JP3755146B2 - Production method of transgenic plants with improved amino acid composition - Google Patents

Description

ａ．ＮＡＤＰ−ＧＤＨ遺伝子特異的プライマー（Ａｌａｓｔａｉｒら、Ｍｏｌ．Ｇｅｎ．Ｇｅｎｅｔ，１９８９，２１８；１０５−１１１，ＰＣＲ産物、約１．４ｋｂｐ）
ｂ．ｌｅｇｄｈ１特異的プライマー（Ｐｕｒｎｅｌｌら、１９９７，Ｇｅｎｅ １８６；２４９−２５４，ＰＣＲ産物、約１．２ｋｂｐ）
＜配列表フリーテキスト＞
配列番号３および４：ＮＡＤＰ−ＧＤＨ特異的ＰＣＲプライマー
配列番号５および６：ｌｅｇｄｈ１特異的ＰＣＲプライマー
【００３５】
（２）ＡＮ−ｇｄｈ−１７遺伝子のＴｉプラスミッド（ｐＭＡＴ０３７）へのサブクローニング
ＰＣＲ２．１ベクターにクローニングしたＡＮ−ｇｄｈ−１７遺伝子を、植物形質転換用ベクターである、Ｔｉプラスミッド（ｐＭＡＴ０３７）（Ｍａｔｓｕｏｋａおよび Ｎａｋａｍｕｒａ，１９９１，Ｐｒｏｃ Ｎａｔｌ．Ａｃａｄ Ｓｃｉ ＵＳＡ ８８：８３４−８３８）にサブクローニングした。直接挿入するするのに適切な制限酵素サイトがｐＭＡＴ０３７に無いため、一度ｐＵＣ１８につなぎ（ＸｂａＩ，ＥｃｏＲＩサイト使用）、Ｅ．ｃｏｌｉ ＪＭ１０９を形質転換した。さらにｐＵＣ１８中のＰｓｔＩサイトとＥｃｏＲＩサイトを用いてＴｉプラスミッドへ連結しプラスミッドｐＡＮ−ｇｄｈ−１７を得（図３、表２）Ｅ．ｃｏｌｉ ＤＨ５αにトランスフォーメーションした。ＡＮ−ｇｄｈ−１７を導入したＴｉプラスミッドｐＡＮ−ｇｄｈ−１７（図３）でグロバクテリウム株ＥＨＡ１０１を形質転換し、得られたアグロバクテリウムをトマトに感染させるために用いた。
【００３６】
（３）ｐＣｔ−ＡＮ−ｇｄｈ、ｐＣｔ−ｄＡＮ−ｇｄｈ、ｐＭｔ−ｄＡＮ−ｇｄｈの構築
Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｒａｎｓ由来ＮＡＤＰ−ＧＤＨ遺伝子には本来、スプライシングサイトが２箇所存在する。ｃＤＮＡを用いてＰＣＲ法によりＧＤＨ遺伝子の増幅を行ったので、これらのスプライシングサイトは除かれ、存在しないはずであるが、本実験で取得したＡＮ−ｇｄｈ−１７遺伝子には、約５０ｂｐのスプライシングサイトが１箇所残ったままであった（図１、塩基配列中に記載）。そこでＰＣＲ法を用いて、残ったままであった約５０ｂｐの塩基配列を取り除いた（表２、図４、５）
【００３７】
これらの遺伝子構築物の構築手順の概略は図５に示した通りである。すなわち、最初に、クローニングされた上記遺伝子配列の５’端を含むプライマーＰ１およびスプライシング領域の５’側と３’側を含むがスプライシング領域を含まないプライマーＰ２、およびスプライシング領域の５’側と３’側を含むがスプライシング領域を含まないプライマーＰ３とＡＮ−ｇｄｈ−１７遺伝子の３’端を含むプライマーＰ４を用いて、それぞれＡＮ−ｇｄｈ−１７遺伝子を鋳型としてＰＣＲによってＤＮＡ断片を増幅した。次に、電気泳動によりＰＣＲ産物のサイズの確認およびゲルからの再抽出を行った。それぞれの再抽出ＰＣＲ産物を混合し、プライマーＰ１およびＰ４を用いて再度ＰＣＲ反応を行った。得られたＰＣＲ産物をクローニングし、シークエンシングを行い、スプライシング領域が正しく除去されていることを確認した。
【００３８】
上述のＰ１〜Ｐ４の配列を以下に示す。

これらの操作により、スプライシングサイトを除去したｄＡＮ−ｇｄｈ−１７遺伝子を得た（表２、図４、５）。
更に導入遺伝子を適所でより機能的に働かせるために、ミトコンドリアまたはクロロプラストへのトランジットペプチド配列をＡＮ−ｇｄｈ−１７遺伝子又はｄＡＮ−ｇｄｈ−１７遺伝子の開始コドンの上流側に連結させた（表２、図４）。用いたペプチド配列はミトコンドリアへのトランジットペプチド配列としてトマト由来ＧＤＨ遺伝子に付加している約７０ｂｐからなる塩基配列を、クロロプラストへのトランジットペプチド配列として、トマト由来ＲｕＢｉｓＣＯの小サブユニット遺伝子に付加している約１２０ｂｐからなる塩基配列を用いた。また、これらの遺伝子とＡＮ−ｇｄｈ−１７遺伝子又はｄＡＮ−ｇｄｈ−１７遺伝子の連結はＰＣＲ法を用いて行った。ミトコンドリアへのトランジットペプチド配列は、２種のプライマー（５’−ＧＧＡＴＣＣＡＴＧＡＡＴＧＣＴＴＴＡＧＣＡＧＣＡＡＣ−３’：配列番号１１、５’−ＴＣＴＡＧＡＴＡＡＡＣＣＡＡＧＡＡＧＣＣＴＡＧＣＴＧ−３’：配列番号１２）を使用しＰＣＲによって得た。クロロプラストへのトランジットペプチド配列は、２種のプライマー（５’−ＣＴＧＣＡＧＡＴＧＧＣＴＴＣＣＴＣＡＡＴＴＧＴＣＴＣＡＴＣＧ−３’：配列番号１３、５’−ＴＣＴＡＧＡＧＣＡＴＣＴＡＡＣＧＣＧＴＣＣＡＣＣＡＴＴＧＣＴ−３’：配列番号１４）を使用しＰＣＲによって得た。
【００３９】
これらのトランジットペプチド配列のＡＮ−ｇｄｈ−１７遺伝子又はｄＡＮ−ｇｄｈ−１７遺伝子への結合は図６に示したように行った。すなわち、トランジットペプチドの５’側に対応するプライマーＰ５とトランジットペプチドの３’端およびＡＮ−ｇｄｈ−１７遺伝子又はｄＡＮ−ｇｄｈ−１７遺伝子の５’端の配列を含むプライマーＰ６、およびトランジットペプチドの３’端およびＡＮ−ｇｄｈ−１７遺伝子またはｄＡＮ−ｇｄｈ−１７遺伝子の５’端の配列を含むプライマーＰ７およびＡＮ−ｇｄｈ−１７遺伝子またはｄＡＮ−ｇｄｈ−１７遺伝子の３’端配列を含むプライマーＰ８を用いてそれぞれのＤＮＡ断片を増幅後、電気泳動によりＰＣＲ産物のサイズの確認およびゲルからの抽出を行った。抽出したそれぞれの断片を混合し、プライマーＰ５とＰ８を用いて再度ＰＣＲを行った。増幅された断片をクローニングしシーケンシングを行い、トランジットペプチドの塩基配列がＡＮ−ｇｄｈ−１７遺伝子またはｄＡＮ−ｇｄｈ−１７遺伝子に正しく付加されていることを確認した（図６）。
【００４０】
ここで、Ｐ５からＰ８の配列は以下に対応する。
ミトコンドリアへのトランジットペプチド配列との連結の際に用いたプライマーの場合は

であり、
クロロプラストへのトランジットペプチド配列との連結の際用いたプライマーの場合は

である。
【００４１】
ここで、ＡＮ−ｇｄｈ−１７遺伝子はＴｉプラスミッドｐＭＡＴ０３７のマルチクローニングサイトにセンス方向に導入したが（図２）、ｐＣｔ−ＡＮ−ｇｄｈ、ｐＣｔ−ｄＡＮ−ｇｄｈ、ｐＭｔ−ｄＡＮ−ｇｄｈの構築に際してはＣａＭＶ３５Ｓプロモーターを用いた場合と後述する果実特異的プロモーター遺伝子（２Ａ１１）を用いた場合との導入遺伝子の効果を比較するためＴｉプラスミッドｐＩＧ１２１−Ｈｍを用いてクローニングを行い、遺伝子導入を行った（表２、図４）。
＜配列表フリーテキスト＞
配列番号７〜１０：スプライシング領域除去のためのＰＣＲプライマー
配列番号１１および１２：ミトコンドリアへのトランジットペプチドをコードする配列増幅用のＰＣＲプライマー
配列番号１３および１４：クロロプラストへのトランジットペプチドをコードする配列増幅用のＰＣＲプライマー
配列番号１５〜１８：ミトコンドリアトランジットペプチド−ＧＤＨをコードする配列の作製のためのＰＣＲプライマー
配列番号１９〜２２：クロロプラストトランジットペプチド−ＧＤＨをコードする配列の作製のためのＰＣＲプライマー
【００４２】
（４）果実特異的プロモーター（２Ａ１１プロモーター）を用いた遺伝子構築
果実特異的発現プロモーター（２Ａ１１）はトマトの幼植物より調製したＴｏｔａｌ ＤＮＡをテンプレートに用いて、ＰＣＲ法により取得した。用いたプライマー（配列番号２３および配列番号２４）にはＴｉプラスミッドに導入する際に用いる制限酵素サイト、ＨｉｎｄＩＩＩおよびＸｂａＩの配列を各々のプライマーに設計した。
プライマーの配列を以下に示す：

得られたＰＣＲ産物はＴＡクローニングキットを用いてクローニングした後、シークエンス分析によって塩基配列の確認を行った。得られた２Ａ１１プロモーターは上記、制限酵素ＨｉｎｄＩＩＩおよびＸｂａＩを用いてＴｉプラスミッドｐＩＧ１２１−ＨｍのＧＵＳ遺伝子前のＣａＭＶ３５Ｓプロモーターと置換した。その後、ＧＵＳ部分とＣｔ−ｄＡＮ−ｇｄｈ遺伝子またはＭｔ−ｄＡＮ−ｇｄｈ遺伝子との置換を行った。Ｃｔ−ｄＡＮ−ｇｄｈ遺伝子またはＭｔ−ｄＡＮ−ｇｄｈ遺伝子の置換手順についてはＣａＭＶ ３５Ｓプロモーターを用いた場合と同様である。これにより、プラスミッドｐ２ＡＣｔ−ｄＡＮ−ｇｄｈおよびＰ２ＡＭｔ−ｄＡＮ−ｇｄｈを得た（図７）。
＜配列表フリーテキスト＞
配列番号２３および２４：２Ａ１１プロモーター配列増幅用ＰＣＲプライマー
上述した手順により作製したプラスミッドの重要な構造を以下の表２にまとめた。
【００４３】
【表２】

【００４４】
（５）Ｔ−ｇｄｈ−４遺伝子のＴｉプラスミッド（ｐＩＧ１２１−Ｈｍ）へのサブクローニング
クローニングしたトマト由来ＧＤＨ遺伝子（Ｔ−ｇｄｈ−４）をＴｉプラスミッド（ｐＩＧ１２１−Ｈｍ）に導入しプラスミッドｐＴ−ｇｄｈ−４を得た。導入に際しては、単離の際に使用したプライマーに予め設けておいたＸｂａＩサイトおよびＳａｃＩサイトを用いた。Ｔ−ｇｄｈ−４遺伝子を導入したＴｉプラスミッド（図８）は、アグロバクテリウム株ＥＨＡ１０１に形質転換した。
【００４５】
（６）変異型トマトＧＤＨ遺伝子のＴｉプラスミッド（ｐＩＧ１２１−Ｈｍ）へのサブクローニング
トマト由来ＮＡＤ−ＧＤＨにおいて、グルタミン酸結合部位の９０番目のアミノ酸リジンをアラニンに置換し、その効果について調査した（図９）。これは、高等植物のＧＤＨ遺伝子がアンモニアイオン濃度や栄養条件によって、グルタミン酸を減少させる方向に働く可能性が有るため、結合できなくすることを目的とした改変である。１アミノ酸の置換はＰＣＲを利用した部位特異的変異導入により、ＬｙｓをコードするＡＡＧをＡｌａをコードするＧＣＧに変換することによって行った。得られた改変遺伝子をＴｄ−ｇｄｈと命名した。次に、Ｔｄ−ｇｄｈ遺伝子配列をＴｉプラスミッドへ導入してプラスミッドｐＴｄ−ｇｄｈを得た（図９）。この手順はＴ−ｄｇｈ−４遺伝子の場合と同様である。
【００４６】
実施例２．トマトの子葉片へのアグロバクテリウム感染による形質転換体の作製
トマト（栽培品種、ミニトマト）の種子を７０％エタノール（３０秒）、２％次亜塩素酸ナトリウム（１５分）を用いて表面殺菌した後、植物ホルモンを含まないＭＳ寒天培地に置床し、１６時間日長、２５℃で１週間培養した。得られた無菌幼植物より子葉を切り取り、２ｍｇ／ｌゼアチンと０．１ｍｇ／ｌインドール酢酸を加えたＭＳ寒天培地（再分化培地、９ｃｍシャーレ使用）に置床し２日間同条件で培養した。構築した遺伝子を含むアグロバクテリウム（ＥＨＡ１０１）はＹＥＰ培地（表−３）で一晩培養したものを感染に用いた。２日間培養した子葉を滅菌シャーレに集めアグロバクテリウム液を加え感染させた。滅菌したろ紙を用いて余分なアグロバクテリウム液を子葉から取り除き、さらに、アグロバクテリウムの急激な増殖を防ぐため、先に用いたシャーレ培地に滅菌ろ紙を敷き、その上に感染させた子葉を乗せ、２４時間共存培養した。
【００４７】
その後、子葉を５０ｍｇ／ｌカナマイシン、５００ｍｇ／ｌクラフォランを含むＭＳ再分化培地（選抜培地）に移し、形質転換体の選抜を行った。再分化したシュートを新しい選抜培地に移し再選抜を行った。緑色で旺盛に生育したシュートを茎の部分で切り取り、植物ホルモンを含まないＭＳ培地（発根培地，試験管）に移した。発根した再分化植物を順次土壌に馴化させた。
【表３】

【００４８】
実施例３．減圧浸潤法によるアラビドプシスの形質転換
アラビドプシスへの遺伝子導入はＢｅｃｈｔｏｌｄ Ｎ（Ｃ．Ｒ．Ａｃａｄ．Ｓｃｉ．Ｐａｒｉｓ，Ｌｉｆｅ Ｓｃｉｅｎｃｅ ３１６；１１９４−１１９９，１９９３）の方法を改編して用いた。培養土にアラビドプシスの種子をまき１０日間、１６時間日長、２２℃栽培した幼植物をロックウール（３ｃｍ×３ｃｍ）１個に１株ずつ移植し、３週間同条件で栽培した。植物が抽だいを始めたら摘心し、さらに一週間栽培を行った。アグロバクテリウムは抗生物質を含むＹＥＰ培地で２４時間、２８℃で培養し、遠心（７０００ｒｐｍ、１０分）によって集菌し、浸潤用懸濁培地（１／２ ＭＳ塩、１／２ Ｇａｍｂｏｒｇ Ｂ５ビタミン、５％ショ糖、０．５ｇ／ｌ ＭＥＳ、０．０４４μＭ ベンジルアミノプリン、ｐＨ ５．７）に懸濁した。すでに開花・結実している花を取り除き、アグロバクテリウム懸濁液に漬け、デシケーター中に入れ、１５分間減圧（４０ｍｍＨｇ）処理を行った。処理した植物は１カ月間栽培した後種子を収穫した。採取した種子は５０ｍｇ／ｌカナマイシン、２００ｍｇ／ｌクラフォランを添加したＭＳ培地にまき、形質転換体の選抜を行った。
【００４９】
実施例４．導入遺伝子の確認
ＡＮ−ｇｄｈ−１７遺伝子を含むアグロバクテリウムを感染させて得られた選抜個体４個体、Ｔ−ｇｄｈ−４遺伝子を含むアグロバクテリウムを感染させて得られた選抜個体４個体、目的遺伝子を含まないＴｉプラスミッドのみのアグロバクテリウムを感染させて得られた植物各々３個体、およびアグロバクテリウムで処理せずに子葉より直接再分化させた植物体２個体からそれぞれＴｏｔａｌ ＤＮＡを本田らの方法に従い抽出した（ＨｏｎｄａおよびＨｉｒａｉ、１９９０、Ｊｐｎ Ｊ Ｂｒｅｅｄ ４０，３３９−３４８）。抽出したＤＮＡはＲＮＡａｓｅ処理、フェノール／クロロホルム処理、さらにＰＥＧ沈殿させ精製した。０．０１μｇ／μｌになるように希釈し、ＰＣＲ用のテンプレートとした。ＰＣＲはＮｏｓ−ＰｒｏｍｏｔｅｒからＮＰＴＩＩの領域を増幅するプライマーＰ９およびＰ１０（ＰＣＲ産物、１．０ｋｂｐ）を用いて行なった。反応条件は、９４℃−１分、５５℃−１分、７２℃−２分、３５サイクルで行った。ＰＣＲ産物は１％アガロースゲルを用いて電気泳動し、エチジウムブロマイドで染色した（図１０および１１）。
使用したプライマーは以下の通りである。

【００５０】
その結果、ＡＮ−ｇｄｈ−１７遺伝子を感染させた４系統に、Ｔ−ｇｄｈ−４遺伝子を感染させた４系統に目的サイズのバンド（１．０ｋｂ）が観察され、非形質転換体２系統には検出されなかった。以上の結果から、ＡＮ−ｇｄｈ−１７遺伝子を含むＴｉプラスミッドを用いて感染させた４系統およびＴ−ｇｄｈ−４遺伝子を含むＴｉプラスミッドを用いて感染させた４系統に各々の遺伝子が導入されていることが確認された。
＜配列表フリーテキスト＞
配列番号２５、２６：Ｎｏｓ−Ｐｒｏｍｏｔｅｒ−ＮＰＴＩＩ領域増幅用ＰＣＲプライマー
【００５１】
実施例５． 導入遺伝子の発現の確認
次に、目的の遺伝子が導入されたことが確認できた形質転換トマトにつき、導入遺伝子が発現されていることをＲＴ−ＰＣＲによって確認した。ＡＮ−ｇｄｈ−１７またはＴ−ｇｄｈ−４遺伝子を含むアグロバクテリウムを感染させ、実施例５において遺伝子導入が確認された系統、および非形質転換トマトの葉または果実から全ＲＮＡを抽出し、Ｆｉｒｓｔ−Ｓｔｒａｎｄ ｃＤＮＡを作製した。次にＦｉｒｓｔ−Ｓｔｒａｎｄ ｃＤＮＡをテンプレートとして各々の遺伝子を単離する際に用いたプライマー（配列番号３と４、及び５と６）を用いてＰＣＲを行なった。反応条件は９４℃−１分、５５℃−１分、７２℃−２分、３０サイクルとした。その結果、それぞれにおいて、導入した遺伝子が葉および果実のいずれにおいても発現していることが確認された（図１２、１３）。
【００５２】
実施例６． 遊離アミノ酸の抽出と定量
馴化させた形質転換トマトの開花後６週目の果実を収穫し、−８０℃に保存した。果実は約１／６にカットし、重さを測定した後、乳鉢に入れ液体窒素で凍らせすりつぶした。さらに３ｍｌの８０％エタノールを加え、丁寧にすりつぶした後、遠心チューブに移し、８０℃で２０分間インキュベートした。１０，０００ｒｐｍで２０分間遠心し、上澄を新しいチューブに移し、残ったペレットに２ｍｌの８０％エタノールを加え、再度乳鉢ですりつぶし、８０℃で２０分間インキュベートした。遠心後、上澄をチューブに移し、先の上澄と併せ、８０％エタノールを用いて総量を５ｍｌに調製した。良く混合した後、２０μｌ取り乾燥させた後、０．０２Ｎ塩酸に溶解した。０．４５μｍのフィルターでろ過し、分析用サンプルとした。アミノ酸分析は日立高速アミノ酸分析計（Ｌ−８８００）を用いて行った。
ＡＮ−ｇｄｈ−１７遺伝子を導入した株の開花後６週間目の果実（赤色）を用いてアミノ酸分析を行った結果を非形質転換体植物（対照植物）の分析結果とともに表４に示した。顕著なグルタミン酸含量の増加が見られた系統においては、Ｎｏ．６で１．７５倍、Ｎｏ．１５で２．５４倍、Ｎｏ．１７で２．４８倍グルタミン酸含量が増加した（図１４）。グルタミン酸以外のアミノ酸、例えば、アスパラギン、アスパラギン酸、アラニン、セリン、スレオニン、ヒスチジン等の増加も見られた。
【００５３】
【表４】

【００５４】
【表５】

【００５５】
トマト由来のＴ−ｇｄｈ−４遺伝子を導入した４株についても同じく開花後６週間目の果実を用いてアミノ酸分析を行った（表５）。顕著なグルタミン酸含量の増加が見られた系統においては、Ｎｏ．２で２．２８倍、Ｎｏ．７−２で３．５２倍、Ｎｏ．９−２で２．７４倍、Ｎｏ．１０で２．５３倍グルタミン酸含量が増加した（図１５）。グルタミン酸含量が高かった系統においては、他のアミノ酸、例えば、アスパラギン酸、アスパラギン、スレオニン、セリン、アラニン、ヒスチジンの含量も高く、Ｔｏｔａｌアミノ酸含量において、Ｎｏ．７−２は４倍も増加していることが分かった。これらの結果を以下の表５にまとめた。
【００５６】
【表６】

【００５７】
【表７】

【００５８】
実施例７．トマト形質転換体後代（Ｔ _１ ）世代の解析
１）Ｔ_１世代の選抜
アグロバクテリウム法により遺伝子導入を行ったトマト形質転換植物（Ｔ_０世代）より得られた種子を、８０％エタノールで３０秒，２％次亜塩素酸ナトリウムで１５分間表面殺菌した後、カナマイシン３５０ｍｇ／ｌを含むＭＳ寒天培地に無菌播種した。１ヵ月後、生育の良い植物を選抜した結果、ＡＮ−ｇｄｈ−１７遺伝子導入系統では、Ｎｏ．１、Ｎｏ．３、Ｎｏ．１５、Ｎｏ．２．１から選抜個体が得られた。Ｔ−ｇｄｈ−４遺伝子導入系統では、Ｎｏ．１、Ｎｏ．３、Ｎｏ．８から選抜個体が得られた。株当たりの果実数を増加させるため、屋外の閉鎖系温室で栽培を行った。栄養条件を同じにするため、馴化時に１ｋｇの培養土（パワーソイル，サカタのタネ）に移植した後は、追肥を行わなかった。また、同化能を同一にするために以下の分析では葉を摘み取らず、腋芽を同一条件で栽培し，その葉組織を用いた。
【００５９】
２）サザン分析による導入遺伝子の確認
馴化させた植物の腋芽の葉組織より全ＤＮＡを抽出した（Ｈｏｎｄａ ａｎｄＨｉｒａｉ １９９０，Ｊｐｎ Ｊ Ｂｒｅｅｄ ４０，３３９−３４８）。１５μｇのＤＮＡを制限酵素ＢａｍＨＩとＥｃｏＲＩの組み合わせとＸｂａＩで処理し、電気泳動後、ナイロンメンブランにトランスファーした。ＤＩＧ−Ｌａｂｅｌｉｎｇ ａｎｄ Ｄｅｔｅｃｔｉｏｎ Ｋｉｔ（Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）を用いて、ＡＮ−ｇｄｈ−１７遺伝子またはＴ−ｇｄｈ−４遺伝子をプローブにし、サザンハイブリダイゼーションを行った。
ＡＮ−ｇｄｈ−１７遺伝子をプローブに用いてサザンハイブリダイゼーションを行った結果、ＡＮ−ｇｄｈ−１７遺伝子導入系統Ｎｏ．１、３、１５、２．１系統に目的サイズのバンド（１．８ｋｂ、０．８ｋｂ）が検出され、導入遺伝子が確認できた（図１６）。同様にＴ−ｇｄｈ−４遺伝子をプローブに用いてサザンハイブリダイゼーションを行った結果、Ｔ−ｇｄｈ−４遺伝子と同じサイズのバンド（１．２ｋｂｐ）が確認された。２０ｋｂｐ付近にバンドが見られるものもあり内在性のｇｄｈ遺伝子と考えられた（図１７）。
【００６０】
３）ＮＡＤＰ−ＧＤＨとＮＡＤ−ＧＤＨ活性の測定
形質転換トマト（Ｔ_１）の腋芽の葉組織（０．２ｇ）を液体窒素で凍結させ、乳鉢で破砕した後、重量の５倍量の抽出緩衝液｛２００ｍＭ Ｔｒｉｓ（ｐＨ８．０），１４ｍＭ β−メルカプトエタノール，１０ｍＭ Ｌ−システイン−ＨＣｌ，０．５ｍＭ ＰＭＳＦ，０．５％ ＴｒｉｔｏｎＸ−１００｝を加えた。遠心チューブに移し、４℃、１２，０００ｒｐｍで１０分間遠心した後、上清を限外ろ過（Ｍｉｌｌｉｐｏｒｅ、ウルトラフリー０．５フィルターユニット、バオイマックス−１０）し、抽出緩衝液で３回洗浄した。
抽出した酵素は反応液｛１００ｍＭ Ｔｒｉｓ（ｐＨ８．０），２０ｍＭ ２−α−ケトグルタル酸，１．０ｍＭ ＣａＣｌ_２，０．２ｍＭ ＮＡＤＰＨ（ＮＡＤＰ−ＧＤＨ活性測定）又は０．２ｍＭ ＮＡＤＨ（ＮＡＤ−ＧＤＨ活性測定），２００ｍＭ塩化アンモニウム｝に混合し、室温で反応させ、３４０ｎｍにおける吸光度の減少を測定した。
【００６１】
ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換トマト（Ｔ_１）の葉組織を用いて、ＮＡＤＰ−ＧＤＨ活性を測定した結果、非形質転換体では活性が認められないのに対し、形質転換体は２３０−４００ｎｍｏｌ／（分・ｍｇタンパク質）の活性が測定できた（表６）。Ｔ−ｇｄｈ−４遺伝子を導入した系統ではＮＡＤ−ＧＤＨ活性が非形質転換体に比べて約２倍以上増加していた（表７）。
【００６２】
【表８】

【００６３】
【表９】

【００６４】
４）果実中のアミノ酸含量の測定
第一果房の開花後６週目の果実３個を分析に用いた。 果実に８０℃に熱した８０％エタノールを重量の３倍量加え、乳鉢ですりつぶした後、再度８０℃で２０分間加熱した。７、０００ｒｐｍで遠心して、上清を回収した後、再度８０％エタノールを加え、８０℃に加温した。エタノールによる抽出を３回行い、総量を８０％エタノールで１００ｍｌに合わせた。良く混合した後、抽出液２００μｌをエッペンドルフチューブに取り、乾燥させ、２００μｌの滅菌水に溶かした。エチルエーテル２００μｌ加え混合後、１２，０００ｒｐｍで遠心し、エーテル層を取り除いた。水層を再度乾燥させた後、２００μｌの０．０２Ｎ ＨＣｌに溶解し、０．４５μｍのろ過フィルターを用いてろ過したろ液をサンプルとし、日立高速アミノ酸計測器（Ｌ−８８００）を用いて分析を行った。
【００６５】
結果は３個の果実の平均値を示した。ＡＮ−ｇｄｈ−１７遺伝子導入系統においては、非形質転換体に比べて、ＡＮ−ｇｄｈ−１７ Ｎｏ．１系統由来のＡＮ１−１−２、ＡＮ１−１−３のグルタミン酸含量がそれぞれ、２．１倍、２．８倍増加し、ＡＮ−ｇｄｈ−１７ Ｎｏ．３系統由来のＡＮ３−１−２、ＡＮ３−１−３が２．８倍，２．５倍それぞれ増加していた。ＡＮ−ｇｄｈ−１７ Ｎｏ．１５系統由来のＡＮ−ｇｄｈ−１７ Ｎｏ．２．１系統由来のＡＮ２．１−１−１も２．１倍、１．９倍増加していた（表８、図１８）。形質転換当代（Ｔ_０）で高いグルタミン酸含量を示したＮｏ．１５系統の後代においても同様な傾向が示された。Ｍｔ−ｄＡＮ−ｇｄｈ遺伝子を導入した系統、ＭｔｄＡＮ−２４−４においては非形質転換体のグルタミン酸含量と有意差は認められなかった。
【００６６】
【表１０】

【００６７】
【表１１】

【００６８】
【表１２】

【００６９】
Ｔ−ｇｄｈ−４遺伝子導入系統では、調査したＴ−ｇｄｈ−４ Ｎｏ．１，３，８系統の後代ＴＧＤＨ１−２、３−１、８−１で、非形質転換体のグルタミン酸含量に比べて、２．３倍，２．１倍、２．４倍増加していた。（表９、図１９）。また、グルタミン酸以外のアミノ酸ではアスパラギン酸、グルタミン、γ−アミノ酪酸含量の顕著な増加が見られ、結果として全遊離アミノ酸含量も非形質転換体と比較して２−３倍増加した。
【００７０】
【表１３】

【００７１】
【表１４】

【００７２】
【表１５】

【００７３】
実施例８．ポテト形質転換体の作出と解析
１）形質転換体の作出（実施例４参照）
茎頂培養によってポテト（バレイショ）の無菌植物を得、茎頂を継代することによって材料を増やした。ＭＳ培地に２％ショ糖を加えた液体培地（１０ｍｌ）に茎頂を入れ、発根を誘導した。発根後、１６％ショ糖を含むＭＳ液体培地を１０ｍｌ加え、暗所培養を行い、マイクロチューバーを誘導した。６−８週後のマイクロチューバーをディスク状に切り、皮をむいた後、２８℃で一晩培養したアグロバクテリウム液（Ｔｉ−プラスミッド、ｐＭｔ−ｄＡＮ−ｇｄｈ又はｐＣｔ−ＡＮ−ｇｄｈを含む）を感染させた。滅菌ろ紙を敷いたＭＳ寒天培地（ＭＳ培地、２．０ｍｇ／ｌゼアチン，０．１ｍｇ／ｌインドール酢酸，０．３％ゲルライト）上に乗せ、２５℃、１６時間日長で２日間共存培養した。その後選抜培地｛ＭＳ培地、２．０ｍｇ／ｌゼアチン，０．１ｍｇ／ｌインドール酢酸，０．３％ゲルライト，５０ｍｇ／ｌカナマイシン，５００ｍｇ／ｌクラフォラン｝に移し、同条件で培養した。１週間ごとに新しい選抜培地に移し、再分化したシュートを植物ホルモンを含まない選抜培地に移し、発根を誘導した。Ｔｉ−プラスミッドｐＭｔ−ｄＡＮ−ｇｄｈおよびｐＣｔ−ＡＮ−ｇｄｈを持つアグロバクテリウムを感染させ、カナマイシン５０ｍｇ／ｌを含む培地で選抜を行った結果、Ｍｔ−ｄＡＮ−ｇｄｈ Ｎｏ．２，５，８，Ｃｔ−ＡＮ−ｇｄｈ Ｎｏ．１の４系統が選抜できた。
【００７４】
２）サザン分析による導入遺伝子の確認
馴化させた植物の葉組織よりＴｏｌａｌ ＤＮＡを抽出した（Ｈｏｎｄａ ａｎｄ Ｈｉｒａｉ １９９０、Ｊｐｎ Ｊ Ｂｒｅｅｄ ４０，３３９−３４８）。１５μｇのＤＮＡを制限酵素ＥｃｏＲＩで処理し、電気泳動後、ナイロンメンブランにトランスファーした。サザンハイブリダイゼーションはＤＩＧ−Ｌａｂｅｌｉｎｇ ａｎｄ Ｄｅｔｅｃｔｉｏｎ Ｋｉｔ （Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）を用いて行った。プローブにＡＮ−ｇｄｈ−１７遺伝子を用いた。
その結果、４系統すべてに導入遺伝子サイズのバンド（約１．５ｋｂ）が確認され（図２０）、トランジェットペプチドを付加したｇｄｈ遺伝子が導入されていることが示唆された。
【００７５】
３）ＮＡＤＰ−ＧＤＨ活性の測定
形質転換ポテトの葉組織（約０．１ｇ）を液体窒素で凍結させ、乳鉢で破砕した後、重量の５倍量の抽出緩衝液｛２００ｍＭ Ｔｒｉｓ（ｐＨ８．０），１４ｍＭ β−メルカプトエタノール、１０ｍＭ Ｌ−システイン−ＨＣｌ，０．５ｍＭ ＰＭＳＦ，０．５％ ＴｒｉｔｏｎＸ−１００｝を加えた。遠心チューブに移し、１２，０００ｒｐｍで１０分間遠心した後、上清を限外ろ過（Ｍｉｌｌｉｐｏｒｅ、ウルトラフリー０．５フィルターユニット、バオイマックス−１０）し、抽出緩衝液で３回洗浄した。抽出した酵素は反応液｛１００ｍＭ Ｔｒｉｓ（ｐＨ８．０），２０ｍＭ ２−α−ケトグルタル酸，１．０ｍＭ ＣａＣｌ_２，０．２ｍＭ ＮＡＤＰＨ，２００ｍＭ 塩化アンモニウム｝に混合し、室温で反応させ、３４０ｎｍにおける吸光度の減少を測定した。
サザン分析で導入遺伝子が確認できた形質転換ポテトおよび非形質転換ポテトの葉組織を用いて、ＮＡＤＰ−ＧＤＨ活性を測定した結果、非形質転換体では活性が認められないのに対し、形質転換体では１５０−３００ｎｍｏｌ／（分ｍｇタンパク質）の活性が測定できた（表１０）。Ｃｔ−ＡＮ−ｇｄｈ系統よりＭｔ−ｄＡＮ−ｇｄｈ系統の方が高い活性を示した。
【００７６】
【表１６】

【００７７】
４）マイクロチュバーのアミノ酸含量の測定
非形質転換体と形質転換体４系統の茎頂を液体培養し、発根誘導した後、１６％ショ糖を加え、暗所処理後６週目のマイクロチューバーを用いて、アミノ酸含量を測定した。
８０℃に熱した８０％エタノールを重量の３倍量加え、乳鉢ですりつぶした後、再度８０℃で２０分間加熱した。７，０００ｒｐｍで遠心して、上清を回収した後、再度８０％エタノールを加え、８０℃に加温した。エタノールによる抽出を３回行い、総量を８０％エタノールで５ｍｌに合わせた。よく混合した後、抽出液２００μｌをエッペンドルフチューブに取り、乾燥させ、２００μｌの滅菌水に溶かした。エチルエーテル２００μｌ加え混合後、１２，０００ｒｐｍで遠心し、エーテル層を取り除いた。水層を再度乾燥させた後、４００μｌの０．０２Ｎ ＨＣｌに溶解し、０．４５μｍのろ過フィルターを用いてろ過したろ液をサンプルとし、日立高速アミノ酸計測器（Ｌ−８８００）を用いて分析を行った。
【００７８】
遺伝子導入系統より誘導したマイクロチューバーを用いてアミノ酸分析を行った。それぞれの株は２個以上のマイクロチュバーを分析し，統計処理を行った。Ｍｔ−ｄＡＮ−ｇｄｈ遺伝子を導入したＮｏ．２，５，８系統由来Ｍｔ２−２，Ｍｔ５−１，Ｍｔ５−２，Ｍｔ８−１，Ｍｔ８−２株はそれぞれグルタミン酸含量が非形質転換体に比べて、１．７倍，２．２倍，２．５倍，３．０倍，２．２倍増加していることが分かった（表１１、図２１）。Ｃｔ−ＡＮ−ｇｄｈ遺伝子導入系統においては、非形質転換体と比べてグルタミン酸含量に有意差は認められなかった。グルタミン酸以外のアミノ酸ではグルタミンやプロリンの顕著な増加が示され、結果として全遊離アミノ酸含量も非形質転換体と比較して２−３倍増加した。
【００７９】
【表１７】

【００８０】
【表１８】

【００８１】
【表１９】

【００８２】
【発明の効果】
本発明により、遊離アミノ酸を高濃度に含有する植物が得られ、付加価値の高い原料作物および食品素材が提供される。本発明により全アミノ酸量が２〜４倍に増加し、特にグルタミン酸、アスパラギン、アスパラギン酸、セリン、スレオニン、アラニンおよびヒスチジンの少なくとも１つについてアミノ酸含量の高い作物が提供され、これらのアミノ酸の後添加を必要としない付加価値の高い原料作物が提供される。また、本発明により、直接調理するような野菜類で高濃度にグルタミン酸を蓄積したもの、すなわち、うま味の優れた食品素材が提供される。
さらに、本発明により、そのような遊離アミノ酸を高度に蓄積する植物を育種するための期間が大幅に短縮される。
【配列表】

【図面の簡単な説明】
【図１】Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ由来グルタミン酸デヒドロゲナーゼ（ＧＤＨ）遺伝子ＡＮ−ｇｄｈ−１７と既知のＮＡＤＰ−ＧＤＨ遺伝子配列を比較したものである。上段がＮＡＤＰ−ＧＤＨ遺伝子の塩基配列、下段がＡＮ−ｇｄｈ−１７遺伝子の塩基配列である。
【図２】Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ由来グルタミン酸デヒドロゲナーゼ（ＧＤＨ）遺伝子ＡＮ−ｇｄｈ−１７と既知のＮＡＤＰ−ＧＤＨ遺伝子配列の比較（図１続き）。上段がＮＡＤＰ−ＧＤＨ遺伝子の塩基配列、下段がＡＮ−ｇｄｈ−１７遺伝子の塩基配列。
【図３】ＡＮ−ｇｄｈ−１７遺伝子のＴｉプラスミッド（ｐＭＡＴ０３７）へのクローニング手順を示したものである。図中、３５Ｓ ＰｒｏはＣａＭＶ ３５Ｓプロモーター、Ｔｅｒｍはターミネータを表す。
【図４】トランジットペプチドを結合したＡｓｐｅｒｇｉｌｌｕｓ由来ＮＡＤＰ−ＧＤＨＡ遺伝子をコードする配列を含む遺伝子構築物、ｐＣｔ−ＡＮ−ｇｄｈ、ｐＣｔ−ｄＡＮ−ｇｄｈ、ｐＭｔ−ｄＡＮ−ｇｄｈの構築手順およびそれらの構造を模式的に示したものである。
【図５】ＮＡＤＰ−ＧＤＨ遺伝子中のスプライシング領域を除去する手順の概略を示したものである。図中、太い線はスプライシング領域、Ｐ１〜Ｐ４はＰＣＲプライマーを表す。
【図６】Ａｓｐｅｒｇｉｌｌｕｓ由来ＮＡＤＰ−ＧＤＨ Ａ遺伝子配列へのトランジットペプチド配列結合手順の概略を示したものである。図中Ａはトランジットペプチドの塩基配列、ＢはＡＮ−ｇｄｈ−１７遺伝子の塩基配列を表す。Ｐ５〜Ｐ８はそれぞれＰＣＲプライマーを表す。
【図７】２Ａ１１プロモーターに接続されたＡｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ由来ＮＡＤＰ−ＧＤＨ遺伝子を有する、遺伝子構築物ｐ２ＡＣｔ−ｄＡＮ−ｇｄｈおよびｐ２ＡＭｔ−ｄＡＮ−ｇｄｈの構造を模式的に示したものである。
【図８】トマト由来ＮＡＤ−ＧＤＨ遺伝子（Ｔ−ｇｄｈ−４）のＴｉプラスミッド（ｐＩＧ１２１−Ｈｍ）へのクローニング手順を示したものである。図中、３５ＳはＣａＭＶ ３５Ｓプロモーター、ＮｏｓはＮｏｓ−ターミネーターを表す。
【図９】トマト由来ＮＡＤ−ＧＤＨ遺伝子、Ｔ−ｇｄｈ−４の改変手順の概略を示したものである。
【図１０】Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ由来ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換体のＰＣＲ解析。レーン２、３：非形質転換トマト、レーン４〜６：プラスミッド（ｐＭＡＴ０３７）遺伝子を導入した形質転換トマト；レーン７〜１０：ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換トマト（Ｎｏ−６，Ｎｏ−８−２，Ｎｏ−１５，Ｎｏ−１７）。
【図１１】トマト由来ＮＡＤ−ＧＤＨ遺伝子ＧＤＨ（Ｔ−ｇｄｈ−４）遺伝子を導入した形質転換体のＰＣＲ解析。レーン２，３：非形質転換トマト；レーン４〜６：プラスミッド（ｐＩＧ１２１−Ｈｍ）遺伝子を導入した形質転換トマト；レーン７〜１０：Ｔ−ｇｄｈ−４遺伝子を導入した形質転換トマト（Ｎｏ．２、Ｎｏ．７−２、Ｎｏ．９−２、Ｎｏ．１０）。
【図１２】Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ由来ＡＮ−ｇｈｄ−１７遺伝子を導入した形質転換体のＲＴ−ＰＣＲ分析。Ｎｏ．６、Ｎｏ．１５は形質転換トマトを表し、（）内は全ＲＮＡを抽出した組織を表す。
【図１３】トマト由来Ｔ−ｇｄｈ−４遺伝子を導入した形質転換体のＲＴ−ＰＣＲ分析。Ｎｏ．２、Ｎｏ．７−２、Ｎｏ．９−２、Ｎｏ．１０は形質転換トマトを表し、（）内は全ＲＮＡを抽出した組織を表す。
【図１４】ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換体（Ｎｏ．６、Ｎｏ．１５、Ｎｏ．１７）のアミノ酸（グルタミン酸−Ｇｌｕ、グルタミン−Ｇｌｎ、γ−アミノ酪酸−ＧＡＢＡ、リジン−Ｌｙｓ）含量を比較したグラフである。
【図１５】Ｔ−ｇｄｈ−４遺伝子を導入した形質転換体（Ｎｏ．２、Ｎｏ．７−２、Ｎｏ．９−２、Ｎｏ．１０）のアミノ酸（グルタミン酸−Ｇｌｕ、グルタミン−Ｇｌｎ、γ−アミノ酪酸（ＧＡＢＡ）、リジン−Ｌｙｓ）含量を比較した結果である。
【図１６】ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換トマトのサザン分析の結果を示したものである。全ＤＮＡ（１５μｇ）をＢａｍＨＩおよびＥｃｏＲＩで消化した（Ａ）、または全ＤＮＡ（１５μｇ）をＸｂａＩで消化した（Ｂ）サンプルを使用した。レーン１：非形質転換トマト、レーン２：ＡＮ−ｇｄｈ−１７ Ｎｏ．１、レーン３：ＡＮ−ｇｄｈ−１７ Ｎｏ．３、レーン４：ＡＮ−ｇｄｈ−１７ Ｎｏ．１５、レーン５：ＡＮ−ｇｄｈ−１７ Ｎｏ．２．１。
【図１７】Ｔ−ｇｄｈ−４遺伝子を導入した形質転換トマト（Ｔ１）のサザン分析の結果を表す。全ＤＮＡ１５μｇをＸｂａＩおよびＳａｃＩで消化したサンプルを使用した。レーン１：非形質転換トマト、レーン２：Ｔ−ｇｄｈ Ｎｏ．１−２、レーン３：Ｔ−ｇｄｈ Ｎｏ．３−１、レーン４：Ｔ−ｇｄｈ Ｎｏ．８−１。矢印は導入遺伝子（１．２ｋｂ）に対応するバンドの位置を示す。
【図１８】ＡＮ−ｇｄｈ−１７遺伝子を導入した形質転換トマトの後代（Ｔ_１）における果実中のアミノ酸含量を比較したグラフである。対照は非形質転換トマトを使用した。測定は３個体ずつ行なった。
【図１９】Ｔ−ｇｄｈ−４遺伝子を導入した形質転換トマトの後代（Ｔ_１）における果実中のアミノ酸含量を比較したグラフである。測定は３個体ずつ行なった。
【図２０】Ｃｔ−ＡＮ−ｇｄｈとＭｔ−ｄＡＮ−ｇｄｈ遺伝子を導入したポテトのサザン分析の結果を示したものである。全ＤＮＡ（１５μｇ）のＥｃｏＲＩ消化物を使用した。レーン１：非形質転換ポテト−１、レーン２：非形質転換ポテト−２、レーン３〜６はそれぞれの遺伝子構築物を導入した形質転換ポテトに対応する。レーン３：Ｃｔ−ＡＮ−ｇｄｈ Ｎｏ．１、レーン４：Ｍｔ−ｄＡＮ−ｇｄｈ Ｎｏ．２、レーン５：Ｍｔ−ｄＡＮ−ｇｄｈ Ｎｏ．５、レーン６：Ｍｔ−ｄＡＮ−ｇｄｈ Ｎｏ．８。矢印は導入遺伝子の断片に対応するバンドの位置を示す。
【図２１】Ｃｔ−ＡＮ−ｇｄｈとＭｔ−ｄＡＮ−ｇｄｈ遺伝子を導入したポテトのマイクロチューバー中のグルタミン酸（Ｇｌｕ）含量を示したグラフである。対照としては非形質転換ポテトを使用した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a transgenic plant with enhanced free amino acids and a method for producing the same. More specifically, the present invention relates to a transgenic plant that highly accumulates at least one of asparagine, aspartic acid, serine, threonine, alanine, histidine, and glutamic acid, and a method for producing the same.
[0002]
[Prior art]
The world's first technology to introduce a specific gene into a plant and transform it was the study of introducing a gene into tobacco using the soil bacterium, Agrobacterium tumefaciens. Attempts have been made to make plants produce useful ingredients. Plant breeding methods using such transgenic plant production techniques are promising as alternatives to conventional traditional breeding by crossing and the like. Among them, research on improving plant characteristics related to nitrogen assimilation has also been promoted, and amino acids are particularly important components in fruits, root vegetables roots and seeds among nitrogen metabolites. Research has been actively conducted because it has a great influence.
[0003]
As a study on the biosynthesis of amino acids, for example, a report that an E. coli-derived DHDPS gene was introduced into tobacco and free lysine was increased 200-fold (US Pat. No. 5,258,300, Molecular Genetics Res. & Development), by introduction of the AK gene. A report that free lysine increased (EP485970, WO9319190), an AS gene introduced into tobacco, and a report that the asparagine content increased 100-fold (WO95099911, Univ New York, WO9015333, Univ Rockfeller), anthranilate synthase There has been a report (WO 9726366, DEKALB Genetic Corp) that the tryptophan content has been increased by 90-fold when introduced into rice. Plants targeted for gene transfer are not limited to model plants such as tobacco and Arabidopsis, but plants having fruits such as tomatoes are also used. For example, for tomatoes, transformants were created in 1986 using the Agrobacterium method (S. McCorick, J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Freley, Plant Cell Reports, 5, 81-84 (1986); Y. S. Chyi, R. A. Jorgenson, D. Goldstern, S. D. Marksley and F. Loaiza- Figueroe, Mol. Gen. Genet., 204, 64-69 ( 1986)), and since that time the transformation system has been improved. In addition to the above, many genes involved in amino acid biosynthesis and nitrogen assimilation are known, including asparaginase, GOGAT and the like, and their base sequences have also been reported.
[0004]
Here, in particular, glutamic acid, which is a kind of α-amino acid, is widely distributed in proteins and is used as a flavoring ingredient in tomatoes, and in soybeans in brewed foods (for example, soy sauce and miso). The umami component is known to be glutamic acid, and is known to be synthesized at the first stage of nitrogen metabolism in higher plants. In addition, it is known that glutamine and asparagine generated from glutamic acid are distributed to each tissue via a sieve tube and used for synthesis of other amino acids and proteins. In plants, there are reports of examples of high concentrations in sieve tubes, which are transport pathways for photosynthetic products such as sucrose and amino acids (Mitsuo Kanno et al., Plant Nutrition and Fertilizer Science p125 (1993)). Examples of high concentrations in the portion include 0.25 g / 100 g in tomato fruit f. w. An example (Tokimeki No. 2, Journal of the Japan Food Industry Association, Vol. 39, p64-67 (1992)) is known. However, in the case of glutamic acid, even if the biosynthetic ability in the source organ can be improved, it is a starting material for donating an amino group and is metabolized by various biosynthetic pathways as described above. Accumulating at a high concentration is not easy. Regardless of crossbreeding or genetic manipulation, there have been no examples that have so far succeeded in dramatically increasing glutamate concentration in plants.
[0005]
The first step of assimilating inorganic nitrogen into an organism is the incorporation of ammonia into glutamate to produce glutamine, as described above, which is catalyzed by a glutamine synthetase enzyme (GS). This glutamine is then catalyzed by glutamate synthase (GOGAT) to produce two molecules of glutamate from α-ketoglutarate. This GS / GOGAT cycle is considered to be the main pathway of nitrogen assimilation in plants (literature: Miflin and Lea, 1976, Phytothermistry, 15; 873-885). On the other hand, it is known that the uptake of ammonia proceeds through metabolic pathways other than those catalyzed by GS (Knight and Langston-Unkefer, 1988, Science, 241: 951-954). That is, the incorporation of ammonia into α-ketoglutarate to produce glutamic acid, which is catalyzed by glutamate dehydrogenase (GDH). However, since plant GDH has a high Km value for ammonia, while ammonia is toxic and intracellular ammonia is usually at low concentrations, this nitrogen assimilation pathway under normal growth conditions. Has not yet been fully elucidated, but one study reports that it contributes to nitrogen assimilation when intracellular ammonium concentrations rise above normal levels (Knight and Langston) -Unkefer, supra).
[0006]
Glutamate dehydrogenase enzyme (GDH) in plants is reversible when ammonia is dissociated from glutamic acid to produce α-ketoglutaric acid as opposed to the case of incorporating ammonia into α-ketoglutaric acid to produce glutamic acid as described above. Catalyze any reaction. The former is considered to function when the amount of ammonia is high, and the latter when the nitrogen content is high (Robinson et al., 1991, Plant Physiol. 95; 809-816: Robinson et al., 1992, Plant Physiol. 98; 1190 -1195), the directionality is not clear, such as the enzyme GDH-A that functions in the direction of synthesizing glutamic acid found in microorganisms and the enzyme GDH-B that functions in the direction of degradation. In plants, it is thought that there are two types, NADP-dependent GDH that functions in chloroplasts and NAD-dependent GDH that functions in mitochondria, but GDH shows a high Km value for ammonia, and photorespiration NAD-dependent GDH localized in mitochondria is thought to play an important role in assimilation of ammonia because of its high correlation with ammonia level at the time (Srivastava and Singh RP, 1987, Phytothermistry, 26 597-610).
[0007]
Plant GDH is known to bind to two different polypeptides (α-subunit, β-subunit) randomly to form a hexamer, and to show seven isozyme patterns depending on the degree of binding. Yes. In a study using Grapevine's callus cells, callus cultured in a medium containing nitrate or glutamic acid increased in the isozyme consisting of β-subunit on the cathode side when electrophoresed, and cultured in a medium containing ammonia or glutamine. In the callus, α-subunit isozyme increased to the anode side, and when transferred from nitrate medium to ammonia source medium, GDH activity increased three-fold (α-subunit increased four-fold, β- (Subunit reduction), and it was reported that the activity shifted from the cathode side to the anode side (Lourakakis and Poubelakis-Angelakis, 1996, Plant Physiol. 97; 104-1111), and the α-subunit plays an important role in assimilation of ammonia. It is thought that
[0008]
In 1995, Sakakibara et al. (Plant Cell Physiol, 33; 1193-1198) isolated GDH genes from corn roots based on two isozyme bands on the cathode side, and then isolated grapevine (Syntakiki et al. , 1996, Gene 168; 87-92), Arabidopsis (Melo-Olivera et al., 1996, Proc Natl Acad Sci USA 93; 4718-4723), tomato (Purnell et al., 1997, Gene 186; 249-254) Have been isolated. In particular, a gene isolated from Grapevine callus has been isolated based on an isozyme expressed in ammonia-treated cells, and is considered to be a gene encoding an α-subunit. All genes have a transit peptide for functioning in mitochondria, but GDH genes of corn and tomato are expressed in large amounts in roots, whereas Arabidopsis has high expression in leaves and flowers . In maize, Arabidopsis, and Grapevine, the presence of multiple genes is suggested, whereas in tomato, it is reported that there is a single copy, and there is a difference in the gene structure and function in plants. Is expected to be complex.
[0009]
A transgenic plant into which this GDH gene has been introduced has also been prepared. When glutamic acid dehydrogenase enzyme GDH (NADP-GDH) derived from Escherichia coli was introduced into tobacco and corn for the purpose of conferring resistance to the herbicide phosphinothricin, the glutamic acid content in the roots (Lightfoot David et al., CA2180786 (1996). In this report, the glutamic acid content in tobacco roots was 14.7 mg / 100 gf.w. In 20.6 mg / 100 gf.w, the glutamic acid content, which was 16.2 mg / 100 gf.w in corn roots, was increased to 19.1 mg / 100 gf.w. There are reports, but examples are That is not (WO9509911, α derived from Chlorella, beta-subunit (WO9712983)). Although not shown also analyzed values for glutamate family amino acids.
[0010]
[Problems to be solved by the invention]
The object of the present invention is to provide at least one of the free amino acid content of the plant storage organ, in particular free amino acids in the edible part of the plant including roots, fruits and seeds, in particular glutamic acid, asparagine, aspartic acid, serine, threonine, alanine and histidine. It is a method to enhance the accumulation of two and to provide a transgenic plant with a high accumulation of free amino acids.
[0011]
[Means for Solving the Problems]
The object of the present invention is achieved by providing a plant in which the expression level and / or cell-specific expression balance of major enzymes involved in assimilation and utilization of nitrogen are changed and a method for producing the same. Such a plant is produced by introducing one or more genes encoding nitrogen assimilation or utilization enzymes together with appropriate control sequences, and overexpressing or suppressing the expression thereof.
A transgenic plant that highly accumulates free amino acids according to the method of the present invention, particularly a plant that highly accumulates at least one of glutamic acid, asparagine, aspartic acid, serine, threonine, alanine and histidine, especially glutamic acid, is derived from a eukaryotic organism. Of glutamate dehydrogenase (GDH) gene together with appropriate regulatory sequences into plants and overexpressed.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to genetic manipulation of nitrogen metabolism in plants. In particular, the present invention relates to the expression level of enzymes involved in nitrogen assimilation and utilization in order to achieve high accumulation of free amino acids in the edible part of useful plants such as fruits, root vegetable roots and seeds, particularly glutamic acid, an umami component. Concerning changing. Expression of these enzymes is enhanced, modified, or suppressed, and a plant having desired properties is produced.
The target gene used in the present invention is a gene encoding an enzyme involved in assimilation of ammonia into an amino acid. Examples of the target gene include glutamate dehydrogenase (GDH). The expression of this enzyme is enhanced, and other modifications (for example, ectopic expression by addition of transit sequences) produce plants having desired properties. The manipulation can be performed by transforming a plant with the nucleic acid construct described herein. Transformed plants or their progeny express the desired modified enzyme and are screened for corresponding mRNA expression changes, nitrogen assimilation or altered availability, and / or increased free amino acid content of the plant.
[0013]
Briefly, the method of the present invention comprises the following procedure, and the transgenic plant of the present invention is a transgenic plant produced by such a method:
a) cloning the gene of interest;
b) recloning the resulting gene into an appropriate vector if necessary;
c) introducing the vector into a plant cell to obtain a transformant;
d) regenerating and cultivating the obtained transformant into a plant;
[0014]
In one embodiment of the invention, one or several genes encoding nitrogen assimilation or utilization enzymes are placed under strong constitutive promoter control and are overexpressed in the plant. Such modification of expression is, for example,
a) a transgene in which the coding sequence of the enzyme is operably linked to a strong constitutive promoter,
b) a high copy number natural gene encoding the desired enzyme,
c) a regulatory gene that activates the expression of the gene of interest for nitrogen assimilation or utilization;
d) a copy of the native gene modified to enhance expression and having a regulatory region, and
e) a transgene expressing a mutant, modified or chimeric nitrogen assimilation or utilization enzyme,
Can be achieved by genetically engineering the plant with at least one of the following.
[0015]
In another embodiment of the invention, the nitrogen assimilation or utilization enzyme expression pattern is altered. Such modification of the expression pattern is, for example,
a) a transgene in which the coding sequence of the enzyme is operably linked to a promoter having a desired expression pattern (eg, a promoter exhibiting an organ-specific or growth stage-specific expression pattern);
b) a modified regulatory gene that activates the expression of the gene encoding the enzyme in a preferred pattern;
c) a copy of the natural gene having regulatory regions modified to be expressed in a preferred pattern;
Can be achieved by genetically engineering the plant with at least one of the following.
[0016]
In yet another embodiment of the invention, an enzyme modified in a nitrogen assimilation or utilization pathway or a different type of enzyme is expressed. In this type of embodiment, a gene construct that expresses in a plant cell that encodes a corresponding enzyme having a catalytic action different from that of the host plant's nitrogen assimilation or utilization enzyme is generated, thereby Includes manipulating. By taking such measures, plants containing enhanced free amino acids are obtained.
Moreover, in order to grow such a plant, the traditional crop breeding method requires screening of a large segregating population, which takes a lot of time. According to the present invention, such measures are taken. It can be avoided.
[0017]
The following are definitions of terms and abbreviations used in this specification.
CaMV35S = Cauliflower Mosaic Virus 35S Promoter
NADP-GDH = NADP-dependent glutamate dehydrogenase
NAD-GDH = NAD-dependent glutamate dehydrogenase
Gene fusion = gene construct comprising a promoter linked to a heterologous gene (the promoter regulates transcription of the heterologous gene)
Heterologous gene = In gene construction, a heterologous gene means that the gene is linked to a promoter that is not naturally linked. The heterologous gene may or may not be from an organism that contributes the promoter.
GABA = γ-aminobutyric acid
[0018]
The enzyme gene that can be used in the present invention may be derived from bacteria, yeast, algae, animals and plants, but is not limited thereto, and can be obtained from various sources. Sequences obtained from such sources can be operably linked to appropriate promoters that function in plant cells, to increase their translation efficiency in the host plant, or to catalyze the encoded enzyme. May be modified by in vitro mutagenesis or de novo synthesis to alter. These modifications include, but are not limited to, modification of residues involved in substrate and / or catalysis. Furthermore, the transgene may be modified to have an optimal codon depending on the codon usage of the host or organelle to be expressed. Moreover, you may connect the nucleic acid sequence which codes an appropriate transit peptide to these gene sequences as needed.
Preferred modifications also include the construction of hybrid enzymes. For example, enzymes having novel properties may be created by combining different domains of related enzymes obtained from the same or different organisms.
Furthermore, nucleic acid fragments that hybridize under stringent conditions to the various nucleic acid sequences described above can be used in the present invention as long as they have the desired activity. Thus, nucleic acid fragments encoding proteins in which one or more amino acids have been deleted, added, or substituted are included. “Stringent conditions” refers to general conditions well known to those skilled in the art, as described in Sambrook et al. (1989, supra). Nucleic acid sequences that hybridize under such conditions will generally have 60%, preferably 80%, particularly preferably 90% or more homology to each other.
[0019]
The nitrogen assimilation or utilization enzyme genes that can be used in the present invention include various genes as described above. One example of a preferable gene that can be used for accumulating glutamate is a glutamate dehydrogenase (GDH) gene. Can be mentioned. When the GDH gene is used, it is expressed in the sense direction. When a GDH gene is selected, it is preferably expressed as a fusion gene in which a transit peptide is bound on the 5 'side, and particularly preferred transit peptides are a transit peptide to mitochondria and a transit peptide to chloroplast.
In one preferred embodiment of the present invention, NADP-dependent GDH gene derived from mold (Aspergillus nidulans) (Mol. Gen. Genetics, 218, 105, 1989) or NAD-dependent GDH gene derived from tomato (Purnell et al., 1997, Gene 186; 249-254) using a recombinant construct encoding the cauliflower mosaic virus (CaMV) 35S promoter, a powerful constitutive plant promoter operably linked to the sequence encoding tomato plants. Illustrated by examples engineered genetically. The strain overexpressing GDH shows an increase in free amino acid content, particularly an increase in glutamic acid content (2 to 3 times), compared to the parent plant of interest.
[0020]
Nucleic acid constructs that can be used in the present invention can be made using methods known to those skilled in the art. For example, see Sambrook et al., Molecular cloning-Laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, for isolating, characterizing, manipulating the components of the construct, and recombinant DNA methods that can be used to make the construct itself. You can refer to the following sources. When the base sequence of the desired component is known, it is advantageous to synthesize it rather than isolate it from biological sources. In such a case, those skilled in the art will be familiar with Caruthers et al. Acids. Res. Symp. Ser. 7: 215-233 and Chow and Kempe, 1981, Nuc. Acids. Res. 9: 2807-2817 can be consulted. In other cases, the desired component can be advantageously produced by polymerase chain reaction (PCR) amplification. For the PCR method, those skilled in the art will know that Gelfan, 1989, PCR technology, principles and applications for DNA amplification, H.C. A. Edited by Errich, Stockton Press, N.C. Y. Reference may be made to the current protocol in molecular biology, Volume 2, Chapter 15, edited by Ausubel et al., John Willy & Sons, 1988.
[0021]
In general, the gene construct used in the present invention includes an appropriate promoter that functions in a plant cell in addition to a target gene, an appropriate terminator such as a nopaline synthase gene terminator, other elements useful for expression control, and An appropriate marker gene for selecting a transformant, for example, a drug resistance gene such as kanamycin resistance, G418 resistance, hygromycin resistance and the like is included. The promoter contained in this construct may be a constitutive promoter, organ-specific or growth-specific, depending on the host used, gene, required expression level, organ to be expressed, growth stage, etc. You can choose.
According to the present invention, a plant exhibiting nitrogen assimilation or overexpression of a utilization enzyme can be produced by transforming a plant cell with a gene construct comprising a plant promoter linked to a sequence encoding the desired enzyme. . Related promoters used in preferred embodiments of the invention are strong and non-organ-specific or non-growth stage-specific promoters (eg, promoters that are strongly expressed in many or all tissues). An example of such a strong constitutive promoter is the CaMV35S promoter.
[0022]
In another embodiment of the invention, it may be advantageous to manipulate the plant with a genetic construct in which an organ-specific or growth stage-specific promoter is linked to a sequence encoding the desired enzyme. For example, when expression in photosynthetic tissues and organs is desired, the ribulose bisphosphate carboxylase (RuBisCO) gene or chloroplast a / b binding protein (CAB) gene promoter can be used. When expression in seeds is desired, promoters of various seed storage protein genes can be used, and when expression in fruits is desired, fruit-specific promoters (eg tomato 2A11) are used. If expression in tubers is desired, a tuber storage protein gene promoter (eg, potato patatin) can be used.
[0023]
In yet another embodiment of the invention, it may be advantageous to transform a plant with a genetic construct in which an inducible promoter is linked to a sequence encoding the desired enzyme. There are many examples of such promoters. For example, heat shock gene, defense response gene (eg, phenylalanine ammonia lyase gene), injury response gene (eg, cell wall protein gene rich in hydroxyproline), chemical induction gene (eg, nitrate reductase gene, chitinase gene), dark place Inducible genes such as, but not limited to, asparagine synthetase genes (Coruzzi and Tsai, US 5,256,558).
[0024]
The recombinant nucleic acid construct of the present invention may include a selectable marker for tracking the delivery of the construct. For example, constructs transmitted in bacteria preferably include genes that confer resistance to antibiotic resistance genes such as kanamycin, tetracycline, streptomycin, or chloramphenicol. Suitable vectors for transferring the construct include plasmids, cosmids, bacteriophages or viruses. In addition, the recombinant constructs may include plant-expressing selectable marker genes or screenable marker genes for the isolation, identification or tracking of plant cells transformed with these constructs. Selectable markers include genes that confer antibiotic resistance (eg, resistance to kanamycin or hygromycin) or herbicide resistance (eg, resistance to sulfonylurea, phosphinothricin, or glyphosate), but these It is not limited to. As a screenable marker, a gene encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), a gene encoding luciferase (Ow et al., 1986, Science 234: 856-859), Examples include, but are not limited to, the B and C1 gene products that regulate anthocyanin pigment production (Goff et al., 1990, EMBO J, 9: 2517-2522).
[0025]
The gene introduction method that can be used in the present invention is not particularly limited, and any method known to those skilled in the art as a gene introduction method into a plant cell or a plant body may be used. For example, in one embodiment of the present invention, Agrobacterium is used to introduce a genetic construct into a plant. Such transformations include two-component Agrobacterium T-DNA vectors (Bevan, 1984, Nuc. Acid Res. 12: 8711-8721) and co-culture operations (Horsch et al., 1985, Science, 227: 1229-1231). It is desirable to use In general, the Agrobacterium transformation system is used to manipulate dicotyledonous plants (Bevans et al., 1982, Ann. Rev. Genet., 16: 357-384; Rogers et al., 1986, Methods Enzymol., 118: 627-641). The Agrobacterium transformation system can also be used to transform monocotyledonous plants and plant cells (Hernalstein et al., 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984 Nature). , 311: 763-764; Grimsley et al., 987, Nature, 325: 167-1679; Bolton et al., 1989, Plant Mol. Biol., 12: 31-40; Gould et al., 1991, Plant Physiol., 95: 426 434). If an Agrobacterium system is used to transform a plant, the recombinant DNA construct further includes at least a right border sequence of the T-DNA region at a position adjacent to the DNA sequence to be introduced into the plant cell. In a preferred embodiment, the transferred sequence is inserted between the left and right T-DNA border sequences. Appropriate design and construction of such T-DNA based transformation vectors are known to those skilled in the art.
[0026]
In other embodiments, various alternative methods for introducing recombinant nucleic acid constructs into plants and plant cells can be used. Alternative gene transfer and transformation methods include protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation-mediated uptake (Paszkowski et al., 1984, EMBO J., 3: 2717-2722; Potrykus et al., 1985, Mol. Gen. Genet., 199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA, 82: 5824-5828; Shimamoto et al., 1989, Nature, 338: 274- 276). According to the present invention, a wide variety of plants and plant cell lines can be manipulated for the desired physiological properties described herein using the nucleic acid constructs of the present invention and the transformation methods described above. These methods are particularly beneficial when the target is a monocotyledonous plant or plant cell. In preferred embodiments, target plants and plant cells for manipulation include, but are not limited to, tomatoes, potatoes, beets, soybeans, arabidopsis, corn, wheat, rice, sugar cane and the like.
[0027]
In accordance with the present invention, gene constructs as disclosed herein can be used in a variety of plant cell types including, but not limited to, protoplasts, tissue culture cells, tissue and organ explants, pollen, embryos and whole plants. The desired plant can be obtained by introducing and manipulating it. In an embodiment of the invention, the engineered plants are selected or screened for transformants according to the following approaches and methods. Next, the isolated transformant may be regenerated into a plant individual. Methods for regenerating a plant individual from plant cells, tissues or organs are known to those skilled in the art for many plant species.
[0028]
A transformed plant cell, callus, tissue or plant can be identified and isolated by selecting or screening for a trait encoded by a marker gene present in the gene construct used for transformation. For example, the selection can be made by growing a plant that has been manipulated in a medium containing a suppressive amount of antibiotic or herbicide that the transformed gene construct provides resistance. Further transformed plant cells and plants are screened for the activity of a visible marker gene (eg, β-glucuronidase gene, luciferase gene, B gene or C1 gene) that may be present in the recombinant nucleic acid construct of the present invention. Can be identified. Such selection methods and screening methods are known to those skilled in the art.
[0029]
In addition, physical and biochemical methods can be used to identify plants or plant cell transformants containing the gene construct of the present invention. As such,
1) Southern analysis or PCR amplification to detect and measure the structure of the recombinant DNA insert;
2) Northern blot, S1 RNase protection, primer extension PCR amplification or reverse transcriptase PCR (RT-PCR) amplification to detect and measure RNA transcripts of gene constructs;
3) When the gene construction product is a protein, protein gel electrophoresis, Western blot, immunoprecipitation, or enzyme immunoassay can be used, but not limited thereto. All of these assay methods are known to those skilled in the art.
[0030]
According to the present invention, transformed plants may be screened for desired physiological changes in order to obtain plants with improved component characteristics. For example, when manipulated for GDH enzyme overexpression, transformed plants will be tested for plants that express the GDH enzyme at the desired level and in the desired tissue and growth stages. The plants that exhibit the desired physiological changes, eg, GDH gene overexpression, can then be subsequently screened for the desired component changes.
According to the present invention, plants engineered with changes in nitrogen assimilation processes or utilization processes have improved component characteristics, i.e. high free amino acid content, in particular glutamic acid, asparagine, aspartic acid, serine, threonine, alanine, histidine, In particular, it can show a high content of glutamic acid, which is an umami component. Engineered plants and plant lines with such improved properties can be identified by measuring the free amino acid content of the plant. The procedures and methods for this analysis are known to those skilled in the art.
Plants obtained by the present invention are plants that have an increased free amino acid content relative to control plants (non-transformed plants). In a preferred embodiment, the desired plant has a free amino acid content in its edible parts such as fruits, roots, seeds, etc., in particular, an increase in the content of glutamic acid, which is an umami component, more than twice, and the total amino acid content is also 2-4. Doubled. As for amino acids other than glutamic acid, the increase in aspartic acid, asparagine, alanine, serine, threonine and histidine is particularly remarkable.
[0031]
【Example】
The present invention is illustrated in detail and in detail by the following examples relating to the production of plants engineered for overexpression of NADP-GDH gene or NAD-GDH gene.
Example 1: Isolation of Aspergillus nidulans and tomato GDH gene and construction of Ti plasmid
(1) Isolation of Aspergillus nidulans-derived NADP-dependent GDH gene (AN-gdh-17) and tomato-derived NAD-dependent GDH gene (T-gdh-4)
A. Nidulans was seeded on a potato dextrose agar medium and cultured overnight at 30 ° C., and the obtained colonies were further cultured in a dextrose liquid medium for 2 days. Total RNA was prepared from the grown bacteria.
Tomato seeds surface-sterilized with 70% ethanol (30 seconds), 2% sodium hypochlorite (15 minutes), MS agar medium without plant hormones (Murashige and Skoog, 1962, Physiol. Plant. 15: 473) -479) and cultivated for 16 hours at 25 ° C. for 1 week to obtain a sterile plant. Total RNA was prepared from the roots of the obtained seedlings.
[0032]
Total RNA was purified using Poly (A) Quick mRNA Isolation Kit (Stratagene), and then First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) was used to create FirstStrad using First-Strand cDNA. PCR reaction was performed using the prepared First-Strand cDNA as a template. The reaction conditions were 94 ° C-3 minutes; 94 ° C-45 seconds, 59 ° C-30 seconds, 72 ° C-90 seconds, 35 cycles; 72 ° C- 10 minutes, and performed using a Perkin Elmer PCR system 2400. The primers used are shown in Table 1. As a result, A. A band of about 1.4 kbp derived from nidulans and a band of about 1.2 kb derived from tomato were amplified, which were consistent with the predicted target gene size. The obtained PCR product was cloned using TA-Cloning Kit (Invitrogen).
[0033]
A. The sequences of plasmid 2 clones in which genes of the desired size derived from nidulans were cloned and plasmids 5 clones in which the genes of desired size derived from tomato roots were cloned were determined using a sequencer (ABI 377A) and known. A. Nidulans-derived NADP-dependent GDH gene (Alastair et al., Mol. Gen. Genet, 1989, 218; 105-111) and tomato-derived GDH gene (Purnell et al., 1997, Gene 186; 249-254) were investigated.
A. The base sequence of 1 clone (AN-gdh-17) out of 2 clones derived from nidulans matched the known NADP-GDH gene sequence (FIGS. 1 and 2). However, it was found that one splicing site of about 50 bp remained among the two splicing sites present in the gene. A. Since nidulans has a eukaryotic splice site recognition site, in this experiment, it was decided to proceed to the next operation while leaving the remaining splicing site. On the other hand, the base sequence of 2 clones (T-gdh-4, T-gdh-22) out of 5 clones derived from tomato roots was consistent with the known legdh1 sequence. The base sequence of AN-gdh-17 is shown in SEQ ID NO: 1 in the sequence listing, and the base sequence of T-gdh-4 is shown in SEQ ID NO: 2 in the sequence listing.
[0034]
[Table 1]

a. NADP-GDH gene specific primer (Alastair et al., Mol. Gene. Genet, 1989, 218; 105-111, PCR product, about 1.4 kbp)
b. legdh1 specific primer (Purnell et al., 1997, Gene 186; 249-254, PCR product, approximately 1.2 kbp)
<Sequence Listing Free Text>
SEQ ID NOs: 3 and 4: NADP-GDH specific PCR primers
SEQ ID NOs: 5 and 6: legdh1-specific PCR primers
[0035]
(2) Subcloning of AN-gdh-17 gene into Ti plasmid (pMAT037)
The AN-gdh-17 gene cloned into the PCR2.1 vector was subcloned into Ti plasmid (pMAT037) (Matsuoka and Nakamura, 1991, Proc Natl. Acad Sci USA 88: 834-838), a plant transformation vector. did. Since there is no restriction enzyme site suitable for direct insertion in pMAT037, it is once connected to pUC18 (XbaI, using EcoRI site). E. coli JM109 was transformed. Furthermore, the plasmid pAN-gdh-17 was obtained by ligation to Ti plasmid using the PstI site and EcoRI site in pUC18 (FIG. 3, Table 2). E. coli DH5α was transformed. Groplasma strain EHA101 was transformed with Ti plasmid pAN-gdh-17 (FIG. 3) into which AN-gdh-17 was introduced, and the resulting Agrobacterium was used to infect tomatoes.
[0036]
(3) Construction of pCt-AN-gdh, pCt-dAN-gdh, pMt-dAN-gdh
The Aspergillus nidurans-derived NADP-GDH gene originally has two splicing sites. Since the GDH gene was amplified by PCR using cDNA, these splicing sites should be removed and should not exist. However, the AN-gdh-17 gene obtained in this experiment has a splicing site of about 50 bp. Remained in place (FIG. 1, described in the base sequence). Therefore, the PCR method was used to remove the remaining base sequence of about 50 bp (Table 2, FIGS. 4 and 5).
[0037]
The outline of the construction procedure of these gene constructs is as shown in FIG. That is, first, the primer P1 including the 5 ′ end of the cloned gene sequence, the primer P2 including the 5 ′ side and 3 ′ side of the splicing region but not including the splicing region, and the 5 ′ side and 3 of the splicing region are included. A DNA fragment was amplified by PCR using primer P3 containing the 'side but not containing the splicing region and primer P4 containing the 3' end of the AN-gdh-17 gene, respectively, using the AN-gdh-17 gene as a template. Next, the size of the PCR product was confirmed by electrophoresis and re-extraction from the gel was performed. Each re-extracted PCR product was mixed, and PCR reaction was performed again using primers P1 and P4. The resulting PCR product was cloned and sequenced to confirm that the splicing region was correctly removed.
[0038]
The arrangement of the above P1 to P4 is shown below.

By these operations, the dAN-gdh-17 gene from which the splicing site was removed was obtained (Table 2, FIGS. 4, 5).
Furthermore, in order to make the transgene work more functionally in place, a transit peptide sequence to mitochondrion or chloroplast was linked upstream of the start codon of AN-gdh-17 gene or dAN-gdh-17 gene (Table 2). , FIG. 4). The used peptide sequence is a base sequence consisting of about 70 bp added to the tomato-derived GDH gene as a transit peptide sequence to mitochondria, and added to the small subunit gene of tomato-derived RuBisCO as a transit peptide sequence to chloroplast. A base sequence consisting of about 120 bp was used. Moreover, these genes and the AN-gdh-17 gene or the dAN-gdh-17 gene were linked using the PCR method. The transit peptide sequence to the mitochondria was obtained by PCR using two kinds of primers (5'-GGATCCCATGAATGCTTTAGCAGCAC-3 ': SEQ ID NO: 11, 5'-TCTAGATAAAACCAAGAAGCCTAGCTG-3': SEQ ID NO: 12). The transit peptide sequence to chloroplasts was obtained by PCR using two kinds of primers (5'-CTGCAGATGGCTTCCTCAATTGTTCCATCG-3 ': SEQ ID NO: 13, 5'-TCTAGAGCATCTACACCGTCCCACCCATTGCT-3': SEQ ID NO: 14).
[0039]
Binding of these transit peptide sequences to the AN-gdh-17 gene or dAN-gdh-17 gene was performed as shown in FIG. That is, the primer P5 corresponding to the 5 ′ side of the transit peptide, the primer P6 containing the sequence of the 3 ′ end of the transit peptide and the 5 ′ end of the AN-gdh-17 gene or the dAN-gdh-17 gene, and 3 of the transit peptide Primer P7 containing 'end and 5' end sequence of AN-gdh-17 gene or dAN-gdh-17 gene and primer P8 containing 3 'end sequence of AN-gdh-17 gene or dAN-gdh-17 gene After amplification of each DNA fragment using the PCR, the size of the PCR product was confirmed by electrophoresis and extracted from a gel. The extracted fragments were mixed and PCR was performed again using primers P5 and P8. The amplified fragment was cloned and sequenced to confirm that the nucleotide sequence of the transit peptide was correctly added to the AN-gdh-17 gene or dAN-gdh-17 gene (FIG. 6).
[0040]
Here, the arrangement from P5 to P8 corresponds to the following.
For primers used for ligation to transit peptide sequence to mitochondria

And
For primers used for ligation of transit peptide sequence to chloroplast

It is.
[0041]
Here, the AN-gdh-17 gene was introduced into the multi-cloning site of Ti plasmid pMAT037 in the sense direction (FIG. 2), but when pCt-AN-gdh, pCt-dAN-gdh, and pMt-dAN-gdh were constructed. Was cloned using Ti plasmid pIG121-Hm in order to compare the effect of the transgene in the case of using the CaMV35S promoter and in the case of using the fruit-specific promoter gene (2A11) described later, and the gene was introduced. (Table 2, FIG. 4).
<Sequence Listing Free Text>
SEQ ID NOs: 7 to 10: PCR primers for removing the splicing region
SEQ ID NOs: 11 and 12: PCR primers for sequence amplification encoding transit peptides into mitochondria
SEQ ID NOs: 13 and 14: PCR primers for sequence amplification encoding transit peptide to chloroplast
SEQ ID NOs: 15-18: PCR primers for the generation of sequences encoding mitochondrial transit peptide-GDH
SEQ ID NOs: 19-22: PCR primers for the generation of sequences encoding chloroplast transit peptide-GDH
[0042]
(4) Gene construction using a fruit-specific promoter (2A11 promoter)
A fruit-specific expression promoter (2A11) was obtained by PCR using Total DNA prepared from tomato seedlings as a template. For the primers (SEQ ID NO: 23 and SEQ ID NO: 24), the restriction enzyme sites used for introduction into Ti plasmid, HindIII and XbaI were designed for each primer.
Primer sequences are shown below:

The obtained PCR product was cloned using a TA cloning kit, and the nucleotide sequence was confirmed by sequence analysis. The obtained 2A11 promoter was replaced with the CaMV35S promoter in front of the GUS gene of Ti plasmid pIG121-Hm using the restriction enzymes HindIII and XbaI. Thereafter, replacement of the GUS portion with the Ct-dAN-gdh gene or the Mt-dAN-gdh gene was performed. The procedure for replacing the Ct-dAN-gdh gene or the Mt-dAN-gdh gene is the same as that using the CaMV 35S promoter. Thereby, plasmid p2ACt-dAN-gdh and P2AMt-dAN-gdh were obtained (FIG. 7).
<Sequence Listing Free Text>
SEQ ID NOS: 23 and 24: PCR primers for 2A11 promoter sequence amplification
The important structures of the plasmids produced by the procedure described above are summarized in Table 2 below.
[0043]
[Table 2]

[0044]
(5) Subcloning of T-gdh-4 gene into Ti plasmid (pIG121-Hm)
The cloned tomato-derived GDH gene (T-gdh-4) was introduced into Ti plasmid (pIG121-Hm) to obtain plasmid pT-gdh-4. For the introduction, the XbaI site and the SacI site previously provided for the primers used for the isolation were used. Ti plasmid introduced with the T-gdh-4 gene (FIG. 8) was transformed into Agrobacterium strain EHA101.
[0045]
(6) Subcloning of mutant tomato GDH gene into Ti plasmid (pIG121-Hm)
In tomato-derived NAD-GDH, the 90th amino acid lysine at the glutamate binding site was substituted with alanine, and the effect was investigated (FIG. 9). This is a modification aiming to prevent the GDH gene of higher plants from being able to bind because it may act in the direction of decreasing glutamic acid depending on the ammonia ion concentration and nutrient conditions. The substitution of one amino acid was performed by converting AAG encoding Lys into GCG encoding Ala by site-directed mutagenesis using PCR. The obtained modified gene was named Td-gdh. Next, the plasmid pTd-gdh was obtained by introducing the Td-gdh gene sequence into the Ti plasmid (FIG. 9). This procedure is the same as that for the T-dgh-4 gene.
[0046]
Example 2 Production of transformants by Agrobacterium infection of tomato cotyledons
Tomato (cultivar, cherry tomato) seeds were sterilized with 70% ethanol (30 seconds) and 2% sodium hypochlorite (15 minutes), then placed on MS agar medium without plant hormones, The cells were cultured for 16 hours at 25 ° C. for 1 week. The cotyledons were cut from the obtained sterile seedlings, placed on MS agar medium (regeneration medium, using 9 cm petri dish) added with 2 mg / l zeatin and 0.1 mg / l indoleacetic acid, and cultured under the same conditions for 2 days. Agrobacterium (EHA101) containing the constructed gene was cultured overnight in YEP medium (Table 3) and used for infection. The cotyledons cultured for 2 days were collected in a sterile petri dish and infected with Agrobacterium solution. Remove excess Agrobacterium solution from the cotyledons using sterilized filter paper.In addition, in order to prevent rapid growth of Agrobacterium, place sterile filter paper on the petri dish previously used, and infect the cotyledons on it. Placed and co-cultured for 24 hours.
[0047]
Thereafter, the cotyledons were transferred to an MS regeneration medium (selection medium) containing 50 mg / l kanamycin and 500 mg / l claforan, and transformants were selected. The redifferentiated shoots were transferred to a new selection medium and reselected. Shoots that were vigorously grown in green were cut off at the stem and transferred to MS medium (rooting medium, test tube) containing no plant hormones. The rooted redifferentiated plants were sequentially acclimated to the soil.
[Table 3]

[0048]
Example 3 Transformation of Arabidopsis by vacuum infiltration
For introduction of the gene into Arabidopsis, the method of Bechold N (CR Acad. Sci. Paris, Life Science 316; 1194-1199, 1993) was used. Seedlings of Arabidopsis seedlings were cultivated in culture soil for 10 days, 16 hours long, and grown at 22 ° C. One plant was transplanted to one rock wool (3 cm × 3 cm) and cultivated under the same conditions for 3 weeks. When the plant started drawing, it was pinched and cultivated for another week. Agrobacterium is cultured in YEP medium containing antibiotics for 24 hours at 28 ° C., collected by centrifugation (7000 rpm, 10 minutes), and suspended in suspension medium (1/2 MS salt, 1/2 Gamburg B5 vitamin) 5% sucrose, 0.5 g / l MES, 0.044 μM benzylaminopurine, pH 5.7). The already flowered and fruited flowers were removed, dipped in an Agrobacterium suspension, placed in a desiccator, and subjected to a reduced pressure (40 mmHg) treatment for 15 minutes. The treated plants were cultivated for one month and the seeds were harvested. The collected seeds were seeded in MS medium supplemented with 50 mg / l kanamycin and 200 mg / l claforan, and transformants were selected.
[0049]
Example 4 Confirmation of transgene
4 selected individuals obtained by infecting Agrobacterium containing AN-gdh-17 gene, 4 selected individuals obtained by infecting Agrobacterium containing T-gdh-4 gene, including target gene The method of Honda et al. Was obtained from total DNA obtained from 3 plants each obtained by infecting only Agrobacterium with only Ti plasmid and 2 plants directly redifferentiated from cotyledons without treatment with Agrobacterium. (Honda and Hirai, 1990, Jpn J Breed 40, 339-348). The extracted DNA was purified by RNAase treatment, phenol / chloroform treatment, and PEG precipitation. It diluted to 0.01 microgram / microliter, and it was set as the template for PCR. PCR was performed using primers P9 and P10 (PCR product, 1.0 kbp) that amplify the region of NPTII from Nos-Promoter. The reaction conditions were 94 ° C-1 min, 55 ° C-1 min, 72 ° C-2 min, 35 cycles. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide (FIGS. 10 and 11).
The used primers are as follows.

[0050]
As a result, a target size band (1.0 kb) was observed in 4 lines infected with AN-gdh-17 gene and 4 lines infected with T-gdh-4 gene, and 2 non-transformant lines were observed. Was not detected. From the above results, each gene was introduced into 4 strains infected with Ti plasmid containing AN-gdh-17 gene and 4 strains infected with Ti plasmid containing T-gdh-4 gene. It has been confirmed.
<Sequence Listing Free Text>
SEQ ID NOs: 25 and 26: PCR primers for amplification of Nos-Promoter-NPTII region
[0051]
Example 5 FIG. Confirmation of transgene expression
Next, it was confirmed by RT-PCR that the transgene was expressed in the transformed tomato that had been confirmed to have the target gene introduced. Infecting Agrobacterium containing AN-gdh-17 or T-gdh-4 gene, extracting total RNA from the line or the non-transformed tomato leaf or fruit in which the gene transfer was confirmed in Example 5, First -Strand cDNA was prepared. Next, PCR was performed using the primers (SEQ ID NOs: 3 and 4, and 5 and 6) used in isolating each gene using First-Strand cDNA as a template. The reaction conditions were 94 ° C-1 min, 55 ° C-1 min, 72 ° C-2 min, 30 cycles. As a result, it was confirmed that the introduced gene was expressed in both the leaves and the fruits (FIGS. 12 and 13).
[0052]
Example 6 Extraction and quantification of free amino acids
The fruit at the 6th week after flowering of the conditioned transformed tomato was harvested and stored at −80 ° C. The fruit was cut into about 1/6, weighed, placed in a mortar, frozen with liquid nitrogen and ground. Further, 3 ml of 80% ethanol was added and carefully ground, then transferred to a centrifuge tube and incubated at 80 ° C. for 20 minutes. The mixture was centrifuged at 10,000 rpm for 20 minutes, the supernatant was transferred to a new tube, 2 ml of 80% ethanol was added to the remaining pellet, ground again in a mortar, and incubated at 80 ° C. for 20 minutes. After centrifugation, the supernatant was transferred to a tube, combined with the previous supernatant, and adjusted to a total volume of 5 ml using 80% ethanol. After mixing well, 20 μl was taken and dried, and then dissolved in 0.02N hydrochloric acid. It filtered with the 0.45 micrometer filter and was set as the sample for analysis. Amino acid analysis was performed using a Hitachi high-speed amino acid analyzer (L-8800).
The results of amino acid analysis using the fruit (red) 6 weeks after flowering of the strain into which the AN-gdh-17 gene was introduced are shown in Table 4 together with the analysis results of the non-transformant plant (control plant). In lines where a marked increase in glutamic acid content was observed, no. 6 1.75 times, No. 15, 2.54 times, No. 15 17, the glutamic acid content increased 2.48 times (FIG. 14). An increase in amino acids other than glutamic acid, such as asparagine, aspartic acid, alanine, serine, threonine, histidine, etc. was also observed.
[0053]
[Table 4]

[0054]
[Table 5]

[0055]
Amino acid analysis was also performed on the 4 strains into which the tomato-derived T-gdh-4 gene was introduced, using the fruits at 6 weeks after flowering (Table 5). In lines where a marked increase in glutamic acid content was observed, no. 2 is 2.28 times. 7-2, 3.52 times. 9-2, 2.74 times, No. 10 increased the glutamic acid content 2.53 times (FIG. 15). In the line having a high glutamic acid content, the content of other amino acids such as aspartic acid, asparagine, threonine, serine, alanine, and histidine is also high. 7-2 was found to increase 4 times. These results are summarized in Table 5 below.
[0056]
[Table 6]

[0057]
[Table 7]

[0058]
Example 7 Tomato transformant progeny (T ₁ Generation analysis
1) T₁Generation selection
Tomato transgenic plant (T₀The seeds obtained from the generation) were surface sterilized with 80% ethanol for 30 seconds and 2% sodium hypochlorite for 15 minutes and then aseptically sown on an MS agar medium containing 350 mg / l kanamycin. One month later, as a result of selecting plants having good growth, the AN-gdh-17 gene-introduced line was No. 1, no. 3, no. 15, no. Individuals selected from 2.1 were obtained. In the T-gdh-4 transgenic line, No. 1, no. 3, no. Individuals selected from 8 were obtained. In order to increase the number of fruits per strain, cultivation was conducted in an outdoor closed greenhouse. In order to make the nutrition conditions the same, no additional fertilization was performed after transplanting to 1 kg of culture soil (power soil, Sakata seed) at the time of acclimatization. In addition, in order to make the assimilation ability the same, in the following analysis, the leaves were not picked, but the buds were cultivated under the same conditions and the leaf tissue was used.
[0059]
2) Confirmation of transgene by Southern analysis
Total DNA was extracted from the leaf tissue of acclimatized buds (Honda and Hirai 1990, Jpn J Breed 40, 339-348). 15 μg of DNA was treated with a combination of restriction enzymes BamHI and EcoRI and XbaI, electrophoresed, and transferred to a nylon membrane. Southern hybridization was performed using the DIG-Labeling and Detection Kit (Roche Molecular Biochemicals) with the AN-gdh-17 gene or the T-gdh-4 gene as a probe.
As a result of Southern hybridization using the AN-gdh-17 gene as a probe, AN-gdh-17 gene introduction strain No. Bands of the desired size (1.8 kb, 0.8 kb) were detected in lines 1, 3, 15, and 2.1, and the transgene was confirmed (FIG. 16). Similarly, as a result of Southern hybridization using the T-gdh-4 gene as a probe, a band (1.2 kbp) having the same size as the T-gdh-4 gene was confirmed. There was a band around 20 kbp, which was considered to be an endogenous gdh gene (FIG. 17).
[0060]
3) Measurement of NADP-GDH and NAD-GDH activity
Transformed tomato (T₁), And the leaf tissue (0.2 g) of buds was frozen in liquid nitrogen, crushed in a mortar, and then extracted buffer 5 times the weight {200 mM Tris (pH 8.0), 14 mM β-mercaptoethanol, 10 mM L -Cysteine-HCl, 0.5 mM PMSF, 0.5% Triton X-100} was added. After transferring to a centrifuge tube and centrifuging at 12,000 rpm for 10 minutes at 4 ° C., the supernatant was ultrafiltered (Millipore, Ultrafree 0.5 filter unit, Baoimax-10) and washed 3 times with extraction buffer.
The extracted enzyme was a reaction solution {100 mM Tris (pH 8.0), 20 mM 2-α-ketoglutarate, 1.0 mM CaCl.₂, 0.2 mM NADPH (NADP-GDH activity measurement) or 0.2 mM NADH (NAD-GDH activity measurement), 200 mM ammonium chloride} and reacted at room temperature, and the decrease in absorbance at 340 nm was measured.
[0061]
Transformed tomato introduced with AN-gdh-17 gene (T₁) As a result of measuring NADP-GDH activity using leaf tissue, the non-transformant showed no activity, whereas the transformant was able to measure 230-400 nmol / (min / mg protein) activity. (Table 6). In the line into which the T-gdh-4 gene was introduced, the NAD-GDH activity was increased by about 2 times or more compared to the non-transformant (Table 7).
[0062]
[Table 8]

[0063]
[Table 9]

[0064]
4) Measurement of amino acid content in fruits
Three fruits 6 weeks after flowering of the first fruit bunches were used for analysis. 80% ethanol heated to 80 ° C. was added to the fruit three times the weight, ground in a mortar, and then heated again at 80 ° C. for 20 minutes. After centrifugation at 7,000 rpm and collecting the supernatant, 80% ethanol was added again and the mixture was heated to 80 ° C. Extraction with ethanol was performed three times, and the total amount was adjusted to 100 ml with 80% ethanol. After mixing well, 200 μl of the extract was placed in an Eppendorf tube, dried and dissolved in 200 μl of sterile water. After adding 200 μl of ethyl ether and mixing, the mixture was centrifuged at 12,000 rpm to remove the ether layer. After drying the aqueous layer again, the filtrate was dissolved in 200 μl of 0.02N HCl and filtered using a 0.45 μm filter, and analyzed using a Hitachi high-speed amino acid measuring instrument (L-8800). Went.
[0065]
The result showed the average value of 3 fruits. In the AN-gdh-17 transgenic line, AN-gdh-17 No. was compared to the non-transformant. The glutamic acid contents of AN1-1-2 and AN1-1-3 derived from one strain increased 2.1-fold and 2.8-fold, respectively, and AN-gdh-17 No. 1 increased. AN3-1-2 and AN3-1-3 derived from the three lines increased 2.8 times and 2.5 times, respectively. AN-gdh-17 No. AN-gdh-17 No. 15 derived from 15 lines. 2.1-derived AN2.1-1-1 also increased 2.1-fold and 1.9-fold (Table 8, FIG. 18). Transformation generation (T₀No.) which showed a high glutamic acid content in No. A similar trend was shown in the 15 progenies. In the Mt-dAN-gdh gene-introduced line, MtdAN-24-4, there was no significant difference from the glutamic acid content of the non-transformants.
[0066]
[Table 10]

[0067]
[Table 11]

[0068]
[Table 12]

[0069]
In the T-gdh-4 gene-introduced line, the investigated T-gdh-4 No. The progeny TGDH1-2, 3-1, and 8-1 of lines 1, 3, and 8 increased 2.3 times, 2.1 times, and 2.4 times the glutamic acid content of the non-transformants. . (Table 9, FIG. 19). In addition, amino acids other than glutamic acid showed a marked increase in aspartic acid, glutamine, and γ-aminobutyric acid content. As a result, the total free amino acid content also increased 2-3 times compared to the non-transformant.
[0070]
[Table 13]

[0071]
[Table 14]

[0072]
[Table 15]

[0073]
Example 8 FIG. Production and analysis of potato transformants
1) Production of transformant (see Example 4)
Potato (potato) aseptic plants were obtained by shoot tip culture, and the material was increased by passage of the shoot tips. The shoot apex was introduced into a liquid medium (10 ml) obtained by adding 2% sucrose to MS medium to induce rooting. After rooting, 10 ml of MS liquid medium containing 16% sucrose was added and cultured in the dark to induce a microtuber. After 6-8 weeks, the microtuber was cut into a disk, peeled, and then cultured at 28 ° C. overnight. Agrobacterium solution (containing Ti-plasmid, pMt-dAN-gdh or pCt-AN-gdh) ). Placed on MS agar medium (MS medium, 2.0 mg / l zeatin, 0.1 mg / l indoleacetic acid, 0.3% gellite) with sterilized filter paper and co-cultured for 2 days at 25 ° C. for 16 hours. . Thereafter, the cells were transferred to a selective medium {MS medium, 2.0 mg / l zeatin, 0.1 mg / l indoleacetic acid, 0.3% gellite, 50 mg / l kanamycin, 500 mg / l claforan}, and cultured under the same conditions. Every week, the cells were transferred to a new selection medium, and the redifferentiated shoots were transferred to a selection medium not containing plant hormones to induce rooting. As a result of infection with Agrobacterium having Ti-plasmid pMt-dAN-gdh and pCt-AN-gdh and selection in a medium containing 50 mg / l of kanamycin, Mt-dAN-gdh No. 2, 5, 8, Ct-AN-gdh No. 2 Four lines of 1 were selected.
[0074]
2) Confirmation of transgene by Southern analysis
Tolal DNA was extracted from the leaf tissue of the acclimated plant (Honda and Hirai 1990, Jpn J Breed 40, 339-348). 15 μg of DNA was treated with the restriction enzyme EcoRI, electrophoresed, and transferred to a nylon membrane. Southern hybridization was performed using DIG-Labeling and Detection Kit (Roche Molecular Biochemicals). AN-gdh-17 gene was used as a probe.
As a result, a band of a transgene size (about 1.5 kb) was confirmed in all four strains (FIG. 20), suggesting that the gdh gene added with a transit peptide was introduced.
[0075]
3) Measurement of NADP-GDH activity
The leaf tissue (about 0.1 g) of the transformed potato was frozen with liquid nitrogen, crushed in a mortar, and then extracted buffer 5 times the weight {200 mM Tris (pH 8.0), 14 mM β-mercaptoethanol, 10 mM L-cysteine-HCl, 0.5 mM PMSF, 0.5% Triton X-100} was added. After transferring to a centrifuge tube and centrifuging at 12,000 rpm for 10 minutes, the supernatant was subjected to ultrafiltration (Millipore, Ultrafree 0.5 filter unit, Baoimax-10), and washed 3 times with extraction buffer. The extracted enzyme was a reaction solution {100 mM Tris (pH 8.0), 20 mM 2-α-ketoglutarate, 1.0 mM CaCl.₂, 0.2 mM NADPH, 200 mM ammonium chloride} and reacted at room temperature, and the decrease in absorbance at 340 nm was measured.
As a result of measuring NADP-GDH activity using the transformed potato and non-transformed potato leaf tissues in which the transgene was confirmed by Southern analysis, the non-transformant showed no activity, whereas the transformant Then, the activity of 150-300 nmol / (min mg protein) could be measured (Table 10). The Mt-dAN-gdh line showed higher activity than the Ct-AN-gdh line.
[0076]
[Table 16]

[0077]
4) Measurement of amino acid content of microtuber
The shoot apex of the non-transformant and 4 transformant lines was cultured in liquid, and after rooting was induced, 16% sucrose was added, and the amino acid content was measured using a microtuber 6 weeks after dark treatment. .
80% ethanol heated to 80 ° C. was added 3 times the weight, ground in a mortar, and then heated again at 80 ° C. for 20 minutes. After centrifuging at 7,000 rpm and collecting the supernatant, 80% ethanol was added again and the mixture was heated to 80 ° C. Extraction with ethanol was performed three times, and the total amount was adjusted to 5 ml with 80% ethanol. After mixing well, 200 μl of the extract was placed in an Eppendorf tube, dried and dissolved in 200 μl of sterile water. After adding 200 μl of ethyl ether and mixing, the mixture was centrifuged at 12,000 rpm to remove the ether layer. The water layer was dried again, dissolved in 400 μl of 0.02N HCl, and filtered using a 0.45 μm filtration filter as a sample and analyzed using a Hitachi high-speed amino acid meter (L-8800). Went.
[0078]
Amino acid analysis was performed using a microtuber derived from the transgenic line. Each strain analyzed two or more microtubers and performed statistical processing. No. in which the Mt-dAN-gdh gene was introduced. The Mt2-2, Mt5-1, Mt5-2, Mt8-1, and Mt8-2 strains derived from lines 2, 5, and 8 have a glutamic acid content of 1.7 times, 2.2 times that of the non-transformant, respectively. It was found that the increase was 2.5 times, 3.0 times, and 2.2 times (Table 11, FIG. 21). In the Ct-AN-gdh gene-introduced line, no significant difference was found in the glutamic acid content compared to the non-transformant. Amino acids other than glutamic acid showed a significant increase in glutamine and proline, and as a result, the total free amino acid content was also increased 2-3 times compared to the non-transformant.
[0079]
[Table 17]

[0080]
[Table 18]

[0081]
[Table 19]

[0082]
【The invention's effect】
According to the present invention, a plant containing a high concentration of free amino acids is obtained, and a raw material crop and a food material with high added value are provided. According to the present invention, a total amino acid amount is increased 2 to 4 times, and a crop having a high amino acid content is provided for at least one of glutamic acid, asparagine, aspartic acid, serine, threonine, alanine and histidine. A high value-added raw material crop that does not need to be provided. In addition, the present invention provides a vegetable material that is directly cooked and has accumulated glutamic acid at a high concentration, that is, a food material with excellent umami taste.
Furthermore, the present invention significantly reduces the period for breeding plants that accumulate such free amino acids to a high degree.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a comparison of Aspergillus nidulans-derived glutamate dehydrogenase (GDH) gene AN-gdh-17 and a known NADP-GDH gene sequence. The upper row is the base sequence of the NADP-GDH gene, and the lower row is the base sequence of the AN-gdh-17 gene.
FIG. 2: Comparison of Aspergillus nidulans-derived glutamate dehydrogenase (GDH) gene AN-gdh-17 and known NADP-GDH gene sequences (continuation of FIG. 1). The upper row shows the nucleotide sequence of NADP-GDH gene, and the lower row shows the nucleotide sequence of AN-gdh-17 gene.
FIG. 3 shows the cloning procedure of AN-gdh-17 gene into Ti plasmid (pMAT037). In the figure, 35S Pro represents a CaMV 35S promoter, and Term represents a terminator.
FIG. 4 is a schematic diagram showing the construction procedures and structures of gene constructs, pCt-AN-gdh, pCt-dAN-gdh, and pMt-dAN-gdh containing sequences encoding the Aspergillus-derived NADP-GDHA gene linked with transit peptides. It is shown as an example.
FIG. 5 shows an outline of a procedure for removing a splicing region in a NADP-GDH gene. In the figure, thick lines represent splicing regions, and P1 to P4 represent PCR primers.
FIG. 6 shows an outline of a procedure for binding a transit peptide sequence to an Aspergillus-derived NADP-GDH A gene sequence. In the figure, A represents the base sequence of the transit peptide, and B represents the base sequence of the AN-gdh-17 gene. P5 to P8 each represent a PCR primer.
FIG. 7 schematically shows the structures of gene constructs p2ACt-dAN-gdh and p2AMt-dAN-gdh having an Aspergillus nidulans-derived NADP-GDH gene connected to the 2A11 promoter.
FIG. 8 shows the cloning procedure of tomato-derived NAD-GDH gene (T-gdh-4) into Ti plasmid (pIG121-Hm). In the figure, 35S represents a CaMV 35S promoter, and Nos represents a Nos-terminator.
FIG. 9 shows an outline of a procedure for modifying a tomato-derived NAD-GDH gene, T-gdh-4.
FIG. 10 shows a PCR analysis of a transformant into which an Aspergillus nidulans-derived AN-gdh-17 gene was introduced. Lanes 2 and 3: untransformed tomatoes, lanes 4 to 6: transformed tomatoes into which the plasmid (pMAT037) gene was introduced; lanes 7 to 10: transformed tomatoes into which the AN-gdh-17 gene was introduced (No-6, No-8-2, No-15, No-17).
FIG. 11 shows a PCR analysis of a transformant introduced with a tomato-derived NAD-GDH gene GDH (T-gdh-4) gene. Lanes 2 and 3: Non-transformed tomatoes; Lanes 4 to 6: Transformed tomatoes into which the plasmid (pIG121-Hm) gene was introduced; Lanes 7 to 10: Transformed tomatoes into which the T-gdh-4 gene was introduced (No. 2, No. 7-2, No. 9-2, No. 10).
FIG. 12 shows RT-PCR analysis of a transformant introduced with an Aspergillus nidulans-derived AN-ghd-17 gene. No. 6, no. 15 represents a transformed tomato, and the parentheses represent the tissue from which total RNA was extracted.
FIG. 13 shows RT-PCR analysis of a transformant introduced with a tomato-derived T-gdh-4 gene. No. 2, no. 7-2, no. 9-2, no. 10 represents a transformed tomato, and () represents the tissue from which total RNA was extracted.
14 shows amino acids (glutamic acid-Glu, glutamine-Gln, γ-aminobutyric acid-GABA, lysine-Lys) of transformants (No. 6, No. 15, No. 17) into which the AN-gdh-17 gene has been introduced. ) Is a graph comparing the contents.
15 shows amino acids (glutamate-Glu, glutamine-Gln, γ-) of transformants (No. 2, No. 7-2, No. 9-2, No. 10) into which T-gdh-4 gene has been introduced. It is the result of comparing aminobutyric acid (GABA) and lysine-Lys content.
FIG. 16 shows the results of Southern analysis of transformed tomatoes introduced with the AN-gdh-17 gene. Samples were used where total DNA (15 μg) was digested with BamHI and EcoRI (A) or total DNA (15 μg) was digested with XbaI (B). Lane 1: Non-transformed tomato, Lane 2: AN-gdh-17 No. 1, Lane 3: AN-gdh-17 No. 1 3, Lane 4: AN-gdh-17 No. 3 15, Lane 5: AN-gdh-17 No. 2.1.
FIG. 17 shows the result of Southern analysis of transformed tomato (T1) introduced with T-gdh-4 gene. A sample obtained by digesting 15 μg of total DNA with XbaI and SacI was used. Lane 1: Non-transformed tomato, Lane 2: T-gdh No. 1-2, Lane 3: T-gdh No. 3-1, Lane 4: T-gdh No. 3 8-1. The arrow indicates the position of the band corresponding to the transgene (1.2 kb).
FIG. 18: Progeny of transformed tomato introduced with AN-gdh-17 gene (T₁It is the graph which compared the amino acid content in the fruit in). The control used non-transformed tomato. Measurements were made in triplicate.
FIG. 19: Progeny of transformed tomato introduced with T-gdh-4 gene (T₁It is the graph which compared the amino acid content in the fruit in). Measurements were made in triplicate.
FIG. 20 shows the results of Southern analysis of potatoes into which Ct-AN-gdh and Mt-dAN-gdh genes were introduced. An EcoRI digest of total DNA (15 μg) was used. Lane 1: non-transformed potato-1, lane 2: non-transformed potato-2, lanes 3-6 correspond to transformed potatoes into which the respective gene constructs were introduced. Lane 3: Ct-AN-gdh No. 1, Lane 4: Mt-dAN-gdh No. 1 2, Lane 5: Mt-dAN-gdh No. 2 5, Lane 6: Mt-dAN-gdh No. 5 8. The arrow indicates the position of the band corresponding to the transgene fragment.
FIG. 21 is a graph showing glutamic acid (Glu) content in microtubers of potatoes introduced with Ct-AN-gdh and Mt-dAN-gdh genes. Non-transformed potato was used as a control.

Claims (20)

A method for producing transgenic tomatoes that accumulate more of one or more free amino acids in fruits than natural tomatoes cultivated under the same conditions, comprising NAD- dependent glutamate dehydrogenase in tomatoes containing transit peptides to mitochondria A tomato is transformed with a gene construct to be overexpressed, the transformed tomato is selected or identified based on a trait conferred by a marker gene linked to the gene construct, and one or more amino acids are accumulated in the fruit. for tomato for screening the transformed tomato, comprising selecting the transformed tomato, said method. 2. The method of claim 1, wherein the genetic construct comprises a gene encoding a tomato NAD- dependent glutamate dehydrogenase comprising a mitochondrial transit peptide operably linked to a strong constitutive or fruit-specific promoter.   The method according to claim 2, wherein the constitutive promoter is the CaMV35S promoter and the fruit-specific promoter is the 2A11 promoter. The method of claim 1, wherein the gene construct is any of the following:
i) a gene construct comprising a nucleic acid having the sequence of SEQ ID NO: 2 connected downstream of the CaMV 35S promoter;
ii ) a gene construct comprising a nucleic acid having the sequence described in SEQ ID NO: 2 connected downstream of the CaMV 35S promoter, wherein the sequence aag located at positions 267-269 in SEQ ID NO: 2 is replaced with the sequence gcg The gene construct.   The method according to any one of claims 1 to 4, wherein the free amino acid is selected from the group consisting of asparagine, aspartic acid, serine, threonine, alanine, histidine and glutamic acid.   6. The method of claim 5, wherein one of the free amino acids is free glutamic acid. A transgenic tomato produced by the method according to any one of claims 1 to 6, and a progeny of the tomato containing a gene construct that overexpresses a NAD- dependent glutamate dehydrogenase of the tomato containing a transit peptide to the mitochondria . Claim is produced by the method according to any one of 1-6, transgenic tomato glutamic acid content of the fruit is more than doubled and glutamic acid content of the fruit is increased to more than twice its the tomato progeny. A transgenic tomato produced by the method according to any one of claims 1 to 6, wherein the total free amino acid content of the fruit is increased by 2 times or more, and the total free amino acid content of the fruit is increased by 2 times or more. Offspring of the tomato . A transgenic tomato or seed progeny thereof tomato according to any one of claims 7-9, wherein containing the gene construct overexpressing NAD-dependent glutamate dehydrogenase tomato including transit peptide for mitochondria seed. Relative to the native potato cultivated under the same conditions, a method of producing a transgenic potatoes for more store one or more free amino acids in the tubers, Aspergillus nidulans, including transit peptide to mitochondria (Aspergillus nidulans) A potato is transformed with a gene construct that overexpresses NADP- dependent glutamate dehydrogenase , and the transformed potato is selected or identified based on a trait conferred by a marker gene linked to the gene construct. more about potatoes to accumulate in tubers screening the transformed potatoes, comprising selecting the transformed potatoes, the method. 12. The method of claim 11, wherein the genetic construct comprises a gene encoding an Aspergillus nidulans NADP- dependent glutamate dehydrogenase comprising a transit peptide to the mitochondria operably linked to a strong constitutive promoter.   The method according to claim 12, wherein the constitutive promoter is the CaMV35S promoter. 12. The method of claim 11, wherein the gene construct is any of the following:
i ) CaMV 35S Downstream of the promoter, Five From Three A gene construct comprising a nucleic acid having a sequence encoding a transit peptide to mitochondria derived from tomato glutamate dehydrogenase on the 'side and a sequence of SEQ ID NO: 1, 323-375 The gene construct in which the base is deleted,
ii )Tomato 2A11 Downstream of the promoter, Five From Three A gene construct comprising a nucleic acid having a transit peptide sequence to mitochondria derived from tomato glutamate dehydrogenase and a sequence to which the sequence described in SEQ ID NO: 1 is connected to 323-375 The gene construct having a deleted base number.   The method according to any one of claims 11 to 14, wherein the free amino acid is selected from the group consisting of asparagine, aspartic acid, serine, threonine, alanine, histidine and glutamic acid.   16. A method according to claim 15, wherein one of the free amino acids is free glutamic acid. Transgenic potatoes and were generated by the method according to any one of claims 11 to 16, wherein the potato containing a gene construct overexpressing NADP-dependent glutamate dehydrogenase of Aspergillus nidulans including transit peptide for mitochondria Descendants of. Was produced by the method according to any one of claims 11 to 16, tubers transgenic potato and glutamic acid content is increased to more than 2 times, the potato offspring glutamic acid content of tubers was more than doubled . A transgenic potato produced by the method according to any one of claims 11 to 16, wherein the total free amino acid content of the tuber is increased by 2 times or more, and the total free amino acid content of the tuber is increased by 2 times or more. A descendant of the potato . 20. A seed of the transgenic potato according to any one of claims 17 to 19 or a progeny potato thereof , comprising a gene construct that overexpresses an NADP- dependent glutamate dehydrogenase of Aspergillus nidulans comprising a transit peptide to the mitochondria Said seed.

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