JP4654561B2 - Plant with improved productivity and production method thereof - Google Patents

Description

【００５２】
得られたＦＳＡＭ２４と既知の植物由来のＳ−アデノシルメチオニン脱炭酸酵素遺伝子とアミノ酸比較を行った（表２）。表２の結果からクロダネカボチャ根由来のＦＳＡＭ２４は他の植物由来のＳＡＭＤＣ遺伝子とアミノ酸レベルで高い相同性を示した。
【００５３】
【表２】

【００５４】
得られたＦＡＤＣ７６と既知の植物由来のアルギニン脱炭酸酵素遺伝子とアミノ酸比較を行った（表３）。表３の結果からクロダネカボチャ根由来のＦＡＤＣ７６は他の植物由来のＡＤＣ遺伝子とアミノ酸レベルで高い相同性を示した。
【００５５】
【表３】

【００５６】
実施例２：トランスジェニックサツマイモの作製
（１）発現コンストラクトの作製
配列番号１に示したポリアミン代謝関連遺伝子ＦＳＰＤ１の塩基配列よりオープンリーディングフレームをすべて含むように、ＸｈｏＩで切断し、グラスミルク法で精製した。次にｐＧＥＭ−７Ｚｆ（Ｐｒｏｍｅｇａ社製）をＸｈｏＩ切断して、ＦＳＰＤ１断片をセンスとアンチセンス方向にそれぞれサブクローニングした。ｐＧＥＭ−７Ｚｆのマルチクローニングサイトの制限酵素ＸｂａＩとＫｐｎＩで再度ＦＳＰＤ１断片を切り出して、３５Ｓプロモーターが連結しているバイナリーベクターｐＢＩ１０１−Ｈｍ２にサブクローニングした。これらのプラスミドをｐＢＩ３５Ｓ−ＦＳＰＤ１＋／−と命名した。その発現コンストラクトの構造を図１に示した。なお、形質転換された大腸菌ＪＭ１０９を、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＢＩ３５Ｓ−ＦＳＰＤ１＋／−と命名した。
配列番号３に示したポリアミン代謝関連遺伝子ＦＳＡＭ２４の塩基配列よりオープンリーディングフレームをすべて含むように、ＮｏｔＩで切断し、それぞれ平滑末端化した。これらの断片を平滑末端化した３５Ｓプロモーターが連結しているバイナリーベクターｐＢＩ１０１−Ｈｍ２にセンス方向とアンチセンス方向にサブクローニングした。これらのプラスミドをｐＢＩ３５Ｓ−ＦＳＡＭ２４＋／−と命名した。その発現コンストラクト（センス方向のみ）の構造を図１に示した。なお、形質転換された大腸菌ＪＭ１０９を、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＢＩ３５Ｓ−ＦＳＡＭ２４＋／−と命名した。
配列番号５に示したポリアミン代謝関連遺伝子ＦＡＤＣ７６の塩基配列よりオープンリーディングフレームをすべて含むように、ＮｏｔＩで切断し、それぞれ平滑末端化した。これらの断片を平滑末端化した３５Ｓプロモーターが連結しているバイナリーベクターｐＢＩ１０１−Ｈｍ２にセンス方向とアンチセンス方向にサブクローニングした。これらのプラスミドをｐＢＩ３５Ｓ−ＦＡＤＣ７６＋／−と命名した。その発現コンストラクト（センス方向のみ）の構造を図１に示した。なお、形質転換された大腸菌ＪＭ１０９を、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＢＩ３５Ｓ−ＦＡＤＣ７６＋／−と命名した。
（２）プラスミドのアグロバクテリウムへの導入
（１）で得られた大腸菌ｐＢＩ３５Ｓ−ＦＳＰＤ１＋／−、大腸菌ｐＢＩ３５Ｓ−ＦＳＡＭ２４＋／−、大腸菌ｐＢＩ３５Ｓ−ＦＡＤＣ７６＋／−とヘルパープラスミドｐＲＫ２０１３を持つ大腸菌ＨＢ１０１株を、それぞれ５０ｍｇ／ｌのカナマイシンを含むＬＢ培地で３７℃で１晩、アグロバクテリウムＥＨＡ１０１株を５０ｍｇ／ｌのカナマイシンを含むＬＢ培地で３７℃で２晩培養した。各培養液１．５ｍｌをエッペンドルフチューブに取り集菌したのち、ＬＢ培地で洗浄した。これらの菌体を１ｍｌのＬＢ培地に懸濁後、３種の菌を１００μｌずつ混合し、ＬＢ培地寒天培地にまき、２８℃で培養してプラスミドをアグロバクテリウムに接合伝達（三者接合法）させた。１から２日後に一部を白金耳でかきとり、５０ｍｇ／ｌカナマイシン、２０ｍｇ／ｌハイグロマイシン、２５ｍｇ／ｌクロラムフェニコールを含むＬＢ寒天培地上に塗布した。２８℃で２日間培養した後、単一コロニーを選択した。得られた形質転換体をＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＳＰＤ１＋／−、ＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＳＡＭ２４＋／−、ＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＡＤＣ７６＋／−と命名した。
（３）形質転換用サツマイモカルスの作製
サツマイモ高系１４号（石川県立農業短期大学農業資源研究所より提供、以下「高系１４号」又は「野生株」という）を鉢植えにて通常の栽培管理により栽培し、茎頂を含む５ｃｍ程度の茎先端部数十本を採取した。これを滅菌した３００ｍｌビーカーに入れた７０％エタノール１５０ｍｌに２分浸漬の後、同様に滅菌したビーカーに入れた滅菌液（５％次亜塩素酸ナトリウム、０．０２％Ｔｒｉｔｏｎ Ｘ−１００）１５０ｍｌに２分浸漬して滅菌を行った。滅菌した茎先端部は、滅菌ビーカーに入れた滅菌水で３回洗浄を行った。洗浄後、実体顕微鏡下で無菌的に茎頂分裂組織を含む０．５ｍｍ程度の組織を摘出した。これを胚性（Ｅｍｂｒｙｏｇｅｎｉｃ）カルス誘導培地〔４Ｆ１プレート：ＬＳ培地（１．９ｇ／ｌ ＫＮＯ₃、１．６５ｇ／ｌ ＮＨ₄ＮＯ₃、０．３２ｇ／ｌ ＭｇＳＯ₄・７Ｈ₂Ｏ、０．４４ｇ／ｌ ＣａＣｌ₂・２Ｈ₂Ｏ、０．１７ｇ／ｌ ＫＨ₂ＰＯ₄、２２．３ｍｇ／ｌ ＭｎＳＯ₄・４Ｈ₂Ｏ、８．６ｍｇ／ｌ ＺｎＳＯ₄・７Ｈ₂Ｏ、０．０２５ｍｇ／ｌ ＣｕＳＯ₄・５Ｈ₂Ｏ、０．０２５ｍｇ／ｌ ＣｏＣｌ₂・６Ｈ₂Ｏ、０．８３ｍｇ ＫＩ、６．２ｍｇ Ｈ₃ＢＯ₃、２７．８ｍｇ ＦｅＳＯ₄・７Ｈ₂Ｏ、３７．３ｍｇ／ｌ Ｎａ₂・ＥＤＴＡ、１００ｍｇ／ｌ ｍｙｏ−イノシトール、０．４ｍｇ／ｌ 塩酸チアミン）、１ｍｇ／ｌ ４−ｆｌｕｏｒｏｐｈｅｎｏｘｙａｃｅｔｉｃ ａｃｉｄ（４ＦＡ）、３０ｇ／ｌ ショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８〕上に置床し、植物インキュベーター（サンヨー社製、ＭＬＲ−３５０ＨＴ）中で２６℃、暗黒条件下にて培養した。約１ヶ月後、増殖した組織より植物体への再分化能を持つ胚性カルスを選抜した。選抜した胚性カルスは以後、１ヶ月毎に新しい４Ｆ１プレートに移植し、増殖させた。
（４）アグロバクテリウムの感染
形質転換アグロバクテリウム株ＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＳＰＤ１＋／−、ＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＳＡＭ２４＋／−、ＥＨＡ１０１／ｐＢＩ３５Ｓ−ＦＡＤＣ７６＋／−を５０ｍｇ／ｌカナマイシン及び５０ｍｇ／ｌハイグロマイシンを含むＬＢ寒天培地にて２７℃、２晩培養後、菌体を飯粒２粒程度掻き取り、感染培地（ＬＳ培地、２０ｍｇ／ｌ アセトシリンゴン、１ｍｇ／ｌ ４ＦＡ、３０ｇ／ｌ ショ糖、ｐＨ５．８）５０ｍｌに懸濁し、１００ｒｐｍ、２６℃、暗黒条件下にて１時間振とうした。この菌体懸濁液を、滅菌したステンレスネット製バスケットを入れた３００ｍｌ滅菌ビーカーに移した。前記（３）で移植後２週間〜３週間培養した胚性カルスをこのビーカーのバスケットに入れて２分間浸したのち、２枚重ねた滅菌濾紙上にバスケットごと乗せて余分な水分を除き、共存培養培地（４Ｆ１Ａ２０プレート：ＬＳ培地、１ｍｇ／ｌ ４ＦＡ、２０ｍｇ／ｌ アセトシリンゴン、３０ｇ／ｌ ショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８）に移して、２２℃、暗黒条件下にて３日間共存培養した。
（５）除菌
前記（４）にて３日間共存培養した胚性カルスを、除菌液（滅菌水にカルベニシリンを終濃度５００ｍｇ／ｌとなるように加えたもの）５０ｍｌを入れた滅菌したステンレスバスケット入り３００ｍｌビーカーのバスケットに移し、ピンセットでバスケットをつまみ、数分間良く洗浄した。次に胚性カルスを、バスケットごと除菌液を入れた３００ｍｌ滅菌ビーカーに移し、再び洗浄を行った。同じ操作を再度繰り返した後、滅菌濾紙上で余分な水分を除き、選抜培地（４Ｆ１ＨｍＣａｒプレート：ＬＳ培地、１ｍｇ／ｌ ４ＦＡ、２５ｍｇ／ｌ ハイグロマイシン、５００ｍｇ／ｌ カルベニシリン、３０ｇ／ｌ ショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８）に並べて２６℃、暗黒条件下にて培養した。
（６）形質転換カルスの選択
前記（５）で２週間培養した胚性カルスは、以後２週間毎に新しい４Ｆ１ＨｍＣａｒプレートに移植し、培養した。非形質転換カルスは褐変したが、一部の形質転換された細胞は淡黄色の胚性カルスを形成した。
（７）形質転換植物の再生
形質転換された胚性カルスは、選抜培地置床後６０日で、体細胞胚形成培地（Ａ４Ｇ１ＨｍＣａｒプレート：ＬＳ培地、４ｍｇ／ｌ ＡＢＡ、１ｍｇ／ｌ ＧＡ３、２５ｍｇ／ｌ ハイグロマイシン、５００ｍｇ／ｌ カルベニシリン、３０ｇ／ｌ ショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８）に移植し２６℃、弱光（３０〜４０μｍｏｌ／ｍ²／ｓ）、全日長条件下にて２週間培養の後、植物体形成培地（Ａ０．０５ＨｍＣａｒプレート：ＬＳ培地、０．０５ｍｇ／ｌ ＡＢＡ、２５ｍｇ／ｌ ハイグロマイシン、５００ｍｇ／ｌ カルベニシリン、３０ｇ／ｌショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８）に移植し、同条件で培養した。以後２週間毎に新しいＡ０．０５ＨｍＣａｒプレートに移植した。形質転換された細胞は緑色を呈する胚性カルス由来体細胞胚を形成するので、これを植物生育培地（０Ｇプレート：ＬＳ培地、３０ｇ／ｌ ショ糖、３．２ｇ／ｌ ゲランガム、ｐＨ５．８）に移植し、シュートを形成させた。
（８）ＤＮＡ抽出とサザンハイブリダイゼーション
前記で得られた形質転換体から葉を切り取り、直ちに液体窒素で凍結した。これを液体窒素存在下乳鉢で細かく粉砕し、１ｇ当たり、３mlのＤＮＡ抽出用緩衝液〔２００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、１００ｍＭ ＥＤＴＡ−２Ｎａ、１％ Ｎ−ラウロイルサルコシンナトリウム、１００μg／ml ＰｒｏｔｅｉｎａｓｅＫ〕を加え十分撹拌した。６０℃で１時間インキュベート後、遠心（１０，０００×ｇ、１０分間）して、その上清をミラクロスで濾渦し新しいチューブに移した。フェノール：クロロホルム：イソアミルアルコール（２５：２４：１）抽出を３回行った後、エタノール沈殿を行った。沈殿をＴＥ緩衝液に溶解した。それぞれ植物体約２．０ｇから、２０μgずつのゲノムＤＮＡが得られた。このうち１μgのＤＮＡを用いて、それぞれを制限酵素ＥｃｏＲＩ、ＨｉｎｄIIIで切断し、１％アガロース電気泳動及びサザンハイブリダイゼーションに供した。
【００５７】
また、形質転換されていない野生株についても同様な条件下で栽培し、同様にＤＮＡを抽出し、制限酵素ＥｃｏＲＩ、ＨｉｎｄIIIによる消化を行ない、１％アガロースゲル電気泳動及びサザンハイブリダイゼーションに供した。ハイブリダイゼーション用プローブは各ＦＳＰＤ１、ＦＳＡＭ２４、ＦＡＤＣ７６遺伝子断片を用いた。
【００５８】
サザンハイブリダイゼーションは、モレキュラー クローニング，ア ラボラトリー マニュアル（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ）、第９章、第３１〜５８頁〔コールド スプリング ハーバー（Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｅｒ）社、１９８９年刊〕に記載の方法に従って行った。すなわち、それぞれのＤＮＡ試料について１％アガロースゲル電気泳動を行ない、泳動後、アルカリ変性を行ないナイロンメンブレン（ハイボンド−Ｎ、アマシャム社製）に一晩サザンブロットした。紫外線トランスイルミネーター（２５４ｎｍ）に３分間照射させ、ＤＮＡを固定した。このメンブレンをプレハイブリダイゼーション緩衝液（５×デンハルト液、６×ＳＳＣ、０．１％ＳＤＳ、１０μg／mlサケ精子ＤＮＡ）５ml中で５０℃、２時間プレハイブリダイゼーションを行った。プローブを加え、５０℃で一晩ハイブリダイゼーションを行った。ハイブリダイゼーションの後、メンブレンを２×ＳＳＣ、０．１％ＳＤＳを含む洗浄液で室温１０分間２回洗浄し、続いて同じ洗浄液で５０℃、３０分間で２回洗浄した。メンブレンは乾燥させた後、Ｘ線フィルム（コダック社製）を入れたカセット内で−８０℃一晩感光させ、オートラジオグラフィーをとった。形質転換を行っていない株（１）、ＦＳＰＤ１、ＦＳＡＭ２４、ＦＡＤＣ７６を導入した形質転換体（２）、ベクターのみを導入した形質転換体（３）について、サザンハイブリダイゼーションにより検出されたシグナルのパターンを比較した。
（２）には、（１）、（２）、（３）共通の内在性シグナルのほかに、ＥｃｏＲＩで切断したサンプルと、ＨｉｎｄIIIで切断したサンプルでは特異的なシグナルが観察され、目的遺伝子が（２）に組み込まれていることが観察された。
実施例３：ノーザンブロット解析
実施例２で得られた形質転換体で目的の遺伝子が実際に遺伝子発現しているかを確かめるために、ノーザンブロッティングを下記に示す様に行った。
【００５９】
形質転換を行っていない野生株（ＷＴ）と形質転換体（ＴＳＰ）の若葉から全ＲＮＡを抽出した。ＲＮＡ抽出は定法に従って行った。得られた全ＲＮＡ10μgを1.5%ホルムアルデヒドアガロースゲルで電気泳動した後、ハイボンドNナイロンメンブランに一晩ブロッティングした。UVクロスリンカーでＲＮＡを固定した後、プレハイブリダイゼーションバッファー（50% Formamide、5X SSPE、5X Denhardt's、0.1% SDS、80μg/ml Salmon sperm DNA、pH7.0）で、42℃、2時間プレハイブリダイゼーションを行った。形質転換を行ったクロダネカボチャＳＰＤＳ遺伝子断片、ＳＡＭＤＣ遺伝子断片、ＡＤＣ遺伝子断片のcDNAを³²P-dCTPとランダムラベルキット（アマシャム社製）を用いて、プローブを作製した。このプローブをプレハイブリダイゼーションに加え、42℃で一晩ハイブリダイゼーションを行った。ハイブリダイゼーション後、メンブレンを2×SSC、0.1% SDSを含む洗浄液からスタートし、最終的には0.1× SSC、0.1% SDSを含む洗浄液で55℃・30分2回まで洗浄した。メンブレンをX線フィルム（Kodak社製）を用いて、オートラジオグラフィーを行った。その結果、野生株（ＷＴ）では外因性クロダネカボチャＳＰＤＳ遺伝子、ＳＡＭＤＣ遺伝子、ＡＤＣ遺伝子の発現は検出されなかったが、形質転換を行った全ての形質転換体では非常に高いレベルでクロダネカボチャＳＰＤＳ遺伝子（ＦＳＰＤ１）、ＳＡＭＤＣ遺伝子（ＦＳＡＭ２４）、ＡＤＣ遺伝子（ＦＡＤＣ７６）が発現していることが確認された。
実施例４：ポリアミン含量の評価
（１）系統（セルライン）の選抜
実施例２で作製した形質転換体について、ＰＣＲ（またはサザン解析）とノーザン解析による目的遺伝子の導入確認でセルラインの選抜を行った。その結果、確実にポリアミン代謝関連酵素遺伝子が導入され、且つ該遺伝子を安定的に発現している系統についてポリアミン分析を行った。ＦＳＰＤ１がセンス方向（＋）で導入されているセルライン、ＴＳＰ−ＳＳ−１、ＴＳＰ−ＳＳ−２、ＴＳＰ−ＳＳ−３、ＴＳＰ−ＣＳ−１、ＴＳＰ−ＣＳ−３、ＴＳＰ−ＣＳ−４を選抜し、ＴＳＰ−ＳＳ−１、ＴＳＰ−ＳＳ−２、ＴＳＰ−ＳＳ−３はプロモーターとしてCaMV 35Sプロモーターが導入されている系統、ＴＳＰ−ＣＳ−１、ＴＳＰ−ＣＳ―３、ＴＳＰ−ＣＳ−４は西洋わさび由来のペルオキシダーゼプロモーター（C2プロモーター）が導入されている系統である。ＦＳＰＤ１がアンチセンス方向（−）で導入されているセルライン、ＴＳＰ−ＳＡ−１、ＴＳＰ−ＳＡ−２を選抜し、いずれもCaMV 35Sプロモーターが導入されている系統である。ＦＳＡＭ２４がセンス方向（＋）で導入されているセルライン、ＴＳＰ−ＳＭ−１、ＴＳＰ−ＳＭ−２、ＴＳＰ−ＳＭ−５を選抜し、ＴＳＰ−ＳＭ−１、ＴＳＰ−ＳＭ−２、ＴＳＰ−ＳＭ−５はプロモーターとしてCaMV 35Sプロモーターが導入されている系統である。
（２）ポリアミン含量の分析
同時に栽培を行った野生株（ＷＴ）と形質転換体（ＴＳＰ）から約０．３〜０．９ｇの若葉をサンプリングして凍結保存した。サンプリングした試料に希釈内部標準液（１，６−ｈｅｘａｎｅｄｉａｍｉｎｅ、内部標準量＝７．５ｎｍｏｌ）と５％過塩素酸水溶液（試料生体重１．０ｇ当たり５〜１０ｍｌ）を加え、オムニミキサーを用いて室温下で十分に磨砕抽出した。磨砕液を、４℃・３５，０００×ｇで２０分間遠心分離して上清液を採取し本液を遊離型ポリアミン溶液とした。スクリューキャップ付きのマイクロチューブに４００μｌの遊離型ポリアミン溶液、２００μｌの飽和炭酸ナトリウム水溶液、２００μｌのダンシルクロライド／アセトン溶液（１０ｍｇ／ｍｌ）を加えて軽く混和した。チューブの栓をしっかりと閉めたのちアルミ箔で多い、６０℃のウォーターバスで１時間加温してダンシル化を行った。チューブを放冷した後、プロリン水溶液（１００ｍｇ／ｍｌ）を２００μｌ加えて混和した。アルミ箔で覆ってウォーターバスで３０分間再加温した。放冷後、窒素ガスを吹き付けてアセトンを除いた後に、６００μｌのトルエンを加えて激しく混和した。チューブを静置して２相に分かれた後に、上層のトルエン層を３００μｌマイクロチューブに分取した。分取したトルエンに窒素ガスを吹き付けてトルエンを完全除去した。チューブに２００μｌのメタノールを加えてダンシル化遊離型ポリアミンを溶解させた。プトレシン、スペルミジン、スペルミンの遊離型ポリアミン量の定量は蛍光検出器を接続した高速液体クロマトグラフィーを用いて内部標準法で分析した。ＨＰＬＣカラムはμＢｏｎｄａｐａｋ Ｃ１８（Ｗａｔｅｒｓ社製：０２７３２４、３．９×３００ｍｍ、粒子径１０μｍ）を使用した。試料中のポリアミン含量は標準液と試料のＨＰＬＣチャートから、それぞれ各ポリアミンと内部標準のピーク面積を求めて算出した。その結果を表４に示す。
【００６０】
【表４】

【００６１】
表４より、明らかなようにポリアミン代謝関連酵素遺伝子をセンス方向に導入したセルラインは、プトレシン含量、スペルミジン含量、スペルミン含量が野生株（ＷＴ）より有意に増大し、総ポリアミン含量も野生株（ＷＴ）より有意に増大していることが明らかとなった。特にスペルミジンとスペルミン含量の増加が顕著であった。さらに、ポリアミン代謝関連酵素遺伝子をアンチセンス方向に導入したセルラインは、特にスペルミジン含量とスペルミン含量が野生株（ＷＴ）より有意に減少し、総ポリアミン含量も野生株（ＷＴ）より有意に減少していることが明らかとなった。
【００６２】
以上の結果から、ポリアミン代謝関連酵素遺伝子を植物にセンス方向又はアンチセンス方向で遺伝子導入することで、植物内のポリアミン含量を増加又は減少させることが可能であることが明らかとなった。これらのことから、ポリアミン代謝関連酵素遺伝子を植物に遺伝子導入することで、ポリアミン代謝を操作してポリアミン含量を制御できることが示された。
実施例５：生産性の評価
（１）茎（蔓）・葉・根（塊根）の評価
実施例２で得られた形質転換体（ＴＳＰ）と野生株（ＷＴ：高系１４号）の幼苗を培養土（サンサン床土：タキイ種苗社製）を詰めたプランターに定植して７日間、２３℃、長日条件（１６時間日長・８時間暗黒）、多湿条件下で活着させた。その後、２３℃、弱光長日条件下で約１ヶ月間順化育成した後に自然光型グロースチャンバー（コイトトロンＳＢＨ−３０３０、小糸工業社製）に移して、温度（昼／夜：２６℃／２４℃）、湿度（５０〜６０％）、光（なりゆき）条件下で通常栽培を開始した。通常栽培から１ヶ月後に蔓（茎）と葉について調査を行った。その結果を図２に示した。図２の結果から、形質転換体（ＴＳＰ）は野生株（ＷＴ）に比べて、茎（蔓）が長く、葉数が増大していることが明らかとなった。さらに、蔓を詳細に調べたところ、形質転換体（ＴＳＰ）は野生株（ＷＴ）比べて節間が短く、野生株（ＷＴ）の葉柄（葉）数が５個（５枚）であるのに対して、形質転換体（ＴＳＰ）の葉柄（葉）数は１７個（１７枚）であり顕著に増加していた。さらに、通常栽培から３ヶ月後に塊根（イモ）の調査を行った。その結果を図３Ａ及び図３Ｂに示した。形質転換体（ＴＳＰ）と野生株（ＷＴ）の一株当たりの塊根数を比較したところ、形質転換体（ＴＳＰ）の方がイモの数が多かった。さらに、イモの収量を調べたところ、形質転換体（ＴＳＰ）は一株当たりのイモの新鮮重が３２５．９９ｇと２００．１８ｇであるのに対して、野生株（ＷＴ）の新鮮重は７９．０８ｇと１０１．３６ｇであり、形質転換体（ＴＳＰ）の方がイモの収量が顕著に増大していることが明らかとなった。
（２）根（塊根）の評価
実施例２で得られた形質転換体（ＴＳＰ−ＳＳ−１，ＴＳＰ−ＣＳ−３）と野生株（ＷＴ：高系１４号）の苗から挿し穂（一節挿し木法）を培養土（サンサン床土：タキイ種苗社製）を詰めたプランターに挿し込んで７日間、２３℃、長日条件（１６時間日長・８時間暗黒）、多湿条件下で発根させた。その後、２３℃、弱光長日条件下で約１ヶ月間順化育成した後に自然光型グロースチャンバー（コイトトロンＳＢＨ−３０３０、小糸工業社製）に移して、温度（昼／夜：２６℃／２４℃）、湿度（５０〜６０％）、光（なりゆき）条件下で通常栽培を開始した。通常栽培から２ヶ月後に塊根（イモ）の肥大形成について調査を行った。その結果を図４に示した。図４の結果から、野生株（ＷＴ）は全株において塊根の肥大形成が見られなかったが、形質転換体（ＴＳＰ−ＳＳ−１，ＴＳＰ−ＣＳ−３）は全株で塊根の肥大形成が観察された。
以上の結果から、サツマイモにポリアミン代謝関連酵素遺伝子を導入することで、葉、茎（蔓）、根（塊根）等の生産性が増加し、植物の生産性が改良できることが立証された。
【００６３】
【配列表】

【図面の簡単な説明】
【図１】ポリアミン代謝関連酵素遺伝子を含む発現コンストラクトの構造を示す図である。
【図２】ポリアミン代謝関連酵素遺伝子を導入したサツマイモと野生株との茎・葉・蔓の比較を示す写真である。
【図３Ａ】ポリアミン代謝関連酵素遺伝子を導入したサツマイモと野生株との根（塊根、イモ）の比較を示す写真である。
【図３Ｂ】ポリアミン代謝関連酵素遺伝子を導入したサツマイモと野生株との根（塊根、イモ）の比較を示す写真である。
【図４】ポリアミン代謝関連酵素遺伝子を導入したサツマイモと野生株との根（塊根、イモ）の肥大形成の比較を示す写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to plants having improved productivity or traits, in particular improved stems, tubers, leaves, roots, tuberous roots, buds, flowers, petals, ovary, fruits, pods, berries, seeds, fibers And a plant having an ovule or the like.
The present invention also relates to a method for producing the plant.
[0002]
Furthermore, an object of the present invention is to obtain a useful substance (eg starch) or a derivative thereof (eg biodegradable plastic) from the plant.
[0003]
[Prior art]
The food crisis has become a problem on a global scale, and it is an important issue to improve plant productivity or traits (production potential, hereinafter simply referred to as “productivity” in the column of conventional technology). Yes.
[0004]
In order to improve the productivity of plants, cross breeding, breeding using recent genetic engineering techniques, methods utilizing the action of plant hormones and plant regulators, and the like have been carried out. In particular, attempts have been made to improve productivity by cross breeding centering on rice, and many high-yielding varieties with dramatically improved productivity have been developed so far. In breeding by genetic engineering technology, productivity is improved by using genes of key enzymes related to photosynthesis and carbohydrate metabolism.
[0005]
For example, by expressing the fructose-1,6-bisphosphatase / sedheptulose-1,7-bisphosphatase gene derived from cyanobacteria in tobacco chloroplasts, the photosynthetic ability is enhanced and the photosynthetic metabolic intermediate (hexose, The content of sucrose and starch was increased, the growth was promoted, and the productivity was improved (Patent No. 3357909). Furthermore, sucrose phosphate synthase (SPS), which is a carbohydrate metabolism, is thought to affect not only carbon distribution but also production capacity, and SPS genes have been introduced into tomatoes, potatoes and tobacco. It was confirmed that by introducing the SPS gene in tomato, the dry matter weight of the above-ground part during the vegetative growth period increased (Plant Physiol., 101, 535, 1993, Planta, 196, 327, 1995). In potatoes, it was confirmed that the dry matter weight of the above-ground part (leaves and stems) and tubers increased (Jpn. J. Crop Sci., 65, 102, 1996, Jpn. J. Crop Sci, 65, 143, 1996). However, many of the plants transformed with these genes have not yet obtained a sufficient effect for practical use in the industry and have not yet been put into practical use.
[0006]
Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body, and more than 20 types of polyamines have been found. Typical polyamines include putrescine, spermidine and spermine. The main physiological functions of polyamines are as follows: (1) Nucleic acid stabilization and structural changes by interaction with nucleic acids, (2) Promoting action on various nucleic acid synthesis systems, (3) Activation of protein synthesis systems, (4) ▼ Stabilization of cell membrane and enhancement of membrane permeability of substances are known.
The role of polyamines in plants has been reported to promote nucleic acid and protein biosynthesis and cell protection during cell growth and division, but recently, the relationship between polyamines and environmental stress tolerance has also attracted attention. To date, no reports have been found on productivity improvements and their involvement with polyamines.
[0007]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a technique for enhancing the productivity or character of a practical plant.
[0008]
[Means for Solving the Problems]
As a result of diligent efforts to achieve the above-mentioned object, the present inventors have isolated polyamine metabolism-related enzyme genes involved in polyamine biosynthesis, introduced the genes into plants, and overexpressed to manipulate polyamine metabolism. Thus, it was found that by changing the polyamine concentration, productivity or trait parameters were improved regardless of the cultivation environment, in other words, with or without environmental stress.
A polyamine is a basic substance containing a large amount of amine in the molecule, and typical polyamines include putrescine containing a bimolecular amine, spermidine containing a trimolecular amine, spermine containing a tetramolecular amine, and the like. In plants, ADCs and ODCs for putrescine, SAMDC and SPDS for spermidine, and SAMDC and SPMS for spermine are found as polyamine metabolism-related enzymes involved in polyamine biosynthesis. Polyamine metabolism-related enzyme genes encoding these polyamine metabolism-related enzymes have already been isolated in several plants. For example, polyamine metabolism-related enzymes involved in plant polyamine biosynthesis include arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), Spermine synthase (SPMS) and the like are known. Several polyamine metabolism-related genes encoding these polyamine metabolism-related enzymes have already been isolated from plants. ADC genes are oats (Mol. Gen. Genet., 224, 431-436, 1990), tomatoes (Plant Physiol., 103, 829-834, 1993), Arabidopsis (Plant Physiol., 111, 1077-1083, 1996) Pea (Plant Mol. Biol., 28, 997-1009, 1995), ODC gene is Datura (Biocem. J., 314, 241-248, 1996), SAMDC gene is potato (Plant Mol. Biol , 26, 327-338, 1994), spinach (Plant Physiol., 107, 1461-1462, 1995), tobacco, SPDS genes are Arabidopsis (Plant cell Physiol., 39 (1), 73-79, 1998), etc. Has been isolated from. Furthermore, some polyamine metabolism-related enzyme genes have been attempted to be introduced into plants, but no improvement in productivity or traits has been reported in the resulting transformed plants.
[0009]
Under these circumstances, the present inventors have intensively studied to improve the productivity and traits of plants, and as a result, it has been found that the content of spermidine and spermine, which are polyamines, is important in improving the productivity and traits. As a result, spermidine and polyamine metabolism-related genes (SPDS, SAMDC, ADC) involved in spermidine and spermine biosynthesis were actually isolated and identified from plant tissues. Further, by introducing the gene into a plant and overexpressing it, it has been found that productivity or trait parameters are improved by manipulating polyamine metabolism and changing the polyamine concentration, thereby completing the present invention. It came.
[0010]
The present invention provides the following inventions.
1. A nucleic acid sequence that regulates the amount of polyamine under the control of a promoter that can function in a plant is stably retained, and the productivity or trait is independent of the cultivation environment compared to a control plant that does not have the nucleic acid sequence. Improved transgenic plants and their progeny.
17. A nucleic acid sequence comprising the step of stably retaining a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant and transforming a plant cell not having the nucleic acid sequence; A method for producing a plant having improved productivity or traits regardless of the cultivation environment compared to a comparative control plant that has not been used.
19. An expression vector comprising a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant, comprising the step of transforming a plant cell not having the nucleic acid sequence, the nucleic acid sequence comprising A method for producing a plant having improved productivity or traits regardless of the cultivation environment compared to a non-comparative control plant.
31. The following steps:
(1) transforming a plant cell not having the nucleic acid sequence with an expression vector comprising a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant;
(2) regenerating a plant having improved productivity or trait from the transformed cell compared to a plant not having the nucleic acid sequence;
(3) collecting seeds from the plant body by pollination; and
(4) selecting the homozygote of the nucleic acid sequence by examining the nucleic acid sequence in the seed obtained by pollination from the plant obtained by cultivating the seed
A method for producing a plant having a fixed trait having improved productivity or trait that is homozygous for the nucleic acid sequence, and that is independent of the cultivation environment compared to a control plant not having the nucleic acid sequence .
32. The following steps:
(1) transforming a plant cell not having the nucleic acid sequence with an expression vector containing a nucleic acid sequence inhibitor that regulates the amount of polyamine under the control of a promoter that can function in the plant;
(2) Inducing callus from the transformed cells
A method for producing a callus to which a trait having improved productivity or trait, which is homozygous for the nucleic acid sequence, and has improved productivity or trait compared to a comparative control plant not having the nucleic acid sequence .
33. Item 33. The method according to any one of Items 19 to 32, wherein the plant having improved productivity or trait is a sweet potato plant.
34. A sweet potato plant having improved productivity or traits regardless of the presence or degree of environmental stress as compared with a comparative control sweet potato characterized by cultivating a sweet potato plant obtained by the method of item 33 and harvesting the sweet potato Production method.
35. The step of cultivating the sweet potato plant obtained by the method of item 33 and harvesting the sweet potato,
Process for separating sweet potato starch from the obtained sweet potato
A process for producing sweet potato starch.
36. The step of cultivating the sweet potato plant obtained by the method of item 33 and harvesting the sweet potato,
Process for separating sweet potato starch from the obtained sweet potato
Converting the sweet potato starch into a biodegradable plastic
A method for producing a biodegradable plastic comprising:
[0011]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, “productivity or trait is improved” means any organ (tissue) of a plant, such as stem, tuber, leaf, root, tuberous root, bud, flower, petal, ovary, fruit, pod, saku Increase in size, total weight, quantity, etc. of fruits, seeds, fibers, ovules, etc., shorten growth period (improvement of productivity), and traits related to the organ (tissue) (for example, tomato, cucumber, banana Increase the number and size of fruits, potatoes and cassava increase the number and size of tubers, sweet potatoes increase the number and size of tuberous roots, corn, rice, soybean and so on seeds (endosperm) ) Increase in the number and size of cotton, cotton balls for cotton, and increase in the number and size of ovules, increase in the amount of fiber, petunia, torenia, etc. increase in the number and size of flowers, and other changes in shape , Coloring (eg between pigments Increase changes and chromogenic amount of balance), etc.) refers to be improved.
[0012]
The plant obtained by the present invention can improve the productivity by increasing the amount of polyamine expressed regardless of the cultivation environment (for example, environmental stress).
Examples of the nucleic acid sequence that regulates the amount of polyamine include exogenous polyamine metabolism-related enzyme genes or suppressors of endogenous polyamine metabolism-related enzyme genes. The exogenous polyamine metabolism-related enzyme gene can increase the amount of polyamine in the plant body, and the suppressor of the endogenous polyamine metabolism-related enzyme gene can decrease and increase the amount of polyamine in the plant body.
In the present invention, “a comparative control plant that does not have a nucleic acid sequence that regulates the amount of polyamine” refers to the nucleic acid sequence (for example, an exogenous polyamine metabolism-related enzyme gene or an inhibitor of an endogenous polyamine metabolism-related enzyme gene). Means every plant before introduction. Therefore, in addition to so-called wild species, all cultivars established by normal mating, natural or artificial mutants thereof, and transgenic plants into which exogenous genes other than polyamine metabolism-related enzyme genes have been introduced are included.
[0013]
The “polyamine” referred to in the present invention is a general natural product that exists universally in living organisms, and is an aliphatic hydrocarbon compound having two or more primary amino groups. For example, 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropyl cadaverine, theremin, spermine, thermospermine, canabalmin, aminopentylnorspermidine, N, N-bis (aminopropyl) cadaverine, homo Examples include spermine, cardopentamine, homocardopentamine, cardohexamine, and homocardohexamine.
Polyamine metabolism-related enzyme gene
In the present invention, the “polyamine metabolism-related enzyme gene” is a gene encoding an amino acid of an enzyme involved in biosynthesis of polyamine in plants. For example, arginine decarboxylase (ADC) gene for putrescine, which is a typical polyamine And ornithine decarboxylase (ODC) gene, for spermidine, S-adenosylmethionine decarboxylase (SAMDC) gene and spermidine synthase (SPDS) gene, for spermine, S-adenosylmethionine decarboxylase (SAMDC) gene The spermine synthase (SPMS) gene is involved and is thought to be rate limiting.
[0014]
Arginine decarboxylase (ADC) is an enzyme that catalyzes the reaction of producing agmatine and carbon dioxide from L-arginine. Ornithine decarboxylase (ODC: ornithine decarboxylase EC4.1.1.17.) Is an enzyme that catalyzes the reaction of producing putrescine and carbon dioxide from L-ornithine. S-adenosylmethionine decarboxylase (SAMDC) is an enzyme that catalyzes the reaction of producing adenosylmethylthiopropylamine and carbon dioxide from S-adenosylmethionine decarboxylase EC4.1.1.50. Spermidine synthase (SPDS) is an enzyme that catalyzes the reaction of producing spermidine and methylthioadenosine from putrescine and adenosylmethylthiopropylamine.
[0015]
These genes may be derived from any source, but can be isolated from various plants, for example. Specifically, it is selected from the group consisting of dicotyledonous plants such as cucurbitaceae; solanaceae; Brassicaceae such as Arabidopsis; legumes such as alfalfa and cowpy (Vigna unguiculata); mallow; asteraceae; Or monocotyledonous plants such as rice, wheat, barley, corn and the like. Preferably, a cucurbitaceae plant, more preferably a black pumpkin.
[0016]
The plant tissue for isolating the plant-derived polyamine metabolism-related enzyme gene of the present invention is in the form of seeds or in the growth process. Growing plants can be isolated from whole or partial tissues. The site that can be isolated is not particularly limited, but is preferably a whole plant tree, an oak, a flower, an ovary, a fruit, a leaf, a stem, a root, or the like.
Preferable examples of the polyamine metabolism-related enzyme gene used in the present invention include spermidine synthase gene, S-adenosylmethionine decarboxylase gene, and arginine decarboxylase gene. In particular,
-DNA having the base sequence represented by base numbers 77 to 1060 in the base sequence represented by SEQ ID NO: 1
-DNA having the base sequence represented by base numbers 456 to 1547 in the base sequence represented by SEQ ID NO: 3
A DNA having a base sequence represented by base numbers 541 to 2661 in the base sequence represented by SEQ ID NO: 5;
Is mentioned. further,
-A DNA encoding a polypeptide having a base sequence capable of hybridizing with any of the above sequences under stringent conditions and having a polyamine metabolism-related enzyme activity equivalent to that sequence. Furthermore,
-In any one of the above sequences, DNA encoding a polypeptide having a base sequence in which one or more bases are deleted, substituted, inserted or added and having a polyamine metabolism-related enzyme activity equivalent to the sequence is mentioned. It is done.
The term “stringent conditions” as used herein means that only a base sequence encoding a polypeptide having a polyamine metabolism-related enzyme activity equivalent to the polyamine metabolism-related enzyme encoded by the specific polyamine metabolism-related enzyme gene sequence hybridizes with the specific sequence. A base sequence that encodes a polypeptide that forms a (so-called specific hybrid) and does not have an equivalent activity means a condition that does not form a hybrid (so-called non-specific hybrid) with the specific sequence. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, 6 × SSC (0.9M NaCl, 0.09M trisodium citrate) or 6 × SSPE (3M NaCl, 0,2M NaH)₂PO_Four, 20 mM EDTA · 2Na, pH 7.4) at 42 ° C., and further washed with 0.5 × SSC at 42 ° C. is one example of the stringent conditions of the present invention. It is not limited.
The “base sequence in which one or more bases have been deleted, substituted, inserted or added” as used herein generally means that one or more amino acids are substituted, deleted or inserted in the amino acid sequence of a protein having physiological activity. It is widely recognized by those skilled in the art that even when added, the physiological activity may be maintained. Such a modification is added to the present invention, and a gene encoding a polyamine metabolism-related enzyme is also included in the scope of the present invention. For example, the polyA tail or the 5 ′, 3 ′ terminal untranslated region may be “deleted”, or the base may be “deleted” within a range that deletes amino acids. Further, the base may be “substituted” within a range where no frame shift occurs. In addition, a base may be “added” in such a range that an amino acid is added. However, even with such modifications, it is necessary to have polyamine metabolism-related enzyme activity. Preferably, “a gene in which one or several bases are deleted, substituted or added” is used.
Such modified DNA can be substituted, deleted, inserted, or added at a specific site by, for example, site-specific mutagenesis (Nucleic Acid Research, Vol. 10, No. 20, 6487-6500, 1982). Thus, it is obtained by modifying the base sequence of the DNA of the present invention.
The “antisense gene” in the present invention means a gene having a sequence complementary to the base sequence of a polyamine metabolism-related enzyme gene. Antisense DNA is complementary to, for example, the nucleotide sequences of SEQ ID NOs: 1, 3, and 5, and antisense RNA is produced from them.
Inhibitor of endogenous polyamine metabolism-related enzyme gene
The suppressor is not particularly limited as long as it suppresses expression of the gene, and examples thereof include antisense DNA or double-stranded RNA (dsRNA) having a sequence selected from the gene and its upstream or downstream. The antisense DNA may be complementary to either an intron or exon of the gene, a regulatory region upstream of the 5 ′ containing the promoter of the gene, or a region downstream of the stop codon and affecting gene expression. That's fine. The length of the antisense DNA has at least 20 bases, preferably at least 100 bases, more preferably at least 300 bases, in particular at least 500 bases. The transcript of the antisense DNA hybridizes with the mRNA of the endogenous polyamine metabolism-related enzyme gene, or a non-coding region (eg, promoter, intron, transcription termination) of the endogenous polyamine metabolism-related enzyme gene or its upstream or downstream sequence. To the factor).
Plants with improved productivity and their progeny
In the present invention, as described above, “productivity” refers to all organs (tissues) of plants, such as stems, tubers, leaves, roots, tuberous roots, buds, flowers, petals, ovaries, fruits, pods, and berries. , Seeds, fibers, ovules, etc., and examples of the traits of the organ (tissue) include quantity, growth period, shape, coloration, properties, and characteristics.
In the present invention, “plants with improved productivity” and “plants with improved productivity” are defined by introducing an exogenous polyamine metabolism-related enzyme gene or an inhibitor of an endogenous polyamine metabolism-related enzyme gene. This refers to a plant to which a quantity, growth period, shape, color, property, or characteristic, which is a trait related to productivity as compared to before introduction, is imparted or improved or suppressed. For example, by introducing a polyamine metabolism-related enzyme gene or the suppressor into a plant, a trait related to a stem, leaf, root, flower, ovary, or seed has the exogenous polyamine metabolism-related enzyme gene or the suppressor. Examples include, but are not limited to, plants that are improved compared to plants that are not.
[0017]
This can be expected to improve plant (organ, tissue, etc.) productivity, yield, yield, quality, and merchandise. In addition, in edible plants such as vegetables and fruits, there is a possibility that the properties such as quality may be favorably affected by reducing the amount of polyamines in the edible part (leaves, fruits, tubers, rhizomes, etc.). Accordingly, it is possible to improve the yield while improving the quality by expressing a suppressor in these edible parts and expressing a polyamine-related enzyme gene in the other parts.
Furthermore, in one of the preferred embodiments of the present invention, the plant has little difference in appearance during the vegetative growth period (the leaves become dark green), but the productivity of leaves, flowers, stems, roots, etc. during the reproductive growth period. This improvement is more noticeable, and is understood to have improved the plant's potential.
[0018]
The plant of the present invention is not limited to the whole plant (whole tree), but its callus, seeds, all plant tissues, leaves, stems, tubers, roots, tuberous roots, buds, flowers, petals, ovary, fruit, pods, Includes ovules, fibers, etc. Furthermore, the progeny are also included in the plant of the present invention.
[0019]
In the present invention, “a useful substance obtained from a plant and its progeny” refers to a useful substance produced in a plant and its progeny whose productivity is improved by introducing an exogenous polyamine metabolism-related enzyme gene. Examples of useful substances include amino acids, fats and oils, starches, proteins, phenols, hydrocarbons, celluloses, natural rubbers, pigments, enzymes, antibodies, vaccines, pharmaceuticals, biodegradable plastics, and the like.
[0020]
The plant of the present invention is one in which an exogenous polyamine metabolism-related enzyme gene is introduced into a plant not having the exogenous polyamine metabolism-related enzyme gene by a genetic engineering technique and stably maintained. Here, “stablely held” means that the polyamine metabolism-related enzyme gene is expressed at least in the present plant into which the polyamine metabolism-related enzyme gene has been introduced, and the organ formation is thereby improved. It is held in the plant cell. Therefore, practically, it is preferable that the polyamine metabolism-related enzyme gene is integrated into the host plant chromosome. More preferably, the polyamine metabolism-related enzyme gene is stably inherited in the next generation.
[0021]
In addition, the term “exogenous” means that the plant is not naturally present and is introduced from the outside. Therefore, the “exogenous polyamine metabolism-related enzyme gene” of the present invention may be a polyamine metabolism-related enzyme gene of the same kind as the host plant (ie, derived from the host plant) introduced from the outside by genetic manipulation. Considering the identity of codon usage, the use of host-derived polyamine metabolism-related enzyme genes is also preferred.
[0022]
The exogenous polyamine metabolism-related enzyme gene may be introduced into the plant by any genetic engineering technique, for example, a protoplast fusion with a heterologous plant cell having a polyamine metabolism-related enzyme gene, a gene to express a polyamine metabolism-related enzyme gene. Examples include infection with a plant virus having an engineered viral genome, or transformation of a host plant cell with an expression vector containing a polyamine metabolism-related enzyme gene.
[0023]
Preferably, the plant of the present invention is an expression vector containing an exogenous polyamine metabolism-related enzyme gene under the control of a promoter capable of functioning in the plant, and the plant cell does not have the exogenous polyamine metabolism-related enzyme gene It is a transgenic plant obtained by transforming.
[0024]
Examples of promoters that can function in plants include cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase gene (NOS) promoter, octopine synthase gene (OCS) promoter, phenylalanine ammonia, which are constitutively expressed in plant cells. Examples thereof include lyase (PAL) gene promoter and chalcone synthase (CHS) gene promoter. Furthermore, the well-known plant promoter which is not limited to these is also mentioned.
[0025]
By using not only a promoter that is constitutively expressed throughout the organ, such as the 35S promoter, but also an organ or tissue-specific promoter, the target gene can be expressed only in a specific organ or tissue. Only the productivity can be improved. For example, by using a polyamine metabolism-related enzyme gene and a promoter that specifically acts on a flower organ (for example, WO99 / 43818, JP-A-11-178572, JP-A-2000-316582), the number of flowers and the flower The size can be improved. Furthermore, the number and size of ovaries and fruits can be improved by using a promoter that specifically acts on fruits and ovaries (for example, Plant Mol. Biol., 11, 651-662, 1988).
[0026]
If a time-specific promoter is used, a target gene can be expressed only at a specific time, and productivity can be improved only at a specific time. For example, by using a polyamine metabolism-related enzyme gene and a promoter that works in the vegetative growth period, productivity can be improved only in the vegetative growth period.
[0027]
In the expression vector of the present invention, the exogenous polyamine metabolism-related enzyme gene is placed downstream of the promoter so that its transcription is controlled by a promoter that can function in plants. It is preferable that a transcription termination signal (terminator region) that can function in plants is further added downstream of the polyamine metabolism-related enzyme gene. Examples thereof include a terminator NOS (nopaline synthase) gene.
[0028]
The expression vector of the present invention may further contain a cis-regulatory element such as an enhancer sequence. In addition, the expression vector is a marker gene for selecting transformants such as drug resistance gene markers, such as neomycin phosphotransferase II (NPTII) gene, phosphinothricin acetyltransferase (PAT) gene, glyphosate resistance gene, etc. May further be included. Under the condition that the selection pressure is not applied, the incorporated gene may drop off. Therefore, if the herbicide tolerance gene is allowed to coexist on the vector, it is always selected by using the herbicide during cultivation. There is also an advantage that the condition under pressure can be realized.
[0029]
Furthermore, in order to facilitate mass preparation and purification, the expression vector contains an origin of replication that allows autonomous replication in E. coli and a selectable marker gene in E. coli (eg, ampicillin resistance gene, tetracycline resistance gene, etc.). Is desirable. The expression vector of the present invention can be constructed simply by inserting an expression cassette of the above polyamine metabolism-related enzyme gene and, if necessary, a selection marker gene into the cloning site of a pUC or pBR E. coli vector. .
[0030]
When an exogenous polyamine metabolism-related enzyme gene is introduced using infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes, on the Ti or Ri plasmid retained by the bacterium The polyamine metabolism-related enzyme gene expression cassette can be inserted into a T-DNA region (region transferred to a plant chromosome) and used. Currently, the standard method for transformation by the Agrobacterium method uses a binary vector system. The functions required for T-DNA transfer are supplied independently from both the T-DNA itself and the Ti (or Ri) plasmid, and each component can be divided on separate vectors. The binary plasmid has 25 bp border sequences at both ends necessary for excision and integration of T-DNA, the plant hormone gene causing crown gall (or hairy root) has been removed, and at the same time, room for insertion of foreign genes is provided. Yes. As such a binary vector, for example, pBI101 and pBI121 (manufactured by Clontech) are commercially available. The Vir region that acts on T-DNA integration is on a separate Ti (or Ri) plasmid called a helper plasmid and acts on trans.
[0031]
Various conventionally known methods can be used for plant transformation. For example, protoplasts are isolated from plant cells by treatment with cell wall degrading enzymes such as cellulase and hemicellulase, and polyethylene glycol is added to a suspension of the protoplasts and an expression vector containing the above-mentioned polyamine metabolism-related enzyme gene expression cassette. A method of incorporating the expression vector into a protoplast in an endocytosis-like process (PEG method), placing the expression vector into a lipid membrane vesicle such as phosphatidylcholine by sonication, etc., and the vesicle and protoplast in the presence of PEG Fusion method (liposome method), minicell fusion method in the same process, electric pulse applied to suspension of protoplast and expression vector to incorporate vector in external solution into protoplast (electro Poration method). However, these methods are complicated in that they require a culture technique for redifferentiation from protoplasts to plants. As a means for gene introduction into intact cells with cell walls, a micropipette is inserted into the cell, and vector DNA in the pipette is injected into the cell by hydraulic pressure or gas pressure, and micro gold particles coated with DNA There are a direct introduction method such as a particle gun method that accelerates the explosives using explosives and gas pressure, and introduces them into cells, and a method that uses infection by Agrobacterium. Microinjection requires the skill of operation and has the disadvantage that the number of cells that can be handled is small. Therefore, considering the ease of operation, it is preferable to transform plants by the Agrobacterium method and the particle gun method. The particle gun method is further useful in that a gene can be directly introduced into the apical meristem of a plant under cultivation. Further, in the Agrobacterium method, a plant vector, for example, a genomic DNA of a gemini virus such as tomato golden mosaic virus (TGMV) is simultaneously inserted into a binary vector between border sequences, so that an arbitrary site of a plant being cultivated can be detected. By simply inoculating cells with a bacterial suspension using a syringe or the like, viral infection spreads throughout the plant body, and the target gene is also introduced into the entire plant body. These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by the person skilled in the art.
[0032]
The transformed plant produced by the method described above can be subjected to Southern analysis or Northern analysis for gene expression analysis of polyamine metabolism-related enzyme genes, polyamine content analysis, and productivity evaluation.
[0033]
For example, the polyamine is quantified by sampling a sample of 0.05 to 1 g and adding a 5% aqueous perchloric acid solution to extract the polyamine. Quantification of the extracted polyamine can be analyzed by an internal standard method using high performance liquid chromatography (HPLC) connected with a fluorescence or UV detector after labeling with dansylation or benzoylation.
[0034]
For example, as an evaluation of productivity, for stems, transgenic plants or T2 seeds or T3 seeds obtained by self-pollination of transgenic plants are planted or sown in an appropriate growth medium or soil, Day / night: 16 hours / 8 hours day length) Evaluated by examining the number, size, shape, etc. of stems (eg main flower stems, side flower stems, branches, vines, tubers, etc.) grown at 20-30 ° C. can do. For the leaves, transgenic plants, or T2 seeds or T3 seeds obtained by self-pollination of the transgenic plants were planted or sown in an appropriate growth medium or growth soil, and long-day conditions (day / night: 16 hours / day) It can be evaluated by examining the number, size, shape, etc. of leaves (for example, rosette leaves, true leaves, etc.) grown at 20-30 ° C. For flowers, transgenic plants, or T2 seeds or T3 seeds obtained by self-pollination of the transgenic plants, were planted or sown in an appropriate growth medium or soil, and long-day conditions (day / night: 16 hours / day) It can be evaluated by growing at 20-30 ° C. and examining the number, size, shape, color, etc. of the flowers. For the ovary, transgenic plants, or T2 seeds or T3 seeds obtained by self-pollination of the transgenic plants, were planted or sown in an appropriate growth medium or growth soil, and long-day conditions (day / night: 16 hours) It can be evaluated by growing at 20-30 ° C. and examining the number, size, shape, color, etc. of ovaries (eg, pods, fruits, etc.). For seeds, transgenic plants or T2 seeds or T3 seeds obtained by self-pollination of the transgenic plants were planted or sown in an appropriate growth medium or soil, and long-day conditions (day / night: 16 hours / day) It can be evaluated by growing at 20-30 ° C. and examining the number, size, shape, etc. of seeds. For the roots, transgenic plants, or T2 seeds or T3 seeds obtained by self-pollination of the transgenic plants are planted or sown in an appropriate growth medium or soil, and long-day conditions (day / night: 16 hours / day) It can be evaluated by growing at 20-30 ° C. and examining the number, size, shape, etc. of roots (tuberous root, enlarged root, fine root, etc.).
In the present invention, a method for improving (improving) productivity is a method in which a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant is stably retained compared to a control plant. Yields or useful substances (eg starch) such as fruits (eg tomatoes, cucumbers, bananas), tubers (eg potatoes, cassava), tuberous roots (eg sweet potatoes), seeds (eg rice, corn, soybeans, wheat), fibers (eg cotton) , Oils, fats, proteins, cellulose, etc.). Preferably, the taste of edible substances such as fruits and vegetables is not deteriorated by the method and is equal to or higher than that of the comparative control plant.
The plant to be transformed of the present invention is not particularly limited, and examples thereof include dicotyledonous plants, monocotyledonous plants, herbaceous plants, woody plants, and the like. For example, sweet potato, tomato, cucumber, pumpkin, melon, watermelon, tobacco, Arabidopsis, pepper, eggplant, bean, taro, spinach, carrot, strawberry, potato, rice, corn, alfalfa, wheat, barley, soybean, rapeseed, sorghum, Eucalyptus, poplar, kenaf, sugarcane, sugarcane, red lizard, orchid, carnation, rose, petunia, torenia, snapdragon, cyclamen, gypsophila, geranium, sunflower, shiba, cotton, cassava, sago palm, matsutake, shiitake mushroom, mushroom, ginseng Citrus fruits, bananas, kiwi and the like. Preferred are sweet potato, tomato, cucumber, rice, corn, soybean, wheat, petunia, torenia, eucalyptus, cotton and cassava.
The present invention can be suitably implemented, for example, for sweet potatoes.
A large amount of sugar such as sweet potato starch is obtained from sweet potato, and the sugar can be used as a raw material for producing biodegradable plastics. Biodegradable plastics include polyhydroxybutyrate, polyhydroxyvalerate, poly-β-hydroxybutyric acid, polycaprolactone, polybutylene succinate, polybutylene adipate, polyethylene succinate, poly (D, L, DL) lactic acid ( Polylactide), polyglycolic acid (polyglycolide), cellulose acetate, chitosan / cellulose / starch, modified starch and the like, and binary copolymers and ternary copolymers thereof are exemplified. These biodegradable plastics are known and can be produced using known fermentation methods, chemical synthesis methods, and the like.
[0035]
【The invention's effect】
According to the present invention, plant productivity can be improved, and improvement in the quality, value, productivity, and yield of plant organs and tissues can be expected. Specifically, the organ is produced as an agricultural crop by increasing the number and size of stems (including tubers), leaves, roots (including rhizomes), flowers, ovary, fruits, pods, and seeds. If so, an increase in quality, productivity and yield is expected. For example, rice can be expected to increase the yield of harvestable rice as the number of seeds per strain increases. The fruit tree is expected to improve productivity by increasing the number of fruit (ovicles) that have reached fruiting and extending the development period. Ornamental plants are expected to look better and increase in commercial value as the number and size of flowers, stems and leaves, especially flowers, increase. Furthermore, various environmental adaptability is improved by changing the shape and form of the plant, so that stabilization of cultivation, improvement of productivity, and expansion of cultivation areas can be expected.
[0036]
【Example】
EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.
Example 1: Cloning of plant-derived polyamine metabolism-related enzyme genes
(1) Poly (A)⁺RNA preparation
Kurodabita pumpkin (Cucurbita ficifolia Bouche) was sown in vermiculite and transplanted to a pot filled with commercially available floor soil (Sunsan floor soil; manufactured by Takii Seed Co., Ltd.) at the time of cotyledon development. The potted black pumpkin was placed in an incubator for plant cultivation (temperature 26 ° C./night 22 ° C., 13 hours long). When the second true leaf was developed, the temperature in the incubator was lowered to 18 ° C./14° C. in the daytime, and the low temperature treatment was started. Three days after the low-temperature treatment, sampling was carried out separately for roots, stems and leaves. Stored in a -80 ° C freezer until RNA extraction.
[0037]
About 4 g of Kurodane pumpkin root tissue was immediately frozen in liquid nitrogen and finely ground in a mortar in the presence of liquid nitrogen. Then, 10 ml of 0.2 M Tris acetate buffer (5 M guanidine thiocyanate, 0.7% β-mercaptoethanol, 1% polyvinylpyrrolidone (MW 360,000), 0.62% N-Lauroylsarcosine Sodium Salt, pH 8.5) was added and Polytron homogenizer (KINEMATICA For 2 minutes under ice cooling. However, β-mercaptoethanol and polyvinylpyrrolidone were added immediately before use. Thereafter, the pulverized liquid was centrifuged at 17,000 × g for 20 minutes, and the supernatant was recovered.
[0038]
The supernatant is vortexed with Miracloth, and the filtrate is gently layered on 1.5 ml of a 5.7 M cesium chloride solution in an ultracentrifuge tube, centrifuged at 155,000 xg, 20 ° C for 20 hours, and the supernatant is discarded. The RNA precipitate was collected. This precipitate is dissolved in 3 ml of 10 mM Tris-HCl, 1 mM EDTA · 2Na, pH 8.0 (referred to as TE buffer), and an equal amount of phenol: chloroform: isoamyl alcohol (volume ratio, 25: 24: 1) is added. After mixing well, the mixture was centrifuged to recover the upper aqueous layer. To the obtained aqueous layer, 1/10 volume of 3M sodium acetate (adjusted to pH 6.2 with glacial acetic acid) and 2.5 volumes of ethanol were added and mixed well, and allowed to stand at -20 ° C overnight. . Thereafter, the mixture was centrifuged at 17,000 × g for 20 minutes, and the resulting precipitate was washed with 70% ethanol and dried under reduced pressure.
[0039]
This dried preparation was dissolved in 500 μl of the above-mentioned TE buffer to obtain a total RNA solution. This RNA solution was incubated at 65 ° C. for 5 minutes and then rapidly cooled on ice. To this, add 2 × binding buffer (10 mM Tris-HCl, 5 mM EDTA · 2Na, 1 M NaCl, 0.5% SDS, pH 7.5) to the RNA solution in an equal volume, and equilibrate buffer (10 mM Tris-HCl). , 5 mM EDTA · 2Na, 0.5 M NaCl, 0.5% SDS, pH 7.5) and pre-equilibrated with an oligo dT cellulose column (manufactured by Clontech). The column is then washed with about 10 volumes of the equilibration buffer described above and then poly (A) with elution buffer (10 mM Tris-HCl, 5 mM EDTA · 2Na, pH 7.5).⁺RNA was eluted.
[0040]
The obtained eluate was mixed with 1/10 volume of the above-mentioned 3M aqueous sodium acetate solution and 2.5 volumes of ethanol, and allowed to stand at -70 ° C. Thereafter, centrifugation was performed at 10,000 × g, and the resulting precipitate was washed with 70% ethanol and dried under reduced pressure. This dried preparation was dissolved again in 500 μl of TE buffer, and purification with oligo dT cellulose column was repeated. Poly (A) derived from the roots of the resulting low-temperature treated black-headed pumpkin⁺RNA was used to prepare a cDNA library for PCR and a cDNA library for full-length gene isolation.
(2) Preparation of cDNA library for PCR
A cDNA library was prepared using Marathon cDNA Amplification Kit (Clontech). Poly (A) derived from the roots of the black pumpkin obtained in (1)⁺Using RNA as a template and a modified lock-docking oligo (dT) primer with two degenerate nucleotide positions at the 3 ′ end and reverse transcriptase, 2 according to the method of Gubler and Hoffman et al. (Gene, 25, 263-269, 1983) Single-stranded cDNA was synthesized.
[0041]
Marathon cDNA adapters (5 'end phosphorylated so that they can be easily bound to both ends of ds cDNA by T4 DNA ligase) were ligated to both ends of the obtained cDNA. The obtained adapter-bound cDNA was used as a cDNA library for PCR derived from the black root pumpkin root.
(3) PCR primer design
The determined base sequences of the arginine decarboxylase gene, the S-adenosylmethionine decarboxylase gene, and the spermidine synthase gene that were already isolated from plants and mammals were compared. Then, a highly conserved and conserved region was selected and DNA oligomers were synthesized (sequence primers I to VI).
SPDS primer I (SEQ ID NO: 7): 5'-GTTTTGGATGGAGGTGATTCA-3 '
SPDS primer II (SEQ ID NO: 8): 5'-GTGAATCTCAGCGTTGTA-3 '
SAMDC primer III (SEQ ID NO: 9): 5'-TATGTGCTGTCTGAGTCGAGC-3 '
SAMDC primer IV (SEQ ID NO: 10): 5'-GCTAAAACCCATTCTCAGGGGT-3 '
ADC primer V (SEQ ID NO: 11): 5'-GGGGCT (T / G) GGA (G / A) T (G / C) GACTA (C / T) -3 '
ADC primer VI (SEQ ID NO: 12): 5 '-(T / C) CC (A / G) TC (A / G) CTGTC (G / A) CA (G / C) GT-3'
(4) PCR amplification
Using the PCR cDNA library obtained in (2) as a template, PCR was performed using the sequence primers designed in (3). The PCR step is initially 94 ° C, 30 seconds, 45 ° C, 1 minute, 72 ° C, 2 minutes, 5 cycles, followed by 94 ° C, 30 seconds, 55 ° C, 1 minute, 72 ° C, 2 minutes, 30 cycles. It was.
(5) Agarose gel electrophoresis
The PCR amplification product was electrophoresed with 1.5% agarose, the gel after electrophoresis was stained with ethidium bromide, and the amplified band was detected on a UV transilluminator.
(6) Confirmation and collection of PCR products
The detected amplification band was confirmed and cut out from the agarose gel using a razor blade. The excised gel was transferred to a 1.5 ml microtube, and a DNA fragment was isolated and purified from the gel using QIAEXII Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was subcloned into a pGEMT cloning vector (Promega), transformed into E. coli, and plasmid DNA was prepared according to a conventional method.
(7) Determination of nucleotide sequence
The nucleotide sequence of the obtained plasmid insert was determined by the dideoxy method (Messing, Methods in Enzymol., 101, 20-78, 1983). Three genes were isolated for the SPDS gene, one gene for the SAMDC gene, and two genes for the ADC gene.
(8) Homology search
When the homology search of the nucleotide sequences of these genes with a database of known gene nucleotide sequences was performed, the SPDS gene showed 70% homology with known plant-derived SPDS genes. The SAMDC gene showed a homology of 70% or more with a known plant-derived SAMDC gene. The ADC gene showed 67% or more homology with known plant-derived ADC genes.
(9) Acquisition of full-length genes
The full-length gene was obtained by plaque hybridization. A ZAP-cDNA Synthesis Kit (Stratagene) was used for the preparation of the cDNA library. Poly (A) derived from the black root pumpkin root obtained in (1)⁺Double-stranded cDNA was synthesized according to the method of Gubler and Hoffman et al. (Gene, 25, 263-269, 1983) using an oligo (dT) primer and reverse transcriptase as a template.
[0042]
An EcoRI adapter (with Xho I and Spe I sites inside) was ligated to both ends of the obtained cDNA, digested with Xho I, and then ligated to the EcoRI and Xho I sites of the λ phage vector and λZAP II arm. Subsequently, packaging was performed using an in vitro packaging kit (Stratagene, GIGAPACK Gold), and infection with E. coli SURE strain (OD660 = 0.5) yielded a large number of recombinant λ phages. This was used as a cDNA library derived from the black root pumpkin root. The size of this library is 8.0 × 10⁶Met.
[0043]
The probe was prepared by isolating and preparing insert cDNA from the plasmid DNA of SPDS, SAMDC, and ADC genes prepared in (6), and using the obtained cDNA as a template, using a Random Primed DNA Labeling Kit (manufactured by USB). ,³²A P-labeled probe was prepared. Obtained³²P-labeled cDNA was used as a probe.
[0044]
The phages constituting the cDNA library derived from the black root pumpkin root were infected with E. coli and grown on LB agar medium, and about 50,000 phage DNAs were copied onto a nylon membrane (High Bond-N, manufactured by Amersham). .
[0045]
Transfer the nylon membrane with the phage DNA onto a filter paper containing alkaline denaturing solution (0.5M NaOH, 1.5M NaCl), leave it for 4 minutes, and then neutralize it (0.5M Tris-HCl, 1.5M NaCl, pH8). .0) and left for 5 minutes. After washing with 2 × SSC (0.3 M NaCl, 0.03 M trisodium citrate), DNA was immobilized using Stratalinker (Stratagene). Immobilize the nylon membrane with the hybridization solution (50% formamide, 0.5% SDS, 6 x SSPE (3M NaCl, 0.2M NaH₂PO_Four, 20 mM EDTA · 2Na, pH 7.4), 5 × Denhardt's solution (0.1% Ficoll, 0.1% Polyvinylpyrrolidone, 0.1% bovine serum albumin), 50 μg / ml denatured salmon sperm DNA, prehybridized at 42 ° C. for 3 hours, The prepared cDNA probe was added and hybridized at 42 ° C. for 18 hours. Thereafter, the membrane was taken out and washed with a solution containing 2 × SSC, 1 × SSC, 0.5 × SSC and 0.1 × SSC at 42 ° C. for 1 to 2 hours. After drying this membrane, an X-ray film was adhered and exposed overnight.
[0046]
As a result, positive clones hybridized with probes obtained from SPDS, SAMDC, and ADC gene fragments could be selected.
[0047]
A plasmid clone having a cDNA insert was prepared from each positive clone phage DNA by the in vivo excision method. The in vivo excision method followed the method of ZAP-cDNA Synthesis Kit (Stratagene).
[0048]
200 μl of each phage solution containing SPDS gene, SAMDC gene, ADC gene, 200 μl of Escherichia coli XL1-Blue suspension and 1 μl of helper phage R408 suspension were mixed and incubated at 37 ° C. for 15 minutes, and then 3 ml of 2 × YT medium (1.6 % Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) and shake cultured at 37 ° C. for 2 hours, treated at 70 ° C. for 20 minutes, centrifuged (4,000 × g, 10 minutes), and the supernatant was recovered. . 30 μl of the resulting supernatant and 30 μl of E. coli SURE suspension were mixed and incubated at 37 ° C. for 15 minutes, then several μl on LB (1% Bacto Tripton, 0.5% Yeast Extract, 1% NaCl) agar medium containing 50 ppm of ampicillin Inoculated and cultured overnight at 37 ° C. The colonies that formed colonies contained plasmid clones with cDNA inserts. The nucleotide sequences of the inserted sequences of these plasmids were determined by the dideoxy method (Messing, Methods in Enzymol., 101, 20-78, 1983). As a result, the plasmid was found to contain the initiation codon.
[0049]
The spermidine synthase gene derived from the full-length black poppy pumpkin was FSPD1 (SEQ ID NO: 1, 2), S-adenosylmethionine decarboxylase gene was FSAM24 (SEQ ID NO: 3, 4), and arginine decarboxylase gene was It was named FADC76 (SEQ ID NOs: 5 and 6).
[0050]
Amino acid comparison was performed with the obtained FSPD1 and known plant-derived spermidine synthase genes (Table 1). From the results of Table 1, FSPD1 derived from black root pumpkin root showed high homology at the amino acid level with SPDS genes derived from other plants.
[0051]
[Table 1]

[0052]
Amino acid comparison was performed with the obtained FSAM24 and a known plant-derived S-adenosylmethionine decarboxylase gene (Table 2). From the results shown in Table 2, FSAM24 derived from black root pumpkin roots showed high homology at the amino acid level with SAMDC genes derived from other plants.
[0053]
[Table 2]

[0054]
Amino acid comparison was performed with the obtained FADC76 and known plant-derived arginine decarboxylase genes (Table 3). From the results shown in Table 3, FADC76 derived from the black root pumpkin root showed high homology at the amino acid level with ADC genes derived from other plants.
[0055]
[Table 3]

[0056]
Example 2: Production of transgenic sweet potato
(1) Production of expression construct
The polyamine metabolism-related gene FSPD1 shown in SEQ ID NO: 1 was cleaved with XhoI so as to contain the entire open reading frame from the base sequence of FSPD1, and purified by the glass milk method. Next, pGEM-7Zf (Promega) was cut with XhoI, and the FSPD1 fragment was subcloned in the sense and antisense directions, respectively. The FSPD1 fragment was excised again with restriction enzymes XbaI and KpnI at the multicloning site of pGEM-7Zf and subcloned into the binary vector pBI101-Hm2 to which the 35S promoter was linked. These plasmids were named pBI35S-FSPD1 +/−. The structure of the expression construct is shown in FIG. The transformed E. coli JM109 was named Escherichia coli JM109 / pBI35S-FSPD1 +/−.
The polyamine metabolism-related gene FSAM24 shown in SEQ ID NO: 3 was cleaved with NotI so as to include all open reading frames from the base sequence, and each was blunt-ended. These fragments were subcloned in the sense and antisense directions into a binary vector pBI101-Hm2 to which a 35S promoter with blunt ends was linked. These plasmids were named pBI35S-FSAM24 +/−. The structure of the expression construct (sense direction only) is shown in FIG. The transformed E. coli JM109 was named Escherichia coli JM109 / pBI35S-FSAM24 +/−.
The polyamine metabolism-related gene FADC76 shown in SEQ ID NO: 5 was cleaved with NotI so as to include all open reading frames from the base sequence, and each was blunt-ended. These fragments were subcloned in the sense and antisense directions into a binary vector pBI101-Hm2 to which a 35S promoter with blunt ends was linked. These plasmids were named pBI35S-FADC76 +/−. The structure of the expression construct (sense direction only) is shown in FIG. The transformed E. coli JM109 was named Escherichia coli JM109 / pBI35S-FADC76 +/−.
(2) Introduction of plasmid into Agrobacterium
Escherichia coli pBI35S-FSPD1 +/-, Escherichia coli pBI35S-FSAM24 +/-, Escherichia coli pBI35S-FADC76 +/- obtained in (1), and E. coli HB101 strain having the helper plasmid pRK2013 were each 37% in LB medium containing 50 mg / l kanamycin. The Agrobacterium EHA101 strain was cultured at 37 ° C. overnight at 37 ° C. in LB medium containing 50 mg / l kanamycin. 1.5 ml of each culture solution was collected in an Eppendorf tube and then washed with LB medium. These cells are suspended in 1 ml of LB medium, 100 μl of 3 types of bacteria are mixed, spread on LB medium agar, cultured at 28 ° C., and plasmid is transferred to Agrobacterium. ) One to two days later, a portion was scraped with a platinum loop and coated on an LB agar medium containing 50 mg / l kanamycin, 20 mg / l hygromycin, and 25 mg / l chloramphenicol. After culturing at 28 ° C. for 2 days, a single colony was selected. The obtained transformants were named EHA101 / pBI35S-FSPD1 +/−, EHA101 / pBI35S-FSAM24 +/−, and EHA101 / pBI35S-FADC76 +/−.
(3) Production of sweet potato callus for transformation
Sweet potato line 14 (provided by Agricultural Resource Research Institute, Ishikawa Prefectural Agricultural College, hereinafter referred to as “high line 14” or “wild strain”) is cultivated in a potted plant under normal cultivation management, and includes about 5 cm including the stem Dozens of stem tips were collected. This was immersed in 150 ml of 70% ethanol in a sterilized 300 ml beaker for 2 minutes, and then added to 150 ml of sterilized liquid (5% sodium hypochlorite, 0.02% Triton X-100) in a similarly sterilized beaker. Sterilized by immersion for 2 minutes. The sterilized stem tip was washed three times with sterilized water in a sterile beaker. After washing, a tissue of about 0.5 mm including the shoot apical meristem was aseptically removed under a stereomicroscope. This was used as an embryonic callus induction medium [4F1 plate: LS medium (1.9 g / l KNO_Three1.65 g / l NH_FourNO_Three0.32 g / l MgSO_Four・ 7H₂O, 0.44 g / l CaCl₂・ 2H₂O, 0.17 g / l KH₂PO_Four, 22.3 mg / l MnSO_Four・ 4H₂O, 8.6 mg / l ZnSO_Four・ 7H₂O, 0.025 mg / l CuSO_Four・ 5H₂O, 0.025 mg / l CoCl₂・ 6H₂O, 0.83 mg KI, 6.2 mg H_ThreeBO_Three27.8 mg FeSO_Four・ 7H₂O, 37.3 mg / l Na₂EDTA, 100 mg / l myo-inositol, 0.4 mg / l thiamine hydrochloride), 1 mg / l 4-fluorophenoacid acid (4FA), 30 g / l sucrose, 3.2 g / l gellan gum, pH 5.8] Then, it was cultured in a plant incubator (Sanyo Co., Ltd., MLR-350HT) at 26 ° C. under dark conditions. About one month later, embryonic callus having the ability to regenerate into a plant body was selected from the grown tissue. The selected embryonic callus was then transplanted to a new 4F1 plate every month and allowed to grow.
(4) Agrobacterium infection
Transformed Agrobacterium strains EHA101 / pBI35S-FSPD1 +/−, EHA101 / pBI35S-FSAM24 +/−, EHA101 / pBI35S-FADC76 +/− in LB agar medium containing 50 mg / l kanamycin and 50 mg / l hygromycin at 27 ° C., After culturing for 2 nights, the cells were scraped about 2 grains of rice and suspended in 50 ml of infection medium (LS medium, 20 mg / l acetosyringone, 1 mg / l 4FA, 30 g / l sucrose, pH 5.8), 100 rpm, The mixture was shaken at 26 ° C. under dark conditions for 1 hour. This cell suspension was transferred to a 300 ml sterilized beaker containing a sterilized stainless steel net basket. Embryo callus cultured for 2 to 3 weeks after transplantation in (3) above is placed in this beaker basket and soaked for 2 minutes. Then, the entire basket is placed on two sterilized filter papers to remove excess water and coexist. Transfer to culture medium (4F1A20 plate: LS medium, 1 mg / l 4FA, 20 mg / l acetosyringone, 30 g / l sucrose, 3.2 g / l gellan gum, pH 5.8) at 22 ° C. under dark conditions Co-cultured for 3 days.
(5) Disinfection
The embryonic callus co-cultured for 3 days in (4) above was sterilized using a 300 ml beaker containing a sterilized stainless steel basket containing 50 ml of a sterilization solution (sterile water added to a final concentration of 500 mg / l carbenicillin). Moved to basket, picked up basket with tweezers and washed well for several minutes. Next, the embryonic callus was transferred to a 300 ml sterilized beaker containing a sterilization solution together with the basket and washed again. After the same operation was repeated again, excess water was removed on sterile filter paper, and the selection medium (4F1HmCar plate: LS medium, 1 mg / l 4FA, 25 mg / l hygromycin, 500 mg / l carbenicillin, 30 g / l sucrose, 3 .2 g / l gellan gum, pH 5.8) and cultured at 26 ° C. under dark conditions.
(6) Selection of transformed callus
The embryonic callus cultured for 2 weeks in the above (5) was transplanted to a new 4F1HmCar plate every 2 weeks and cultured. Non-transformed callus turned brown, but some transformed cells formed pale yellow embryo callus.
(7) Regeneration of transformed plants
The transformed embryonic callus was 60 days after placement on the selective medium, somatic embryogenesis medium (A4G1HmCar plate: LS medium, 4 mg / l ABA, 1 mg / l GA3, 25 mg / l hygromycin, 500 mg / l carbenicillin, Transplanted into 30 g / l sucrose, 3.2 g / l gellan gum, pH 5.8) at 26 ° C., faint light (30-40 μmol / m)²/ S), cultured for 2 weeks under full day length conditions, plant body formation medium (A0.05 HmCar plate: LS medium, 0.05 mg / l ABA, 25 mg / l hygromycin, 500 mg / l carbenicillin, 30 g / l Sucrose, 3.2 g / l gellan gum, pH 5.8) and cultured under the same conditions. Thereafter, it was transplanted to a new A0.05HmCar plate every two weeks. Since the transformed cells form somatic embryos derived from embryonic callus showing a green color, this is used as a plant growth medium (0G plate: LS medium, 30 g / l sucrose, 3.2 g / l gellan gum, pH 5.8). Transplanted to form shoots.
(8) DNA extraction and Southern hybridization
Leaves were cut from the transformants obtained above and immediately frozen in liquid nitrogen. This was finely ground in a mortar in the presence of liquid nitrogen, and 3 ml of DNA extraction buffer [200 mM Tris-HCl (pH 8.0), 100 mM EDTA-2Na, 1% N-lauroyl sarcosine sodium, 100 μg / ml ProteinaseK per gram] ] Was sufficiently stirred. After incubation at 60 ° C. for 1 hour, the mixture was centrifuged (10,000 × g, 10 minutes), and the supernatant was vortexed with Miracloth and transferred to a new tube. After extraction with phenol: chloroform: isoamyl alcohol (25: 24: 1) three times, ethanol precipitation was performed. The precipitate was dissolved in TE buffer. From about 2.0 g of each plant, 20 μg of genomic DNA was obtained. Of these, 1 μg of DNA was used, and each was cleaved with restriction enzymes EcoRI and HindIII and subjected to 1% agarose electrophoresis and Southern hybridization.
[0057]
Untransformed wild strains were also grown under the same conditions, DNA was extracted in the same manner, digested with restriction enzymes EcoRI and HindIII, and subjected to 1% agarose gel electrophoresis and Southern hybridization. As the probe for hybridization, each FSPD1, FSAM24, and FADC76 gene fragment was used.
[0058]
Southern hybridization was performed according to the method described in Molecular Cloning, A Laboratory Manual, Chapter 9, pages 31-58 (Cold Spring Harbor, 1989). . That is, each DNA sample was subjected to 1% agarose gel electrophoresis, and after electrophoresis, subjected to alkali denaturation, and Southern blotted overnight on a nylon membrane (High Bond-N, manufactured by Amersham). An ultraviolet transilluminator (254 nm) was irradiated for 3 minutes to immobilize DNA. This membrane was prehybridized in 5 ml of a prehybridization buffer (5 × Denhardt's solution, 6 × SSC, 0.1% SDS, 10 μg / ml salmon sperm DNA) at 50 ° C. for 2 hours. Probes were added and hybridization was performed overnight at 50 ° C. After hybridization, the membrane was washed twice with a washing solution containing 2 × SSC and 0.1% SDS for 10 minutes at room temperature, and then washed twice with the same washing solution at 50 ° C. for 30 minutes. After the membrane was dried, it was exposed to −80 ° C. overnight in a cassette containing an X-ray film (manufactured by Kodak Co.), and autoradiography was performed. For the untransformed strain (1), the transformant introduced with FSPD1, FSAM24 and FADC76 (2), and the transformant introduced with only the vector (3), the signal pattern detected by Southern hybridization Compared.
In (2), in addition to (1), (2), and (3) a common endogenous signal, a specific signal was observed in the sample cleaved with EcoRI and the sample cleaved with HindIII. Incorporation into (2) was observed.
Example 3: Northern blot analysis
In order to confirm whether the target gene was actually expressed in the transformant obtained in Example 2, Northern blotting was performed as shown below.
[0059]
Total RNA was extracted from young leaves of wild strains (WT) and transformants (TSP) that were not transformed. RNA extraction was performed according to a standard method. 10 μg of the total RNA obtained was electrophoresed on a 1.5% formaldehyde agarose gel and then blotted overnight on a high bond N nylon membrane. After immobilizing RNA with UV crosslinker, prehybridization with prehybridization buffer (50% Formamide, 5X SSPE, 5X Denhardt's, 0.1% SDS, 80μg / ml Salmon sperm DNA, pH7.0) at 42 ℃ for 2 hours Went. The transformed black pumpkin SPDS gene fragment, SAMDC gene fragment, and ADC gene fragment cDNA³²A probe was prepared using P-dCTP and a random label kit (Amersham). This probe was added to prehybridization, and hybridization was performed overnight at 42 ° C. After hybridization, the membrane was started with a washing solution containing 2 × SSC and 0.1% SDS, and finally washed twice with a washing solution containing 0.1 × SSC and 0.1% SDS at 55 ° C. for 30 minutes. The membrane was subjected to autoradiography using an X-ray film (manufactured by Kodak). As a result, in the wild strain (WT), expression of exogenous black pumpkin SPDS gene, SAMDC gene, and ADC gene was not detected, but all the transformants transformed had a very high level of black pumpkin pumpkin. It was confirmed that the SPDS gene (FSPD1), SAMDC gene (FSAM24), and ADC gene (FADC76) were expressed.
Example 4: Evaluation of polyamine content
(1) Selection of system (cell line)
About the transformant produced in Example 2, the cell line was selected by confirming introduction of the target gene by PCR (or Southern analysis) and Northern analysis. As a result, polyamine analysis was performed on strains in which the polyamine metabolism-related enzyme gene was reliably introduced and the gene was stably expressed. Cell lines in which FSPD1 is introduced in the sense direction (+), TSP-SS-1, TSP-SS-2, TSP-SS-3, TSP-CS-1, TSP-CS-3, TSP-CS-4 TSP-SS-1, TSP-SS-2, and TSP-SS-3 are strains in which a CaMV 35S promoter is introduced as a promoter, TSP-CS-1, TSP-CS-3, TSP-CS- 4 is a strain into which a horseradish peroxidase promoter (C2 promoter) has been introduced. A cell line in which FSPD1 is introduced in the antisense direction (-), TSP-SA-1, and TSP-SA-2 are selected, and all are strains in which the CaMV 35S promoter is introduced. Cell lines, TSP-SM-1, TSP-SM-2, TSP-SM-5, in which FSAM 24 is introduced in the sense direction (+), are selected, and TSP-SM-1, TSP-SM-2, TSP- SM-5 is a strain in which a CaMV 35S promoter is introduced as a promoter.
(2) Analysis of polyamine content
About 0.3 to 0.9 g of young leaves were sampled from the wild strain (WT) and the transformant (TSP) that were simultaneously cultivated and stored frozen. Diluted internal standard solution (1,6-hexaneamine, internal standard amount = 7.5 nmol) and 5% aqueous perchloric acid solution (5-10 ml per 1.0 g of sample weight) are added to the sampled sample, and an omni mixer is used. Fully ground and extracted at room temperature. The milled solution was centrifuged at 4 ° C., 35,000 × g for 20 minutes, and the supernatant was collected to obtain a free polyamine solution. To a microtube with a screw cap, 400 μl of a free polyamine solution, 200 μl of a saturated sodium carbonate aqueous solution and 200 μl of dansyl chloride / acetone solution (10 mg / ml) were added and mixed gently. After tightly closing the tube stopper, dansylation was carried out by heating for 1 hour in a 60 ° C. water bath, which is often made of aluminum foil. After allowing the tube to cool, 200 μl of an aqueous proline solution (100 mg / ml) was added and mixed. It was covered with aluminum foil and reheated in a water bath for 30 minutes. After allowing to cool, nitrogen gas was blown to remove acetone, and then 600 μl of toluene was added and mixed vigorously. After the tube was allowed to stand and separated into two phases, the upper toluene layer was separated into 300 μl microtubes. Toluene was completely removed by blowing nitrogen gas to the collected toluene. 200 μl of methanol was added to the tube to dissolve the dansylated free polyamine. The amount of free polyamines of putrescine, spermidine and spermine was analyzed by an internal standard method using high performance liquid chromatography connected with a fluorescence detector. As the HPLC column, μ Bondapak C18 (manufactured by Waters: 0273324, 3.9 × 300 mm, particle diameter 10 μm) was used. The polyamine content in the sample was calculated by obtaining the peak areas of each polyamine and internal standard from the standard solution and the HPLC chart of the sample, respectively. The results are shown in Table 4.
[0060]
[Table 4]

[0061]
As is apparent from Table 4, the cell line in which the polyamine metabolism-related enzyme gene was introduced in the sense direction showed significantly higher putrescine content, spermidine content, and spermine content than the wild strain (WT), and the total polyamine content was also increased in the wild strain (WT). It became clear that it increased significantly from (WT). In particular, the increase in spermidine and spermine content was remarkable. Furthermore, cell lines in which polyamine metabolism-related enzyme genes have been introduced in the antisense direction have significantly reduced spermidine content and spermine content compared to the wild strain (WT), and total polyamine content also significantly decreased compared to the wild strain (WT). It became clear that.
[0062]
From the above results, it was revealed that the polyamine content in the plant can be increased or decreased by introducing the polyamine metabolism-related enzyme gene into the plant in the sense direction or the antisense direction. These results indicate that polyamine metabolism can be controlled by introducing polyamine metabolism-related enzyme genes into plants to control polyamine metabolism.
Example 5: Productivity evaluation
(1) Evaluation of stem (vine), leaf, root (tuberous root)
The seedlings of the transformant (TSP) obtained in Example 2 and the wild strain (WT: High Line No. 14) were planted in a planter packed with culture soil (Sunsun floor soil: manufactured by Takii Seed Co., Ltd.) for 7 days. It was allowed to survive under conditions of 23 ° C., long day conditions (16 hours day length, 8 hours dark), and humid conditions. Then, after acclimatizing and growing for about one month at 23 ° C. under low light long day conditions, the sample was transferred to a natural light type growth chamber (Kitotron SBH-3030, manufactured by Koito Kogyo Co., Ltd.), and temperature (day / night: 26 ° C./24 Normal cultivation was started under the conditions of (° C.), humidity (50 to 60%), and light. One month after normal cultivation, vines (stems) and leaves were investigated. The results are shown in FIG. From the results of FIG. 2, it was revealed that the transformant (TSP) has a longer stem (vine) and an increased number of leaves compared to the wild type (WT). Furthermore, when the vines were examined in detail, the transformant (TSP) had shorter internodes than the wild strain (WT), and the number of petiole (leaves) in the wild strain (WT) was 5 (5). On the other hand, the number of petiole (leaf) of the transformant (TSP) was 17 (17), which was remarkably increased. Furthermore, tuber root (potato) was investigated 3 months after normal cultivation. The results are shown in FIGS. 3A and 3B. When the number of tuberous roots per strain of the transformant (TSP) and the wild strain (WT) was compared, the transformant (TSP) had more potatoes. Furthermore, when the yield of potato was examined, the fresh weight of potatoes per strain was 325.99 g and 200.18 g, whereas the fresh weight of wild strain (WT) was 79. 0.08 g and 101.36 g, and it was revealed that the yield of potato was significantly increased in the transformant (TSP).
(2) Evaluation of root (tuberous root)
Transformant (TSP-SS-1, TSP-CS-3) obtained in Example 2 and wild-type (WT: high system No. 14) seedlings and cutting ears (one-node cutting method) are cultured in soil (Sun-Sunbed) Soil: Inserted into a planter packed with Takii Seed & Seed Co., Ltd. and rooted for 7 days at 23 ° C., long day conditions (16 hours day length, 8 hours dark), and humid conditions. Then, after acclimatizing and growing for about one month at 23 ° C. under low light long day conditions, the sample was transferred to a natural light type growth chamber (Kitotron SBH-3030, manufactured by Koito Kogyo Co., Ltd.), and temperature (day / night: 26 ° C./24 Normal cultivation was started under the conditions of (° C.), humidity (50 to 60%), and light. Two months after normal cultivation, the tuberous root (potato) hypertrophy formation was investigated. The results are shown in FIG. From the results of FIG. 4, the wild strain (WT) did not show tuberous root hypertrophy formation in all strains, but the transformants (TSP-SS-1, TSP-CS-3) showed tuber root hypertrophy formation in all strains. Was observed.
From the above results, it was proved that by introducing a polyamine metabolism-related enzyme gene into sweet potato, the productivity of leaves, stems (vines), roots (tuberous roots) and the like increased, and the productivity of plants could be improved.
[0063]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the structure of an expression construct containing a polyamine metabolism-related enzyme gene.
FIG. 2 is a photograph showing a comparison of stems, leaves, and vines between a sweet potato introduced with a polyamine metabolism-related enzyme gene and a wild strain.
FIG. 3A is a photograph showing a comparison of roots (tuberous root, potato) between a sweet potato and a wild strain into which a polyamine metabolism-related enzyme gene has been introduced.
FIG. 3B is a photograph showing a comparison of roots (tuberous root, potato) between a sweet potato and a wild strain into which a polyamine metabolism-related enzyme gene has been introduced.
FIG. 4 is a photograph showing a comparison of hypertrophy formation of roots (tuberous root, potato) between a sweet potato introduced with a polyamine metabolism-related enzyme gene and a wild strain.

Claims (9)

Transforming a plant cell not having the nucleic acid sequence with an expression vector comprising a nucleic acid sequence encoding spermidine synthase (SPDS) under the control of a promoter capable of functioning in the plant. A method for producing a plant having an increased number of tuberous roots irrespective of the cultivation environment compared to a control plant having no sequence. Further comprising The method of claim 1, wherein the step of regenerating plants from the transformed cell. The method according to claim 1, further comprising the step of cultivating the plant body and evaluating the number of tuberous roots. The nucleic acid sequence is a nucleic acid sequence of spermidine synthase gene having the base of the following (a) or (b) or (c), The method of claim 1, wherein.
(A) a base sequence represented by base numbers 77 to 1060 in the base sequence represented by SEQ ID NO: 1,
(B) a base sequence that encodes a protein that hybridizes with the base sequence of (a) above under stringent conditions and has spermidine synthase activity;
(C) A base sequence encoding a protein consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence of (a) or (b) and having spermidine synthase activity. The method according to claim 1 , wherein the number of tuberous roots is significantly increased in reproductive growth period rather than vegetative growth period. The following steps:
(1) An expression vector containing a nucleic acid sequence encoding spermidine synthase (SPDS) under the control of a promoter capable of functioning in a plant, transforming a plant cell not having the nucleic acid sequence,
(2) regenerating a plant having an increased number of tuberous roots from the transformed cell compared to a plant not having the nucleic acid sequence;
(3) collecting seeds from the plant by pollination; and (4) examining the nucleic acid sequence in the seed obtained by pollination from the plant obtained by cultivating the seed to obtain a homozygote of the nucleic acid sequence. Selection of a plant with a fixed trait that is homozygous for the nucleic acid sequence and that has an increased number of tuberous roots regardless of the cultivation environment compared to a control plant that does not have the nucleic acid sequence. Production method. The method according to any one of claims 1 to 6 , wherein the plant having improved productivity or trait is a sweet potato plant. A method for producing a sweet potato having an increased number of tuberous roots as compared with a comparative control sweet potato, wherein the sweet potato plant obtained by the method according to claim 7 is cultivated and the sweet potato is harvested. Cultivating a sweet potato plant obtained by the method according to claim 8 and harvesting the sweet potato,
The manufacturing method of the sweet potato starch including the process of isolate | separating a sweet potato starch from the obtained sweet potato.

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