JP2022081655A - 植物調節エレメント及びその使用 - Google Patents
植物調節エレメント及びその使用 Download PDFInfo
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Abstract
Description
本出願は、2016年5月24日に出願された米国仮出願第62/340,656号の利益を主張し、前記文献はその全体を参照として本明細書に援用する。
「MONS419WO-sequence_listing.txt」という名称の、2017年5月22日に作成された14.1KB(Microsoft Windows(登録商標)での測定による)のファイルに含まれる配列表を、電子提出によりファイル化し、参照として本明細書に援用する。
本発明は、植物において使用するための新規遺伝子調節エレメントを提供する。本発明はまた、調節エレメントを含むDNA構築物を提供する。本発明はまた、調節エレメントを含むトランスジェニック植物細胞、植物、及び種子を提供する。一実施形態では、調節エレメントを、転写可能なDNA分子に作動可能に連結させる。特定の実施形態では、転写可能なDNA分子は、調節配列に関して異種であってもよい。したがって、本発明によって提供する調節エレメント配列は、特定の実施形態では、異種の転写可能なDNA分子に作動可能に連結されていると定義され得る。本発明はまた、調節エレメントを作製及び使用する方法、調節エレメントを含むDNA構築物、ならびに転写可能なDNA分子に作動可能に連結した調節エレメントを保有するトランスジェニック植物細胞、植物、及び種子を提供する。
配列番号1は、その天然の第一のイントロンの5′に作動可能に連結する天然のリーダーの5′に作動可能に連結するブルーグラマグラス(Bouteloua gracilis)推定ユビキチン遺伝子由来プロモーターを含む調節発現エレメント群(EXP)のDNA配列である。
本明細書中で使用する場合、用語「DNA」または「DNA分子」とは、5′(上流)末端から3′(下流)末端に向けて読み取られる、ゲノムまたは合成由来の二本鎖DNA分子、すなわち、デオキシリボヌクレオチド塩基のポリマーまたはDNA分子を指す。本明細書中で使用する場合、用語「DNA配列」とは、DNA分子のヌクレオチド配列を指す。本明細書中で使用する命名法は、米国連邦規則集§1.822のTitle 37の名称に対応し、WIPO標準ST.25(1998)、付録2の表、すなわち表1及び3に記載されている。
プロモーター、リーダー(5′UTRとしても知られる)、エンハンサー、イントロン、及び転写終結領域(3′UTRとしても知られる)などの調節エレメントは、生細胞における遺伝子の全体的な発現に不可欠な要素である。本明細書中で使用する場合、用語「調節エレメント」とは、遺伝子調節活性を有するDNA分子を指す。本明細書中で使用する場合、用語「遺伝子調節活性」とは、例えば作動可能に連結する転写可能なDNA分子の転写及び/または翻訳に影響を及ぼすことによって、作動可能に連結する転写可能なDNA分子の発現に影響を及ぼす能力を指す。したがって、植物において機能するプロモーター、リーダー、エンハンサー、イントロン及び3′UTRなどの調節エレメントは、遺伝子改変を介した植物の表現型の改変に有用である。
本明細書中で使用する場合、用語「構築物」とは、少なくとも1つのDNA分子が別のDNA分子に機能的に作動的な様式で連結している、すなわち作動可能に連結したDNA分子を含む、ゲノム組み込みまたは自律複製が可能な任意の供給源に由来する任意の組換えDNA分子、例えば、プラスミド、コスミド、ウイルス、ファージ、または線状もしくは環状DNAもしくはRNA分子などを意味する。本明細書中で使用する場合、用語「ベクター」とは、形質転換、すなわち、異種DNAまたはRNAの宿主細胞への導入のために使用し得る任意の構築物を意味する。構築物は、通常、1つ以上の発現カセットを含む。本明細書中で使用する場合、「発現カセット」とは、1つ以上の調節エレメント、一般的には少なくともプロモーター及び3′UTRに作動可能に連結した少なくとも転写可能なDNA分子を含むDNA分子を指す。
本明細書中で使用する場合、用語「転写可能なDNA分子」とは、タンパク質コード配列を有するもの及び遺伝子の抑制に有用な配列を有するRNA分子を産生するものを含むがこれらに限定されない、RNA分子に転写可能な任意のDNA分子を指す。DNA分子のタイプは、同じ植物由来のDNA分子、別の植物由来のDNA分子、異なる生物由来のDNA分子、または合成DNA分子、例えば、遺伝子のアンチセンスメッセージを有するDNA分子、または導入遺伝子の人工的、合成的、もしくは別の方法で修飾されたバージョンをコードするDNA分子を含み得るが、これらに限定されない。本発明の構築物に組み込むための例示的な転写可能なDNA分子として、例えば、DNA分子を組み込む種以外の種由来のDNA分子もしくは遺伝子、または同じ種に由来もしくは存在するが、古典的な育種技術ではなく遺伝子工学的手法によりレシピエント細胞に組み込む遺伝子が挙げられる。
転写可能なDNA分子は、農学的関心対象遺伝子であってもよい。本明細書中で使用する場合、用語「農学的関心対象遺伝子」とは、特定の植物組織、細胞、または細胞型で発現する場合に、所望の特性を付与する転写可能なDNA分子を指す。農学的関心対象遺伝子の産物は、植物の形態、生理、生長、発生、収量、穀物組成、栄養特性、病害もしくは害虫抵抗性、環境耐性もしくは化学耐性に影響を及ぼすために植物内で作用し得るか、または植物に与える肥料中の害虫の殺虫剤として作用し得る。本発明の一実施形態では、本発明の調節エレメントを、調節エレメントが、農学的関心対象遺伝子である転写可能なDNA分子に作動可能に連結するように、構築物に組み込む。そのような構築物を含むトランスジェニック植物では、農学的関心対象遺伝子の発現は有益な農業形質を付与することができる。有益な農業形質は、例えば、除草剤耐性、昆虫防除、収量の改変、病害抵抗性、病原体耐性、植物生長及び植物発生の改変、デンプン含量の改変、油含量の改変、脂肪酸含量の改変、タンパク質含量の改変、果実成熟の改質、動物及びヒトの栄養向上、バイオポリマー製品、環境ストレス耐性、ペプチド医薬品、加工品質の改良、風味の改良、ハイブリッド種子生産の有用性、繊維生産の向上、及び望ましいバイオ燃料生産が挙げられるが、これらに限定されない。
選択可能なマーカー導入遺伝子もまた、本発明の調節エレメントとともに使用してもよい。本明細書中で使用する場合、用語「選択可能なマーカー導入遺伝子」とは、トランスジェニック植物、組織または細胞における発現、またはその欠如を何らかの方法でスクリーニングまたはスコア評価できる任意の転写可能なDNA分子を指す。本発明の実施に使用するための選択マーカー遺伝子及びそれらに関連する選択及びスクリーニング技術は、当該技術分野で公知であり、β-グルクロニダーゼ(GUS)、緑色蛍光タンパク質(GFP)、抗生物質耐性を付与するタンパク質、及び除草剤耐性を付与するタンパク質をコードする転写可能なDNA分子が挙げられるが、これらに限定されない。選択可能なマーカー導入遺伝子の例を、配列番号6として提供する。
本発明はまた、転写可能なDNA分子に作動可能に連結した1つ以上の調節エレメントを含む形質転換細胞及び植物の製造方法に関する。
調節エレメントの同定及びクローニング
新規転写調節エレメント及び調節発現エレメント群(EXP)を同定し、単子葉植物Bouteloua gracilisのゲノムDNAからクローニングした。
表1:Bouteloua gracilisから単離した転写調節発現エレメント群、プロモーター、リーダー、イントロン、及び3′UTR。
安定的に形質転換したトウモロコシ植物におけるGUS発現を駆動する調節エレメントの分析
トウモロコシ植物を、ベクター、具体的には、β-グルクロニダーゼ(GUS)導入遺伝子の発現を駆動する試験調節エレメントを含む植物発現ベクターで形質転換した。得られた植物をGUSタンパク質発現について分析し、選択した調節エレメントの発現に対する効果を評価した。
表2.Bouteloua gracilis由来の調節エレメントによって駆動される安定形質転換トウモロコシ植物における平均定量的GUS発現。
調節エレメントに由来するエンハンサーエレメント
エンハンサーは、配列番号2として示すプロモーターエレメントに由来する。エンハンサーエレメントは、プロモーターエレメントの5′もしくは3′に作動可能に連結するか、またはプロモーターに作動可能に連結するさらなるエンハンサーエレメントの5′または3′に作動可能に連結する場合に、転写可能なDNA分子の発現レベルを増強または調節するか、あるいは特定の細胞型または植物器官または発生もしくは概日リズムの特定の時点での転写可能なDNA分子の発現を提供することができる1つ以上のシス調節エレメントからなり得る。エンハンサーは、TATAボックスまたは機能的に類似のエレメント及び配列番号2またはその断片として示されるプロモーターからの転写を開始させることを可能にする、プロモーターからの下流の配列を除去することによって作製される。
植物由来プロトプラストを用いたGUS活性のイントロン増強の解析。
イントロンは、導入遺伝子の最適な発現のために、ベクター転写DNA(T-DNA)エレメント配置内のイントロン及び配置を経験的に選択するための実験及びイントロンレス発現ベクター対照との比較に基づいて選択される。例えば、グリホサートに対する耐性を付与するCP4などの除草剤耐性遺伝子の発現においては、除草剤を適用する際の収量の損失を防ぐために、生殖組織及び栄養組織内に導入遺伝子発現を有することが望ましい。この場合、イントロンを、構成的プロモーターに作動可能に連結する場合、特にトランスジェニック植物の生殖細胞及び組織内で導入遺伝子を付与する除草剤抵抗性の発現を増強するその能力に基づいて選択し、したがって、除草剤を噴霧する場合、植物性及び生殖耐性の両方をトランスジェニック植物に提供するであろう。ほとんどのユビキチン遺伝子において、5′UTRはリーダーからなり、リーダーはその内部に組み込まれたイントロン配列を有する。したがって、そのような遺伝子に由来する調節エレメントは、プロモーター、リーダー、及びイントロンを含む5′UTR全体を用いてアッセイする。異なる発現特性を達成するために、または導入遺伝子発現のレベルを調節するために、そのような調節エレメント由来のイントロンを、除去するか、または異種イントロンで置換してもよい。
Claims (21)
- a)配列番号5と少なくとも85%の配列同一性を有する配列;
b)配列番号5を含む配列;及び
c)遺伝子調節活性を有する、配列番号5の断片;
からなる群から選択されるDNA配列を含む組換えDNA分子であって、
前記配列が、異種の転写可能なDNA分子に作動可能に連結している、前記組換えDNA分子。 - 前記配列が、配列番号5のDNA配列に対して少なくとも90%の配列同一性を有する、請求項1に記載の組換えDNA分子。
- 前記配列が、配列番号5のDNA配列に対して少なくとも95%の配列同一性を有する、請求項1に記載の組換えDNA分子。
- 前記配列が、配列番号5のDNA配列を含む、請求項1に記載の組換えDNA分子。
- 前記DNA配列が遺伝子調節活性を含む、請求項1に記載の組換えDNA分子。
- 前記異種の転写可能なDNA分子が、農学的関心対象遺伝子を含む、請求項1に記載の組換えDNA分子。
- 前記農学的関心対象遺伝子が、植物における除草剤耐性を付与する、請求項6に記載の組換えDNA分子。
- 前記農学的関心対象遺伝子が、植物における害虫抵抗性を付与する、請求項6に記載の組換えDNA分子。
- a)配列番号5と少なくとも85%の配列同一性を有する配列;
b)配列番号5を含む配列;及び
c)遺伝子調節活性を有する、配列番号5の断片;
からなる群から選択される配列を含む組換えDNA分子を含むトランスジェニック植物細胞であって、
前記配列が、異種の転写可能なDNA分子に作動可能に連結している、前記トランスジェニック植物細胞。 - 前記トランスジェニック植物細胞が単子葉植物細胞である、請求項9に記載のトランスジェニック植物細胞。
- 前記トランスジェニック植物細胞が双子葉植物細胞である、請求項9に記載のトランスジェニック植物細胞。
- 請求項1に記載の組換えDNA分子を含む、トランスジェニック植物またはその部分。
- 子孫植物またはその部分が前記組換えDNA分子を含む、請求項12に記載のトランスジェニック植物の子孫植物またはその部分。
- 種子が、請求項1に記載の組換えDNA分子を含む、トランスジェニック種子。
- コモディティ製品の製造方法であって、請求項12に記載のトランスジェニック植物またはその部分を取得し、そこから前記コモディティ製品を製造することを含む、前記方法。
- 前記コモディティ製品が、タンパク質濃縮物、タンパク質単離物、穀物、デンプン、種子、ミール、小麦粉、バイオマス、または種子油である、請求項15に記載の方法。
- 請求項12に記載のトランスジェニック植物を取得し、植物を栽培することを含み、転写可能なDNAを発現させる、転写可能なDNA分子の発現方法。
- 請求項1に記載の組換えDNA分子を植物細胞に導入することを含む、トランスジェニック植物細胞の製造方法。
- 前記組換えDNA分子を前記植物細胞に導入することが、形質転換を含む、請求項18に記載の方法。
- 前記植物細胞からトランスジェニック植物を再生することをさらに含む、請求項19に記載の方法。
- 前記組換えDNA分子を前記植物細胞に導入することが、請求項12に記載のトランスジェニック植物を別の植物と交雑して、前記植物細胞を含む子孫植物を産生することを含む、請求項18に記載の方法。
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AU2017269292B2 (en) | 2021-07-22 |
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US10351866B2 (en) | 2019-07-16 |
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NZ748198A (en) | 2023-05-26 |
UA126798C2 (uk) | 2023-02-08 |
CL2018003356A1 (es) | 2019-01-11 |
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RU2774709C2 (ru) | Регуляторные элементы растений и варианты их применения | |
OA19276A (en) | Plant regulatory elements and uses thereof. | |
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