JP7038177B2 - 高いタンパク質含有量及びストレスに対する抵抗性のためのnf-yc4プロモーター中の転写リプレッサー結合部位の修飾 - Google Patents
高いタンパク質含有量及びストレスに対する抵抗性のためのnf-yc4プロモーター中の転写リプレッサー結合部位の修飾 Download PDFInfo
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Description
RAV1(リバース)モチーフ及びERF(フォワード)モチーフ(TGTTGACT;配列番号1中の下線付き太字を参照されたい)の重複配列が欠失されているイネ(Oryza sativa)のNF-YC4遺伝子のプロモーター領域を配列番号3として示し、ここでRAV1モチーフ(リバース)を太字で、ERFモチーフ(リバース)をイタリック体太字で示す。
2つのERFモチーフ(リバース)、RAV1(リバース)及びERF(フォワード)モチーフ、及び介在配列(
ERFモチーフ(リバース)、RAV1モチーフ(フォワード)及び介在配列(
2つのRAV1モチーフ及び介在配列(
トウモロコシ(Zea mays)のNF-YC4遺伝子のプロモーター領域を配列番号20として示し、ここで2つのRAV1モチーフ(フォワード)を太字で示す。
本実施例は、35S::QQS及び35S::AtNF-YC4融合構築物の作成及び形質転換を説明する。
本実施例は、植物組成の分析を説明する。
本実施例は、酵母ツーハイブレッドアッセイを説明する。
本実施例は、二分子蛍光補完(BiFC)アッセイを説明する。
本実施例は、HIS-MBPタグ付き組換え体NF-Yの精製を説明する。
本実施例は、GSTタグ付きQQSの発現及び精製を説明する。
本実施例は、プルダウンアッセイを説明する。
本実施例は、統計的設計及び分析を説明する。
本実施例は、系統発生的推定を説明する。
本実施例は、QQS導入遺伝子が種々の種子タンパク質含有量を有するエリートダイズ系統中のタンパク質含有量及び炭水化物含有量に影響を与えることを立証する;よって、QQSの効果はダイズ系統ウイリアムズ82に限定されない。
本実施例は、QQS導入遺伝子がイネ及びトウモロコシ中のタンパク質含有量に影響を与えることを立証する。
本実施例は、QQSと相互作用する遺伝子を同定するための、アラビドプシス実生由来のcDNAライブフリーに対してつり餌としてQQSを用いる酵母ツーハイブリッドスクリーニングを説明する。
本実施例は、QQS結合のために必要なAtNF-YC4中の領域を同定するために精製組換えAtNF-YC4-GST融合タンパク質を用いるグルタチオン-S-トランスフェラーゼ(GST)プルダウンアッセイを説明する。
本実施例は、QQSが通常ヒストン折りたたみ様ドメインタンパク質に結合するかどうかを調べるための精製組換えアラビドプシス核因子YB7(AtNF-YB7)-GST融合タンパク質を用いるGSTプルダウンアッセイを説明する。
本実施例は、タバコ葉におけるQQS及びAtNF-YC4のインビボ共発現を説明する。
本実施例は、QQSとAtNF-YC4のアミノ酸73-162に類似のアミノ酸配列を有するイネ及びダイズタンパク質との相互作用を説明する。
本実施例は、アラビドプシスにおけるAtNF-YC4及びOsNF-YC4の過剰発現を説明する。
本実施例は、NF-YC4遺伝子のプロモーター領域中の同定されたリプレッサーモチーフの標的化除去を説明する。
本実施例は、転写リプレッサー結合部位を含み得る領域を同定するためのNF-YC4遺伝子のプロモーター領域のスクリーニングを説明する。
本実施例は、植物防御に関与する遺伝子の発現に対するQQSの過剰発現及び過小発現の影響を説明する。
本実施例は、NF-YC4またはQQSを過剰発現するトランスジェニックシロイヌナズナ(Arabidopsis thalian)植物ではウイルス感染が減少したことを立証する。
本実施例は、NF-YC4またはQQSを過剰発現するトランスジェニックシロイヌナズナ(Arabidopsis thalian)植物では細菌増殖が減少したことを立証する。
本実施例は、NF-YC4を過剰発現する、またはQQSを発現するトランスジェニックダイズ植物では細菌増殖が低下したことを立証する。
本実施例は、NF-YC4を過剰発現する、またはQQSを発現するトランスジェニックダイズ植物ではアブラムシが減少したことを立証する。
本実施例は、NF-YC4を過剰発現するトランスジェニックダイズ植物の種子中のタンパク質またはタンパク質+油含有量が増加したことを立証する。
本実施例は、NF-YC4を過剰発現するトランスジェニックトウモロコシ植物の種子中のタンパク質含有量が増加したことを立証する。
本実施例は、NF-YC4を過剰発現するトランスジェニックイネ植物の種子中のタンパク質含有量が増加したことを立証する。
Claims (23)
- 植物又はその一部中の総タンパク質含有量を増加させる方法であって、(i)1つ以上の植物又は1つ以上のその部分を、植物由来のNF-YC4(核因子YサブユニットC4)タンパク質をコードするヌクレオチド配列を含む組換え核酸で形質転換し、ここで、NF-YC4タンパク質をコードする前記ヌクレオチド配列は、プロモーターの制御下にあり、NF-YC4タンパク質は、NF-YC4タンパク質を過剰発現しない対応する野生型(WT)植物又はその対応するWT部分と比較して、前記1つ以上の植物又は1つ以上のその部分において過剰発現され、及び(ii)NF-YC4タンパク質を過剰発現しない対応するWT植物又はその対応するWT部分中の総タンパク質含有量と比較して、総タンパク質含有量が増加している(i)の1つ以上の形質転換植物又は1つ以上のその形質転換部分を選択することを含む、方法。
- 前記プロモーターが、NF-YC4タンパク質をコードする前記ヌクレオチド配列の転写を増加させる修飾NF-YC4プロモーターである、請求項1に記載の方法。
- 前記NF-YC4プロモーター中に位置する転写リプレッサー部位が、転写リプレッサーがNF-YC4タンパク質をコードする前記ヌクレオチド配列の転写を防止できないように修飾されている、請求項2に記載の方法。
- NF-YC4タンパク質をコードする前記ヌクレオチド配列が、シロイヌナズナ(Arabidopsis thaliana)、イネ(Oryza sativa)、及びダイズ(Glycine max)からなる群から選択される植物に由来する、請求項1に記載の方法。
- 前記プロモーターが、カリフラワーモザイクウイルスプロモーターである、請求項1に記載の方法。
- 前記カリフラワーモザイクウイルスプロモーターが、p35sプロモーターである、請求項5に記載の方法。
- 前記組換え核酸が、ベクターである、請求項1に記載の方法。
- 前記組換え核酸が、アグロバクテリウム媒介形質転換により、前記1つ以上の植物又は1つ以上のその部分に導入される、請求項1に記載の方法。
- 前記植物が、作物である、請求項1~8のいずれか一項に記載の方法。
- 前記作物が、トウモロコシ、ダイズ、アルファルファ、カノーラ、コムギ、イネ、ジャガイモ、及びレタスからなる群から選択される、請求項9に記載の方法。
- 転写リプレッサー結合部位を含むプロモーターを含むNF-YC4遺伝子を含む植物においてタンパク質含有量を増加させる方法であって、前記植物は、ダイズ(Glycine max)であり、前記方法は、前記転写リプレッサー結合部位を修飾することを含み、それにより前記転写リプレッサーは、前記NF-YC4遺伝子の転写を防止することができず、それにより前記ダイズ(Glycine max)細胞中のタンパク質含有量が増加し、前記NF-YC4遺伝子は、Glyma06gl7780及びGlyma04g37291からなる群から選択される、方法。
- ERFモチーフ及びRAVIモチーフを欠失させる、請求項2又は11に記載の方法。
- 配列番号10に記載の配列を欠失させる、請求項12に記載の方法。
- 2つのRAVIモチーフを欠失させる、請求項2又は11に記載の方法。
- 配列番号14に記載の配列を欠失させる、請求項14に記載の方法。
- TALENSを用いて、ERFモチーフを欠失させ、RAVIモチーフを欠失させ、又はERFモチーフ及びRAVIモチーフの両方を欠失させる、請求項2又は11に記載の方法。
- CRISPR/Cas9を用いて、ERFモチーフを欠失させ、RAVIモチーフを欠失させ、又はERFモチーフ及びRAVIモチーフの両方を欠失させる、請求項2又は11に記載の方法。
- 前記総タンパク質含有量が、前記植物の葉又は種子中で増加している、請求項1又は11に記載の方法。
- 少なくとも植物部分中の総タンパク質含有量が増加した植物を生産する方法であって、請求項1又は11の方法に従って得た植物を同種の第2植物と交雑させて子孫トランスジェニック植物を生産し、少なくとも選択したトランスジェニック子孫部分中の総タンパク質含有量が増加したトランスジェニック子孫植物を選択して、前記少なくともその部分中の総タンパク質含有量が増加した植物を生産することを含む、方法。
- 前記方法は更に、選択した子孫植物を前記第2植物と交雑して、戻し交雑子孫植物を生産し、タンパク質含有量が増加した第1の戻し交雑子孫植物を選択して、選択した戻し交雑子孫植物を生産し、交雑及び選択を3回以上繰り返して、タンパク質含有量が増加した戻し交雑子孫植物を生産することを含む、請求項19に記載の方法。
- 植物由来のNF-YC4(核因子YサブユニットC4)タンパク質をコードするヌクレオチド配列を含む組換え核酸を含む、植物細胞、植物細胞の集まり、植物組織、植物器官、又は植物であって、NF-YC4タンパク質をコードする前記ヌクレオチド配列は、プロモーターの制御下にあり、NF-YC4タンパク質は、前記植物細胞、植物細胞の集まり、植物組織、植物器官、若しくは植物又はそれらの一部において過剰発現され、前記植物細胞、植物細胞の集まり、植物組織、植物器官、又は植物生物は、NF-YC4タンパク質が過剰発現されていない対応する植物細胞、植物細胞の集まり、植物組織、植物器官、又は植物生物と比較してタンパク質含有量が増加している、植物細胞、植物細胞の集まり、植物組織、植物器官、又は植物。
- 植物又はそのハイブリッド植物である、請求項21に記載の植物。
- 植物由来のNF-YC4(核因子YサブユニットC4)タンパク質をコードするヌクレオチド配列を含む組換え核酸を含む、種子であって、NF-YC4タンパク質をコードする前記ヌクレオチド配列は、プロモーター制御下にあり、NF-YC4タンパク質は、前記種子において過剰発現され、前記種子は、NF-YC4タンパク質が過剰発現されていない対応する種子と比較してタンパク質含有量が増加している、種子。
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JP7453999B2 (ja) | 2015-02-18 | 2024-03-21 | アイオワ・ステイト・ユニバーシティ・リサーチ・ファウンデイション・インコーポレイテッド | 高いタンパク質含有量及びストレスに対する抵抗性のためのnf-yc4プロモーター中の転写リプレッサー結合部位の修飾 |
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