JP2007533310A - トウモロコシにおける脂肪酸デサチュラーゼの発現 - Google Patents
トウモロコシにおける脂肪酸デサチュラーゼの発現 Download PDFInfo
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Abstract
Description
本発明は、一般的に、トウモロコシにおける長鎖ポリ−不飽和脂肪酸(LC−PUFA)中の二重結合の数および位置を変調するデサチュラーゼ酵素の発現およびそれから派生する組成物に関する。
大部分の生物における脂肪酸生合成の一次産物は16−および18−炭素化合物である。これらの脂肪酸の鎖長および不飽和度の相対比は種の中で広く変化する。例えば、哺乳動物は一次的に飽和およびモノ不飽和脂肪酸を産生する一方で、大部分の高等植物は1、2または3の二重結合を有する脂肪酸を産生し、後者の2はポリ不飽和脂肪酸(PUFA)を含んでいる。
1の態様において、本発明は、ステアリドン酸を含有する内因性トウモロコシ種子油を提供する。本発明のある種の形態において、トウモロコシ種子油は約.1%ないし約33%のステアリドン酸を含み、さらなる形態において、すべての中間の値ならびに後記する表中に示す値を含む、約5%ないし約15%;約5%ないし約10%;約7.5%ないし約12%;約10%ないし約15%;約12%ないし約15%;約10%ないし約33%、約15%ないし約33%、約15%ないし約32%、約20%ないし約33%、約20%ないし約30%、約25%ないし約30%、および約25%ないし約33%のステアリドン酸を含み得る。本発明により提供される内因性トウモロコシ種子油は、さらに、ガンマ−リノレン酸を含み得る。本発明のある種の形態において、油のガンマ−リノレン酸含量は、約5%未満および約3%未満を含み、特にすべての中間の値および後記する表中に示す値を含む、約.01%ないし約7.5%および約.01%ないし約5%とし得る。本発明のある種の形態において、α−リノレン酸含量は約5、10、15または20%未満とし得る。
本発明は、改良されたPUFA含量を有する植物の創製のための方法および組成物、ならびにそれによって産生される種子油を提供することによって先行技術の制限を克服する。本発明の1の形態において、出願人らは、ステアリドン酸(SDA)を含有する内因性トウモロコシ種子油を生産し、γ−リノレン酸(GLA)も含み得るトランスジェニック・トウモロコシ(Zea mays)を提供する。このことは極めて重要である。なぜなら、トウモロコシ種子油は、通常これらの成分を欠いており、その各々が重要な健康上の利益を有することが示されているからである。トウモロコシ種子油は、それを、例えばSDAの外的な添加の必要なしにトウモロコシ種子によって生産し得る点において内因性である。かかる内因性の油は、食物および飼料の成分として使用し得、それによってヒトまたは動物の健康が恩恵を受ける抽出油組成物とし得る。したがって、植物のごとき生物の脂肪酸含量の改良は、改善された栄養および健康の恩恵のような多くの恩恵を示す。本発明による脂肪酸含量の修飾を用いて、トウモロコシのような植物、植物部分および植物種子油を含む植物生成物において目的のPUFAの有益なレベルまたはプロフィールを達成することができる。例えば、目的のPUFAを植物の種子組織で生産する場合、油は、典型的に目的のPUFAを多量に含む油、またはついで食物材料および他の生成物に有益な特徴を提供するために使用し得る望ましい脂肪酸含量またはプロフィールを有する油を生じる種子から単離し得る。特に、本発明は、SDAを有する内因性トウモロコシ種子油を提供する。
以下の実施例は、発明の形態を説明するために含めるものである。つづく実施例に開示する技術が、本発明者らによって発見された本発明の実施においてよく機能する技術を表すことは当業者によって理解されるべきである。しかしながら、本明細書の開示の観点から、当業者であれば、本発明の趣旨、意図および範囲から逸脱することなく、開示したおよびなお類似または同様の結果が得られる特定の形態に多くの変化を作成し得ることは理解する。さらに詳細には、化学的にも生理学的にも関係するある種の因子を、同一または同様の結果を達成しつつ本明細書に記載した因子と置き換え得ることは明らかであろう。当業者に明らかなすべてのかかる同様の置換および修飾は、添付する特許請求の範囲によって定義される発明の意図、範囲および趣旨の中にあると考えられる。
トウモロコシにおいてΔ15−およびΔ6−デサチュラーゼを発現させるためのベクター
バイナリーベクターを構築して、トウモロコシの胚およびアリューロン組織においてΔ15−デサチュラーゼおよびΔ6−デサチュラーゼを発現させた。この構築物は、突然変異導入してトウモロコシのような単子葉植物における発現を増大させたノイロスポーラ・クラッサ(Neurospora crassa)のΔ15−デサチュラーゼ(配列番号:5)およびモルティエレラ・アルピナ(Mortierella alpina)のΔ6−デサチュラーゼ(配列番号:6、bp71−1444)(米国特許第6,075,183号)の発現を駆動するグロブリン・プロモーターを用いて構築した。エム・アルピナのΔ6−デサチュラーゼをグロブリン・プロモーターを含むシャトルベクターpMON67624にクローニングして、pMON82809を得た。突然変異導入したエヌ・クラッサのΔ15−デサチュラーゼをグロブリン・プロモーターを含むシャトルベクターpMON67624にクローニングして、pMON82810を得た。
トウモロコシにおいて単子葉植物配列最適化したプリミュラ・ジュリアのΔ6−デサチュラーゼを発現させるためのベクター
本実施例では、単子葉植物において発現させるために修飾したプリミュラ・ジュリアのΔ6−デサチュラーゼ・ポリヌクレオチド分子の設計および構築を記載する。非−内因性のタンパク質をコードする配列を植物において良好に発現させ得ないことはよく知られている(出典明示して本明細書の一部とみなす、米国特許第5,880,275号)。したがって、本来のPjD6Dポリペプチド配列(配列番号:3)を用いて、1)高度に発現される単子葉植物タンパク質のものと同様の同義語コドン使用の偏りを用いることによって、および2)イン・プランタ(in planta)でmRNA安定性に影響することが以前に特徴付けられ、知られているRNA不安定化エレメント(米国特許第5,880,275号)を除去することによって、人工PjD6Dタンパク質をコードするポリヌクレオチド配列を設計および構築した。得られた修飾PjD6Dポリヌクレオチド配列は、PjD6Dnno(配列番号:8)と命名し、これは本来のPjD6Dポリペプチドと配列において同一であるポリペプチドをコードしている(配列番号:3)。
脂肪酸分析
pMON82812を発現している成熟穀粒の脂肪酸組成を、トウモロコシ穀粒を挽きつぶし、ホモジネートをヘプタンで抽出することによって決定した。ヘプタン抽出物を、0.25mg/mlでトリヘプタデカノインを含有するトルエンおよびメタノール中のナトリウムメトキシド(0.6N)で処理した。塩化ナトリウム水溶液(10% wt/vol)を用いて反応を終結した。室温にて分配させた後に、その有機相をGLC(スプリット/スプリットレスのキャピラリー流入口(250℃)および炎イオン化検出器(270℃)を備えたHewlett Packard model 6890 (120ボルト)によって分析した。カラムは、0.25μmを有するSupelco 24077(外径0.25mm×長さ15m)に結合したポリエチレングリコール固定相とした。脂肪酸メチルエステルは、市販されている標準物と比較した保持時間によって同定する。定性的な重量%組成は、同定したピークの面積%として算出する。SDAおよびGLAを示した穀粒について分析した結果を表1に掲載する。GLAのみ含有する部分的に無効な穀粒は見出されなかった。全体として、分析した穀粒の2/3を超えるものがGLAおよびSDAを含有していた。pMON82812で形質転換した事象ZM 103111:@からの成熟穀粒の分析は、9.68%のSDAを示した。
以下に掲載する参考文献は、本明細書で用いた方法、技術および/または組成物を補充し、説明し、背景を提供し、または教示する範囲において参照して本明細書に取り込まれる。
米国特許第4,826,877号
米国特許第4,910,141号
米国特許第5,011,770号
米国特許第5,158,975号
米国特許第5,302,523号
米国特許第5,378,619号
米国特許第5,384,253号
米国特許第5,464,765号
米国特許第5,538,877号
米国特許第5,538,880号
米国特許第5,550,318号
米国特許第5,563,055号
米国特許第5,591,616号
米国特許第5,952,544号
米国特許第6,603,061号
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Claims (33)
- 約0.1%ないし約33%のステアリドン酸(18:4 n−3)含量を有する内因性トウモロコシ種子油。
- ステアリドン酸含量が、約12%ないし約15%、約10%ないし約33%、約15%ないし約32%、約20%ないし約30%、および約25%ないし約30%よりなる群から選択される請求項1記載のトウモロコシ種子油。
- 約7.5%未満、約5%未満、および約3%未満よりなる群から選択される含量でガンマ−リノレン酸を含む請求項1記載のトウモロコシ種子油。
- ガンマ−リノレン酸に対するステアリドン酸の比が、約1:1ないし約10:1、約2:1ないし約10:1、約3:1ないし約5:1、および少なくとも約3:1よりなる群から選択される請求項1記載のトウモロコシ種子油。
- 油中のオメガ−6脂肪酸に対するオメガ−3脂肪酸の比が、約0.5%:1ないし約10:1、約5:1ないし約10:1、および少なくとも約5:1よりなる群から選択される請求項1記載のトウモロコシ種子油。
- 請求項1記載の種子油を含むトウモロコシ種子。
- 同系繁殖種子である請求項6記載のトウモロコシ種子。
- 雑種種子である請求項6記載のトウモロコシ種子。
- 請求項1記載の種子油を産生するトウモロコシ植物。
- 同系繁殖植物である請求項6記載のトウモロコシ植物。
- 雑種植物である請求項6記載のトウモロコシ植物。
- (a) 配列番号:8の核酸配列を含むポリヌクレオチド;
(b)5×SSC、50%ホルムアミドおよび42℃の条件下で配列番号:8にハイブリダイズし、炭素6で脂肪酸分子を脱飽和化するデサチュラーゼ活性を有するポリペプチドをコードするポリヌクレオチド;および
(c)配列番号:8の核酸配列に対して少なくとも90%の配列同一性を有し、炭素6で脂肪酸分子を脱飽和化するデサチュラーゼ活性を有するポリペプチドをコードするポリヌクレオチド;および
(d)(a)、(b)または(c)の配列の相補体
よりなる群から選択される単離されたポリヌクレオチド。 - さらに、配列番号:9の核酸配列を含む請求項12記載のポリヌクレオチド。
- 請求項12記載の単離されたポリヌクレオチドを含む組換え構築物。
- 請求項14記載の組換え構築物で形質転換したトランスジェニック植物。
- 請求項9記載の植物が該種子油を産生するまで、該植物を該植物の成長条件下で生育することを含む種子油を生産する方法。
- 請求項1記載のトウモロコシ種子油を食用製品に添加することを含む、ヒトまたは非−ヒト動物の消費用の食用製品の栄養価を上昇する方法。
- 食用製品が、ヒト食物、動物飼料および食物補充物よりなる群から選択される請求項17記載の方法。
- トウモロコシ種子油が食用製品のステアリドン酸含量を増加する請求項17記載の方法。
- トウモロコシ種子油が、食用製品のオメガ−6脂肪酸に対するオメガ−3脂肪酸の比を上昇する請求項17記載の方法。
- 食用製品が、トウモロコシ種子油を添加する前にステアリドン酸を欠いている請求項17記載の方法。
- 出発成分に請求項1記載のトウモロコシ種子油を添加して食物および/または飼料を生産することを含む、食物および/または飼料を製造する方法。
- 請求項22記載の方法によって製造された食物または飼料。
- 請求項1記載のトウモロコシ種子油をヒトまたは非−ヒト動物に投与することを含む、ヒトまたは非−ヒト動物にステアリドン酸を提供する方法。
- トウモロコシ種子油を食用組成物で投与する請求項24記載の方法。
- 食用組成物が食物または飼料である請求項25記載の方法。
- 食物が、飲料、注入食物、ソース、香辛料、サラダドレッシング、フルーツジュース、シロップ、デザート、アイシングおよびフィリング(icing and filling)、軟質冷凍製品、菓子または中間水分食物を含む請求項26記載の方法。
- 食用組成物が伴侶動物用の食物または飼料である請求項26記載の方法。
- 食用組成物が実質的に液体または固体である請求項25記載の方法。
- 食用組成物が食物補充物および/またはニュートラシューティカルである請求項25記載の方法。
- トウモロコシ種子油をヒトに投与する請求項24記載の方法。
- トウモロコシ種子油を非−ヒト動物に投与する請求項24記載の方法。
- トウモロコシ種子油を家畜または家禽に投与する請求項32記載の方法。
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CA2562548A1 (en) | 2005-11-03 |
CN1968688A (zh) | 2007-05-23 |
EP1734947A1 (en) | 2006-12-27 |
US11034983B2 (en) | 2021-06-15 |
EP2902021B1 (en) | 2018-11-14 |
EP1734947B1 (en) | 2015-04-15 |
BRPI0509944A (pt) | 2007-09-25 |
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EP2902021A1 (en) | 2015-08-05 |
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WO2005102310A1 (en) | 2005-11-03 |
AU2005235081B2 (en) | 2011-06-16 |
US8378186B2 (en) | 2013-02-19 |
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