JP6093303B2 - Alsインヒビター除草剤耐性ベータ・ブルガリス突然変異体 - Google Patents
Alsインヒビター除草剤耐性ベータ・ブルガリス突然変異体 Download PDFInfo
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Classifications
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- A01H6/024—Beta vulgaris [beet]
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Description
(%配列同一性×%最大BLASTスコア)/100
として定義される積スコアであり、それは、2つの配列間の類似性の度合および配列マッチの長さの両方を考慮する。例えば、40の積スコアにおいて、該マッチは1〜2%の誤差内で完全であり、70においては、該マッチは完全であろう。類似分子は通常、15〜40の積スコアを示すものを選択することにより特定されるが、より低いスコアが関連分子を特定することもある。
(a)カルス、好ましくはテンサイ由来のカルスを、約10−7M〜10−9MのALSインヒビター除草剤、好ましくはホラムスルフロン(foramsulfuron)にさらし、
(b)3×10−6MまでのALSインヒビター除草剤、好ましくはホラムスルフロン[CAS RN 173159−57−4]の存在下で成長しうる細胞コロニーを選択し、
(c)ALSインヒビター除草剤、好ましくはホラムスルフロンの存在下でシュートを再生させ、
(d)ALSインヒビター除草剤、好ましくはホラムスルフロン、ヨードスルフロン(iodosulfuron)−メチル−ナトリウム[CAS RN 144550−36−7]および/またはそれらの両方の混合物(ここで、ホルムスルフロンの用量は、好ましくは、7〜70g a.i./haと同等であり、ヨードスルフロン−メチル−ナトリウムの用量は、好ましくは、1〜10g a.i./haと同等である)で、再生された小植物を選択することを含む、ベータ・ブルガリス植物およびその部分の製造方法に関する。
(e)各ALSインヒビター除草剤耐性小植物細胞系(クローン)を樹立することによる、種々の陽性変異体をレスキューするための、工程(d)の個々の小植物の栄養微小繁殖(vegetative micropropagation)、
(f)栄養(vegetative)状態における各樹立クローンの長期貯蔵、
(g)該長期貯蔵からの各クローンのクローン化植物の、温室内への移動、
(h)開花を誘導するための春化室内での春化および適応、
(i)成長室(制御された温度および光)への春化植物の移動、
(j)工程(d)の小植物の生殖稔性(generative fertility)(雄および雌)に対する体細胞クローン変異の負の影響に対処するための、優良(elite)であるがALSインヒビター除草剤感受性である系の除雄植物との交配のための、最良開花クローンの最良花粉放出植物の選択、
(k)稔性が回復するまでは優良系への、そして最終的には、ホモ接合状態を得るための自家ヘテロ接合植物への戻し交配、
(l)圃場評価のための各戻し交配系の自殖種子およびALSインヒビター除草剤感受性相手との検定交雑、
(m)最良性能系(好ましくは、そのホモ接合状態のもの)を選択するための、農業的に適切な用量の種々のALSインヒビター除草剤の施用。
(a)内因性アセト乳酸シンターゼ(ALS)遺伝子の突然変異を含むALSインヒビター除草剤耐性ベータ・ブルガリス植物(親A)を得(ここで、該ALS遺伝子は、ALSポリペプチドの569位にトリプトファンとは異なるアミノ酸を含有するALSポリペプチドをコードしている)、
(b)アミノ酸569位とは異なる位置における内因性ALS遺伝子内の1以上の他の突然変異を含有するベータ・ブルガリス植物(親B)と親Aを交配させ、
(c)アミノ酸569位のALS遺伝子突然変異に関して、および親Bによりコードされるいずれかの他のALS遺伝子突然変異の1以上に関してヘテロ接合であるベータ・ブルガリス後代を得、
(d)ここで、該育種法は、
(i)標識支援育種および/もしくはミクロシークエンシング技術の適用、ならびに/または
(ii)工程(c)により得られた後代が耐性である1以上のALSインヒビター除草剤の農業的に適切な用量の施用により制御される。
テンサイ細胞培養を2倍体テンサイ遺伝子型7T9044の実生から開始した(例えば、Alexander Dovzhenko,PhD Thesis,題名:“Towards plastid transformation in rapeseed(Brassica napus L.)and sugarbeet(Beta vulgaris L.)”,Ludwig−Maximilians−Universitat Munchen,Germany,2001に記載されているとおり)。
SB574TLに基づいて、ヘテロ接合状態で耐性対立遺伝子を付与する実験的ハイブリッドのF2およびF3種子、ならびにホモ接合状態で突然変異対立遺伝子を付与するF4〜F6種子を圃場に播種し、該植物が3〜5個のロセット葉を生成したら、ホラムスルフロン、ヨードスルフロン−メチル−ナトリウムおよび両方のALSインヒビター除草剤の混合物で処理した。該ホモ接合実生は、成長遅延または視認可能な損傷のいずれの徴候をも伴うことなく、35gのホラムスルフロン/ha+7gのヨードスルフロン−メチル−ナトリウム/haの混合物に耐えた。ヘテロ接合系は、これらの比率において遅延成長および何らかの葉クロロシスの徴候を示したが、それらは3〜5週間以内に回復した。一方、通常のテンサイ実生は該ALSインヒビター除草剤により枯死した。
得られた突然変異体の抽出および核酸配列解析は、修正された標準的プロトコールに従いLGC Genomics GmbH,Berlin,Germanyにより行われた。
ベータ・ブルガリス野生型およびW574L−突然変異体(SB574TL)ALS遺伝子のコード配列をNovagen pET−32a(+)ベクター内にクローニングし、該ベクターを、大腸菌(Escherichia coli)AD494内に、該製造業者の説明に従い形質転換した。細菌を、100mg/ml カルベニシリンおよび25mg/l カナマイシンを含有するLB−培地(ルリア−ブロス−培地)内で37℃で増殖させ、1mM イソプロピル−b−D−チオガラクトピラノシドで0.6のOD600で誘導し、18℃で16時間培養し、遠心分離により回収した。細菌ペレットを、0.1mM チアミン−ピロリン酸、1mM MgCl2および1μM FADを含有する100mM リン酸ナトリウムバッファー(pH7.0)に、25mlのバッファー当たり1グラムの湿潤重量の濃度で再懸濁させ、音波処理により破壊した。遠心分離後に得られた粗タンパク質抽出物をALS活性の測定に使用する。
Ray(1984),Plant Physiol 75:827−831に記載されているとおりに、テンサイ葉またはテンサイ組織培養からALSを抽出した。
SB574TLに基づいて、ホモ接合状態で内因性ALS遺伝子の突然変異対立遺伝子を付与するF4〜F6種子を更なる試験のために施用した。ホモ接合SB574TL突然変異植物の植物種子ならびに伝統品種KLARINA(ALSタンパク質の569位にそれぞれの突然変異を有さない、一般的に入手可能なALSインヒビター感受性基準テンサイ品種)の種子を圃場に播き、BBCH標準(monographie“Entwicklungsstadien mono− und dikotyler Pflanzen”,2nd edition,2001,Uwe Meier編,Biologische Bundesanstalt fur Land und Forstwirtschaftに定義されているとおり)に従い種々の成長段階まで成長させた。ついで該植物を、選択法において使用されてる以下の表1に特定されているそれぞれのALSインヒビター除草剤で試験した。種々の施用において施用した水量は200 l/haに等しかった。それぞれのALSインヒビター除草剤の施用の8、14および28日後(DAA)(表1に示されているとおり)、種々のテンサイ植物に対する損傷(植物毒性)を0%から100%までの尺度に従い評価した。この場合、「0%」は「植物毒性無し」を意味し、「100%」は、植物が完全に枯死したことを意味する。
Claims (10)
- ヌクレオチド配列番号1に示されている内因性アセト乳酸シンターゼ(ALS)遺伝子の1705−1707位に対応する位置に突然変異を含むALSインヒビター除草剤耐性ベータ・ブルガリス(Beta vulgaris)植物およびその器官であって、該ALS遺伝子が、ALSポリペプチドの569位にロイシンであるアミノ酸を含有するALSポリペプチドをコードし、内因性アセト乳酸シンターゼ(ALS)遺伝子の突然変異に関してホモ接合である、該ベータ・ブルガリス植物およびその器官。
- 該ALS遺伝子が、配列番号2のALSポリペプチド配列の569位にロイシンであるアミノ酸を含有するALSポリペプチドをコードしている、請求項1記載のベータ・ブルガリス植物およびその器官。
- 該アミノ酸がロイシンであり、該内因性ALS遺伝子が、配列番号3のヌクレオチド配列と同一である、請求項1記載のベータ・ブルガリス植物およびその器官。
- スルホニル尿素除草剤、スルホニルアミノカルボニルトリアゾリノン除草剤、イミダゾリノン除草剤、トリアゾロピリミジン除草剤およびピリミジニル(チオ)ベンゾアート除草剤からなる群に属する1以上のALSインヒビター除草剤に耐性である、請求項1記載のベータ・ブルガリス植物およびその器官。
- 該器官が該植物の種子である、請求項1記載のベータ・ブルガリス植物の器官。
- (a)ベータ・ブルガリス由来のカルスを、10−7M〜10−9MのALSインヒビター除草剤にさらし、
(b)3×10−6MまでのALSインヒビター除草剤の存在下で成長しうる細胞コロニーを選択し、
(c)ALSインヒビター除草剤の存在下でシュートを再生させ、
(d)ALSインヒビター除草剤で、再生された小植物を選択する工程を含む、
請求項1記載のベータ・ブルガリス植物およびその器官の製造方法、
ここで、該小植物は、ヌクレオチド配列番号1に示されている内因性アセト乳酸シンターゼ(ALS)遺伝子の1705−1707位に対応する位置の突然変異を含み、該ALS遺伝子が、ALSポリペプチドの569位にロイシンであるアミノ酸を含有するALSポリペプチドをコードしている。 - 前記ステップ(a)のALSインヒビター除草剤がホラムスルフロンである、請求項6記載のベータ・ブルガリス植物およびその器官の製造方法。
- 前記ステップ(b)のALSインヒビター除草剤がホラムスルフロンである、請求項6又は7記載のベータ・ブルガリス植物およびその器官の製造方法。
- 前記ステップ(c)のALSインヒビター除草剤がホラムスルフロンである、請求項6から8のいずれか一項に記載のベータ・ブルガリス植物およびその器官の製造方法。
- 前記ステップ(d)のALSインヒビター除草剤がホラムスルフロン、ヨードスルフロン−メチル−ナトリウムまたはそれらの両方の混合物である、請求項6から9のいずれか一項に記載のベータ・ブルガリス植物およびその器官の製造方法、ここで、ホルムスルフロンの用量は、7〜70g a.i./haであり、ヨードスルフロン−メチル−ナトリウムの用量は、1〜10g a.i./haである。
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- 2017-02-10 JP JP2017022680A patent/JP6375398B2/ja active Active
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2018
- 2018-07-23 JP JP2018137782A patent/JP2018183168A/ja active Pending
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2020
- 2020-11-06 US US17/091,776 patent/US20210054360A1/en not_active Abandoned
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