JP2004500044A - Flowering time modification - Google Patents

Abstract

Provided are recombinant polynucleotides and methods for modifying the flowering time of a plant. Plants transformed with the recombinant polynucleotides may have an accelerated, delayed or induced flowering time under certain conditions. Further, the transformed plants may have altered vernalization requirements.

Description

【００８０】
ＦＬＣのアミノ酸残基３０およびＧ１５７、Ｇ８５９、Ｇ１８４２、Ｇ１８４３、およびＧ１８４４に関しては酸性残基（ＥまたはＤ）である一方、これまでに同定された他のすべてのアラビドプシス（Ａｒａｂｉｄｏｐｓｉｓ）ＭＡＤＳドメインタンパク質においては、その位置はプラスに帯電したリジン残基で占められている。ＤＮＡに結合したヒトＳＲＦ　ＭＡＤＳドメインの結晶構造ではリジン残基（これは、酵母ＭＣＭ１およびヒトＭＥＦ２Ａタンパク質においても保存されている）がＤＮＡターゲットサイトのリン酸バックボーンと接触していることが示されている（Ｐｅｌｌｅｇｒｉｎｉ　ら、（１９９５）Ｎａｔｕｒｅ　３７６：４９０−４９８）。そのアミノ酸の違いはしたがって、ＦＬＣおよびＧ１５７、Ｇ８５９、Ｇ１８４２、Ｇ１８４３、およびＧ１８４４に関してのＤＮＡ結合は、他のアラビドプシス（Ａｒａｂｉｄｏｐｓｉｓ）ＭＡＤＳドメインタンパク質とは異なる性質を与えるしれない。したがって、位置３０に酸性残基を持つＭＡＤＳドメインタンパク質は特に植物の開花の調整および春化処理の応答に有用であってよい。
【００８１】
これらの遺伝子からの転写物は３’ＲＡＣＥ（Ｒａｐｉｄ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　ｃＤＮＡ　Ｅｎｄｓ）によって解析し、対応するｃＤＮＡはアラビドプシス（Ａｒａｂｉｄｏｐｓｉｓ）組織（Ｃｌｕｍｂｉａ生態型）の混合サンプル中からＲＴ−ＰＣＲにより単離した。この分析の間、Ｇ８５９、Ｇ１８４２、およびＧ１８４４の転写物はマルチプルアルタネイティブリースプライストフォームとして検出された。
【００８２】
実施例ＩＩ　開花時期関連遺伝子
同定された各配列のコード領域内の遺伝子特異的プライマーを用い、リバーストランスクリプターゼＰＣＲを行った。可能な場合は、プライマーは最初に同定された各コード配列の３’領域の近くにデザインした。
【００８３】
全ＲＮＡを植物組織から単離して、ＣＴＡＢ法で抽出した。抽出した全ＲＮＡは全ての組織タイプについて濃度を標準化し、２８Ｓのバンドを参照して、各組織のＰＣＲ反応に同じ量のｃＤＮＡテンプレートが使われるようにした。Ｐｏｌｙ Ａ^＋ は、Ｑｉａｇｅｎ Ｏｌｉｇｏｔｅｘ キットバッチのプロトコールを修正したプロトコールを用いて精製した。ｃＤＮＡは標準的プロトコールを用いて合成した。第一鎖ｃＤＮＡ合成の後、アクチン２ のプライマーを用いて、組織タイプにわたってｃＤＮＡの濃度を標準化した。アクチン２ は、Ａｒａｂｉｄｏｐｓｉｓ の組織タイプにわたって、ほぼ同じレベルで構成的に発現していることが分かっている。
【００８４】
ＲＴ　ＰＣＲのために、ｃＤＮＡテンプレートは、対応するプライマーおよびＴａｑポリメラーゼと混合した。各反応は、０．２μｌ　 ｃＤＮＡテンプレート、２μｌ　 １０Ｘ　トリシンバッファー、および１６．８μｌ 水、５ｐｍｏｌプライマー１、５ｐｍｏｌプライマー２、０．３μｌ　 Ｔａｑポリメラーゼ、２００μＭ　ｄＮＴＰおよび８．６μｌ の水で構成した。
【００８５】
９６穴のプレートをマイクロフィルムで覆い、熱循環装置にセットして、下記の反応サイクルを開始した。ステップ１　９３℃　３分、ステップ２　９３℃　３０秒、ステップ３　６０−６５℃　１分、ステップ４　７２℃　２分。ステップ２、３および４は２０〜３５サイクル繰り返した。ステップ５　７２℃　５分およびステップ６　４℃。ある場合にはＰＣＲプレートを熱循環装置に戻して、非常に発現の低い遺伝子の同定のために５〜１５回の追加サイクルで、反応産物の量を多くした。その反応サイクルは次のとおりとした。ステップ２　９３℃　３０秒、ステップ３　６５℃　１分、およびステップ４　７２℃　２分，８サイクルの繰り返し、およびステップ４　４℃。　２１および３６サイクルの間に、８マイクロリットルのＰＣＲ産物および１．５μｌ の負荷染料を１．２％アガロースゲルに負荷して分析した。特定の転写物が、ＰＣＲの３５サイクルより遅くやっと検出されたなら、それらの発現レベルは低いと考えられる。観察されたアクチン２ の転写レベルと比較した転写レベルに応じて、発現レベルは中または高と考えられる。
【００８６】
一例として、Ｇ１５７植物中でのＧ１５７　ｍＲＮＡレベルを評価するため、プライマー５’−ＧＧＣＡＴＡＡＣＣＣＴＴＡＴＣＧＧＡＧＡＴＴＴＧＡＡＧＣ−３’ （ＳＥＱ ＩＤ Ｎｏ． ５７）および ５’−ＡＣＡＣＡＡＡＣＴＣＴＧＡＴＣＴＴＧＴＣＴＣＣＧＡＡＧＧ−３’ （ＳＥＱ ＩＤ Ｎｏ． ５８） を用いて、２５サイクルにわたるＰＣＲを行った。野生型植物から抽出した別々の組織のｍＲＮＡレベルを評価するため、プライマー５’−ＧＣＡＴＡＡＣＣＣＴＴＡＴＣＧＧＡＧＡＴＴＴＧＡＡＧＣＣＡＴ−３’ （ＳＥＱ ＩＤ Ｎｏ． ５９）および ５’−ＡＡＣＡＴＴＣＣＴＣＴＣＴＣＡＴＣＡＴＣＴＧＴＴＧＣＣＡＧＣ−３’ （ＳＥＱ ＩＤ Ｎｏ． ６０） を用いて、２５または３０サイクルのＰＣＲを行った。ＦＬＣのＰＣＲは、プライマー５’−ＡＡＣＧＣＴＴＡＧＴＡＴＣＴＣＣＧＧＣＧＡＣＴＴＧＡＡＣ−３’ （ＳＥＱ ＩＤ Ｎｏ． ６１）および５’−ＣＴＣＡＣＡＣＧＡＡＴＡＡＧＧＴＡＣＡＡＡＧＴＴＣＡＴＣ−３’ （ＳＥＱ ＩＤ Ｎｏ． ６２）を用いて３５回にわたり、または５’−ＴＴＡＧＴＡＴＣＴＣＣＧＧＣＧＡＣＴＴＧＡＡＣＣＣＡＡＡＣＣ−３’ （ＳＥＱ ＩＤ Ｎｏ． ６３）および５’ ＡＧＡＴＴＣＴＣＡＡＣＡＡＧＣＴＴＣＡＡＣＡＴＧＡＧＴＴＣＧ−３’ （ＳＥＱ ＩＤ Ｎｏ． ６４）を用いて３０回にわたり行った。プライマーの特異性は、ＲＴ−ＰＣＲ産物の配列決定によた確認された。サンプルは、アクチンプライマーを用いた２０〜２５サイクルのＰＣＲにより標準化した。
【００８７】
実施例ＩＩＩ ．発現ベクターの構築
ゲノムライブラリーまたはｃＤＮＡライブラリーからの配列を、コード領域の上流および下流の配列に特異的なプライマーを用いて、増幅した。発現ベクターはｐＭＥＮ２０またはｐＭＥＮ６５であり、両者ともｐＭＯＮ３１６ （Ｓａｎｄｅｒｓ ｅｔ ａｌ， （１９８７） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ １５：１５４３−５８） に由来して、トランス遺伝子を発現するためのＣａＭＶ ３５Ｓプロモーターを含んでいる。配列をベクター中にクローニングするため、ｐＭＥＮ２０および増幅したＤＮＡ断片の両者を別々にＳａｌＩおよびＮｏｔＩ制限酵素で３７℃２時間消化した。消化産物を０．８％アガロースゲルで電気泳動し、エチジウムブロマイド染色で可視化した。この配列および直線化プラスミドを含むＤＮＡ断片を切り出し、Ｑｉａｑｕｉｃｋ ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ （Ｑｉａｇｅｎ，ＣＡ） を用いて精製した。興味対象の断片を３：１（ベクター対挿入片）の比率で連結した。Ｔ４　ＤＮＡリガーゼ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ， ＭＡ）を用いて、連結反応を１６℃１６時間行った。連結したＤＮＡを、熱ショック法を用いて、Ｅ． ｃｏｌｉ ＤＨ５ アルファ株のコンピテント細胞に形質転換した。形質転換体は、５０ｍｇ／ｌのスペクチノマインシ（Ｓｉｇｍａ） を含むＬＢプレートにまいた。
【００８８】
個々のコロニーを、５０ｍｇ／ｌのスペクチノマインシを含む５ｍｌのＬＢブロスで一夜、３７℃で増殖させた。プラスミドＤＮＡはＱｉａｑｕｉｃｋ Ｍｉｎｉ Ｐｒｅｐ ｋｉｔｓ （Ｑｉａｇｅｎ，ＣＡ）を用いて精製した。
【００８９】
実施例 ＩＶ　発現ベクターによるＡｇｒｏｂａｃｔｅｒｉｕｍの形質転換
遺伝子を含有するプラスミドベクターを構築した後、このベクターを使用して遺伝子産物を発現するＡｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ細胞を形質転換した。Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ細胞の形質転換用保存株は、Ｎａｇｅｌ ｅｔ ａｌ． ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌ Ｌｅｔｔｓ ６７： ３２５−３２８ （１９９０）の記載に従って作製した。吸光度（Ａ_６００）が０．５〜１．０に達するまで、Ａｇｒｏｂａｃｔｅｒｉｕｍ ＧＶ３１０１株を２５０ｍｌのＬＢ培地（Ｓｉｇｍａ）中にて一晩２８℃で振盪培養した。４，０００×ｇで１５分間４℃にて遠心分離を行うことにより細胞を回収した。次いで細胞を、２５０μｌの氷冷バッファー（１ｍＭ ＨＥＰＥＳ、ＫＯＨにてｐＨを７．０に調整）へ再懸濁させた。細胞を上述のように再度遠心分離し、１２５μｌの氷冷バッファーへ再懸濁させた。次いで遠心分離と再懸濁をさらに２回繰り返した。その際、上述と同一のＨＥＰＥＳバッファー１００μｌおよび７５０μｌへ細胞を懸濁させた。次いで、再懸濁した細胞を４０μｌずつ分注し、液体窒素中にて急速冷凍させて−８０℃で保存した。
【００９０】
Ｎａｇｅｌ ｅｔ ａｌ． ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌ Ｌｅｔｔｓ ６７： ３２５−３２８ （１９９０）に記載のプロトコールに従い、上述のように調製したプラスミドでＡｇｒｏｂａｃｔｅｒｉｕｍ細胞を形質転換した。形質転換すべき各ＤＮＡ構築物当たり、５０〜１００ｎｇのＤＮＡ（通常、１０ｍＭ Ｔｒｉｓ−ＨＣｌ、１ｍＭ ＥＤＴＡ、ｐＨ８．０へ再懸濁）をＡｇｒｏｂａｃｔｅｒｉｕｍ細胞４０μｌと混合した。次いで、氷冷キュベット（電極間隔２ｍｍ）へＤＮＡ／細胞混合物を移し、Ｇｅｎｅ Ｐｕｌｓｅｒ ＩＩ装置（Ｂｉｏ−Ｒａｄ）を用いて２５μＦおよび２００μＦにて２．５ｋＶの電圧を印加した。エレクトロポレーション後、細胞を１．０ｍｌのＬＢ培地へ直ちに再懸濁させ、抗生物質による選別を行わずに、振盪インキュベーター内で２〜４時間２８℃にて細胞を回復させた。回復後、スペクチノマイシン（Ｓｉｇｍａ）を１００μｇ／ｍｌ含有するＬＢブロス選択培地へ細胞を播種し、２４〜４８時間２８℃にてインキュベートした。次いで単一コロニーを拾い、新鮮な培地へ接種した。プラスミド構築物の完全性をＰＣＲ増幅および配列分析によって確認した。
【００９１】
実施例 Ｖ　 発現ベクターを保有するＡｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓによるＡｒａｂｉｄｏｐｓｉｓ植物体の形質転換
遺伝子を含有するプラスミドベクターでＡｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓを形質転換した後、単一のＡｇｒｏｂａｃｔｅｒｉｕｍコロニーを同定および培養し、Ａｒａｂｉｄｏｐｓｉｓ植物体の形質転換に使用した。簡単に言うと、スペクチノマイシンを５０ｍｇ／ｌ含有するＬＢ培地の５００ｍｌ培養物にコロニーを接種し、吸光度（Ａ_６００）が２．０を超えるまで２８℃で２日間振盪培養を行った。次いで、４，０００×ｇにて１０分間遠心分離を行うことにより細胞を回収し、浸潤用培地（１／２×Ｍｕｒａｓｈｉｇｅ ａｎｄ Ｓｋｏｏｇ塩 （Ｓｉｇｍａ）、１×Ｇａｍｂｏｒｇ’ｓ Ｂ−５ビタミン（Ｓｉｇｍａ）、５．０％（ｗ／ｖ）スクロース（Ｓｉｇｍａ）、０．０４４μＭ ベンジルアミノプリン（Ｓｉｇｍａ）、２００μｌ／Ｌ Ｓｉｌｗｅｔ Ｌ−７７ （Ｌｅｈｌｅ Ｓｅｅｄｓ））中に、吸光度（Ａ_６００）が０．８になるまで再懸濁させた。
【００９２】
形質転換を行う前に、ガラス繊維メッシュ（１８ｍｍ×１６ｍｍ）で覆ったＰｒｏ−Ｍｉｘ ＢＸ注封材（Ｈｕｍｍｅｒｔ Ｉｎｔｅｒｎａｔｉｏｎａｌ）上へ、４インチ鉢１つ当たり植物体が約１０体となるような密度にてＡｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａの種子（生態型：コロンビア）を蒔いた。連続照明下（５０〜７５μＥ／ｍ^２／秒）、２２〜２３℃および相対湿度６５〜７０％にて植物体を栽培した。約４週間後、一次花序茎（ｐｒｉｍａｒｙ ｉｎｆｌｏｒｅｓｃｅｎｃｅ ｓｔｅｍ）を刈り取り、二次茎が多く育つようにした。成熟二次茎が開花した後、長角果と開花した花を全て取り除くことにより植物体を形質転換用に調整した。
【００９３】
次いで、上述のＡｇｒｏｂａｃｔｅｒｉｕｍ浸潤培地混合物中へ鉢を上下逆さにして３０秒間浸漬し、プラスチック製のラップで覆った１フィート×２フィートの平面上で鉢を横にして水分を切った。２４時間後、プラスチックラップを外し、鉢を起こした。１週間後に浸漬手順を繰り返し、１鉢につき合計で２回浸漬を行った。次いで、形質転換の済んだ各鉢から種子を回収し、後述のプロトコールに従って分析を行った。
【００９４】
実施例 ＶＩ　 Ａｒａｂｉｄｏｐｓｉｓの一次形質転換体の同定
形質転換の済んだ鉢から回収した種子を、基本的に下記のようにして滅菌した。０．１％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００（Ｓｉｇｍａ）と滅菌水を含む溶液へ種子を分散させ、２０分間懸濁液を振盪させることで種子を洗浄した。次いで、洗浄溶液を捨てて新しい洗浄溶液と交換し、振盪させながら種子を２０分間洗浄した。２回目の洗浄溶液を除去した後、０．１％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００と７０％エタノール（Ｅｑｕｉｓｔａｒ）を含む溶液を種子へ添加し、懸濁液を５分間振盪した。エタノール／洗剤溶液を除去した後、０．１％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ−１００と３０％（ｖ／ｖ）漂白剤（Ｃｌｏｒｏｘ）を含む溶液を種子へ添加し、懸濁液を１０分間振盪した。漂白剤／洗剤溶液を除去した後、次いで種子を滅菌蒸留水中で５回洗浄した。最終洗浄水中にて種子を４℃で２日間、暗所にて保存した後、抗生物質選択培地（１×Ｍｕｒａｓｈｉｇｅ ａｎｄ Ｓｋｏｏｇ塩（１Ｍ ＫＯＨにてｐＨを５．７に調整）、１×Ｇａｍｂｏｒｇ’ｓ Ｂ−５ビタミン、０．９％ｐｈｙｔａｇａｒ （Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）、および５０ ｍｇ／Ｉカナマイシン）へ播種した。連続照明下（５０〜７５μＥ／ｍ^２／秒）、２２〜２３℃にて種子を発芽させた。このような条件で７〜１０日間育成した後、カナマイシン耐性一次形質転換体（Ｔ_１世代）を黙視で確認して取得した。これらの実生を、まず、新しい選択プレートへ移してさらに３〜５日間育成し、次いで土壌（Ｐｒｏ−Ｍｉｘ ＢＸ注封材）へ移した。
【００９５】
一次形質転換体を自家受粉させ、後代種子（Ｔ２）を回収した。Ｔ２後代種子を上述のようにカナマイシン上で発芽させ、カナマイシン耐性実生を選別して土壌へ移し、分析を行った。
【００９６】
実施例 ＶＩＩ　トランスジェニックＡｒａｂｉｄｏｐｓｉｓ植物体の分析
第１の実験では、Ｇ１５７植物体（即ち、Ｇ１５７トランスジーンを発現する植物体）を１２時間照明下にて栽培した。４０系統のうち３０系統が、対照ベクターで形質転換した対照植物よりも早く開花した。早咲きのＴ１系統の平均ロゼット葉数は１２．４±０．８であり、対照系統のロゼット葉数は２７±１．２であった。４０系統のＴ１植物体のうち２系統が対照と同時に開花し、４０系統のうち７系統は遅咲きであって、野生型よりも２〜３週間遅れて花序が確認された。
【００９７】
さらなる実験では、２４時間照明下、２０〜２５℃にて植物体を栽培した。このような条件下では、非形質転換対照植物体は、花芽形成前の一次苗条の平均合計葉数が１４．３±０．７であった。花芽は、種を蒔いてから平均で２１．１±０．５日後に初めて確認された（誤差の値は、９５％信頼限界を与える標準誤差を表す）。Ｇ８５９の場合は、Ｔ１植物体の１４／１９が早咲きであり（平均合計葉数は６．４±０．７、花芽は播種後１２．９±０．７日目に確認）、３／１９が野生型であり、２／１９が野生型に比べて若干遅咲きであった（平均合計葉数は１９、花芽は２７日目に確認）。ＲＴ発現研究からは、遅咲きの個体のトランスジーン発現量が最も高いことが判明した。これらの結果はＧ１５７の場合と非常に似通っている。Ｇ１８４２の場合には、Ｔ１植物体の７／１０が早咲きであり（平均合計葉数は７．９±０．６、花芽は１３．９±１．０日目に確認）、３／１０が野生型であった。また、Ｇ１８４２の短鎖スプライス変異体をコードするｃＤＮＡを用いて過剰発現研究を行った。Ｇ１８４２．２（１８５のアミノ酸からなるスプライス変異体をコード）の場合には、Ｔ１植物体の１５／１８が早咲きであり（平均合計葉数は６．９±０．９、花芽は１４．５±０．６日目に確認）、３／１８が野生型であった。Ｇ１８４２．６（７７のアミノ酸からなるスプライス変異体をコード）の場合には、Ｔ１植物体の８／１０が早咲きであり（平均合計葉数は６．８±１．６、花芽は１３．９±０．９日目に確認）、２／１０が野生型であった。Ｇ１８４２．７（１１８のアミノ酸からなるスプライス変異体をコード）の場合には、Ｔ１植物体の８／１０が早咲きであり（正確な葉数は未計測）、２／１０が野生型であった。このように、Ｇ１８４２スプライス変異体では、過剰発現した場合には、完全長ｃＤＮＡクローンに匹敵する効果が得られた。Ｇ１８４３の場合には、７／１１が早咲きであり（平均合計葉数は６．４±０．５、花芽は１６．０±１．６日目に確認）、２／１１が野生型の開花期を示した。しかしながら、Ｇ１８４３のＴ１植物体は生育が悪く、一部の器官の発達が遅れていた。このことから、Ｇ１８４３は、過剰発現した場合には不測の毒性作用を示すことが示唆される。Ｇ１８４４の場合には、Ｔ１植物体の６／１０が早咲きであり（平均合計葉数は６．８±１．７、花芽は１４．７±１．３日目に確認）、４／１０が野生型であった。同様に、Ｇ１８４４の短鎖スプライス変異体をコードするｃＤＮＡを用いて過剰発現研究を行った。Ｇ１８４４．２（１８４のアミノ酸からなるスプライス変異体をコード）を過剰発現させた場合には、Ｔ１植物体の６／１９が早咲きであり（平均合計葉数は７．８±１．７、花芽は１５．７±１．３日目に確認）、１３／１９が野生型であった。Ｇ８５９、Ｇ１８４２、Ｇ１８４３およびＧ１８４４の過剰発現データは、これらが開花期を制御する役割を担っているとの仮説を裏付けるものである。
【００９８】
ＲＴ−ＰＣＲはＧ１５７特異的プライマーを用いて約２５サイクルにてＧ１５７植物からの材料上において実施した。高いレベルのＧ１５７発現が、遅く開花する個々の植物または遅く開花する個々を含んだ保存された苗からのサンプル中に検出された。野生型に比較して中度または低いレベルの過剰発現しか示さなかった植物はわずかに開花が早いかまたは正常であた。
【００９９】
Ｇ１５７における増加がＡｒａｂｉｄｏｐｓｉｓの遅い開花生態型における開花時期に影響するか否かを試験するため、我々は、遅い開花生態型のＳｔｏｃｋｈｏｌｍおよびＰｉｚｔａｌにおいてＧ１５７を過剰発現させた。この実験において、各生態型からの３２の第一期形質転換体を連続光条件下で対照を散りばめて生育させた。両生態型において、約５０％の形質転換体が対処よりも早く開花し、そして少数のＰｉｚｔａｌおよびＳｔｏｃｋｈｏｌｍ形質転換体が対照に比較して明らかに遅い開花であった。
【０１００】
Ｇ１５７トランス遺伝子発現と開花時期の相関もＧ１５７　ＳｔｏｃｋｈｏｌｍおよびＰｉｚｔａｌ　Ｔ１植物において観察された。ＲＴ−ＰＣＲを、各バックグラウンドにおいて２つの早い開花系列及び２つの遅い開花系列を用いて実施した。再び、遅い開花系列は高いレベルのＧ１５７発現を含んだ。即ち、上記因子は定量性様式において開花時期に影響するらしい；中レベルの過剰発現が早い開花を誘因し、開花の遅延が大きく増加する。
【０１０１】
結論として、Ｇ１５７の過剰発現または同族の遺伝子の何れかが植物において開花時期を修飾する：中レベルの過剰発現が早い開花を誘引し、多くが開花を遅延させる。
【０１０２】
上記実施例に記載されたのと類似または同一の方法論を用いて、その変化した発現が開花の遅延または加速に相関した別のＡｒａｂｉｄｏｐｓｉｓ遺伝子を同定した。これらの遺伝子を表２にそれらの特定の配列番号およびそれらの開花時期への影響と共に図表作成した。
【０１０３】
【表２】

【０１０４】
春化処理の応答も調査した。遅い開花の春化処理応答性の生態型および変異体は高い定常レベルのＦＬＣ転写物を有し、春化処理による開花の促進の間は低下する（Ｍｉｃｈａｅｌｓ　ａｎｄ　Ａｍａｓｉｎｏ，（１９９９）Ｐｌａｎｔ　Ｃｅｌｌ　１１：９４９−９５６；Ｓｈｅｌｄｏｎ　ｅｔ　ａｌ．，（１９９９）Ｐｌａｎｔ　Ｃｅｌｌ　１１：４４５−４５８；Ｓｈｅｌｄｏｎ　ｅｔ　ａｌ．，（２０００）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．９７：３７３５−３７５８）。ＦＬＣとは対照的に、Ｇ１５７転写物レベルは遅い開花のＳｔｏｃｋｈｏｌｍおよびＰｉｚｔａｌ生態型における春化処理の応答と一致した相関は示さない。さらに、我々は、Ｇ１５７の過剰発現がＦＬＣレベルに影響しなかったことを見いだした。Ｇ８６１，Ｇ（５９，Ｇ１８４３，およびＧ１８４４の発現に対する春化処理の効果も試験した。Ｃｏｌｕｍｂｉａ、Ｐｉｚｔａｌ，Ｓｔｏｃｋｈｏｌｍ、ｃｏｎｓｔａｎｓ−１、およびｆｃａ−９の発芽種子をＭＳ寒天プレート上で４℃コールドルームにおいて８週間春化処理し、そして次に連続光生育ルームに移した。春化処理した種子および新たに撒いた非春化処理の対照からの全組織を移し換えから９日後に採集した。ＲＴ−ＰＣＲをＦＬＣ，Ｇ１５７，Ｇ８５９，Ｇ１８４２，Ｇ１８４３，Ｇ１８４４およびＧ８６１及びアクチンに関して実施した。ＦＬＣとＧ１５７を比較すると、どの遺伝子も５つの別々のサンプルセットにおける春化処理に際して明確な一致した低下を示さなかった。しかしながら、Ｇ１８４４はＦＬＣに対して発現の逆パターンを示した：Ｇ１８４４レベルは春化処理と一致して増加した。これは、春化処理の応答の対照においてＧ１８４４を直接に連座させるように、特別顕著である。即ち、Ｇ１８４４は開花を活性化し、そしてＦＬＣと反対の役割を有する可能性がある。
【０１０５】
Ｇ１５７の過剰発現が春化処理に匹敵する効果を有するか否かを探索するため、野生型ＰｉｚｔａｌおよびＳｔｏｃｋｈｏｌｍ苗のバッチを６週間４℃において冷却処理し、次にＧ１５７　Ｔ１　Ｐｉｚｔａｌ、Ｇ１５７　Ｔ１　Ｓｔｏｃｋｈｏｌｍおよび非春化処理の野生型植物の第２選択に対して生育させた。予測されたとおり、春化処理はＰｉｚｔａｌおよびＳｔｏｃｋｈｏｌｍ野生型植物の両方において開花時期を顕著且つ一律に縮めた。Ｇ１５７　Ｓｔｏｃｋｈｏｌｍ系列の間では、もっとも早い開花のＴ１グループ（８／２３系列）は春化処理された植物と識別不可能であった。Ｐｉｚｔａｌに関しては、しかしながら、早い開花のＴ１植物は春化処理した植物よりも平均して下限に近いほど遅かった。よって、Ｇ１５７の過剰発現は遅い開花の生態型における春化処理の要求を実質上減少させる。
【０１０６】
さらに、我々は、遅い開花のＧ１５７系列がＦＬＣ発現とは独立しており、そして春化処理に応答しないことを観察した。しかしながら、遅い開花のＧ１５７植物は光周期に応答性である。８時間の光の短日条件下で実施した実験においては、我々は、野生型対照より１カ月遅くに及んで開花した、多数のＧ１５７　Ｃｏｌｕｍｂｉａ　Ｔ１植物を得た（データは示さず）。Ｇ１５７発現により引き起こされた遅い開花の効果がＦＬＣ転写とは独立であったのか否かを確認するため、我々は、遅い開花のＧ１５７　Ｃｏｌｕｍｂｉａ植物が春化処理に応答性であったか否かを試験した。開花時期における顕著な変化は記録されなかった：連続光条件下で系列４の春化処理したＴ２植物は、全体で、春化処理した場合の３０．１＋／−１．３に比べて３１．３＋／−１．８を有した。対照のｆｃａ植物は、上記の処理が有効であったことを立証した：春化処理した植物は非春化処理の対照の４０より多い葉に比較して、たった１０．３＋／−０．９の葉の後に開花した。即ち、Ｇ１５７により引き起こされた遅い開花の表現型は、ＦＬＣ発現における変化とは独立して遅延が起こったか否かが予測されたように、春化処理により克服することができなかった。
【０１０７】
実施例ＩＸ．相同配列の同定
Ａｒａｂｉｄｏｐｓｉｓ以外の植物種の相同体をデータベースサーチツール、例えばＢａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ（ＢＬＡＳＴ）（Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．（１９９０）Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５：４０３−４１０；およびＡｌｔｓｃｈｕｌ　ｅｔ　ａｌ．（１９９７）Ｎｕｃｌ．Ａｃｉｄ　Ｒｅｓ．２５：３３８９−３４０２）を用いて同定した。ｔｂｌａｓｔｘ配列分析プログラムはＢＬＯＳＵＭ−６２スコアリングマトリックスを用いて実施した（Ｈｅｎｉｋｏｆｆ，Ｓ．ａｎｄ　Ｈｅｎｉｋｏｆｆ，Ｊ．Ｇ．（１９９２）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８９：１０９１５−１０９１９）。
【０１０８】
全部のＮＣＢＩジェンバンクデータベースをＡｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ以外の全ての植物からの配列に関してフィルターを通したが、ＮＣＢＩ分類ＩＤ３３０９０（Ｖｉｒｉｄｉｐｌａｎｔａｅ；全ての植物）を付随したＮＣＢＩジェンバンクデータベース中の全てを選択し、そして３７０１（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ）に付随した全体を排除することによった。これらの配列をワシントン大学ＴＢＬＡＳＴＸ計算式（バージョン２．０ａ１９ＭＰ）を用いてＳＥＱ　ＩＤｓ　１−５６の遺伝子を表す配列と２０００年９月２６日に比較した。ＳＥＱ　ＩＤｓ　１−５６の各遺伝子に関して個々の比較を確率スコア（Ｐ−値）により整列させたが、但し上記スコアは特定の整列が偶然に起こった確率を反映する。例えば、３．６ｅ−４０のスコアは３．６　ｘ　１０^−４０である。１０種までに関しては、もっとも低いＰ値を有する遺伝子（および、従って、最もありそうな相同体）を図２に掲載する。
【０１０９】
Ｐ値に加え、パーセンテージ同一性による比較もスコアした。パーセンテージ同一性は、２つのＤＮＡまたは蛋白質のセグメントが特定の長さにわたり同一である程度を反映する。図２に示す非Ａｒａｂｉｄｏｐｓｉｓ遺伝子と配列表の中のＡｒａｂｉｄｏｐｓｉｓ遺伝子の間のパーセント同一性の範囲は、ＳＥＱ　ＩＤ　Ｎｏ．１：５４％−６７％；ＳＥＱ　ＩＤ　Ｎｏｓ．３，５，７：３７％−４７％；ＳＥＱ　ＩＤ　Ｎｏｓ．９，１１，１３，１５：５４％−６２％；ＳＥＱ　ＩＤ　Ｎｏ．１７：６２％−７１％；ＳＥＱ　ＩＤ　Ｎｏｓ．１９，２１：５０％−６７％；ＳＥＱ　ＩＤ　Ｎｏｓ．２３，２５：７５％−９１％；ＳＥＱ　ＩＤ　Ｎｏ．２７：４６％−６９％；ＳＥＱ　ＩＤ　Ｎｏ．２９：４４％−９０％；ＳＥＱ　ＩＤ　Ｎｏ．３１：５７％−８９％；ＳＥＱ　ＩＤ　Ｎｏ．３３：３７％−７９％；ＳＥＱ　ＩＤ　Ｎｏ．３５：５０％−７１％；ＳＥＱ　ＩＤ　Ｎｏ．３７：３９％−６３％；ＳＥＱ　ＩＤ　Ｎｏ．３９：５８％−７０％；ＳＥＱ　ＩＤ　Ｎｏ．４１：４５％−７３％；ＳＥＱ　ＩＤ　Ｎｏ．４３：４２％−８４％；ＳＥＱ　ＩＤ　Ｎｏ．４５：４７％−８１％；ＳＥＱ　ＩＤ　Ｎｏ．４７：３１％−７１％；ＳＥＱ　ＩＤ　Ｎｏ．４９：４０％−６７％；ＳＥＱ　ＩＤ　Ｎｏ．５１：６９％−５１％；ＳＥＱ　ＩＤ　Ｎｏ．５３：４３％−８６％；そしてＳＥＱ　ＩＤ　Ｎｏ．５５：７９％−８９％。
【０１１０】
表２の中の遺伝子のＡｒａｂｉｄｏｐｓｉｓの相同体もＢＬＡＳＴを使用して同定した。これらの遺伝子は、以下のＡｒａｂｉｄｏｐｓｉｓ　ＢＡＣ配列において見いだされ。それらのジェンバンク配列ＮＩＤ番号：２８２７６９８（２３４相同体）、３２４１９１７（Ｇ７４８相同体）、２６１８６０４（Ｇ９９４相同体）、６５９８５４８（Ｇ１３３５相同体）、７３４０３３１（Ｇ７３６相同体）、６５２３０５１（Ｇ１４３５相同体）、６５９８４９１（Ｇ２０８相同体）および３１７２１５６（Ｇ２０８相同体）により確認される。
【０１１１】
全ての文献（刊行物および特許）はアユるもくてきの為にそれらの全体を引用により本明細書に取り込む。
発明は上記態様および実施例を参照して記載されてきたが、発明の精神を逸脱することなく様々な修飾がなされ得ることは理解されるべきである。従って、発明は以下の請求の範囲のみにより限定される。
【図面の簡単な説明】
【図１】図１は、本発明の典型的なポリヌクレオチドおよびポリペプチド配列の表を提供する。この表には、左から右に、それぞれの配列について、ＳＥＱ ＩＤ Ｎｏ、内部コード参照番号、配列がポリヌクレオチドまたはポリペプチド配列のいずれであるか、そしてポリペプチド配列についてのいずれかの保存ドメインの同定、が含まれる。
【図２】図２は、配列表中に提供する配列と相同な配列の表を提供する。この表には、左から右に、ＳＥＱ ＩＤ Ｎｏ、内部コード参照番号、独自のＧｅｎｂａｎｋ配列番号（ＮＩＤ）、比較が偶然に生成される蓋然性（Ｐ−値）、そして相同遺伝子が同定された種、が含まれる。[0001]
The present invention relates to US Provisional Application Serial Nos. 60 / 159,464 (filed October 12, 1999); 60 / 164,132 (filed November 8, 1999); 60 / 166,228 (11 November 1999). 60 / 197,899 (filed on April 17, 2000); and Plant Tray Modification III (filed on August 22, 2000).
[0002]
Field of the Invention
The present invention belongs to the field of plant molecular biology and relates to compositions and methods for modifying the flowering time or vernalization requirements of plants.
[0003]
Background of the Invention
In order to maximize reproductive success, plants have evolved complex mechanisms to ensure that they bloom under favorable conditions. Analysis of recent flowering mutants and ecotypes in Arabidopsis indicates that such a mechanism is based on several genetic pathways that may contain 80 or more genes. (Martinez-Zapater and Somerville, (1990) Plant Physiol. 92: 770-776; Koorneef et al. (1991) Mol. Gen. Genet. 229: 57-66; ) Arabidopsis pp 403-433 Cold Spring Harbor Laboratory Press, New York). At the same time, these loci regulate flowering time by environmental variables (eg, sunshine duration, temperature, light quality, and availability), and by the stage of plant development.
[0004]
Arabidopsis blooms rapidly when grown under long-day conditions or continuous light conditions for 16 hours, but much more slowly under short-day conditions at 8 or 10 hours light. Genes controlling this response constitute the photoperiodic pathway and have been identified by mutants that cause a delay in flowering under long-day conditions but do not alter flowering under short-day conditions. Examples from this group that promote flowering in response to long days include CONSTANS (CO), GIGANTEA (GI), FT, FWA, FE, FD, and FHA. A second group of genes, including LUMINIDEPENDENS (LD), FCA, FVE, FY, and FPA, form an autonomous pathway that is active under all sunshine conditions. Variants for this second group of genes flower more slowly than wild-type controls, irrespective of sunshine conditions (Koorneef et al. (1991) Mot Gen. Genet, 229: 57-66; EM Meyerwitz and CR) Somerville Eds (1994) Arabidopsis pp 403-433 Cold Spring Harbor Laboratory Press, New York).
[0005]
In addition to their differing responsiveness to sunshine conditions, mutants derived from the photoperiodic and autonomous pathways show differential responsiveness to prolonged cold (vernalization) treatments (Vence-Prue, (1975) Vernalization, In Photoperiodism in Plants pp 263-291, McGraw Hill, London). Via the vernalization reaction, for example, Arabidopsis ecotypes from higher north latitudes, such as Stockholm, are adapted to spring flowering by exposure to cold and winter conditions. This avoids flowering at the end of summer if seed maturation may be shortened by the onset of winter conditions (Reeves and Coupland, (2000) Curr. Opin. Plant Biol 3:37. -42). When these ecotypes are grown in the laboratory, they flower later, but can cool much earlier if the seeds are allowed to germinate for a cool period of 4-6 weeks. Similarly, mutants derived from the autonomous pathway show a very significant shortening of flowering time when subjected to vernalization. In contrast, mutants derived from the photoperiodic pathway show only a minor response to cold treatment (Chandler et al., (1996) Plant J. 10: 637-644; Koorneef et al., (1998). Genetics 148: 885-892). In this way, the requirement for the autonomous route condition can be overcome by the vernalization process (Reeves and  Coupland, (2000) Curr. Opin. Plant Biol 3: 37-42).
[0006]
The two Arabidopsis genes, FLOWERING LOCUS C (flowering locus C, FLC) (also known as FLOWERING LOCUS F, FLF), and FRIGIDA (FRI) together act to flower when vernalization is not performed. (Napp-Zinn, K. (1957) Indukt. Abstammungs. Verebungsl. 88: 253285; Napp-Zinn. K. (1985) CRC Handbook of Flowing, Vol. 2, Vol. 1, Vol. 2, Vol. 4, No. 1, Vol. Clarke and Dean (1994) Mol. Gen. Genet. 248: 81-89; Koorneef. Et al., (1994) Plant Jo. rnal 6:. 911-919; Lee et al, (1994) Plant Journal 6: 903-909).. Predominant functional alleles of FLC and FRI have been found together in northern European Arabidopsis ecotypes such as Pitztal and Stockholm. These ecotypes flower extremely late if not vernalized. The widely used laboratory ecotype Columbia contains functional alleles at only one of these two loci, and compared to strains such as Landsberg erecta that do not have functional alleles of either gene. Flowering somewhat late. The FRIGIDA protein sequence has not yet been published. However, the FLC gene was recently cloned and shown to encode a MADS box protein (Sheldon C. et al., 1999, Plant Cell 11: 445-458; Michaels S. and Amasino, R., 1999). , Plant Cell 11: 949-956). It was reported that predominant alleles and overexpression of FLC delayed flowering, while null flc mutants flowered earlier (Lee et al., (1994) Plant J. 6: 903-909; Michaels and Amasino, (1999) Plant Cell 11: 949-956; Sheldon et al., (1999) Proc. Natl. Acad. Sci. 97: 3753-3758). Thus, FLC acts to prevent premature flowering.
[0007]
We have found transcription factors that control plant flowering time or vernalization requirements. These transcription factors may therefore be useful for manipulating the flowering properties of plants.
[0008]
Summary of the Invention
In one aspect, the invention relates to a transgenic plant comprising the recombinant polynucleotide. The recombinant polynucleotide comprises a polypeptide comprising at least 6 contiguous amino acids of a sequence selected from the group consisting of SEQ ID No: 2N (where N is 1-28) but excluding SEQ ID No: 28 The presence of a recombinant polynucleotide alters the flowering time or vernalization requirement of a transgenic plant when compared to the same property of another plant that does not contain the recombinant polynucleotide. .
[0009]
In one embodiment, the nucleotide sequence is 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) SEQ ID No: 2N, where N is 1-28. ) Encodes a polypeptide comprising a conserved domain, such as a DNA binding domain. In another embodiment, the recombinant polynucleotide has a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. Encodes the containing polypeptide. In a further aspect, the nucleotide sequence further comprises a promoter operably linked to the nucleotide sequence. Promoters may be constitutive or inducible or tissue activated.
[0010]
In a second aspect, the present invention relates to a method for altering the flowering time or vernalization requirement of a plant. The method comprises: (a) a set comprising a nucleotide sequence encoding a polypeptide comprising at least 6 contiguous amino acids of a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. Transforming the plant with the altered polynucleotide; (b) selecting the transformed plant; and (c) identifying the transformed plant for the desired property.
[0011]
In one embodiment, the nucleotide sequence is 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) SEQ ID No: 2N, where N is 1-28. ) But encodes a polypeptide that contains a conserved domain such as a DNA binding domain except for SEQ ID No: 28. In another embodiment, the recombinant polynucleotide has a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. Encodes a polypeptide comprising In a further aspect, the nucleotide sequence further comprises a promoter operably linked to the nucleotide sequence. Promoters may be constitutive or inducible or tissue activated.
[0012]
In a third aspect, the invention relates to another method for altering plant characteristics associated with flowering time or vernalization requirements of the plant. The method comprises the steps of (a) combining at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID No: 2N-1 (where N is 1-28) but excluding SEQ ID No: 27 Transforming the plant with a recombinant polynucleotide comprising the nucleotide sequence comprising; and (b) selecting the transformed plant.
[0013]
In yet another aspect, the present invention is yet another method of altering the flowering time or vernalization requirement of a plant. The method comprises: (a) preparing a database sequence; (b) comparing the database sequence to a polypeptide selected from SEQ ID No: 2N, where N is 1-28; (c) Selecting a database sequence that matches the selected sequence classification; and (d) transforming said database sequence into a plant. Alternatively, the database sequence may be compared to a polynucleotide selected from SEQ ID No: 2N-1, where N is 1-28.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Definition
A "recombinant polynucleotide" is a nucleotide sequence that comprises the genetic coding sequence or a fragment thereof (comprising at least 18 contiguous nucleotides, preferably at least 30 contiguous nucleotides, and more preferably at least 50 contiguous nucleotides). . In addition, the polynucleotides may include promoters, introns, enhancer regions, polyadenylation sites, translation initiation sites, 5 'or 3' untranslated regions, reporter genes, selectable markers, and the like. A polynucleotide may comprise single-stranded or double-stranded DNA or RNA. A polynucleotide may include a modified base or a modified backbone. A polynucleotide may include a genomic sequence, a transcript sequence (eg, mRNA) or a processed nucleotide sequence (eg, cDNA). A polynucleotide may include a sequence in either the sense or antisense orientation.
[0015]
A "recombinant polynucleotide" is a polynucleotide that does not exist in nature, for example, a polynucleotide sequence that includes a nucleotide sequence not found in nature, or a polynucleotide sequence to which the polynucleotide is typically contiguous. It is separated or typically flanked by non-contiguous nucleotide sequences.
[0016]
A “recombinant polypeptide” is a polypeptide derived from the translation of a recombinant polynucleotide or is increased intracellularly, eg, by more than 5%, in a wild-type cell than a polypeptide that is in a native state. Or more than 10%, or more than 20%, and not as a result of the natural response of the wild-type plant, or Is separated from the constituent materials.
[0017]
“Transgenic plants” are genetic materials that are not normally found in wild-type plants of the same species, or in naturally occurring varieties, or cultivars, and have been introduced into plants by human manipulation. It is a plant containing. A transgenic plant is a plant that may contain an expression vector or cassette. The expression cassette contains the gene coding sequence and allows for expression of the gene coding sequence. The expression cassette can be introduced by transformation or by crossing after transformation of the parent plant.
[0018]
Transgenic plants include not only whole plants, but also, for example, plant parts such as seeds, fruits, leaves, or roots, plant tissues, plant cells, protoplasts, or any other plant material, and their progeny. That means.
[0019]
The phrase "altered expression" with respect to the expression of a polynucleotide or polypeptide refers to an expression pattern in a transgenic plant that is different from that in a wild-type plant; or, for example, the sequence is expressed in a wild-type plant Being expressed in a cell type different from the cell type, or being expressed at a time different from the time when the sequence is expressed in a wild type plant, or being responsive to different inducible substances such as hormones and environmental signals, Or a different expression level (either higher or lower) compared to the expression level found in wild-type plants, and so on. The term also refers to reducing the level of expression to below the level of detection, or eliminating expression altogether. The resulting expression pattern may be transient or stable, constitutive or inducible.
[0020]
A “transcription factor” (TF) directs the expression of one or more genes, either directly by joining one or more nucleotide sequences that join the gene coding sequence, or one or more genes that join the gene coding sequence. A polynucleotide or polypeptide that directly regulates a further nucleotide sequence or is indirectly regulated by affecting the level or activity of another polypeptide that actually binds indirectly. TF in this definition includes any polypeptide that can activate or repress the transcription of a single gene or multiple genes. This group of polypeptides includes, but is not limited to, DNA binding proteins, protein kinases, protein phosphatases, GTP-binding proteins and receptors.
[0021]
A transcription factor sequence may include the entire coding sequence or a fragment or domain of the coding sequence. A “fragment or domain” when referring to a polypeptide, refers to at least one biological function of the intact polypeptide, in substantially the same way or to a similar extent as the intact polypeptide performs its function. It may be a part of the polypeptide that exerts its effect. Fragments may include, for example, a DNA binding domain that binds to a specific DNA promoter region, an activation domain, or a domain for protein-protein interaction. Fragments may vary in size from as little as 6 amino acids to the length of the intact polypeptide, but are preferably at least 30 amino acids in length, and more preferably at least 60 amino acids in length. When referring to nucleotide sequences, a "fragment" refers to any sequence of at least 18 contiguous nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, of any of the sequences provided herein. I mean that.
[0022]
Exemplary polynucleotides and polypeptides include the following sequences provided in the Sequence Listing: SEQ ID No: 1: G157 (cDNA); SEQ ID No: 2: G157 (protein); SEQ ID No : 3: G859 (cDNA); SEQ ID No: 4: G859 (protein); SEQ ID No: 5: G859.1 (cDNA); SEQ ID No: 6: G859.1 (protein); SEQ ID No: 7 : G859.2 (cDNA); SEQ ID No: 8: G859.2 (protein); SEQ ID No: 9: G1842 (cDNA); SEQ ID No: 10: G1842 (protein); SEQ ID No: 11: G1842 .2 (cDNA); SEQ ID No: 12: G1842.2 (protein) SEQ ID No: 13: G1842.6 (cDNA); SEQ ID No: 14: G1842.6 (protein); SEQ ID No: 15: G1842.7 (cDNA); SEQ ID No: 16: G1842.7 (protein) SEQ ID No: 17: G1843 (cDNA); SEQ ID No: 18: G1843 (protein); SEQ ID No: 19: G1844 (cDNA); SEQ ID No: 20: G1844 (protein); SEQ ID No: 21: G1844.2 (cDNA); SEQ ID No: 22: G1844.2 (protein); SEQ ID No: 23: G861 (cDNA); SEQ ID No: 24: G861 (protein); SEQ ID No: 25: G861.1 (cDNA); SEQ D No: 26: G861.1 (protein); SEQ ID No: 27: G1759 (cDNA); SEQ ID No: 28: G1759 (protein); SEQ ID No: 29: G192 (cDNA); SEQ ID No: 30 SEQ ID No: 31: G234 (cDNA); SEQ ID No: 32: G234 (protein); SEQ ID No: 33: G361 (cDNA); SEQ ID No: 34: G361 (protein); SEQ ID No: 35: G486 (cDNA); SEQ ID No: 36: G486 (protein); SEQ ID No: 37: G748 (cDNA); SEQ ID No: 38: G748 (protein); SEQ ID No: 39: G994 (cDNA); SEQ D No: 40: G994 (protein); SEQ ID No: 41: G1335 (cDNA); SEQ ID No: 42: G1335 (protein); SEQ ID No: 43: G562 (cDNA); SEQ ID No: 44: G562 (Protein); SEQ ID No: 45: G736 (cDNA); SEQ ID No: 46: G736 (protein); SEQ ID No: 47: G1073 (cDNA); SEQ ID No: 48: G1073 (protein); No: 49: G1435 (cDNA); SEQ ID No: 50: G1435 (protein); SEQ ID No: 51: G180 (cDNA); SEQ ID No: 52: G180 (protein); SEQ ID No: 53: G592 ( cDNA); SE ID No: 54: G592 (protein); SEQ ID No: 55: G208 (cDNA); and SEQ ID No: 56: G208 (protein).
[0023]
A "conserved domain" refers to a polynucleotide or polypeptide fragment that is more conserved at the sequence level than other fragments when comparing the polynucleotide or polypeptide to homologous genes or proteins from other plants. The conserved domains are: 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) a dimeriZation or an oligomeriZation domain, 5) It may be a DNA binding domain or a combination thereof. For MADS proteins, the conserved domain is typically a DNA binding domain.
[0024]
A nucleotide sequence is "operably linked" when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or enhancer is operably linked to a sequence encoding a gene when the presence of the promoter or enhancer increases the expression level of the sequence encoding the gene.
[0025]
"Trait" refers to the physiological, morphological, biochemical, or physical properties of a plant or particular plant material or cell. This property is visible to the human, such as the size of a seed or plant, or by biochemical techniques, such as the protein, starch, or oil content of the seed or leaf, or by Northern, RTPCR, microarray It may be measured by observing the gene expression level using a gene expression assay or a reporter gene expression system, or may be measured by agricultural observation such as stress tolerance, yield or disease resistance.
[0026]
"Trait modification" refers to a detectable difference in the property of a polynucleotide or polypeptide of the invention in a transformed plant in which the expression has been modified, as compared to a non-plant (such as a wild-type plant). Say. The trait alteration is at least a 5% increase or decrease in the observed trait (difference), at least a 10% difference, at least a 20% difference, at least 30%, at least 50%, at least 70%, at least 100%, or even more. It may result in a big difference. It is known that altered traits may have natural variability. Thus, the observed trait modification involves a change in the normal distribution of the trait in the transformed plant compared to the distribution observed in the wild-type plant.
[0027]
Specific trait modifications are trait modifications of seeds (embryos), fruits, roots, flowers, leaves, stems, stems, saplings, etc., including: frozen, cold, heat, drought, water saturated, Increased resistance to environmental conditions, including radiation, ozone; increased resistance to diseases caused by microorganisms, fungi, and viruses; resistance to nematodes, reduced herbicide susceptibility, increased resistance to heavy metals (or increased Heavy metal uptake capacity), enhanced growth under poor light conditions (eg, weak light / or short daytime), or altered expression levels of certain genes. Other phenotypes that may be modified include taxol, tocopherol, tocotrienol, sterol, phytosterol, vitamins, wax monomers, antioxidants, amino acids, lignin, cellulose, tannins, prenyl lipids (such as chlorophyll and carotenoids), Production of plant metabolites, such as variability in the production of glucosinolates and terpenoids, enhanced or altered protein and lipid production (particularly in seeds), or modified sugars (insoluble or soluble) and / or Or the composition of the starch. Physical plant properties that may be modified include cell development (eg, number of protruding structures), fruit and seed size and number, yield of plant parts such as stems, leaves and roots, storage Seed stability, seed pod properties (eg, fragility), root hair length and quantity, internode distance, or seed shell quality. The growth characteristics of plants that may be modified include growth rate, seed germination rate, plant and young plant momentum, leaf and flower senescence, male inability, apomixis, flowering time, flower organ detachment, nitrogen uptake rate. , Biomass or transpiration characteristics, as well as properties of the plant structure such as apical dominance, branching pattern, number of organs, organ identity, organ shape and abundance.
[0028]
Of particular interest are altered vernalization requirements, such as changes in flowering time in response to daytime, temperature, light quality, nutrient availability, and plant development stages. And traits related to the characteristics of flowering time.
[0029]
1. Array
We have found certain plant transcription factors (TFs) that can be used to alter the flowering time of plants. That is, the use of the TFs of the present invention makes it possible to reduce, increase or induce the flowering time of a plant under specific conditions. In addition, this transcription factor can be used to modify the vernalization requirements of plants.
[0030]
These plant transcription factors will belong to one of the following transcription factor families:
AP2 (APETALA2) domain transcription factor family (Richmann and Meyowitz (1998) Biol. Chem. 379: 633-646); the MYB transcription factor family (Martin and Paz-Ares, (1997) Trends 13-67. The MADS domain transcription factor family (Riechmann and Meyerowitz (1997) Biol. Chem. 378: 1079-1101); the WRKY protein family (Ishiro and Nakamura, 1994; Ganemura, G. 1974; ankyrin-repeat protein family Zhang et al. (1992) Plant Ce 114: 1575-1588); the zinc finger protein (Z) family (Klug and Schwabe (1995) FASEB J. 9: 597-604 (the mobile phone); (1994) Guidebook to the Homebox Genes, Oxford University Press; the CART-element binding proteins (Forsurge and Guarente (1989) 11b. (Klein et al. (1996) Mol. Gen. Genet. 1996 250: 7-16); the NAM protein family (Souer et al. (1996) Cell 85: 159-170); the IAA / AUX proteins al. (1998) Science 279: 13711373); the HLH / MYC protein family (Littlewood et al. (1994) Prot. Profile 1: 639-709); the DNAbinding protein (DByl. (1994) EMBO J .; 13: 2994-3002); the bZIP family of transcription factors (Foster et al. (1994) FASEB J. 8: 192-200); the Box P-Binding (the Belfa sylvan FteBay). al. (1993) Plant J. 4: 125-135); the high mobility group (HMG) family (Bustin and Reeves (1996) Prog. Nucl. Acids Res. Mol. Biol. (SCR) family (Di Laurenzio et al. (1996) Cell 86: 423). -433); the GF14 family (Wu et al. (1997) Plant Physiol. 114: 1421-1431); the polycomb (PCOMB) family (Kennison (1995) Annu. Rev. Genet. 29: 29-29: 29-29). teosint branched (TEO) family (Luo et al. (1996) Nature 383: 794-799; the AB13 family (Giraudat et al. (1992) Plant Cell 4: 1251-1261); et al. (1990) Science 250: 1397-1399); family (Chao et al. (1997) Cell 89: 1133-44); the AT-HOOK family (Reeves and Nissen (1990) Journal of Biochemical Chemistry 265: 8573-Zeo. 1995) Nucleic Acids Res. 23: 11651169); the bZIPT2 family (Lu and Ferl (1995) Plant Physiol. 109: 723); the YABBBY family (Bowman et al. (1999) Development 126: 2387-96); the PAZ family (Bohmert et al. (1998) EMBO J.e.am.e.om. (MISC) transscription factors including the DPBF family (Kim et al. (1997) Plant J. 11: 1237-1251) and the SPF1 family: Ishiguro and Namikawa. golden (GLD) family (Hall et . L (1998) Plant Cell 10: 925-936), and the TUBBY family (Boggin et al, (1999) Science 286: 2119-2125)
In particular, the TFs that we have found related to flowering time and vernalization are members of the MADS transcription family, MYB family, WRKY family, HLH / MYC family, GLD family, AT-HOOK family, CAAT family, bZIP family, and zinc. Includes members of the cooperative protein family (Z-Dof, D-CLDSH, and Z-CH2H2). In fact, we have identified the first members of the WRKY, CAAT, bZIP, AT-HOOK, and HLH / MYC families associated with alterations in plant flowering time: G192 and G190 (WRKY), G486 (CAAT), G562 (bZIP), G1073 (AT-HOOK) and G592 (HLH / MYC).
[0031]
These polynucleotides and polypeptides are listed in the Sequence Listing and tabulated in FIG. FIG. 1 shows the nucleotide sequence number, the corresponding GID number, whether the sequence is a polynucleotide or polypeptide sequence, and the conserved domains. We have also identified domains or fragments derived from each nucleotide sequence in the nucleotide sequence listing. Fragments can be from any location in the sequence, can be of any length up to the length of the sequence, are as short as 6 residues for proteins and as short as 18 bases for DNA. May be. Examples of fragments of a DNA sequence are as follows: 1-50, 51-100, 101-200, 201-218, 218-300, 301-450, and 450-600; It is as follows: 1-50, 51-100, 101-200, 201-206, 206-250, 251-300. For DNA sequences, numbers were determined from the 5 'or 3' end of the DNA. With respect to the protein sequence, the location of the fragment is determined from the N-terminus or C-terminus of the protein and includes the contiguous amino acid sequence, e.g. Includes 20, 40, 60 or 100 amino acids.
[0032]
The identified polypeptide fragments are ligated to fragments or sequences derived from other transcription factors and are referred to as Short, PCT Publication No. WO 98227230, "Methods and Compositions for Polypeptide Engineering", or Patten et al., PCT Publications WO9923236, "Moftho DNA". More novel sequences are generated using the method described in Minshul and Stemmer, US Pat. No. 5,837,458. Alternatively, the identified fragment may be linked to a transcription activation domain. The transcription activation domain helps initiate transcription from the DNA binding site. A common property of some activation domains is that they form an amphipathic alpha helix with an excess of positive or negative charge (Giniger and Ptashne (1987) Nature 330: 670-672,). Gill and Ptashne (1987) Cell 51: 121-126, Estruch et al (1994) Nucl. Acids Res. 22: 3983-3989). For example, the transcriptional activation region of VP16 or GAL4 (Moore et al. (1998) @HYPERLINK http: // Proc. Natl. Acad. Sci. USA)Proc. Natl. Acad. Sci. USA 95: 376-381; and Aoyama et al. (1995) Plant Cell 7: 1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51; 113-119), and synthetic peptides (Giniger and Ptashne, supra).
[0033]
The isolated polynucleotides and polypeptides may be used to modify plant development, physiology, or biochemistry such that the modified plant has more favorable traits than a wild-type plant. The identified polynucleotide fragment is also useful as a nucleic acid probe or primer. Nucleic acid probes are useful in hybridization protocols, including those for microarray experiments. The primer anneals to the complementary target DNA strand by nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and is extended along the target DNA by the DNA polymerase enzyme. Primer pairs can be used, for example, to amplify nucleic acid sequences by the polymerase chain reaction (PCR) or other nucleic acid amplification methods. See Sambrook et al., Molecular Cloning. A Laboratory Manual, Ed. 2, Cold Spring Harbor Laboratory Press, New York (1989) and Ausubel et al. (Eds) Current Protocols in Molecular Biology, John Wiley & Sons (1998).
[0034]
2. Identification of homologous sequences (Homologs)
Arabidopsis thalianaAlternatively, a sequence homologous to those listed in the nucleotide sequence list derived from other plants may be used to modify plant characteristics. The homologous sequence may be derived from any plant including monocotyledonous plants and dicotyledonous plants, and includes, but is not limited to, particularly agriculturally important plant species such as the following, soybean, wheat, corn, Grains such as potatoes, rice, rapeseed (including canola), sunflower, alfalfa, sugarcane and turf; banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grape, Honeyju, lettuce, mango, melon, onion, papaya, beans, pepper, pineapple, spinach, pumpkin, sweet corn, tobacco, tomato, watermelon, rose fruits (apple, peach, none, cherry, plum, etc.), And Brassica vegetables (broccoli, cabbage, cauliflower) Brussels sprouts, kohlrabi, etc.) fruits and vegetables, such as. Other cereals, fruits and vegetables that can change phenotype include barley, currants, avocados, oranges, lemons, grapefruits, citrus fruits such as tangerine, arch chalks, cherries, walnuts and peanuts. Includes nuts, endives, leek, arrow roots, beets, cassava, turnips, radishes, yams, root vegetables such as sweet potatoes, and legumes. Homologous sequences may be derived from tree species such as pine, poplar, eucalyptus.
[0035]
Substitutions, deletions and insertions introduced into the sequences shown in the sequence listing are also included within the scope of the present invention. Such sequence modifications can be engineered by site-directed mutagenesis (Wu (ed.) Meth. Enzymol. (1993) vol. 217, Academic Press). Amino acid substitutions are typically single residue substitutions; insertions are generally on the order of about 1 to 10 amino acid residues; and deletions range from about 1 to 30 residues. In a preferred embodiment, the deletions or insertions are made in adjacent sets, for example a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof, can occur in combination in the sequence. Mutations made in the polynucleotide encoding the transcription factor must not place the sequence out of reading frame and should not create a complementary region that becomes a secondary mRNA structure.
[0036]
Substitution is the removal of at least one residue in the amino acid sequence and the insertion of a different residue in its place. Such substitutions are conservative, having little effect on the function of the gene, for example, by replacing alanine for serine, arginine for lysine, glutamate for aspartate, and the like. Things. Substitutions that would not be conservative and would result in a large change in the properties of the protein include: (B) substitution of cysteine or proline for another residue; (c) an electronegative residue of a residue having an electropositive side chain (eg, lysyl, arginyl, or histidyl) (eg, glutamyl, Or (d) substitution of a residue having a large side chain (eg, phenylalanine) with a residue having no side chain (glycine).
[0037]
Furthermore, the term “homologous sequence” includes polypeptide sequences that have been modified by chemical or enzymatic means. A homologous sequence may be a sequence modified by lipids, sugars, peptides, organic or inorganic compounds, or a sequence modified by the use of modified amino acids. Protein modification techniques are described in Ausubel et al. (Eds) Current Protocols in Molecular Biology, John Wiley & Sons (1998).
[0038]
Homologous sequences also use sequence analysis programs available from database searches, sequence alignments and comparisons (eg, from Wisconsin Package Version 10.0 (GCG, Medicine, WI) such as BLAST, FASTA, PILEUP, FINDPATTERNS, etc.). Two sequences having a percentage that is considered to be substantially sequence identity after alignment as determined by the above. Searches can be made in public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR, or in private sequence databases such as PhytoSeq (Incyte Pharmaceuticals, Palo Alto, CA). Sequence alignments for comparison are described in the local homology algorithm of Smith and Waterman (1981) Adv. App /. Math. 2: 482, by the homology algorithm of Needleman and Wunsch (1970) Mol. Biol. 48: 443, by the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U. S. A. 85: 2444, which can be performed by computer implementation of those algorithms. Following alignment, sequence comparisons between two or more polynucleotides or polypeptides typically involve comparing the entire comparison region with the sequence of the two sequences by identifying and comparing local regions of sequence similarity. It is done by doing. The comparison region may be a segment of at least about 20 contiguous locations, typically about 50 to about 200, and more usually about 100 to 150 contiguous locations. The method is described in Ausubel et al. (Eds) (1999) Current Protocols in Molecular Biology, John Wiley & Sons.
[0039]
Transcription factors that are homologs of the described sequences typically share at least 40% amino acid sequence identity. More closely related TFs have at least 50%, 60%, 65%, 70%, 75% or 80% sequence identity with the described sequences. The factors most closely related to the disclosed sequences have at least 85%, 90% or 95% sequence identity. At the nucleotide level, sequences typically have at least 40% nucleotide sequence identity, preferably at least 50%, 60%, 70% or 80% sequence identity, and more preferably 85% , 90%, 95% or 97% sequence identity. The degeneracy of the genetic code allows for the preservation of major diversity in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.
[0040]
One way to determine whether two nucleic acid molecules are closely related is whether the two molecules hybridize to each other under stringent conditions. In general, the stringent conditions are selected to be about 5 ° C. to 20 ° C. lower than the thermal melting temperature (Tm) of the specific sequence under a predetermined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Nucleic acid hybridization conditions and stringency calculations are described in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual, Ed. 2, Cold Spring Harbor Laboratory Press, New York, and Tijssen (1993) Laboratory Techniques in Biochemistry, Molecular Biologics, Hybrid Biologics-Hybridology-Hybridication-Hybridology Nucleic acid molecules that hybridize under stringent conditions typically include a probe based on the full-length cDNA or a selected portion of the cDNA, with 0.2 × SSC to 2.0 × SSC, 0.1% SDS, 50%. Hybridize under washing conditions at -65 ° C, for example, washing conditions of 0.2 x SSC, 0.1% SDS, 65 ° C. For detection of lower related homologs, washing can also be performed at 50 ° C.
[0041]
For normal hybridization, a hybridization probe is attached to a detection label, such as a radioactive label, and the probe is preferably at least 20 nucleotides in length. As is well known to those skilled in the art, increasing the length of a hybridization probe increases specificity. Labeled probes derived from Arabidopsis nucleotide sequences can hybridize to plant cDNA or genomic libraries and hybridization signals detected by means known to those of skill in the art. The hybridized colonies or plaques are purified (depending on the type of library used), and the cloned sequence containing the colonies or plaques is isolated and characterized. Homologs can be analyzed using denaturing primers by PCR-based techniques (such as inverse PCR or RACE). Ausubel et al. (Eds) (1998) Current Protocols in Molecular Biology, John Wiley & Sons.
[0042]
Furthermore, TF homologs can also be obtained by immunoscreening an expression library. Given the described TF nucleic acid sequence, the polypeptide can be expressed and purified by a heterologous expression system (eg, E. coli), and produce an antibody (monoclonal or polyclonal) specific for the TF. Can also be used for Antibodies can also be raised against synthetic peptides derived from the TF amino acid sequence. Methods for producing antibodies are well known to those of skill in the art and are described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such antibodies can be used to screen an expression library made from the plant from which the TF homolog is to be cloned, using the methods described above. The selected cDNA can be confirmed by sequence analysis and biological activity.
[0043]
3. Improved transcription factor expression
Any identified sequences are inserted into cassettes or vectors for expression in plants. Several expression vectors suitable for the appropriate transformation of plant cells or for the establishment of transformed plants are described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvinet. , (1990), including those described in Plant Molecular Biology Manual, Kluer Academic Publishers. Characteristic examples include those obtained from the Ti plasmid of Agrobacterium tumefaciens, as well as those described in Herrera-Estellla, L. et al. , Et al. , (1983) Nature 303: 209, Bevan, M .; , Nucl. Acids Res. (1984) 12: 8711-8721, Klee, H .; J. , (1985) Biotechnology 3: 637-642, including those for dicotyledonous plants. Ti-derived plasmids can be transformed into both monocots and dicots using Agrobacterium-mediated transformation (Ishida et al (1996) Nat. Biotechnol. 14: 745-50; Barton et al. (1983) Cell 32: 1033-1043).
[0044]
Alternatively, non-Ti vectors can be used to transfer DNA into plants and cells using free DNA transfer techniques. Such methods include, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. Using those techniques, transgenic plants such as wheat, rice (Christou, P., (1991) Biotechnology 9: 957-962), and corn (Gordon-Kamm, W., (1990) Plant Cell 2: 603-618). Direct DNA Transfer Technology Using Particle Guns (Weeks, T. et al., (1993) Plant Physiol. 102: 1077-1084; Vasil, V., (1993) BiolTechnology 10: 667-674; Wan, Y. and In Lemeaux, P., (1994) Plant Physiol. 104: 37-48, and Agrobacterium-mediated DNA transformation (Ishida et al., (1996) Nature Biotech. 14: 745-750), the immature embryo is a monocotyledon. A good target.
[0045]
Typically, a plant transformation vector contains one or more cloned plant coding sequences (genomic or cDNA) under transcriptional control of 5 'and 3' regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically contain a promoter (eg, whether expression is inducible or constitutive, environmentally or developmentally dominant, or cell or tissue specific). Control sites that control DNA), transcription initiation sites, RNA process signals (such as intron splice sites), transcription termination sites, and / or polyadenylation signals.
[0046]
Examples of constitutive plant promoters that can be used to express TF sequences include the cauliflower mosaic virus (CaMV) 35S promoter that provides constitutive and high level expression in most plant tissues (eg, Odel et al., (1985)). Nature 313: 810); nopaline synthetic promoter (An et al., (1988) Plant Physiol. 88: 547); and octopine synthetic promoter (Fromm et al., (1989) Plant Cell 1: 977).
[0047]
A variety of plant gene promoters that respond to environmental, hormonal, chemical, and developmental signals and regulate gene expression in a tissue-active manner can be used for expression of TF in plants, including: and so on. US Pat. No. Seed-specific promoters such as the napin, phaseolin or DC3 promoters described in US Pat. No. 5,773,697. Root-specific promoters as described in US Pat. Nos. 5,618,988, 5,837,848 and 5,905,186; drul promoter (US Pat. No. 5,783,393), or 2A11 promoter (US No. 4,943,674) and a tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol. Biol. 11: 651), a fruit-specific promoter active during the fruit ripening period, US Patent Nos. Pollens such as PTA29, PTA26 and PTA13 (US Pat. No. 5,792,929), root-specific promoters as described in US 5,618,988, 5,837,848 and 5,905,186. Activated promoter, promoter active in vascular tissue (Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), flower-specific (Kaiser et al, (1995) Plant Mol. Biol. 28: 231-). 243), pollen (Baerson et al. (1994) Plant MoI. Biol. 26: 1947-1959), carpel (Ohl et al. (1990) Plant Cell 2: 837-848), pollen and early developmental seeds ( Bae ... Son et al (1993) Plant Mol Biol 22: 255-267), van der Kop et al (1999) Plant Mol. Siol. 39: 979-990 or Baumann et al. (1999) Plant Cell 11: 323-334), which reacts with an auxin-inducible promoter, a cytokinin-inducible promoter (Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), and gibberellin. Promoters (Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmot et al. (1998) 38: 817-825), and the like. Examples of additional promoters include heat (Ainley, et al. (1993) Plant Mol. Biol. 22: 13-23), light (eg, pea rbcS-3A promoter, Kuhlemeier et al., (1989) Plant Cell 1). : 471, and the maize rbcS promoter, Schaffner and Sheen, (1991) Plant Cell 3: 997); trauma (eg, wunl, Siebertz et al., (1989) Plant Cell 1: 961); Promoters whose expression is induced in response to chemicals such as salicylic acid (Gatz et al., (1997) Plant Mol. Biol. 48: 89-108). There is over. In addition, the timing of expression can be controlled by using a promoter that acts late in seed development (Odel et al. (1994) Plant Physiol. 106: 447-458).
[0048]
Plant expression vectors can include RNA processing signals located upstream or downstream of the coding sequence. Furthermore, the expression vector may be a 3 'terminator that increases the stability of the mRNA, such as the 3' untranslated region of a plant gene, such as the PI-II terminator region of potato, or the octopine or nopaline synthase 3 'terminator region. From the region, additional control sequences can be included.
[0049]
Finally, as noted above, plant expression vectors may contain a dominant selectable marker gene that allows for rapid selection of transformants. Such genes include genes encoding antibiotic resistance genes (eg, hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (eg, phosphinothricin acetyltransferase). Including.
[0050]
Reduction of TF expression in transgenic plants to modify plant traits can be obtained by introducing into plants a TF cDNA-based antisense construct. For antisense suppression, the TF cDNA is placed in the opposite direction to the promoter sequence of the expression vector. The sequence introduced need not be the full length TF cDNA or gene, nor need it be the same as the TF cDNA or the gene found in the type of plant to be transformed. However, if the introduced sequence is shorter, effective antisense suppression will usually require higher homology to the native TF sequence. Preferably, the length of the introduced antisense sequence in the vector is at least 30 nucleotides, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. The length of the antisense sequence in the vector is preferably at least 100 nucleotides. As noted above, transcription of the antisense construct results in the production of an RNA molecule, which is reversely complementary to an mRNA molecule transcribed from the endogenous TF gene in plant cells. Suppression of endogenous TF gene expression can also be achieved by using ribozymes. Ribozymes are synthetic RNA molecules with highly specific endoribonuclease activity. The production and use of ribozymes is disclosed in U.S. Patent No. 4,987,071 to Cech and U.S. Patent No. 5,543,508 to Haselhoff. Induction of a ribozyme sequence in the antisense RNA can be used to confer RNA degradation activity on the antisense RNA. Thereby, endogenous mRNA molecules that bind to the antisense RNA are degraded, and the inhibition of endogenous gene expression by antisense is increased.
[0051]
Using a vector that overexpresses the RNA encoded by the TF cDNA (or a variant thereof), co-suppression of the endogenous TF gene can also be achieved by the method described in US Patent No. 5,231,020 to Jorgensen. Such co-suppression (also referred to as sense suppression) does not require that the complete TF cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly the same as the endogenous TF gene. However, as in the case of antisense suppression, suppression efficiency will increase as (1) the introduced sequence is longer and (2) the sequence homology between the introduced sequence and the endogenous TF gene increases.
[0052]
It is also possible to suppress the expression of endogenous TF activity by using a vector that expresses a non-translatable form of TF mRNA and transform the trait. Methods for producing such constructs are described in Dougherty et al., US Pat. No. 5,583,021. Preferably, such constructs are made by introducing an early stop codon into the TF gene. Alternatively, plant traits can be modified by silencing the gene using double-stranded RNA (Sharp (1999) Genes and Development 13: 139-141). This approach allows the vector to be prepared, the cDNA or gene placed in an overlapping fashion, and the generation of a double-stranded RNA molecule with a hairpin structure upon expression. This method has been used to modify the genetic activity of plants (Chuang and Meyerowitz (1999) Proc. Natl. Acad. Sci. 97: 4985-9490).
[0053]
Another way to abolish gene expression is by insertional mutagenesis using Agrobacterium tumefaciens T-DNA. After the generation of the insertion mutant, the mutant can be screened to identify a mutant containing an insertion in the TF gene. Mutants that contain a single mutation event in the desired gene can also be crossed for mutation to produce homozygous plants (Koncz et al. (1992) Methods in Arabidopsis Research. World Scientific).
[0054]
Plant traits can also be modified using the cre-lox system (eg, as described in US Pat. No. 5,658,772). The plant genome can be modified to include first and second lox sites. These sites are then contacted with Cre recombinase. If these lox sites are in the same orientation, the intervening DNA sequence between the two sites will be excised. If these lox sites are inverted, the intervening sequence will be inverted.
[0055]
The polynucleotides and polypeptides of the present invention can also be expressed in plants in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means. For example, by ectopically expressing a gene with a T-DNA activation tag (Ichikawa et al., (1997) Nature 390 698-701, Kakimoto et al., (1996) Science 274: 982-985). This method requires that the plant be transformed with a genetic tag containing multiple transcription enhancers, and when the tag is inserted into the genome, expression of the flanking gene coding sequence is deregulated. In another example, the transcription machinery of a plant can be modified to increase the level of transcription of a polynucleotide of the invention (the DNA binding specificity of a zinc finger protein by changing certain amino acids in the DNA binding motif). See PCT publications WO09606166 and WO9853057, which describe modifications of).
[0056]
The transgenic plant can also include the necessary mechanisms to express or alter the activity of the polypeptide encoded by the endogenous gene, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state. .
[0057]
4. Transgenic plants with altered TF expression
Once an expression cassette comprising a polynucleotide encoding the TF gene of the present invention has been constructed, the polynucleotide can be introduced into plants using standard techniques to modify plant traits. The plant can be any higher plant, including gymnosperms, monocots and dicots. Suitable pros and calls are Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrots, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melon, corn and cucumber, melon and cucumber) Useful for rice, barley, millet, etc.), Solanaceae (potatoes, tomatoes, tobacco, pepper, etc.), and various other crops. Ammirato et al. (1984) Handbook of Plant Cell Culture-Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338: 274-276; Fromm et al. (1990) Bio / Technology 8: 833-839; and Vasil et al. (1990) Bio / Technology 8: 429-434.
[0058]
Transformation and regeneration of monocotyledonous and dicotyledonous cells are now routine, and the choice of optimal transformation technique will be determined by the practitioner. The choice of method will depend on the type of plant to be transformed, and those skilled in the art will recognize the suitability of a particular method for a given plant type. Preferred methods include: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) -mediated transformation; transformation with viruses; microinjection of plant cells; micro-projectile bombardment of plant cells; And Agrobacterium tumeficiens-mediated transformation. Transformation refers to the introduction of a nucleotide sequence into a plant in such a way as to cause stable or transient expression of the sequence.
[0059]
Successful plant modifications characterized by transformation with cloned sequences, which serve to illustrate the current knowledge of the art and are incorporated herein, include US Pat. No. 5,571,706. No. 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; No. 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042. .
[0060]
After transformation, plants are preferably selected using a dominant selectable marker introduced into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plant. Selection of the transformants is then achieved by exposing the plants to appropriate concentrations of antibiotics or herbicides.
[0061]
After transformed plants have been selected and matured, those plants exhibiting the altered trait are identified. The modified trait may be any of the traits described above. Further, by analyzing the expression of mRNA using Northern blot, RT-PCR or microarray, to determine that the modified trait is due to a change in the expression level or activity of the polypeptide or polynucleotide of the present invention, or It can be confirmed by analyzing protein expression using an immunoblot, western blot or gel shift assay.
[0062]
5. Commercial applications of polynucleotides and polypeptides
A specific application for the gene of the invention relates to its potential role in the flowering time of a plant or in the vernalization response. The most modern crop varieties are the result of large breeding programs, and multiple generations of backcrossing may be required to introduce the desired attributes. Systems that promote flowering can have valuable applications in such programs, as they can greatly accelerate generation times. Further, in some cases, earlier generation times allow more crop harvest to be obtained during a given growing season. With the advent of transform systems for tree species such as, for example, oil palm, aspen, pine, and eucalyptus, forest biotechnology is growing as an area of interest.
[0063]
Similarly, in species such as sugar beet where some of the vegetative organs of the plant make up the cereal and reproductive tissue is discarded, it may be advantageous to delay or inhibit flowering. Extending the development of vegetative organs causes a significant increase in yield.
[0064]
In addition, by using an inducible promoter to control the expression of the flowering time regulator gene, flowering can potentially be induced as desired (eg, by using a chemical inducer). . This can, for example, synchronize flowering through the cereal and promote more efficient harvesting. Using such an inducible system, flowering of cereal varieties can be adapted to different latitudes. At present, species such as soybean and cotton are available as a series of mature groups suitable for different latitudes based on their flowering time (which is controlled by daylight hours). A system that can chemically regulate flowering allows a single, high-yielding northern mature group to grow at any latitude. In the southern region, such plants can grow longer, thereby increasing the yield before inducing flowering. In the more northern regions, induction can be used to ensure that the cereals flower before the first winter frosts. Today, the existence of a series of mature groups for different latitudes represents a major barrier to introducing new and valuable attributes.
[0065]
For many cereal species, high-yield winter varieties can only grow in temperate regions, where the winter season persists and is cold enough to cause a vernalization response. Altered expression of the genes of the invention could offset vernalization in the Arabidopsis ecotype, which blooms later. Similar effects can be achieved in cereal plants. For example, subsequently, winter varieties of wheat overexpressing G157 (or wheat orthologs) can be grown in areas other than that, such as southern California, where it is too warm to perform efficient vernalization. A second use of this system is in cherries (Prunus). Locally grown cherries are not available in early spring in California, as winter is too warm for vernalization.
[0066]
Further applications exist in strawberries (Fragaria). Strawberries have a well-defined and permanent cycle of onset of flowering, dormancy, fruit growth and vine growth. In temperate European countries, plants bloom in early spring and fruit is produced in May or June. After the fruit has grown, vines form and carry the seedlings that take root. Next, all plants dormant from late summer to autumn. Flowering cannot be repeated until after spring after the plants have undergone winter cooling. Opening this vernalization requirement may allow strawberries' second fall fruit to be harvested in addition to spring fruit.
[0067]
Finally, in addition to the direct applications of the genes themselves, their regulatory regions may also be of value. If the promoters of these genes are responsive to low temperatures, they can be incorporated into expression systems for the control of genes that confer tolerance to freezing. Such genes are then specifically up-regulated when needed, thereby minimizing any toxic effects caused by their constitutive expression.
[0068]
6. Other uses of polypeptides and polynucleotides
The transcription factor coding provided by the present invention may be used to identify exogenous or endogenous molecules that may affect the expression of the transcription factor and may affect the timing of flowering. These molecules may include organic or inorganic compounds.
[0069]
For example, the methods described above may require the initial placement of a molecule in contact with a plant or plant cell. The molecules described above may be introduced by topical administration, for example, by spraying or plant infiltration, and monitoring the effect of the molecule on TF polypeptide expression or activity or polynucleotide expression. Changes in TF polypeptide expression may be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis, and the like. Changes in the expression of the corresponding polynucleotide sequence may be detected by use of a microarray, Northern or any other technique that monitors changes in mRNA expression. These techniques are described in Ausubel et al. (Eds) Current Protocols in Molecular Molecular Biology, John Wiley & Sons (1998). Such changes in expression levels may be correlated to the characteristics of the modified plant, and thus the identified molecules may be used to modify the characteristics of the plant by infiltrating or spraying fruits, vegetables and cereals. May be used.
[0070]
Transcription factors may be used to identify promoter sequences that may interact. After the promoter sequence has been identified, the interaction between the transcription factor and the promoter sequence is changed by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter plant characteristics. The effect may be modified. Typically, the DNA binding site of the transcription factor is identified by a gel shift assay. After identifying the promoter region, molecules that affect the interaction between TFs and their promoters may be identified by using the sequences of the promoter region in a double-stranded DNA array (Bulyk et al. (1999) Nature Biotechnology). 17: 573-577).
[0071]
The identified transcription factor is also useful for identifying a protein that regulates the activity of the transcription factor. Such modulation may be caused by covalent modifications, such as phosphorylation or protein-protein (homo or heteropolymer) interactions. Any method suitable for detecting protein-protein interactions may be applied. Methods that may be applied include co-immunoprecipitation, cross-purification and co-purification by gradient or chromatography columns, and two-hybrid yeast systems.
[0072]
The two-hybrid system detects protein interactions in vivo, Chien et al., (1991), Proc. Natl. Acad. Sci. USA, 88, 9578-9958, and can be purchased from Clontech (Palo @ Alto, Calif.). In this system, the plasmid is constructed to encode two hybrid proteins: one consisting of the DNA binding domain of the transcriptional activator protein fused to the TF polypeptide, and the other comprising the plasmid as part of a cDNA library. Consists of the activation domain of a transcriptional activator protein fused to an unknown protein encoded by the recombinant cDNA. The DNA binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae having a reporter gene (eg, LacZ) having a transcription activator binding site in the control region. Neither of the hybrid proteins alone can activate transcription of the reporter gene. The interaction of the two hybrid proteins reconstitutes a functional activator protein, resulting in expression of the reporter gene, as detected by reporter gene product assays. Thereafter, a library plasmid capable of effecting reporter gene expression is isolated and sequenced to identify the protein encoded by the library plasmid. After identifying proteins that interact with transcription factors, assays for compounds that inhibit the TF protein-protein interaction may be performed.
[0073]
The following examples illustrate, but do not limit, the invention.
Methods—All experiments were performed using the Arabidopsis ecotype unless otherwise indicated. Stockholm (CS6863) and Pitztal (CS6832) lines were procured from ABRC of Ohio State University. In all experiments, seeds were treated with 30% bleach / 0.01% Tween for 30 minutes following ethanol treatment for 2 minutes, and washed five times with distilled water to sterilize. Seeds were planted on 0.1% agarose MS agar and stored between layers at 4 ° C for 3-5 days before transfer to a growth room at a temperature of 20-25 ° C. MS medium was supplemented with 50 mg / l kanamycin for selection of transformed plants. The plants were cultivated on a plate for 7 days and then replanted in soil. For vernalization treatment, seeds were planted on MS agar plates, sealed with microporous tape, and placed in a low light 4 ° C. cold room for 6-8 weeks. Plates were then lined with plates containing freshly planted unvernalized controls and transferred to the growth room. Whole viable seedlings were harvested after 6 to 9 weeks for gene expression analysis. Rosette leaves were enumerated when a recognizable flowering of about 3 cm was revealed. Rosettes on progeny stems and total leaf numbers correlate well with the timing of flowering (Koorneef et al. (1991) Mol. Gen. Genet. 229: 57-66).
[0074]
Example I.同 定 Identification and cloning of full-length gene
In the following examples, G157 refers to SEQ ID Nos. 1 and 2, G859 refers to SEQ ID Nos. 3-8, G1842 refers to SEQ ID Nos. 9-16, and G1843 refers to SEQ ID Nos. 17 and 18. For reference, G1844 refers to SEQ ID NOs 19-22, G861 refers to SEQ ID NOs 23-26, and FLC or G1759 refers to SEQ ID NOs 27 and 28.
[0075]
Putative transcription factor sequences (in the genome or ESTs) associated with known transcription factors use P-value cutoff thresholds of -4 or -5 or less depending on default parameters and length of the sequence in question. And identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program. Those that hit the putative transcription factor sequence were then examined to identify those that contained the particular sequence strand. If the hit sequence contains such a sequence strand, the sequence was confirmed to be a transcription factor.
[0076]
For example, we identified the MADS @ box gene G157 from BAC @ F22K20 (GenBank Access # AC002291) in chromosome I, which was predicted to encode a protein associated with FLC. The G157 872 bp cDNA clone was identified from clones isolated from a library derived from leaf mRNA. The encoded protein was 196 amino acids long and 62% of the total amino acid sequence matched FLC and 82% matched the MADSＭＡDNA binding domain.
[0077]
G157 also occupies approximately 22 kb, downstream of chromosome V, as a tightly connected cluster, spanning three adjacent clones of MXK3, F1505, and MQN23 (GenBank accession numbers AB01236, AB026333, and AB013395, respectively). Also associated are G859, G1842, G1843, and G1844 located together. G859, G1842, G1843, and G1844 appear to be created in the course of replication; this allows their divergence to different aspects of gene regulation. Their physical proximity indicates that they are working to be units controlled by common control factors.
[0078]
A one-to-one comparison of the 57 amino acid MADS domains of FLC, G157, G859, G1842, G1843, and G1844 is shown in Table 1. The table shows the percentage of amino acid sequence matches, and the figures in parentheses indicate the percentage of sequence matches when conservative amino acid substitutions are also taken into account. The MADS domain of the proteins encoded by G859, G1842, G1843, and G1844 is highly conserved with that of FLC and G157: according to the one-to-one comparison shown below, these proteins have between 75% and 91% Share amino acid sequence identity. When conservative amino substitutions occur, the MADS domains of these proteins are 88% -99% identical to each other (indicated in parentheses).
[0079]
[Table 1]

[0080]
While it is an acidic residue (E or D) for amino acid residues 30 and G157, G859, G1842, G1843, and G1844 of FLC, in all other Arabidopsis MADS domain proteins identified so far, , The position is occupied by a positively charged lysine residue. The crystal structure of the human SRF @ MADS domain bound to DNA shows that lysine residues (which are also conserved in yeast MCM1 and human MEF2A proteins) are in contact with the phosphate backbone of the DNA target site. (Pellegrini et al., (1995) Nature 376: 490-498). The amino acid difference therefore does not allow FLC and DNA binding for G157, G859, G1842, G1843, and G1844 to confer different properties than other Arabidopsis MADS domain proteins. Thus, a MADS domain protein with an acidic residue at position 30 may be particularly useful for regulating flowering of plants and responding to vernalization.
[0081]
Transcripts from these genes were analyzed by 3'RACE (Rapid \ Amplification \ cDNA \ Ends) and the corresponding cDNAs were isolated by RT-PCR from a mixed sample of Arabidopsis tissue (Clumbia ecotype). During this analysis, transcripts of G859, G1842, and G1844 were detected as multiple alternative native splice forms.
[0082]
Example II Flowering time-related genes
Reverse transcriptase PCR was performed using gene-specific primers in the coding region of each identified sequence. Where possible, primers were designed near the 3 'region of each coding sequence initially identified.
[0083]
Total RNA was isolated from plant tissue and extracted by the CTAB method. The extracted total RNA was standardized in concentration for all tissue types, and the 28S band was referenced so that the same amount of cDNA template was used in the PCR reaction for each tissue. Poly A⁺ Was purified using a modified protocol of the Qiagen Oligotex kit batch. cDNA was synthesized using standard protocols. Following first strand cDNA synthesis, actin2 primers were used to normalize cDNA concentrations across tissue types. Actin2 has been found to be constitutively expressed at approximately the same level across Arabidopsis tissue types.
[0084]
For RT @ PCR, the cDNA template was mixed with the corresponding primers and Taq polymerase. Each reaction consisted of 0.2 μl cDNA template, 2 μl 10X Tricine buffer, and 16.8 μl water, 5 pmol primer 1, 5 pmol primer 2, 0.3 μl Taq polymerase, 200 μM dNTP and 8.6 μl water.
[0085]
The 96-well plate was covered with microfilm and set in a thermal circulator to initiate the following reaction cycle. Step 1 @ 93 ° C for 3 minutes, Step 2 @ 93 ° C for 30 seconds, Step 3 @ 60-65 ° C for 1 minute, Step 4 @ 72 ° C for 2 minutes Steps 2, 3 and 4 were repeated for 20-35 cycles. Step 5-72 ° C for 5 minutes and Step 6-4 ° C. In some cases, the PCR plates were returned to the thermocycling device and the reaction product was enriched with 5-15 additional cycles to identify very low expressed genes. The reaction cycle was as follows. Step 2 @ 93 ° C @ 30 seconds, step 3 @ 65 ° C @ 1 minute, and step 4 @ 72 ° C @ 2 minutes, 8 cycle repetition, and step 4 @ 4 ° C. Between 21 and 36 cycles, 8 microliters of PCR product and 1.5 μl of loading dye were loaded on a 1.2% agarose gel and analyzed. If particular transcripts are detected only later than 35 cycles of PCR, their expression levels are considered low. Depending on the transcript level compared to the observed actin2 transcript level, expression levels are considered moderate or high.
[0086]
As an example, to evaluate G157１５ mRNA levels in G157 plants, use primers 5′-GGCATAACCCTTATCGGAGATTTTGAAGC-3 ′ (SEQ ID No. 57) and 5′-ACACAAACTCTGATCTTGTCTCCGAAGG-3 ′ (SEQ ID No. 58). PCR over 25 cycles was performed. To assess mRNA levels in separate tissues extracted from wild-type plants, use primers 5'-GCATAACCCTTATCGGAGATTTTGAAGCCAT-3 '(SEQ ID No. 59) and 5'-AACATTCCTCTCTCATCATCTGTTGCCAGC-3' (SEQ ID No. 60). , 25 or 30 cycles of PCR were performed. The FLC PCR was performed 35 times using the primers 5′-AACGCTTAGTATCTCCGGCGACTTTGAAC-3 ′ (SEQ ID No. 61) and 5′-CTCACACGAGAATAAGGTACAAAGTTCATC-3 ′ (SEQ ID NO. (SEQ ID No. 63) and 5 'AGATTCTCAACAAGCTTCAACATGAGTTCG-3' (SEQ ID No. 64) were performed 30 times. The specificity of the primer was confirmed by sequencing the RT-PCR product. Samples were normalized by 20-25 cycles of PCR using actin primers.
[0087]
Example III. Construction of expression vector
Sequences from genomic or cDNA libraries were amplified using primers specific for sequences upstream and downstream of the coding region. Expression vectors are pMEN20 or pMEN65, both of which are derived from pMON316 (Sanders et al, (1987) Nucleic Acids Research 15: 1543-58) and contain the CaMV 35S promoter for expressing transgenes. To clone the sequence into the vector, both pMEN20 and the amplified DNA fragment were separately digested with SalI and NotI restriction enzymes at 37 ° C. for 2 hours. The digest was electrophoresed on a 0.8% agarose gel and visualized by ethidium bromide staining. A DNA fragment containing this sequence and the linearized plasmid was cut out and purified using a Qiaquick gel extraction kit (Qiagen, CA). Fragments of interest were ligated in a ratio of 3: 1 (vector to insert). The ligation reaction was performed at 16 ° C. for 16 hours using T4 DNA ligase (New England Biolabs, Mass.). The ligated DNA was transformed into E. coli using the heat shock method. E. coli DH5 alpha strain was transformed into competent cells. Transformants were plated on LB plates containing 50 mg / l Spectinomycin (Sigma).
[0088]
Individual colonies were grown overnight at 37 ° C. in 5 ml LB broth containing 50 mg / l spectinomycin. Plasmid DNA was purified using Qiaquick Mini Prep kits (Qiagen, CA).
[0089]
Example IV形 質 Transformation of Agrobacterium with expression vector
After constructing the plasmid vector containing the gene, this vector was used to transform Agrobacterium tumefaciens cells expressing the gene product. A stock for the transformation of Agrobacterium tumefaciens cells is described in Nagel et al. It was prepared as described in FEMS Microbiol Letts 67: 325-328 (1990). Absorbance (A₆₀₀) Reached 0.5 to 1.0, and the Agrobacterium GV3101 strain was shake-cultured overnight at 28 ° C in 250 ml of LB medium (Sigma). The cells were collected by centrifugation at 4,000 × g for 15 minutes at 4 ° C. The cells were then resuspended in 250 μl of ice-cold buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 μl ice-cold buffer. The centrifugation and resuspension were then repeated two more times. At that time, the cells were suspended in 100 μl and 750 μl of the same HEPES buffer as described above. The resuspended cells were then aliquoted in 40 μl aliquots, snap frozen in liquid nitrogen and stored at -80 ° C.
[0090]
Nagel et al. According to the protocol described in FEMS Microbiol Letts 67: 325-328 (1990), Agrobacterium cells were transformed with the plasmid prepared as described above. For each DNA construct to be transformed, 50-100 ng of DNA (usually resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40 μl of Agrobacterium cells. The DNA / cell mixture was then transferred to an ice-cooled cuvette (electrode spacing 2 mm) and a 2.5 kV voltage was applied at 25 μF and 200 μF using a Gene Pulser II instrument (Bio-Rad). After electroporation, cells were immediately resuspended in 1.0 ml of LB medium and cells were allowed to recover for 2-4 hours at 28 ° C. in a shaking incubator without antibiotic selection. After recovery, cells were seeded in LB broth selection medium containing 100 μg / ml spectinomycin (Sigma) and incubated at 28 ° C. for 24-48 hours. A single colony was then picked and inoculated into fresh medium. The integrity of the plasmid construct was confirmed by PCR amplification and sequence analysis.
[0091]
Example V形 質 Transformation of Arabidopsis plants with Agrobacterium tumefaciens having an expression vector
After transforming Agrobacterium tumefaciens with the plasmid vector containing the gene, a single Agrobacterium colony was identified and cultured and used for transformation of Arabidopsis plants. Briefly, a 500 ml culture of LB medium containing 50 mg / l of spectinomycin was inoculated with a colony and the absorbance (A₆₀₀) Was shake-cultured at 28 ° C for 2 days until 2.0 exceeded 2.0. Next, the cells were collected by centrifugation at 4,000 × g for 10 minutes, and the medium for infiltration (１ ／ × Murashige and Skoog salt (Sigma), 1 × Gamborg's B-5 vitamin (Sigma)) was collected. Absorbance (A) in 5.0% (w / v) sucrose (Sigma), 0.044 μM benzylaminopurine (Sigma), 200 μl / L Silwet L-77 (Lehle Seeds)₆₀₀) Was 0.8.
[0092]
Prior to the transformation, onto a Pro-Mix BX potting material (Hummert International) covered with a glass fiber mesh (18 mm × 16 mm), the density was such that there were about 10 plants per 4 inch pot. Arabidopsis thaliana seeds (ecotype: Colombia) were sown. Under continuous illumination (50-75μE / m²/ Sec) at 22-23 ° C and 65-70% relative humidity. After about four weeks, the primary inflorescence stem was cut off so that the secondary stem grew more. After the mature secondary stems flowered, the plants were prepared for transformation by removing all siliques and flowering flowers.
[0093]
The pots were then immersed in the Agrobacterium infiltration medium mixture described above upside down for 30 seconds, and the pots were laid dry on a 1 foot x 2 foot plane covered with plastic wrap to drain the water. After 24 hours, the plastic wrap was removed and the pot was raised. One week later, the immersion procedure was repeated, and immersion was performed twice per pot in total. Next, seeds were collected from each transformed pot and analyzed according to the protocol described below.
[0094]
Example VI同 定 Identification of primary transformants of Arabidopsis
Seeds recovered from the transformed pots were sterilized essentially as follows. Seeds were dispersed in a solution containing 0.1% (v / v) Triton X-100 (Sigma) and sterilized water, and the suspension was shaken for 20 minutes to wash the seeds. The washing solution was then discarded and replaced with a new washing solution, and the seeds were washed for 20 minutes with shaking. After removing the second wash solution, a solution containing 0.1% (v / v) Triton X-100 and 70% ethanol (Equistar) was added to the seeds and the suspension was shaken for 5 minutes. After removing the ethanol / detergent solution, a solution containing 0.1% (v / v) Triton X-100 and 30% (v / v) bleach (Clorox) was added to the seeds and the suspension was allowed to stand for 10 minutes. Shake. After removing the bleach / detergent solution, the seeds were then washed five times in sterile distilled water. After storing the seeds in the final wash water at 4 ° C. for 2 days in the dark, an antibiotic selection medium (1 × Murashige and Skoog salt (pH adjusted to 5.7 with 1M KOH), 1 × Gambor ') sB-5 vitamin, 0.9% phytagar (Life Technologies), and 50 mg / I kanamycin). Under continuous illumination (50-75μE / m²/ Sec) at 22-23 ° C. After growing under such conditions for 7 to 10 days, a kanamycin-resistant primary transformant (T₁Generations) were obtained after confirmation by eyesight. These seedlings were first transferred to a new selection plate and grown for an additional 3-5 days, then transferred to soil (Pro-Mix BX potting material).
[0095]
The primary transformant was self-pollinated and progeny seeds (T2) were collected. T2 progeny seeds were germinated on kanamycin as described above, and kanamycin-resistant seedlings were selected and transferred to soil for analysis.
[0096]
Example VII分析 Analysis of transgenic Arabidopsis plants
In the first experiment, G157 plants (ie, plants expressing the G157 transgene) were grown under illumination for 12 hours. Thirty out of the forty lines flowered earlier than the control plants transformed with the control vector. The average number of rosette leaves of the early blooming T1 line was 12.4 ± 0.8, and that of the control line was 27 ± 1.2. Two out of the forty T1 plants bloomed simultaneously with the control, and seven out of the forty strains bloomed late, and the inflorescence was confirmed 2-3 weeks behind the wild type.
[0097]
In further experiments, plants were grown at 20-25 ° C under 24 hours of illumination. Under these conditions, the non-transformed control plant had an average total number of leaves of primary shoots before flower bud formation of 14.3 ± 0.7. Flower buds were only identified on average 21.1 ± 0.5 days after seeding (error values represent the standard error giving a 95% confidence limit). In the case of G859, 14/19 of the T1 plants bloomed early (the average total leaf number was 6.4 ± 0.7, flower buds were confirmed 12.9 ± 0.7 days after sowing), and 3/19 Was wild type, and 2/19 bloomed slightly later than the wild type (average total leaf number was 19, flower buds were confirmed on day 27). RT expression studies revealed that late-blooming individuals had the highest transgene expression. These results are very similar to those of G157. In the case of G1842, 7/10 of the T1 plants are early blooming (average total leaf number is 7.9 ± 0.6, flower buds are confirmed at 13.9 ± 1.0 days), and 3/10 is It was wild type. In addition, overexpression studies were performed using cDNA encoding a short splice variant of G1842. In the case of G1842.2 (encoding a splice variant consisting of 185 amino acids), 15/18 of the T1 plants bloom early (average total leaf number is 6.9 ± 0.9, flower buds are 14.5). (Confirmed on ± 0.6 day) 3/18 were wild type. In the case of G1842.6 (encoding a splice variant consisting of 77 amino acids), 8/10 of the T1 plants are in early bloom (average total leaf number is 6.8 ± 1.6, flower bud is 13.9). (Confirmed on ± 0.9 days), 2/10 were wild type. In the case of G1842.7 (encoding a splice variant consisting of 118 amino acids), 8/10 of the T1 plants bloomed early (the exact number of leaves was not measured), and 2/10 were wild-type. . Thus, the G1842 splice variant, when overexpressed, had an effect comparable to a full-length cDNA clone. In the case of G1843, early blooming occurred on 7/11 (average total leaf number was 6.4 ± 0.5, flower buds were confirmed on day 16.0 ± 1.6), and 2/11 was wild-type flowering. Period. However, the growth of the G1843 T1 plant was poor, and development of some organs was delayed. This suggests that G1843 exhibits unexpected toxic effects when overexpressed. In the case of G1844, 6/10 of T1 plants are in early bloom (average total leaf number is 6.8 ± 1.7, flower buds are confirmed on 14.7 ± 1.3 days), 4/10 is It was wild type. Similarly, overexpression studies were performed using cDNA encoding a short splice variant of G1844. When G1844.2 (encoding a splice variant consisting of 184 amino acids) was overexpressed, 6/19 of the T1 plants bloomed early (average total leaf number was 7.8 ± 1.7, flower buds). Was confirmed on day 15.7 ± 1.3), and 13/19 was wild type. The overexpression data for G859, G1842, G1843, and G1844 support the hypothesis that they play a role in controlling the flowering period.
[0098]
RT-PCR was performed on material from G157 plants in about 25 cycles using G157-specific primers. High levels of G157 expression were detected in samples from individual plants that bloomed slowly or from preserved seedlings that contained individuals that bloomed slowly. Plants that showed only moderate or low levels of overexpression compared to wild type were slightly earlier or normal in flowering.
[0099]
To test whether the increase in G157 affects flowering time in the slow flowering ecotype of Arabidopsis, we overexpressed G157 in the late flowering ecotypes Stockholm and Piztal. In this experiment, 32 first-stage transformants from each ecotype were grown under continuous light conditions sprinkled with controls. In both ecotypes, about 50% of transformants flowered earlier than coping, and a small number of Piztal and Stockholm transformants flowered significantly later than controls.
[0100]
Correlation between G157 transgene expression and flowering time was also observed in G157 @ Stockholm and Piztal @ T1 plants. RT-PCR was performed with two early flowering lines and two slow flowering lines in each background. Again, the slow flowering line contained high levels of G157 expression. That is, the factors appear to influence flowering time in a quantitative manner; medium levels of overexpression trigger early flowering and greatly increase flowering delay.
[0101]
In conclusion, either G157 overexpression or cognate genes modulate flowering time in plants: moderate levels of overexpression induce early flowering and many delay flowering.
[0102]
Using a similar or identical methodology as described in the above examples, another Arabidopsis gene whose altered expression was correlated with the delay or acceleration of flowering was identified. These genes are tabulated in Table 2 with their particular SEQ ID NOs and their effect on flowering time.
[0103]
[Table 2]

[0104]
The response of vernalization treatment was also investigated. Slow-flowering vernalization-responsive ecotypes and mutants have high steady-state levels of FLC transcripts and are reduced during the promotion of flowering by vernalization (Michaels @ and @ Amasino, (1999) Plant @ Cell @ 11: 949-956; Shelldon et al., (1999) Plant Cell 11: 445-458; Shelldon et al., (2000) Proc. Natl. Acad. Sci. 97: 3735-3758). In contrast to FLC, G157 transcript levels do not show a consistent correlation with the response of vernalization treatment in the Stockholm and Piztal ecotypes of slow flowering. In addition, we found that overexpression of G157 did not affect FLC levels. The effect of vernalization on the expression of G861, G (59, G1843, and G1844 was also tested. Germinated seeds of Columbia, Piztal, Stockholm, constans-1, and fca-9 were plated on MS agar plates in a 4 ° C. cold room. Vernalized for 8 weeks and then transferred to a continuous light growing room, all tissues from vernalized seeds and freshly sown non-vernalized controls were harvested 9 days after transfer. PCR was performed on FLC, G157, G859, G1842, G1843, G1844 and G861 and actin.Comparing FLC with G157, none of the genes showed a clear consistent decrease upon vernalization treatment in five separate sample sets. However, G1844 is not compatible with FLC. Showed an inverse pattern of expression: G1844 levels increased consistently with vernalization treatment, which is particularly striking, such that G1844 is directly articulated in controls of vernalization response. Activates flowering and may have the opposite role as FLC.
[0105]
To explore whether overexpression of G157 has effects comparable to vernalization treatment, batches of wild-type Piztal and Stockholm seedlings were chilled at 4 ° C. for 6 weeks, then G157 @ T1 @ Piztal, G157 @ T1 @ Stockholm and Non-vernalized wild-type plants were grown against a second selection. As expected, vernalization significantly and uniformly shortened flowering time in both Piztal and Stockholm wild-type plants. Among the G157 @ Stockholm lines, the earliest flowering T1 group (8/23 line) was indistinguishable from vernalized plants. With respect to Piztal, however, early flowering T1 plants were on average closer to the lower limit than vernalized plants. Thus, overexpression of G157 substantially reduces the need for vernalization in slow flowering ecotypes.
[0106]
In addition, we observed that the slow-flowering G157 lineage was independent of FLC expression and did not respond to vernalization. However, slow flowering G157 plants are responsive to the photoperiod. In experiments performed under short-day conditions of 8 hours of light, we obtained a large number of G157 {Columbia} T1 plants that flowered one month later than wild-type controls (data not shown). To determine whether the effects of slow flowering caused by G157 expression were independent of FLC transcription, we tested whether slow flowering G157 @ Columbia plants were responsive to vernalization treatment. . No significant change in flowering time was recorded: the vernalized T2 plants of line 4 under continuous light conditions overall had a total of 31. + /-1.3 when vernalized. 3 +/- 1.8. Control fca plants demonstrated that the treatment was effective: vernalized plants only 10.3 +/- 0.9 compared to more than 40 leaves of non-vernalized controls. Flowers after the leaves. That is, the slow flowering phenotype caused by G157 could not be overcome by vernalization treatment, as would be expected if a delay had occurred independent of changes in FLC expression.
[0107]
Example IX. Identification of homologous sequences
Homologs of plant species other than Arabidopsis can be searched using database search tools, for example, Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215: 403-410; and Altschul et al. (1997). Nucl. Acid {Res. 25: 3389-3402). The tblastx sequence analysis program was performed using the BLOSUM-62 scoring matrix (Henikoff, S. and Henikoff, JG (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).
[0108]
All NCBI GenBank databases were filtered for sequences from all plants except Arabidopsis thaliana, but all selected in the NCBI GenBank database with NCBI classification ID 33090 (Viridiplantae; all plants) and 3701 (Arabidopsis thaliana) by eliminating the whole. These sequences were compared to the sequence representing the gene for SEQ IDS 1-56 on September 26, 2000 using the Washington University TBLASTX formula (version 2.0a19MP). Individual comparisons for each gene of SEQ IDs 1-56 were aligned by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6e-40 is 3.6 x 10^-40It is. For up to 10 species, the gene with the lowest P value (and, therefore, the most likely homolog) is listed in FIG.
[0109]
In addition to P values, comparisons by percentage identity were also scored. Percentage identity reflects the degree to which two DNA or protein segments are identical over a particular length. The range of percent identity between the non-Arabidopsis gene shown in FIG. 2 and the Arabidopsis gene in the sequence listing is shown in SEQ ID NO. 1: 54% -67%; SEQ {ID} Nos. 3, 5, 7: 37% -47%; SEQ ID Nos. 9, 11, 13, 15: 54% -62%; SEQ ID No. 17: 62% -71%; SEQ ID Nos. 19, 21: 50% -67%; SEQ ID Nos. 23, 25: 75% -91%; SEQ ID No. 27: 46% -69%; SEQ ID No. 29: 44% -90%; SEQ ID No. 31: 57% -89%; SEQ ID No. 33: 37% -79%; SEQ ID No. 35: 50% -71%; SEQ ID No. 37: 39% -63%; SEQ ID No. 39: 58% -70%; SEQ ID No. 41: 45% -73%; SEQ ID No. 43: 42% -84%; SEQ ID No. 45: 47% -81%; SEQ ID No. 47: 31% -71%; SEQ ID No. 49: 40% -67%; SEQ ID No. 51: 69% -51%; SEQ ID No. 53: 43% -86%; and SEQ {ID} No. 55: 79% -89%.
[0110]
Arabidopsis homologues of the genes in Table 2 were also identified using BLAST. These genes are found in the following Arabidopsis BAC sequences. Their GenBank sequence NID numbers: 2827698 (234 homolog), 3241917 (G748 homolog), 2618604 (G994 homolog), 6598548 (G1335 homolog), 7340331 (G736 homolog), 6523051 (G1435 homolog), 6598491 (G208 homolog) and 3172156 (G208 homolog).
[0111]
All documents (publications and patents) are incorporated herein by reference in their entirety for sweetfish.
Although the invention has been described with reference to the above embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
[Brief description of the drawings]
FIG. 1 provides a table of exemplary polynucleotide and polypeptide sequences of the present invention. The table includes, from left to right, for each sequence, SEQ ID No., an internal code reference number, whether the sequence is a polynucleotide or polypeptide sequence, and any conserved domains for the polypeptide sequence. Identification.
FIG. 2 provides a table of sequences homologous to the sequences provided in the Sequence Listing. The table includes, from left to right, SEQ ID No., internal code reference number, unique Genbank sequence number (NID), probability that a comparison would be generated by chance (P-value), and species in which the homologous gene was identified. , Is included.

Claims (16)

SEQ ID No: nucleotide sequence encoding a polypeptide comprising at least 6 contiguous amino acids of a sequence selected from the group consisting of 2N, where N is 1-28 but excluding SEQ ID No: 28 A transgenic plant comprising a recombinant polynucleotide, comprising:
(I) a vernalization treatment that has a modified flowering time compared to another plant that does not contain the recombinant polynucleotide, or (ii) is modified compared to another plant that does not contain the recombinant polynucleotide. The transgenic plant having the requirement. 2. The transgenic plant of claim 1, wherein the nucleotide sequence encodes a polypeptide comprising a conserved domain selected from the group consisting of the conserved domains of SEQ ID No: 2N, where N is 1-28. . The transgenic plant of claim 1, wherein the recombinant polynucleotide further comprises a promoter operably linked to said nucleotide sequence. 4. The transgenic plant of claim 3, wherein said promoter is constitutive, inducible, or tissue activated. Wherein the recombinant polynucleotide comprises a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. 2. The transgenic plant of claim 1, which encodes a peptide. A method for changing the flowering time or vernalization requirement of a plant,
(A) encodes a polypeptide comprising at least 6 contiguous amino acids of a sequence consisting of SEQ ID No: 2N (where N is 1-28) but selected from the group except SEQ ID No: 28 Transforming a plant with a recombinant polynucleotide comprising a nucleotide sequence that:
(B) selecting the transformed plant; and (c) identifying a transformed plant with an altered flowering time;
The above method, comprising: 7. The method of claim 6, wherein the nucleotide sequence encodes a polypeptide comprising a conserved domain selected from the group consisting of the conserved domains of SEQ ID No: 2N, where N is 1-28. 7. The method of claim 6, wherein the recombinant polynucleotide further comprises a promoter operably linked to said nucleotide sequence. 9. The method of claim 8, wherein said promoter is constitutive, inducible, or tissue activated. Wherein the recombinant polynucleotide comprises a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. 7. The method of claim 6, which encodes a peptide. A method for changing the flowering time or vernalization requirement of a plant,
(A) a nucleotide sequence comprising at least 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID No: 2N-1 (where N is 1-28) but excluding SEQ ID No: 27 Transforming a plant with a recombinant polynucleotide comprising: and (b) selecting the transformed plant;
The above method, comprising: Wherein the recombinant polynucleotide comprises a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. 12. The method of claim 11, which encodes a peptide. A method for changing the flowering time or vernalization requirement of a plant,
(A) preparing a database sequence;
(B) comparing the database sequence to a polypeptide selected from SEQ ID No: 2N, where N is 1-28;
(C) selecting a database sequence that matches the selected sequence classification; and (b) transforming the selected database sequence into a plant;
The above method, comprising: Wherein the recombinant polynucleotide comprises a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. 14. The method of claim 13, which encodes a peptide. A method for changing the flowering time or vernalization requirement of a plant,
(A) preparing a database sequence;
(B) comparing the database sequence to a polynucleotide selected from SEQ ID No: 2N-1, where N is 1-28;
(C) selecting a database sequence that matches the selected sequence classification; and (b) transforming the selected database sequence into a plant;
The above method, comprising: Wherein the recombinant polynucleotide comprises a conserved domain having greater than 84% sequence identity to a sequence selected from the group consisting of SEQ ID No: 2N, where N is 1-28. 16. The method of claim 15, which encodes a peptide.

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