluggoe亜族
Bluggoeバナナ(orinoco(オリノコ)及び「burro」としても知られる)
Silver Bluggoeバナナ
Pelipitaバナナ(Pelipia、Pilipia)
Saba亜族
Sabaバナナ(Cardaba、Dippig)
Cardabaバナナ
Benedettaバナナ
マレーヤマバショウとリュウキュウバショウとの間の交雑種の四倍体栽培品種
Tiparotバナナ
二倍体リュウキュウバショウ(Musa balbisiana)、野生のバナナ
三倍体リュウキュウバショウ、野生のバナナ及び栽培品種
Kluai Lep Chang Kut
>Ma01_g07800.1(配列番号1
>Ma01_g12130.1(配列番号2);
>Ma02_g10500.1(配列番号3);
>Ma03_g12030.1(配列番号4);
>Ma03_g27050.1(配列番号5);
>Ma04_g01260.1(配列番号6);
>Ma04_g24230.1(配列番号7);
>Ma04_g31490.1(配列番号8);
>Ma04_g35640.1(配列番号9);
>Ma04_g37400.1(配列番号10);
>Ma05_g08580.1(配列番号11);
>Ma05_g13700.1(配列番号12);
>Ma09_g19150.1(配列番号13);又は
>Ma10_g27510.1(配列番号14)
>Ma09_g04370.1(配列番号15);
>Ma06_g17160.1(配列番号16);
>Ma11_g05490.1(配列番号17);
>Ma00_g04490.1(配列番号18);
>Ma07_g15430.1(配列番号19);
>Ma01_g11540.1(配列番号20);
>Ma10_g16100.1(配列番号21);
>Ma05_g08170.1(配列番号22);
>Ma06_g14430.1(配列番号23);
>Ma05_g09360.1(配列番号24);
>Ma11_g22170.1(配列番号25);
>Ma05_g31690.1(配列番号26);
>Ma07_g19730.1(配列番号27);
>Ma06_g02600.1(配列番号28);
>Ma10_g05270.1(配列番号29);
>Ma06_g14370.1(配列番号30);
>Ma11_g05480.1(配列番号31);
>Ma06_g14410.1(配列番号32);
>Ma06_g14420.1(配列番号33);
>Ma06_g34590.1(配列番号34);
>Ma02_g21040.1(配列番号35);
>Ma11_g04210.1(配列番号36);
>Ma05_g12600.1(配列番号37);
>Ma04_g23390.2(配列番号38);
>Ma03_g06970.1(配列番号39);
>Ma05_g09980.1(配列番号40);
>Ma04_g36640.1(配列番号41);
>Ma11_g04180.1(配列番号42);
>Ma11_g02650.1(配列番号43);又は
>Ma00_g04770.1(配列番号44)
(a)バナナ植物細胞を、バナナのエチレン生合成経路における成分をコードする核酸配列に向けられたDNA編集剤に供し、上記エチレン生合成経路をコードする核酸配列の中に機能喪失型変異を生じる工程と、
(b)上記植物細胞から植物を再生する工程と
を備える方法が提供される。
GACTCTAAGATCAGGGTTAAAGG(配列番号45);
>Ma09_g19150/Ma04_g35640/Ma04_g31490
GCAGCTAACATCAGGGTTAAAGG(配列番号46)。
(a)バナナ植物の細胞を、ゲノム編集剤(上記のとおり)及び蛍光レポーターを含む核酸構築物で形質転換する工程と、
(b)フローサイトメトリ又は撮像を使用して、蛍光レポーターによって発せられる蛍光を呈する形質転換細胞を選択する工程と、
(c)上記DNA編集剤によって生成されたゲノム編集イベントを含むが上記DNA編集剤をコードするDNAを欠く細胞を得るために、上記DNA編集剤によるゲノム編集イベントを含む形質転換細胞を、上記DNA編集剤の発現を失うのに十分な時間のあいだ培養する工程と
を備える方法にも関する。
胚発生カルス及び細胞懸濁液の生成及び維持
胚発生カルスを、Ma、1988(Ma S.S.1991 Somatic embryogenesis and plant regeneration from cell suspension culture of banana。Proceedings of Symposium on Tissue culture of horticultural crops、台北、台湾、1988年3月8〜9日所収、181−188頁)及びSchoofs、1997(Schoofs H.1997.The origin of embryogenic cells in Musa.PhD thesis、KULeuven、ベルギー)によって記載されているように、未熟な雄花又は茎頂等の初期外植片から発生させる。胚形成細胞懸濁液は、新しく発生した高胚発生のカルスから液体媒体中で開始する。この培地の80%を、最初の細胞懸濁液が十分に樹立するまで(6〜9ヶ月)まで、12〜14日ごとに新しくした。
利用したトランスフェクションプラスミドは、1、G7終結配列によって終結したCaMV35sプロモーターによって駆動されるeGFP;2、Mas終結配列によって終結したCaMV35sプロモーターによって駆動されるCas9(ヒトコドンに最適化済み);3、ガイド1のためにsgRNAを駆動するAtU6プロモーター;4、ガイド2のためにsgRNAを駆動するAtU6プロモーターを含む4つのモジュールから構成された。pCAMBIA又はpRI−201−AN DNA等のバイナリーベクターを使用することができる。
ここで提案する非遺伝子組換えGEシステムを、外来性遺伝子(GFP)のDNAを標的とすることにより検証し最適化した。異なるRNAポリメラーゼIII(pol−III)プロモーターの強さを分析するために、sgRNAを、上記CRISPR Cas9複合体中のeGFPを標的とするために設計し、次いで形質転換細胞においてeGFP発現をノックアウトすることにおける異なるプロモーターの効果を試験した。
一過性発現のために、4つの転写単位を含有するプラスミドを使用した。第1転写単位は、Cas9の発現を駆動するCaMV−35Sプロモーター及びタバコモザイクウイルス(TMV)ターミネーターを含有していた。次の転写単位は、eGFPの発現を駆動する別のCaMV−35Sプロモーター及びnosターミネーターからなっていた。第3及び第4の転写単位は、各々、標的遺伝子に対するsgRNA(上述のように、各ベクターは、2つのsgRNAを含む)を発現するシロイヌナズナU6プロモーターを含有していた。
植物材料(例えば葉、カルス、細胞懸濁液)を、消化溶液(1%セルラーゼ、0.5%マセロザイム(macerozyme)、0.5%ドリセラーゼ(driselase)、0.4Mマンニトール、154mM NaCl、20mM KCl、20mM MES pH5.6、10mM CaCl2)中、室温で4〜24時間、ゆっくりと振盪しながらインキュベーションすることにより、プロトプラストを単離した。消化後、残りの植物材料をW5溶液(154mM NaCl、125mM CaCl2、5mM KCl、2mM MES pH5.6)で洗浄し、プロトプラスト懸濁液を40μm濾過器により濾過した。80gで、室温、3分間の遠心分離の後、プロトプラストを2mlのW5緩衝液に再懸濁し、氷中で重力により沈殿させた。最終のプロトプラストペレットを2mlのMMg(0.4Mマンニトール、15mM MagCl2、4mM MES pH5.6)に再懸濁し、血球計数器を使用してプロトプラスト濃度を決定した。プロトプラスト生存率をトリパンブルー染色を使用して推定した。
関連する遺伝子(上記表2のリストを参照)を標的とするPol2駆動GFP/RFP、Pol2駆動NLS−Cas9及びPol3駆動sgRNAを含有するプラスミドを、電気穿孔(BIORAD−GenePulserII;Miao及びJian 2007 Nature Protocols 2(10):2348−2353)を使用して上記細胞に導入した。500μlのプロトプラストを電気穿孔キュベットに移し、100μlのプラスミド(10〜40μg DNA)と混合した。プロトプラストを130V及び1,000Fで電気穿孔にかけ、室温で30分間インキュベーションした。1mlのプロトプラスト培地を各キュベットに加え、このプロトプラスト懸濁液を小さいペトリ皿の中へと注ぎ込んだ。24〜48時間のインキュベーションの後、蛍光を顕微鏡法により検出した。
プラスミド/RNA送達から48時間後に、細胞を収集し、そしてGFP/編集剤発現細胞について濃縮するためにフローサイトメーターを使用して蛍光タンパク質発現についてソートした[Chiang,T.W.ら、CRISPR−Cas9(D10A) nickase−based genotypic and phenotypic screening to enhance genome editing.Sci Rep,2016.6:24356頁]。この濃縮工程により、抗生物質選択を省略して、蛍光タンパク質、Cas9及びsgRNAを一過性に発現する細胞だけを収集することが可能になる。これらの細胞は、非相同末端結合(NHEJ)による標的遺伝子の編集及び対応する遺伝子発現の喪失についてさらに試験することができる。
蛍光タンパク質陽性細胞を一部サンプリングし、DNA抽出及びゲノム編集(GE)試験のために使用し、一部は液体媒体中において高希釈でプレーティングし、28〜35日間コロニー形成させた。コロニーを採集し、成長させ、2つのアリコートに分けた。1つのアリコートをDNA抽出及びゲノム編集(GE)試験並びにCRISPR DNAフリー試験(下記参照)のために使用し、他方をその状態が検証されるまで培養液中で保った。GE及びCRISPR DNAフリーであることを明確に示すものだけを次の段階へと選択した。
各コロニーから、DNAを、GFPソートしたプロトプラスト(任意の工程)のアリコートから、及びプロトプラスト由来のコロニーから抽出し、標的とした遺伝子に隣接しているプライマーを用いてPCR反応を実施した。陽性コロニーを植物を再生するために使用することになるため、コロニーをサンプリングするために測定を行う。Cas9−sgRNAを用いないこと以外は同じ方法に供したプロトプラスト由来の対照反応物を含め、これを野生型(WT)と考える。PCR産物を、次に、WTと比べて産物サイズの何らかの変化を検出するためにアガロースゲルで分離した。WT産物とは異なるPCR反応産物をpBLUNT又はPCR−TOPO(インビトロジェン(Invitrogen))にクローニングした。あるいは、シークエンシングを使用して、上記編集イベントを検証した。得られたコロニーを採集し、プラスミドを単離し、変異の性質を判定するために配列決定した。CRISPRシステムDNA/RNAを含まないことを検証するために、及びゲノムDNAレベルで変異を検出するために、ドメイン変更又は対応するタンパク質の完全な喪失を生じると予測される変異を有するクローン(コロニー又はカルス)を全ゲノムシークエンシング用に選んだ。
エチレン産生:エチレン生合成は、ガスクロマトグラフィー(GC)又はレーザーベースのアッセイ(Cristescuら、2013、前出)により小さい植物片において測定することができる。
バナナのACS遺伝子及びACO遺伝子におけるゲノム編集並びに植物再生
Claims (28)
- バナナのエチレン生合成経路における成分をコードする核酸配列の中に機能喪失型変異を含むゲノムを含むバナナ植物。
- バナナの貯蔵寿命の延長方法であって、
(a)バナナ植物細胞を、バナナのエチレン生合成経路における成分をコードする核酸配列に向けられたDNA編集剤に供し、前記エチレン生合成経路をコードする核酸配列の中に機能喪失型変異を生じる工程と、
(b)前記植物細胞から植物を再生する工程と
を備える方法。 - 前記植物から果実を収穫する工程をさらに備える請求項2に記載の方法。
- 前記植物が、前記DNA編集剤をコードする導入遺伝子を欠く請求項1又は請求項2に記載の植物又は方法。
- 前記変異がホモ接合型である請求項1から請求項4のいずれか一項に記載の植物又は方法。
- 前記エチレン生合成経路における前記成分をコードする前記ゲノム配列に向けられたDNA編集剤で処理されたものである請求項1、請求項4又は請求項5に記載の植物又はその祖先。
- 前記変異が、欠失、挿入、挿入/欠失(インデル)及び置換からなる群から選択される請求項1から請求項6のいずれか一項に記載の植物又は方法。
- 前記エチレン生合成経路における前記成分が、1−アミノシクロプロパン−1−カルボン酸シンターゼ(ACS)及びACCオキシダーゼ(ACO)からなる群から選択される請求項1から請求項6のいずれか一項に記載の植物又は方法。
- 植物プロモーターに動作可能に連結されているバナナのエチレン生合成経路における成分をコードする核酸配列に向けられたDNA編集剤をコードする核酸配列を含む核酸構築物。
- 前記DNA編集剤が、メガヌクレアーゼ、ジンクフィンガーヌクレアーゼ(ZFN)、転写活性化因子様エフェクターヌクレアーゼ(TALEN)及びCRISPR−Casからなる群から選択されるDNA編集システムのものである請求項2から請求項8のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤がCRISPR−Casを含むDNA編集システムのものである請求項2から請求項8のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記エチレン生合成経路における前記成分が、Ma04_g35640(配列番号9)及びMa07_g19730(配列番号27)からなる群から選択される請求項1から請求項11のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記エチレン生合成経路における前記成分が、Ma09_g19150(配列番号13)、Ma04_g35640(配列番号9)、Ma04_g31490(配列番号8)、Ma01_g11540(配列番号20)及びMa07_g19730(配列番号27)からなる群から選択される請求項1から請求項11のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記エチレン生合成経路における前記成分が、Ma04_g35640(配列番号9)及びMa07_g19730(配列番号27)からなる群から選択される請求項1から請求項11のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記エチレン生合成経路における前記成分が、Ma09_g19150(配列番号13)、Ma04_g31490(配列番号8)及びMa01_g11540(配列番号20)からなる群から選択される請求項1から請求項11のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、前記エチレン生合成経路における前記成分をコードする複数の核酸配列を特異的に標的とする核酸座標に向けられている請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、配列番号47〜配列番号54からなる群から選択される核酸配列に少なくとも99%同一の核酸配列を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、配列番号47に示される核酸配列に少なくとも99%同一の核酸配列を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が配列番号47に示される核酸を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、配列番号47〜配列番号54に示される複数の核酸配列を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、配列番号47、配列番号49又は配列番号50に示される複数の核酸配列を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 前記DNA編集剤が、配列番号51及び配列番号53に示される複数の核酸配列を含む請求項1から請求項13のいずれか一項に記載の植物、方法又は核酸構築物。
- 請求項1、請求項4から請求項8、請求項10及び請求項11のいずれか一項に記載の植物の植物部分。
- 果実である請求項23に記載の植物部分。
- 乾燥している請求項24に記載の果実。
- バナナの製造方法であって、
(a)請求項1、請求項4から請求項8、請求項10及び請求項11のいずれか一項に記載の植物を成長させる工程と、
(b)前記植物から果実を収穫する工程と
を備える方法。 - バナナのエチレン生合成経路における成分をコードする核酸配列の中に機能喪失型変異を含むゲノムバナナDNAを含む加工バナナ製品。
- 前記バナナ植物は非遺伝子組換えのものである請求項1から請求項8及び請求項10から請求項27のいずれか一項に記載の植物又は方法又は加工製品。
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WO2024063076A1 (ja) * | 2022-09-21 | 2024-03-28 | 学校法人玉川学園 | 生産方法、ブロッコリ、及び種子 |
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KR20170026490A (ko) | 2011-11-28 | 2017-03-08 | 아르셀러미탈 인베스티가시온 와이 데살롤로 에스엘 | 1700 ~ 2200 ㎫ 인장 강도를 갖는 마텐자이트 강 |
WO2020249593A1 (en) * | 2019-06-13 | 2020-12-17 | Nunhems B.V. | Tomato plant producing fruit having improved ripening characteristics |
WO2020263561A1 (en) | 2019-06-26 | 2020-12-30 | Eg Crop Science, Inc. | Identification of resistance genes from wild relatives of banana and their uses in controlling panama disease |
CN111073903A (zh) * | 2019-11-15 | 2020-04-28 | 青岛农业大学 | 利用基因枪法和液体培养基抗性筛选的香蕉遗传转化方法 |
CN113755490B (zh) * | 2020-06-01 | 2023-06-13 | 广东省农业科学院果树研究所 | 基于CRISPR/Cas9的香蕉MaACO1基因编辑载体及其构建方法和应用 |
WO2023111130A1 (en) | 2021-12-17 | 2023-06-22 | Tropic Biosciences UK Limited | Modified agrobacteria for editing plants |
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EP3630983A1 (en) | 2020-04-08 |
CN110892074A (zh) | 2020-03-17 |
JP7273033B2 (ja) | 2023-05-12 |
CR20190575A (es) | 2020-01-24 |
PH12019550255A1 (en) | 2020-07-13 |
CO2019014469A2 (es) | 2020-01-17 |
CA3064502A1 (en) | 2018-12-06 |
US20200102570A1 (en) | 2020-04-02 |
AU2018275356A1 (en) | 2020-01-16 |
ECSP19090449A (es) | 2019-12-27 |
BR112019025498A2 (pt) | 2020-06-23 |
JP2023071706A (ja) | 2023-05-23 |
IL271043A (en) | 2020-01-30 |
WO2018220581A1 (en) | 2018-12-06 |
CR20220425A (es) | 2023-01-18 |
US20230365984A1 (en) | 2023-11-16 |
GB201708662D0 (en) | 2017-07-12 |
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